WO1994025572A1 - Mycobacterial species-specific reporter mycobacteriophages - Google Patents

Mycobacterial species-specific reporter mycobacteriophages Download PDF

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Publication number
WO1994025572A1
WO1994025572A1 PCT/US1994/004788 US9404788W WO9425572A1 WO 1994025572 A1 WO1994025572 A1 WO 1994025572A1 US 9404788 W US9404788 W US 9404788W WO 9425572 A1 WO9425572 A1 WO 9425572A1
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Prior art keywords
reporter
mycobacterial
mycobacteriophages
mycobacteriophage
genes
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PCT/US1994/004788
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French (fr)
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William R. Jacobs, Jr.
Barry R. Bloom
Graham F. Hatfull
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Albert Einstein College Of Medicine Of Yeshiva University, A Division Of Yeshiva University
University Of Pittsburgh
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Priority to AU69052/94A priority Critical patent/AU6905294A/en
Publication of WO1994025572A1 publication Critical patent/WO1994025572A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/5695—Mycobacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of making such reporter mycobacteriophages, and the use of such reporter mycobacteriophages, for example, to rapidly diagnose mycobacterial infection and to assess drug susceptibilities of mycobacterial strains in clinical samples.
  • this invention relates to the use of mycobacterial species-specific luciferase reporter mycobacteriophages to diagnose tuberculosis and to assess the drug susceptibilities of the various strains of Mycobacterium tuberculosis (M. tuberculosis).
  • transcriptional promoters and reporter genes are introduced into the genomes of mycobacterial species-specific mycobacteriophages.
  • reporter genes may be the genes for luciferase or the ⁇ -galactosidase gene, and provide the DNA which encodes the production of a gene product.
  • the reporter mycobacteriophages may be used for diagnosing mycobacterial infections by incubating same with samples which may contain the specific mycobacteria of interest. If the mycobacteria of interest is present, then the reporter mycobacteriophages introduce the recombinant nucleic acids which encode expression of the gene product into the mycobacteria of interest, and the mycobacteria then express the gene product.
  • the expressed reporter gene product may be detected by a suitable assay, for example, through the detection of photons or the conversion of an easily assayable chemical reaction. The presence of such gene product indicates that the sample contains the mycobacteria of interest, and hence the mycobacterial species-specific reporter mycobacteriophages may be used to detect and thereby diagnose the specific mycobacterial infection.
  • the mycobacteria species-specific reporter mycobacteriophages of this invention may be used to assess the drug susceptibilities of various strains of mycobacteria. If antibiotic drugs are added to the sample containing the reporter mycobacteriophages and the gene product is detected, the mycobacteria is metabolically active and hence resistant to the antibiotic drug.
  • tuberculosis (and other mycobacterial infections) which is rapid, sensitive and specific, which method is also capable of assessing the drug susceptibilities of the various strains of M. tuberculosis and other mycobacterial strains. It is critical that a mycobacterial strain be assessed for drug resistance rapidly because a patient infected with a strain of M. tuberculosis or another mycobacteria must be treated immediately with the particular antibiotic drug(s) to which the strain is not resistant, and not with antibiotic drug(s) to which the strain is resistant, or the patient may die. Currently, the most rapid test available for the diagnosis of M.
  • tuberculosis is the staining of sputum samples for acid-fast bacilli, which is a tedious procedure, and which procedure has low sensitivity.
  • Alternative methods for diagnosis require cultivation of the bacilli for approximately two to six weeks followed by classification of the cultured organism.
  • Typical diagnostic tools include biochemical tests, analysis of mycolic acids and serotyping. All of these tests are time-consuming.
  • More recently, the use of oligonucleotide probes and Polymerase Chain Reaction have been suggested for the identification of M. tuberculosis species. Although these methods may be useful approaches, their uses in a clinical setting have not yet been determined. Further, these methods do not distinguish between live and dead organisms, and are therefore of limited use in the determination of drug sensitivities of clinical isolates.
  • Mycobacterium avium (M. avium) is a mycobacteria which is often found in immunosuppressed patients. This mycobacteria is typically disseminated throughout the bodies of immunosuppressed patients, such as
  • This invention relates to broad host range and mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages, and the use of such reporter mycobacteriophages to rapidly diagnose mycobacterial infection, such as M. tuberculosis, and to distinguish which strains of the mycobacteria are drug-resistant.
  • reporter mycobacteriophages To produce these reporter mycobacteriophages, reporter genes and transcriptional promoters are introduced into the genomes of mycobacterial species-specific mycobacteriophages.
  • the promoter and reporter gene-containing mycobacteriophages are then incubated with a clinical sample which may contain the mycobacteria of interest, such as M. tuberculosis.
  • the reporter mycobacteriophages are specific for the mycobacteria which is sought to be detected.
  • the reporter mycobacteriophages efficiently introduce the recombinant nucleic acids which encode the expression of the reporter gene's gene product into the mycobacteria of interest, and the mycobacteria then express the gene product.
  • a substrate or other means capable of allowing for the detection of the gene product is then added to the sample. If the gene product or the signal generated by the gene product is detected, the presence of the infectious mycobacteria is known, thereby diagnosing the
  • drugs such as antibiotics may be added to a sample containing the reporter mycobacteriophages of this invention. If the mycobacteria are susceptible to a drug after exposure to the drug, the mycobacteria will be killed. However, drug-resistant mycobacteria will continue to be metabolically active in the presence of the drug, and will continue to express the detectable gene product of the reporter genes.
  • the reporter mycobacteriophages of the invention are temperate, and have increased sensitivity for use in drug screening.
  • the preferred reporter genes of the present invention are the Firefly luciferase lux gene (FFlux), the luciferase lux genes of Vibrio fischeri, the luciferase lux genes of Xenorhabdus luminescens and the E. coli ⁇ -galactosidase gene (lacZ).
  • Some preferred promoters of the present invention are hsp60 and gene 71-70-69 promoters, and the preferred mycobacteriophages are L5, TM4 and DS6A. These reporter mycobacteriophages are preferably used for the rapid diagnosis of tuberculosis and M. avium infection, and the accurate assessment of drug susceptibilities of the various strains of M. tuberculosis and M. avium.
  • FIGURE 1 (which is comprised of Figure 1A and Figure 1B) represents the genome organization of mycobacteriophage L5;
  • FIGURE 2 represents a luciferase shuttle plasmid pYUB180 wherein reporter gene FFlux is fused to the BCG hsp60 promoter;
  • FIGURE 3 represents the amount of luciferase activity of M. smegmatis which contains the pYUB180 shuttle plasmid and the FFlux gene;
  • FIGURE 4 (which is comprised of Figure 4A, Figure 4B and Figure 4C) represents the effect of various antibiotic drugs on the metabolic activity of control mycobacteria and drug resistant mycobacteria in the presence of reporter mycobacteriophages which contain luciferase reporter genes;
  • FIGURE 5 represents shuttle plasmid phAE39 wherein the reported gene is FFlux, the promoter is hsp ⁇ O, the phage is TM4 and the cosmid is pYUB216;
  • FIGURE 6 represents luciferase activity
  • FIGURE 7 represents a flow chart for cloning different promoters into TM4 : : lux shuttle phasmid phAE39;
  • FIGURE 8 represents a schematic diagram of the luciferase reporter mycobacteriophages phAE39 and phAE40;
  • FIGURE 9 represents the production of light (photons) by mycobacteria following infection with the luciferase reporter phage phAE40;
  • FIGURE 10 (which is comprised of Figure 10A, Figure 10B and Figure 10C) represents a comparison of the kinetics of light production following phage infection of drug-sensitive BCG cells to drug-resistant BCG mutant cells;
  • FIGURE 11 (which is comprised of Figure 11A, Figure 11B and Figure 11C) represents a comparison of drug-sensitive M. tuberculosis and drug-resistant M. tuberculosis using the luciferase reporter phage assay;
  • FIGURE 12 represents a schematic diagram of the extrachromosomal plasmid pYUB180 and the integration plasmid pGS16;
  • FIGURE 13 represents the expression of luciferase by
  • FIGURE 14 represents a DNA fragment of the L5 segment defined by the coordinates 3,150-7,143 after cleaving with Xba I and Bcl I;
  • FIGURE 15 represents the DNA fragment of L5 after insertion into plasmid pMV2611acZ and cleaving with Xba I and BAMHI to produce plasmid pGS11;
  • FIGURE 16 represents plasmid pGS12 which was produced by cleaving pGS11 DNA with Xba I and Hind III, and inserting fragment 4,013bp into plasmid pMD31;
  • FIGURE 17 represents plasmid pGS22, which was produced by cutting plasmid pYUB216 with Hind III and converting the sticky ends to blunt ends by Klenow enzyme and dNTP's;
  • FIGURE 18 represents plasmid pGS24, which was produced by inserting plasmid pGS22 into the NHE I site of pGS12 plasmid;
  • FIGURE 19 (which is comprised of Figure 19A and Figure 19B) represents a double crossover event between plasmid pGS24 and L5;
  • FIGURE 20 (which is comprised of Figure 20A and Figure 20B) represents hybridized bands detected by autoradiography
  • FIGURE 21 (which is comprised of Figure 21A-1, Figure 21A-2, Figure 21B-1 and Figure 21B-2) represents a map of expected DNA fragments resulting from a pair of homologous recombination events in common flanking sequences when FFlux is inserted into the L5 genome in a corresponding location to that in pGS24;
  • FIGURE 22 represents restriction enzyme mapping and Southern blot hybridization for phGS1 and phGS5;
  • FIGURE 23 represents determination of the luciferase activity of pGS24, phGS1 and phGS5;
  • FIGURE 24 represents luciferase activity as determined after liquid infection of M. smegmatis mc 2 155 with phGS1 and phGS5;
  • FIGURE 25 represents a comparison of luciferase activity of phGS5 with clear plaque mutant derivatives that are not competent to form lysogens;
  • FIGURE 26 represents the activity of phAE40 and the L5 : :FFlux phages following infection with M. smegmatis mc 2 155;
  • FIGURE 27 represents the sensitivity of phage phGS5 after infecting M. smegmatis mc 2 155;
  • FIGURE 28A represents the detection of luciferase activity after liquid infection of serial dilutions of M. smegmatis with phGS18;
  • FIGURE 28B represents the light produced (RLU);
  • FIGURE 29 represents the result of liquid infection of nonlysogen and lysogen strains of M. smegmatis with phAE40, phGS18 and phGS26;
  • FIGURE 30 represents a list of L5 reporter mycobacteriophages of the invention which have been developed.
  • FIGURE 31 represents an outline of a method which can be used to diagnose tuberculosis and determine drug susceptibility using reporter mycobacteriophage DS6A.
  • This invention is directed to mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages and the use of such reporter mycobacteriophages for the rapid diagnosis of mycobacterial infections and the accurate assessment of mycobacterial drug susceptibilities.
  • mycobacterial species-specific mycobacteriophage genomes are modified by introducing therein transcriptional promoters and reporter genes whose gene product can be sensitively detected.
  • the reporter mycobacteriophages may then be incubated with clinical samples suspected of containing the mycobacteria of interest, either directly of after culture, and the samples tested for the presence of the reporter gene product, thereby diagnosing mycobacterial infection.
  • the method of this invention allows for rapid diagnosis because only the amount of time necessary for the reporter mycobacteriophages to infect their host cells and the amount of time necessary for the host cells to synthesize the reporter gene product are required to allow for diagnosis.
  • the amount of time required for the reporter mycobacteriophages to infect their host cells and for the host cells to synthesize the reporter gene product is between ten minutes and sixteen hours.
  • the assessment of drug susceptibilities with the reporter mycobacteriophages of this invention is accurate because the reporter mycobacteriophages only allow for the detection of metabolically active mycobacterial organisms, the presence of which metabolic activity indicates that a drug has not killed the mycobacteria and that the mycobacteria is resistant to the drug.
  • the L5 reporter mycobacteriophages of this invention are temperate, i.e., they are able to exist in bacterial cells as prophages integrated into mycobacterial genomes without causing cell lysis. Because the L5 reporter mycobacteriophages do not cause cell lysis, they replicate as part of the bacterial genomes in bacterial cells.
  • the integrated reporter phages express high levels of luciferase activity, since the luciferase reporter phages can be stably maintained. This growth causes amplification of photon signal. Because temperate phages possess the ability to site specifically integrate into mycobacterial genomes, they are replicated as part of the mycobacterial chromosome. In addition, the integrated luciferase reporter phages confer to the infected cell the ability to produce amounts of luciferase activity comparable to plasmid transformed cells and 100 to 1000 times more luciferase activity than phage-infected cells.
  • the luciferase lysogens can be readily used to screen for drug activity by simply observing the inhibition of growth measured by proportional luciferase activity.
  • temperate L5 reporter mycobacteriophages results in a more sensitive assay for drug screening, as compared to the use of lytic reporter mycobacteriophages.
  • Mycobacteriophage L5 a temperate virus with a broad host-range among mycobacteria, is the most thoroughly characterized of the mycobacteriophages.
  • L5 particles are morphologically simi lar to the f ami ly of phages that includes phage ⁇ and contain a linear dsDNA genome with cohes ive ends .
  • the inventors have determined the DNA sequence of the entire genome of L5 , as well as several gene functions .
  • DNA sequence of the L5 mycobacteriophage is as follows :
  • ATCTACGCCA CTCCTGACGG GTGGCTGTCA AGGATACTCA CCTTCCCTAC TAATGAGGGG 240
  • CTAAGAGCCC CTCTCTATAG AGCGCCGCAC AGGCGGCGCG ATAAGAGCGC CACCAGGCGC 300
  • GATCCTCAAC TACGAGGGTC CAGGGACCGT CGAGGTCTCC GACGAGAAGC TCGCCGAAGC 2280
  • GTCTCCCCCT CACATCATCG GCCCGTCCTG GCAGAAGACG GTCGATGGTG AGTGGTATCT 6720
  • CTGGCTCACG TCGGATACCC CAGGCTCTAC GTCGACTCAG TCGCCGAGCG CCAGGCCGTC 8640
  • CTGTAGGTGA GTGGCTGCGA CTGCTGCAGG TGCTGTTCCC CGAAATCCAA CGGCGGTATG 10020
  • ACTTCGCCTA CAGCTCAGCG GAAGCCGCAG GGCTCAACCT CGATGACGAG ACCGTGATCG 10440
  • TCGAGGCCAT CAAGGCCGCT GTCGGGATCA AGAAGTAACC CACCCAACAG ATCTCAAGGA 11340
  • CAAGGTCGCG CAGACCGGCG ACTCGATGTT CGAGGGCTAC CTCGAGCCCG AGCAGGCCCA 11460
  • GGCCCAGTCC CAGATCGAGT GGCGAGACCA GCGGTGGGCG CTCTTCGGAG ACGCCACCGT 13440
  • TCGTCATCCT CAACGACGCG AAGCAGGGGC CGCGCTCCTG GCTGTCGCGA GACTCCGAGT 18600
  • CAAGAGGTAA ACCCCTTGAG GTCACGGTGA TGTCGTGCAT CGCGTACGAC CCGTTCTGGT 18780
  • CTACTCCCCC TGGTTCATGG GCACCAGCCC GATAGCACCC CACGTCGTGT TCGAAGAAGG 20400
  • CTGCGCCCGA CCGGGACAGA AATACATAGA GAACCTATGG ATGTAGGAGG CACAAAAAAA 26280
  • CTCCTCGACC GACTCGCGCT CCACGCGGAT CAGCCGGGGA CCGAGCCGAA CGGCCTTGAG 26640
  • GGCACCAGCG ACCGACTCAC GTCGGGCACC AACGGTGTCA GGCTCTTGCA GACCCTCGCG 29880
  • GAGCCTCTTC CTCTTCGACT ACCTCGTCTA CCCGGCGGAA TAACTCCGCT AGTTCTGCGG 30840
  • TAGTAGTAGT CAACGACCTT GTCCCAGTTG AAGGTTCGGG ACGTGCCGTC ATCGAACGCG 31500
  • CTCCATACAT CAGATCCTTT CCAGCAGAGC AGCTTTGCCC TGCGATGTGA CTAGTGAGTT 37860
  • CTACCGCGTC CTCGATGTTC TGCTCGCGAA CAGCCCGCGT AGCTCGTTCG AGCGACCATC 41340
  • GCGCGGCCCA GACCGGGGGT AGCGGATCCC CCGCCGACCC TCGGATGTAG AGCGATTGTC 41580 TAGGTGTGTA CACCTTCCTC CTCGTGGATG TGATTGACCA GGTCATAGAT CTCGTCGCGA 41640
  • CTCCACTGGG TGTGTCGGTC CTCGCTGGTG TGGTCCCCGA CGTATGGGAA GTGGCTCAGC 41820
  • AAGTCACACC TCCAATTCGT GGGGCTTGAT CTCGTTGGTC ACGTCGTAGT CGTTCAGCAG 45420
  • CAGCGTGGAC CACCTTGCGG CGCTCGCGCC GTACCTTGTC GCGGCCGGCC GGCCGAACCA 48360
  • CTCCCTCCAA GGCTTGCACC GAGTACCACG GCTTGCCCTC GCGGTGCGTG CGGTGCAGGT 48480
  • Integration-proficient plasmid vectors have been constructed which efficiently transform both fast-growing and slow-growing mycobacteria through stable integration of the plasmid sequences into the bacterial chromosomal attachment site (attB).
  • L5 sequence is now known, and because L5 has been previously characterized, the use of transcriptional promoters with this mycobacteriophage may be evaluated efficiently, and host synthesis inhibition may also be evaluated efficiently.
  • Figure 1 represents the genome organization of the entire L5 genome. DNA analysis has indicated that the L5 genome is organized into a right and left arm with the attachment site at the center of the genome. The integration functions have been successfully employed to construct integration-proficient vectors for mycobacteria.
  • L5 genome is not essential for mycobacteriophage growth.
  • gene 71-70-69 may be deleted without affecting the lytic cycle of the L5 phage. Therefore, it may be a suitable region in the L5 mycobacteriophage for the insertion of reporter genes. As a general role, it is critical that reporter genes be inserted into non-essential regions of the mycobacteriophage. Otherwise, the mycobacteriophage will be unable to survive and replicate.
  • the L5 mycobacteriophage may have introduced therein promoter gene 71 fused to reporter gene lacZ, and this reporter mycobacteriophage would be capable of rapid diagnosis of mycobacterial infection and accurate assessment of mycobacterial strain drug susceptibilities.
  • TM4 mycobacteriophage which may be successfully used to produce the reporter mycobacteriophages
  • TM4 has been used to construct a first generation reporter mycobacteriophage, and has the ability to discriminate between M. tuberculosis and BCG.
  • a shuttle plasmid may be employed with TM4, and may be useful in the construction of recombinant and other mycobacteriophages.
  • L5 which is a broad host-range mycobacteriophage
  • TM4 is a species-specific mycobacteriophage.
  • TM4 is not as well characterized as the L5 mycobacteriophage, and therefore it is more difficult to analyze its functions.
  • DS6A is a mycobacteriophage that has been found to be specific for the M. tuberculosis complex of mycobacteria. It has been shown to infect both M. tuberculosis and BCG. It has been demonstrated that DS6A can infect over 3,000 different types of M. tuberculosis strains. Current efforts are under way to develop DS6A shuttle phasmids containing Firefly luciferase genes as the reporter molecule.
  • DS6A mycobacteriophage is specific for only M. tuberculosis strains.
  • L5 and TM4 mycobacteriophages are specific for several mycobacteria, including M. tuberculosis and M. smegmatis.
  • mycobacteriophages which are specific for M. tuberculosis strains only. Because DS6A mycobacteriophage is specific for M.
  • tuberculosis strains only, it can be used to narrow the host specificity of L5 and TM4 mycobacteriophages so that L5 and TM4 mycobacteriophages can be used to accurately diagnose tuberculosis.
  • a clinical sample control
  • Another clinical sample from the same source experimental
  • DS6A mycobacteriophages can be used to confer specificity for M. tuberculosis onto L5 mycobacteriophages.
  • Figure 31 represents an outline of a method which can be used to diagnose tuberculosis and determine drug susceptibility using reporter mycobacteriophage DS6A.
  • mycobacteriophages are different, except that one of the mycobacteriophages from France had a considerable similarity to the L5 mycobacteriophage, which was originally isolated in Japan.
  • the host range of the mycobacteriophages varies greatly, some being able to infect only M. smegmatis and others being able to infect M. smegmatis, BCG and M. tuberculosis, but not M. avium.
  • These mycobacteriophages may be developed into reporter mycobacteriophages and cosmid cloning systems, and may provide a source of useful transcriptional translation initiating sequences, transcriptional terminators, or host-range specificity genes.
  • reporter gene In addition, the choice of reporter gene and its method of expression are critical. It is necessary to choose a reporter gene whose product would not normally be found in clinical samples, but whose product is also easily detectable.
  • Luciferase reporter genes have been used in many diversified biological systems, including E. coli, cyanobacteria, phytopathogenic bacteria and Bacillus.
  • the presence of luciferase reporter genes can be detected by the emission of photons in the presence of a substrate, such as luciferin or decanal.
  • Luciferin and decanal can permeate mycobacteria, and thereby allow for the detection of gene products, such as photons. Since one molecule of the luciferase gene product can yield 0.85 photons of light, it is the most sensitive biological reporter molecule known.
  • the preferred reporter genes of this invention are luciferase reporter genes, such as the Firefly lux gene (FFlux), the Vibrio fischeri lux genes and the Xenorhabdus luminescens lux genes, as well as the E. coli ⁇ -galactosidase (lacZ) genes.
  • Luciferase genes especially the Firefly lux gene, generate a high amount of luminescence activity. They generate photons. the detection of which is simple and sensitive, using commercially available luminometers that can detect 100-1000 molecules of luciferase with a linear relationship to enzyme concentration. In addition, it is unlikely that clinical samples will contain significant levels of endogenous luciferase activity.
  • promoter candidates currently available are the BCG hsp60 promoter and the L5 gene 71 promoter, which are of comparable strength.
  • the hsp ⁇ o promoter gives good levels of luciferase expression from plasmid recombinants, but lower levels of luciferase expression where the mycobacteriophage is TM4. It is possible that the reason for this is that the hsp60 promoter is shut off by the TM4 enzymes following infection, thus producing only a modest level of luciferase.
  • the gene 71 promoter may behave in a similar manner with the TM4 phage since the gene 71 product is a good candidate for the L5 repressor and is expressed at high levels in the absence of other mycobacteriophage functions. Knowing the sequence of the mycobacteriophage used will help in identifying, characterizing and cloning the appropriate promoter to be used in the reporter mycobacteriophages of this invention. There are several methods which can be utilized to introduce the reporter genes and transcriptional promoters into mycobacterial species-specific mycobacteriophages. One method is the utilization of shuttle phasmids.
  • the shuttle phasmids which consist of the E. coli cosmid, the reporter genes and mycobacteriophage promoters, may then be characterized.
  • Shuttle phasmids can be propagated in E. coli as plasmids, and propagated in mycobacteria as mycobacteriophages.
  • a second method of introducing the reporter genes and transcriptional promoters into mycobacteriophages is by homologous recombination.
  • non-essential regions of a mycobacteriophage must be determined. Again, in order to do this, it is necessary to know the sequence of the mycobacteriophage. Consequently, L5 is an ideal phage to use with this method as its genome has already been sequenced and characterized by the inventors.
  • plasmids are constructed wherein reporter genes hooked to transcriptional promoters are flanked by mycobacteriophage non-essential region sequences in mycobacterial plasmids.
  • homologous recombination systems may be utilized in M. smegmatis or E. coli to perform gene replacement whereby the plasmid constructs containing the reporter genes are put into mycobacteriophages.
  • a third method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by use of transposons.
  • transposon IS1096 may be utilized.
  • reporter genes and transcriptional promoters are put into transposons, and the transposons containing the reporter genes and transcriptional promoters are delivered on plasmids in mycobacteria.
  • the transposons will hop into non-essential regions of the mycobacteriophages, thereby introducing themselves therein.
  • the mycobacteriophages are still viable, and contain the reporter genes and transcriptional promoters.
  • a fourth method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by debilitated phages packaged into phage heads and tails (phage particles).
  • helper phage systems which allow for pieces of DNA containing pac sites to be packaged.
  • helper phages allow for the synthesis of head and tail genes at will in mycobacteria, prevent themselves from being packaged into phage heads and tails, and facilitate packaging of pacmids into phage heads and tails.
  • Helper phage systems may be generated from the L5 mycobacteriophage. The genome of the helper phage is put into the mycobacterial chromosome, at which time the mycobacteria are grown up.
  • pacmids which comprise phages which have pac sites, reporter genes, transcriptional promoters and mycobacterial replicons are transformed onto the mycobacterial strain.
  • the production of head and tail proteins may be induced, for example, through an increase in temperature, and the pacmids are then packaged into phage heads and tails.
  • the L5 genome has cohesive (cos) termini. This suggests the possibility of constructing L5 cosmid vectors, which could be packaged through the cos sites into L5 particles either in vivo or in vitro. Then, a large number of genes could be easily and efficiently delivered to mycobacteria.
  • Packaging into phage heads and tails may also be utilized in a fifth methodology wherein the pacmid is a plasmid.
  • the methodology is similar to the methodology wherein a debilitated phage is used, however, instead of using phage pacmids, the pacmids comprise plasmids which have pac sites, reporter genes, transcriptional promoters, and plasmid replicons.
  • direct cloning using recombinant DNA techniques in vitro may be used to introduce reporter genes and transcriptional promoters into mycobacteriophages.
  • This methodology consists of ligating a mycobacteriophage, identifying or introducing unique restriction enzyme sites in non-essential regions of the mycobacteriophage, cleaving the mycobacteriophage with the restriction enzyme sites, and cleaving DNA which encodes the promoter and the reporter gene so that it has the unique sites flanking it on either side.
  • ligation is set up in vitro between the cleaved mycobacteriophage with the unique restriction enzyme sites and the reporter gene cassette. The result is a circular DNA molecule which consists of the mycobacteriophage, the reporter genes and the transcriptional promoters.
  • the circular DNA may then be electroporated directly into mycobacteria.
  • a promoter probe vector was constructed which incorporated a truncated E. coli ⁇ -galactosidase (lacZ) gene as a reporter probe into a shuttle plasmid vector that replicated in either mycobacteria or E. coli. Random DNA fragments from the three mycobacteriophages Ll, TM4 and Bxbl were cloned into a unique BamHI site immediately upstream of the lacZ gene and screened for their ability to produce ⁇ -galactosidase. This established that lacZ could be used as a reporter gene in the mycobacteria, and identified the DNA sequences which could effectively express foreign genes in both M. smegmatis and M. tuberculosis.
  • lacZ E. coli ⁇ -galactosidase
  • ⁇ -galactosidase activity could be detected from lysed cells using OMPG, or from unlysed cells using either X-gal or a fluorescent methylumbelliferyl ⁇ -galactosidase derivative.
  • the promoter hsp60 gene highly expressed the lacZ gene in both M. smegmatis and BCG.
  • the FFlux gene was cloned into pMV261 downstream from the hsp60 promoter in plasmid pYUB180 (see Figure 2), which plasmid was shown to express the FFlux gene in M. smegmatis, BCG and M. tuberculosis H37Ra.
  • the expression of the FFlux gene was detected by observing luminescence of mycobacterial clones containing the cloned gene in the dark room, and verified use in photographic film. This demonstrated that the luciferase was expressed in the mycobacteria, and that luciferin, the substrate used, was able to penetrate mycobacterial cell walls and yield photons expressed by the mycobacteria.
  • M. smegmatis provided a model with which to determine a minimal number of individual cells detectable with the luciferase assay.
  • M. smegmatis containing pYUB180 were grown in the presence of kanamycin to ensure that every cell contained the plasmid.
  • the cells were diluted 10-fold serially and the amount of luciferase activity was determined using a luminometer.
  • Figure 3 shows that the amount of luciferase activity from 5 X 10 7 cells approached 108 luciferase units, though at this level of activity the luminometer was unable to yield an accurate measurement.
  • the activity decreased in a linear manner down to 1200 units for 500 cells.
  • 5000 cells expressing the FFlux gene can be clearly discerned above the background measurement, which approaches the number of cells that one would expect to observe in clinical samples.
  • FIG. 12 shows a schematic diagram of the extrachromosomal plasmid pYUB180 and the integration plasmid pGS16. Both of the luciferase constructs were electroporated into the M. smegmatis strain mc 2 155.
  • Kan r transformants were grown to a density of approximately 5 ⁇ 10 8 cells/ml and 10-fold serial dilutions were prepared. 100 ⁇ l samples were mixed with 250 ⁇ l of 0.1 M Na citrate, pH5 in a 13 ⁇ 75 mM polystyrene tube. This mixture was placed in the monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and 100 ⁇ l of 1 mM luciferin (Sigma, St. Louis, MO) was injected into the tube and the luciferase activity was measured as relative light units.
  • monolight 2010 luminometer Analytical Luminescence Laboratory, San Diego, CA
  • 1 mM luciferin Sigma, St. Louis, MO
  • luciferase activity was readily measured from intact mycobacterial cells infected with both the extrachromosomal and the integrating vectors. Serial dilutions indicated that it was possible to detect as few as 500 to 5,000 M. smegmatis cells expressing firefly luciferase, thereby establishing that the luciferase-luciferin system could be developed as a sensitive reporter system for ATP in mycobacteria. Distinguishing Drug-Resistant Mycobacteria From
  • luciferase is a powerful indicator of the metabolic abilities of a bacterial cell. Since anti-tuberculosis drugs are likely to significantly decrease the metabolic activity of a cell, the measurement of luciferase activity should provide a sensitive means of distinguishing drug-resistant mycobacteria from drug-sensitive mycobacteria.
  • the levels of luciferase production were 100 to 1000 times less at eight hours after the addition of the drugs compared to the untreated control.
  • the first vectors developed to introduce recombinant DNA into mycobacteria were shuttle phasmid phage vectors.
  • Shuttle phasmids have the ability to replicate in E. coli as cosmids and then replicate in mycobacteria as phages.
  • Shuttle phasmids of TM4 which contained the FFlux and lacZ genes transcribed from hsp60 and L1 promoters, respectively, were constructed (see Figure 5).
  • FIG. 7 represents a flow chart for cloning different promoters into the TM4::lux shuttle phasmid phAE39.
  • E. coli cosmid pYUB216 was inserted into a non-essential region of the mycobacteriophage TM4.
  • the pYUB216 cosmid contained FFlux in a transcriptional fusion with the hsp60 promoter of BCG, a ColEl origin and an ampicillin-resistant gene (AP) for replication and selection in E. coli, and a bacteriophage lambda cos sequence as well as a unique Bc/l site.
  • the phAE39 shuttle phasmid was constructed with Bc/l-digested pYUB216 being ligated to Sau3A-partially digested TM4 DNA. As shown in Figure 8, the shuttle phasmid phAE39 readily forms plaques of M. tuberculosis, but does not efficiently plaque on BCG. A spontaneous host range mutant of phAE39 was isolated at a frequency of 10 to 10 , and designated phAE40. Mutant shuttle phasmid phAE40 was found to be capable of infecting BCG vaccine strains, in addition to being capable of infecting M. smegmatis and M. tuberculosis strains.
  • the shuttle phasmid phAE40 was deposited with the American Type Culture Collection on April 29, 1993 and catalogued as ATCC No. 75457.
  • the reporter mycobacteriophages were mixed with M. smegmatis cells and then exposed at different times to luciferin.
  • high titers of phAE40 were prepared as described above for TM4 phages. Both M. smegmatis, mc 2 155 cells and
  • BCG-Pasteur cells were grown in roller bottles to approximately 5 ⁇ 10 7 cells per ml in M-ADC-TW broth at 37oC. Either the M. smegmatis or the BCG cells were harvested by centrifugation and washed two times in M-ADC broth, containing no tween. The resulting pellet was resuspended in the original volume of M-ADC broth. The cells were then diluted into fresh M-ADC broth and allowed to incubate overnight standing at 37oC. Tween-80 appeared to remove the receptors, and it was determined that the optimal activities were achieved if the cells were given a chance to grow in the absence of tween.
  • washed cells approximately 5 ⁇ 10 7 cells
  • 0.1 ml phAE40 particles 5 ⁇ 10 8 pfu/ml
  • the cells phage mixture was incubated at 37oC.
  • 0.1 ml samples were removed. Luciferase activity was measured as described in Figure 13. Light signals were detected within minutes following infection using a luminometer and increased 1,000 fold within 2 hours.
  • mutants of BCG were selected that were resistant to streptomycin, isoniazid and rifampicin.
  • Spontaneous mutants of BCG-Pasteur were isolated on Middlebrook 7H10 agar containing either 50 ⁇ g/ml rifampicin, 250 ⁇ g/ml streptomycin or 50 ⁇ g/ml isoniazid.
  • the rifampicin-, streptomycin-, or isoniazid-resistant mutants were purified and designated mc 2 768, mc 2 767 and mc 2 765, respectively. All three mutants and the BCG parent were grown to midlog phase, harvested and washed. As shown in Figure 10C, the mc 2 768 cells and the BCG cells were incubated standing at
  • M. tuberculosis strains were grown in a biological safety level 3 containment facility: (i) the virulent drug-sensitive M. tuberculosis Erdman strain; (ii) strain 92-2025, a singly isoniazid-resistant strain; and (iii) an MDR strain of tuberculosis that has been shown to be resistant to rifampicin, streptomycin, isoniazid, ethambutol and ethionamide and the cause of several nosocomial outbreaks in New York City.
  • the Erdman strain was subcultured from the starter culture by inoculation of 0.4 ml into 20 ml of Middlebrook 7H9 broth containing OADC enrichment (Difco Laboratories, Detroit, MI) plus 0.5 Tween-80 (M-OADC-TW broth).
  • OADC enrichment Difco Laboratories, Detroit, MI
  • M-OADC-TW broth 0.5 Tween-80
  • L5::FFlux phages a plasmid (pGS12) was constructed in which a DNA segment of the L5 genome was inserted into the E. coli-mycobacterial shuttle plasmid pMD31.
  • pMD31 is described by Donnelly-Wu et al. in "Superinfection Immunity of Mycobacteriophage L5: Applications for Genetic Transformation of Mycobacteria", Molecular Microbiology, Vol. 7, No. 3, pages 407-417 (1993).
  • This DNA segment contained the tRNA gene cluster from L5 as described by Hatfull et al. in "DNA Sequence, Structure and Gene Expression of Mycobacteriophage L5: A Phage System for Mycobacterial Genetics", Molecular Microbiology, Vol.
  • this plasmid was further manipulated by insertion of a segment of DNA containing the FFlux gene between the second and third tRNA, to produce pGS24.
  • the resulting plasmid DNA was introduced into M. smegmatis by electroporation, and an L5 lysate was prepared by growth of L5 phage on this plasmid-containing strain.
  • Plasmids pGS11, pGS12 and pGS22 were constructed as described below and then used to construct plasmid pGS24.
  • L5 DNA was cleaved with Xba I and Bcl I and the 3,993bp fragment was purified. This DNA fragment represents the L5 segment defined by the coordinates 3,150-7,143.
  • Figure 14 is a segment of L5 DNA used for FFlux insertion which shows the left arm of the L5 genome with genes 1-33 indicated. The segment of L5 taken to make FFlux inserts is between the Xba I and Bcl I sites indicated. The Nhe I site that defines the position of insertion of FFlux is shown.
  • FIG. 15 is a map of plasmid pGS11 which contains the Xba I - Bcl I segment of L5 inserted into pMV2611acZ.
  • the Bcl I end was inserted into the Bam HI site of the vector and both the Bcl I and Bam HI sites were destroyed.
  • the Hind III and Xba I sites that were used to construct pGS12 are indicated.
  • Plasmid pGS1l DNA was cleaved with Xba I and Hind III and the 4,013bp fragment was purified and inserted into plasmid pMD31 (Donnelly-Wu et al., 1993) cleaved with Xba I and Hind III.
  • This plasmid was named pGS12.
  • Figure 16 is a map of plasmid pGS12 showing the location of the Xba I and Hind III sites used to insert the Xba I - Hind III piece from pGS11 into pMD31. The unique Nhe I site used for the insertion of FFlux is also shown.
  • Plasmid pGS12 contains a unique Nhe I restriction site which corresponds to the Nhe I site at position 4,441 in the L5 genome which is located between the tRNA-trp and tRNA-gln genes (genes 8 and 9).
  • Plasmid pYUB216 was cut with Hind III, the sticky ends converted to blunt ends by Klenow enzyme and dNTP's and the DNA religated. The resulting plasmid was named pGS22.
  • Figure 17 is a map of plasmid pGS22 which shows the two Nhe I sites that flank the FFlux gene. This procedure was followed to generate an additional Nhe I site upstream of the FFlux gene in pYUB216.
  • pGS12 was digested with Nhe I.
  • pGS22 was also digested with Nhe I which produces a fragment of approximately 2.4kb.
  • the DNA's were mixed, ligated and a recombinant recovered in which the Nhe I fragment derived from pGS22 was inserted into the Nhe I site of pGS12.
  • This plasmid was named pGS24.
  • Figure 18 is a map of plasmid pGS24 which contains the Nhe I FFlux DNA fragment inserted into the unique Nhe I site of pGS12. The two Nhe I sites are indicated.
  • the orientation of the inserted DNA was determined by restriction enzyme digestion and found to be in the appropriate orientation for FFlux to be expressed from the same DNA strand as the L5 tRNA's.
  • pGS24 is thus a E. coli-mycobacterial shuttle plasmid that contains the FFlux gene flanked upstream by approximately 1,291bp of L5 DNA and downstream by approximately 2,702b
  • Figure 19 shows the strategy for recombination between pGS24 and L5. Specifically, Figures 19A and 19B show the left arm of L5 and the position of genes 1-33. Figure 19A shows the segment of L5 DNA present in pGS24 and the location of FFlux inserted between the tRNA-trp and tRNA-gln genes.
  • Plasmid pGS24 DNA was introduced into M. smegmatis mc 2 -155 by electroporation, and transformants recovered by selection with kanamycin.
  • a lysate of phage L5 was prepared by infection of approximately 0.5 ml late-log phage M. smegmatis cells containing plasmid pGS24 with approximately 10 L5ts11 particles and incubation on solid media at 37°C. [L5ts11 is a poorly characterized temperature-sensitive mutant of L5].
  • the phages were harvested and shown to have a titer of approximately 10 plaque forming particles/ml (pfu/ml).
  • Phage DNA's were prepared from high titer stocks of phGS1 and phGS5 using standard methods.
  • phGS1 and phGS5 DNA's were digested with several different restriction enzymes (including Bam HI, Nhe I, Bst E II, Asp718, Cla I, Bgl II) and the patterns observed compared with those obtained from wild-type L5, using agarose gel electrophoresis.
  • restriction enzymes including Bam HI, Nhe I, Bst E II, Asp718, Cla I, Bgl II
  • Several differences were observed between phGS1 and phGS5 as compared to L5 DNA. Some of these changes were consistent with a double crossover recombination event inserting FFlux onto the L5 genome as anticipated. Other differences were consistent with deletion of some of the L5 DNA close to the right end of the genome.
  • Figures 21A-1 and 21A-2 maps show the expected restriction products from FFlux insertion - Bam HI.
  • the location of the L5 probe (from pGS12) used for hybridization is shown (labeled 'probe'). This probe is expected to hybridize to two comigrating Bam HI fragments (3.010bp and 3,104bp) in wild-type L5 DNA (shown as 'labeled BAM HI fragments' in the top part of the figure).
  • Figure 21A-2 shows the anticipated structure of the FFlux insertion and the expected fragments resulting from digestion with either Bam HI or BAM HI + EcoRI that hybridize with the probe.
  • Figures 21B-1 and 21B-2 maps show the expected restriction products from FFlux insertion - Asp718/Cla I.
  • the pGS12 probe is anticipated to hybridize to 2,690bp, 1,148bp and 8,078bp fragments resulting from Asp718 digestion of L5 and 2,690bp, 2,981bp and 8,078bp from the FFlux recombinants.
  • This probe is also expected to hybridize to 645bp, 1,148bp and 8,078bp L5 fragments from Asp718 + Cla I digestion and 645bp, 1,078bp and 8,078bp fragments from this digestion of the FFlux recombinants.

Abstract

This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of producing said reporter mycobacteriophages and the use of said reporter mycobacteriophages for the rapid diagnosis of mycobacterial infection and the assessment of drug susceptibilities of mycobacterial strains in clinical samples. In particular, this invention is directed to the production and use of luciferase reporter mycobacteriophages to diagnose tuberculosis. The mycobacterial species-specific reporter mycobacteriophages comprise mycobacterial species-specific mycobacteriophages which contain reporter genes and transcriptional promoters therein. When the reporter mycobacteriophages are incubated with clinical samples which may contain the mycobacteria of interest, the gene product of the reporter genes will be expressed if the sample contains the mycobacteria of interest, thereby diagnosing mycobacterial infection.

Description

MYCOBACTERIAL SPECIES-SPECIFIC REPORTER MYCOBACTERIOPHAGES
STATEMENT OF GOVERNMENT INTEREST
This invention was made with government support under NIH Grant Number AI26170. CROSS-REFERENCE TO RELATED APPLICATIONS
This Application is a Continuation-In-Part of Application Serial No. 07/833,431, filed February 7, 1992, entitled MYCOBACTERIAL SPECIES-SPECIFIC REPORTER MYCOBACTERIOPHAGES, now pending. FIELD OF THE INVENTION
This invention relates to mycobacterial species-specific reporter mycobacteriophages (reporter mycobacteriophages), methods of making such reporter mycobacteriophages, and the use of such reporter mycobacteriophages, for example, to rapidly diagnose mycobacterial infection and to assess drug susceptibilities of mycobacterial strains in clinical samples. Specifically, this invention relates to the use of mycobacterial species-specific luciferase reporter mycobacteriophages to diagnose tuberculosis and to assess the drug susceptibilities of the various strains of Mycobacterium tuberculosis (M. tuberculosis).
To produce the mycobacterial species-specific reporter mycobacteriophages of the invention, transcriptional promoters and reporter genes are introduced into the genomes of mycobacterial species-specific mycobacteriophages. These reporter genes may be the genes for luciferase or the β-galactosidase gene, and provide the DNA which encodes the production of a gene product.
The reporter mycobacteriophages may be used for diagnosing mycobacterial infections by incubating same with samples which may contain the specific mycobacteria of interest. If the mycobacteria of interest is present, then the reporter mycobacteriophages introduce the recombinant nucleic acids which encode expression of the gene product into the mycobacteria of interest, and the mycobacteria then express the gene product. The expressed reporter gene product may be detected by a suitable assay, for example, through the detection of photons or the conversion of an easily assayable chemical reaction. The presence of such gene product indicates that the sample contains the mycobacteria of interest, and hence the mycobacterial species-specific reporter mycobacteriophages may be used to detect and thereby diagnose the specific mycobacterial infection.
Since signals may not be generated by cells which are not metabolically active in the presence of antibiotics, the mycobacteria species-specific reporter mycobacteriophages of this invention may be used to assess the drug susceptibilities of various strains of mycobacteria. If antibiotic drugs are added to the sample containing the reporter mycobacteriophages and the gene product is detected, the mycobacteria is metabolically active and hence resistant to the antibiotic drug.
BACKGROUND OF THE INVENTION
In 1990, there was a 10% increase in the incidence of tuberculosis in the United States. In addition, there has been an increase in the appearance of clinical isolates of tuberculosis that are resistant to antibiotics used to treat the disease. This problem is exacerbated by the length of time that is currently needed both to diagnose tuberculosis, and to determine the drug susceptibilities of various strains of M. tuberculosis. As a result, patients with M. tuberculosis may remain infectious for long periods of time without being treated, or may be treated with a drug to which the bacterial strain is resistant. Therefore, a need has arisen in the field for a method of diagnosis of M. tuberculosis (and other mycobacterial infections) which is rapid, sensitive and specific, which method is also capable of assessing the drug susceptibilities of the various strains of M. tuberculosis and other mycobacterial strains. It is critical that a mycobacterial strain be assessed for drug resistance rapidly because a patient infected with a strain of M. tuberculosis or another mycobacteria must be treated immediately with the particular antibiotic drug(s) to which the strain is not resistant, and not with antibiotic drug(s) to which the strain is resistant, or the patient may die. Currently, the most rapid test available for the diagnosis of M. tuberculosis is the staining of sputum samples for acid-fast bacilli, which is a tedious procedure, and which procedure has low sensitivity. Alternative methods for diagnosis require cultivation of the bacilli for approximately two to six weeks followed by classification of the cultured organism. Typical diagnostic tools include biochemical tests, analysis of mycolic acids and serotyping. All of these tests are time-consuming. More recently, the use of oligonucleotide probes and Polymerase Chain Reaction have been suggested for the identification of M. tuberculosis species. Although these methods may be useful approaches, their uses in a clinical setting have not yet been determined. Further, these methods do not distinguish between live and dead organisms, and are therefore of limited use in the determination of drug sensitivities of clinical isolates.
In addition, Mycobacterium avium (M. avium) is a mycobacteria which is often found in immunosuppressed patients. This mycobacteria is typically disseminated throughout the bodies of immunosuppressed patients, such as
AIDS patients, and causes M. avium infection. Because this mycobacteria often causes death in immunosuppressed patients, it is necessary to be able to diagnose and assess the drug susceptibilities of the various strains of M. avium.
It is therefore an object of this invention to construct broad mycobacterial host range and mycobacterial species-specific reporter mycobacteriophages.
It is another object of this invention to provide mycobacterial species-specific reporter mycobacteriophages which may be used to rapidly diagnose mycobacterial infections.
It is still another object of this invention to provide mycobacterial species-specific reporter mycobacteriophages which may be used to rapidly assess the drug susceptibilities of different strains of mycobacteria in clinical samples.
It is yet another object of this invention to provide mycobacterial species-specific reporter mycobacteriophages wherein the reporter genes are luciferase genes, which mycobacterial species-specific reporter mycobacteriophages may be used to rapidly diagnose mycobacterial infections and to rapidly assess the drug susceptibilities of various strains of mycobacteria.
It is a further object of this invention to provide mycobacterial species-specific luciferase gene reporter mycobacteriophages which may be used to rapidly diagnose tuberculosis and assess the drug susceptibilities of the various strains of M. tuberculosis. SUMMARY OF THE INVENTION
This invention relates to broad host range and mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages, and the use of such reporter mycobacteriophages to rapidly diagnose mycobacterial infection, such as M. tuberculosis, and to distinguish which strains of the mycobacteria are drug-resistant.
To produce these reporter mycobacteriophages, reporter genes and transcriptional promoters are introduced into the genomes of mycobacterial species-specific mycobacteriophages. The promoter and reporter gene-containing mycobacteriophages (reporter mycobacteriophages) are then incubated with a clinical sample which may contain the mycobacteria of interest, such as M. tuberculosis. The reporter mycobacteriophages are specific for the mycobacteria which is sought to be detected. The reporter mycobacteriophages efficiently introduce the recombinant nucleic acids which encode the expression of the reporter gene's gene product into the mycobacteria of interest, and the mycobacteria then express the gene product. A substrate or other means capable of allowing for the detection of the gene product is then added to the sample. If the gene product or the signal generated by the gene product is detected, the presence of the infectious mycobacteria is known, thereby diagnosing the
To assess drug susceptibility of mycobacteria, drugs such as antibiotics may be added to a sample containing the reporter mycobacteriophages of this invention. If the mycobacteria are susceptible to a drug after exposure to the drug, the mycobacteria will be killed. However, drug-resistant mycobacteria will continue to be metabolically active in the presence of the drug, and will continue to express the detectable gene product of the reporter genes. Preferably, the reporter mycobacteriophages of the invention are temperate, and have increased sensitivity for use in drug screening.
The preferred reporter genes of the present invention are the Firefly luciferase lux gene (FFlux), the luciferase lux genes of Vibrio fischeri, the luciferase lux genes of Xenorhabdus luminescens and the E. coli β-galactosidase gene (lacZ). Some preferred promoters of the present invention are hsp60 and gene 71-70-69 promoters, and the preferred mycobacteriophages are L5, TM4 and DS6A. These reporter mycobacteriophages are preferably used for the rapid diagnosis of tuberculosis and M. avium infection, and the accurate assessment of drug susceptibilities of the various strains of M. tuberculosis and M. avium.
BRIEF DESCRIPTION OF THE DRAWING
The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiment of the present invention when taken in conjunction with the accompanying drawing wherein:
FIGURE 1 (which is comprised of Figure 1A and Figure 1B) represents the genome organization of mycobacteriophage L5;
FIGURE 2 represents a luciferase shuttle plasmid pYUB180 wherein reporter gene FFlux is fused to the BCG hsp60 promoter;
FIGURE 3 represents the amount of luciferase activity of M. smegmatis which contains the pYUB180 shuttle plasmid and the FFlux gene;
FIGURE 4 (which is comprised of Figure 4A, Figure 4B and Figure 4C) represents the effect of various antibiotic drugs on the metabolic activity of control mycobacteria and drug resistant mycobacteria in the presence of reporter mycobacteriophages which contain luciferase reporter genes;
FIGURE 5 represents shuttle plasmid phAE39 wherein the reported gene is FFlux, the promoter is hspβO, the phage is TM4 and the cosmid is pYUB216;
FIGURE 6 represents luciferase activity of
M. smegmatis cells infected with shuttle phasmids phAE39;
FIGURE 7 represents a flow chart for cloning different promoters into TM4 : : lux shuttle phasmid phAE39;
FIGURE 8 represents a schematic diagram of the luciferase reporter mycobacteriophages phAE39 and phAE40; FIGURE 9 represents the production of light (photons) by mycobacteria following infection with the luciferase reporter phage phAE40;
FIGURE 10 (which is comprised of Figure 10A, Figure 10B and Figure 10C) represents a comparison of the kinetics of light production following phage infection of drug-sensitive BCG cells to drug-resistant BCG mutant cells;
FIGURE 11 (which is comprised of Figure 11A, Figure 11B and Figure 11C) represents a comparison of drug-sensitive M. tuberculosis and drug-resistant M. tuberculosis using the luciferase reporter phage assay;
FIGURE 12 represents a schematic diagram of the extrachromosomal plasmid pYUB180 and the integration plasmid pGS16;
FIGURE 13 represents the expression of luciferase by
M. smegmatis after plasmids pYUB180 and pGS16 were electroporated therein;
FIGURE 14 represents a DNA fragment of the L5 segment defined by the coordinates 3,150-7,143 after cleaving with Xba I and Bcl I;
FIGURE 15 represents the DNA fragment of L5 after insertion into plasmid pMV2611acZ and cleaving with Xba I and BAMHI to produce plasmid pGS11;
FIGURE 16 represents plasmid pGS12 which was produced by cleaving pGS11 DNA with Xba I and Hind III, and inserting fragment 4,013bp into plasmid pMD31; FIGURE 17 represents plasmid pGS22, which was produced by cutting plasmid pYUB216 with Hind III and converting the sticky ends to blunt ends by Klenow enzyme and dNTP's;
FIGURE 18 represents plasmid pGS24, which was produced by inserting plasmid pGS22 into the NHE I site of pGS12 plasmid;
FIGURE 19 (which is comprised of Figure 19A and Figure 19B) represents a double crossover event between plasmid pGS24 and L5;
FIGURE 20 (which is comprised of Figure 20A and Figure 20B) represents hybridized bands detected by autoradiography;
FIGURE 21 (which is comprised of Figure 21A-1, Figure 21A-2, Figure 21B-1 and Figure 21B-2) represents a map of expected DNA fragments resulting from a pair of homologous recombination events in common flanking sequences when FFlux is inserted into the L5 genome in a corresponding location to that in pGS24;
FIGURE 22 represents restriction enzyme mapping and Southern blot hybridization for phGS1 and phGS5;
FIGURE 23 represents determination of the luciferase activity of pGS24, phGS1 and phGS5;
FIGURE 24 represents luciferase activity as determined after liquid infection of M. smegmatis mc2155 with phGS1 and phGS5;
FIGURE 25 represents a comparison of luciferase activity of phGS5 with clear plaque mutant derivatives that are not competent to form lysogens;
FIGURE 26 represents the activity of phAE40 and the L5 : :FFlux phages following infection with M. smegmatis mc2155; FIGURE 27 represents the sensitivity of phage phGS5 after infecting M. smegmatis mc2155;
FIGURE 28A represents the detection of luciferase activity after liquid infection of serial dilutions of M. smegmatis with phGS18;
FIGURE 28B represents the light produced (RLU); FIGURE 29 represents the result of liquid infection of nonlysogen and lysogen strains of M. smegmatis with phAE40, phGS18 and phGS26;
FIGURE 30 represents a list of L5 reporter mycobacteriophages of the invention which have been developed; and
FIGURE 31 represents an outline of a method which can be used to diagnose tuberculosis and determine drug susceptibility using reporter mycobacteriophage DS6A.
DETAILED DESCRIPTION OF THE- INVENTION
This invention is directed to mycobacterial species-specific reporter mycobacteriophages, (reporter mycobacteriophages), methods of producing such reporter mycobacteriophages and the use of such reporter mycobacteriophages for the rapid diagnosis of mycobacterial infections and the accurate assessment of mycobacterial drug susceptibilities. In order to produce such reporter mycobacteriophages, mycobacterial species-specific mycobacteriophage genomes are modified by introducing therein transcriptional promoters and reporter genes whose gene product can be sensitively detected. The reporter mycobacteriophages may then be incubated with clinical samples suspected of containing the mycobacteria of interest, either directly of after culture, and the samples tested for the presence of the reporter gene product, thereby diagnosing mycobacterial infection.
The method of this invention allows for rapid diagnosis because only the amount of time necessary for the reporter mycobacteriophages to infect their host cells and the amount of time necessary for the host cells to synthesize the reporter gene product are required to allow for diagnosis. Typically, the amount of time required for the reporter mycobacteriophages to infect their host cells and for the host cells to synthesize the reporter gene product is between ten minutes and sixteen hours.
The assessment of drug susceptibilities with the reporter mycobacteriophages of this invention is accurate because the reporter mycobacteriophages only allow for the detection of metabolically active mycobacterial organisms, the presence of which metabolic activity indicates that a drug has not killed the mycobacteria and that the mycobacteria is resistant to the drug. The L5 reporter mycobacteriophages of this invention are temperate, i.e., they are able to exist in bacterial cells as prophages integrated into mycobacterial genomes without causing cell lysis. Because the L5 reporter mycobacteriophages do not cause cell lysis, they replicate as part of the bacterial genomes in bacterial cells. The integrated reporter phages express high levels of luciferase activity, since the luciferase reporter phages can be stably maintained. This growth causes amplification of photon signal. Because temperate phages possess the ability to site specifically integrate into mycobacterial genomes, they are replicated as part of the mycobacterial chromosome. In addition, the integrated luciferase reporter phages confer to the infected cell the ability to produce amounts of luciferase activity comparable to plasmid transformed cells and 100 to 1000 times more luciferase activity than phage-infected cells. The luciferase lysogens can be readily used to screen for drug activity by simply observing the inhibition of growth measured by proportional luciferase activity. Hence, the use of temperate L5 reporter mycobacteriophages results in a more sensitive assay for drug screening, as compared to the use of lytic reporter mycobacteriophages.
Mycobacteriophage L5, a temperate virus with a broad host-range among mycobacteria, is the most thoroughly characterized of the mycobacteriophages. L5 particles are morphologically simi lar to the f ami ly of phages that includes phage λ and contain a linear dsDNA genome with cohes ive ends . The inventors have determined the DNA sequence of the entire genome of L5 , as well as several gene functions . The
DNA sequence of the L5 mycobacteriophage is as follows :
SEQ ID NO : 1 :
GGTCGGTTAT GCGGCCGAGC CATCCTGTAC GGGTTTCCAA GTCGATCAGA GGTAGGGGCC 60
GGCACAGAAA CCACTCACAT CAGGGCTGTG CGCCTCCAGG GCGCGTGAAC TCCCACACCC 120
CGGTGTAGTT ACATCCCGGA ATTGTCTCAG CGCCTCTCAG GGCGCTTCTC ATAAACAGTG 180
ATCTACGCCA CTCCTGACGG GTGGCTGTCA AGGATACTCA CCTTCCCTAC TAATGAGGGG 240
CTAAGAGCCC CTCTCTATAG AGCGCCGCAC AGGCGGCGCG ATAAGAGCGC CACCAGGCGC 300
TCATCTAAAG ACCGGCCTTG AAGGGCCGGT CATAGAGATC TATTCGATCC GGCAACCGCC 360
GGATCTCAAG GCCGCGCCAG TGCGCGGCCC TATAGAGGGG TGACTCAACT GTGCATGGCA 420
CTCGCTCGAG TGCCCACTGG AGCACTCAAC CGGGGAAGTT CGACGTTCTC AACCTGCGAA 480
TGACGTTTGA ATCGTCATCC GCGTACGAAA TCCCCGATCT GCGGCCGACC GACTTCGTGC 540
CGGCCTATCT CGCGGCCTGG AATATGCCGC GTCACCGCGA TTACGCCGCC AAGAACGGCG 600
GCGCGCTGCA CTTCTTCCTT GACGATTACC GGTTTGAGAC CGCGTGGTCG TCCCCCGAGC 660
GCCTTCTCGA CCGCGTAAAG CAGGTCGGCG CTGCACTCAC GCCGGATTTC AGCCTCTGGA 720
CGAACATGCC GAAGGCGGCG CAGCTATGGA ACGTCTACCG CTCCCGCTGG TGTGGCGCGT 780
ATTGGCAGTC GGAAGGAATC GAGGTGATTC CGACGGCGTG TTGGGCGACT CCCGACACGT 840
TCGATTTCTG TTTCGACGGG ATCCCGATGG GATCGACCGT CGCAATTTCT TCGATGGGCA 900
TTCGCTCTTC AAAAGTCGAC CAGGAGCTTT TCCGGTACGG ACTACGCGAA CTCATCGATC 960
GCACTCAACC GCAACTGCTT TTGGCATATG GCCAGCTTCG GCATTGCGAC GACATGGATT 1020
TACCAGAGGT CCGCGAATAC CCGACCTACT GGGACAGACG ACGAAAGTGG GTAACTGCCG 1080
ATGGGAGGCC GGGGAAGTAA AGGCGGCCCC GGTCCCGGAA CCGGAGCACG CAACCGCAGA 1140
GGCGCTGGAG CCCCCGGATC GGGCGGCGTA GGCGGCGTCG GAGGCGGGGG TGGAGCTGCA 1200
GGGAGCAGCG GAGGCGGCAA GGGAACGGCA GCGCCGGTAC CGGAGGCGTC ACCGGTGGCG 1260
GCGGAAGTGG AGCCGGCGGC GGTGGCAGCA GCCCCAACAC CCCGGTGCCC CCCACCGAGC 1320
TGGAGAAGAA GCGCGGCGAA TACAACCAGA TCGCCATCGA CGCCCAGAAA CAGCACGCGC 1380
CCACCGATGA GAAGCGCGAG GCCAAGCGCA AGCAACTGAT GGATCGAGTC GGAGGAGACT 1440
GGCAGGCTTT GGACCCGGAT CACCACGACG CCATCAAGGT GGCGATGGAT GACGCCATGC 1500
GGAAGATCCT CTCCGAGGAG GAGATCGTCC ACCGCACCAA GCACTTCGGC GACCTACTCG 1560
ACTCCGGTCG ACTCAAGTCG CTGTTCGAGG TCGGCTTCTC AGCCGGTGGC GACACCCCGA 1620
CCGAACGCGC CCTCCTCGAG GACGCCTGGT TCGGCGCAGG CAAGGTTCCC CCGATCTACT 1680
CGGCAATCGA GTTCAACGGC GCTCCGACAG CCGGCCTCGG CATGTACGGC GGCACCAAGC 1740
TCTACATGAA GGACTCGGTC AAGGACCGCG TCACCGTGAC CATCGGCGAC TCGCTGATGT 1800
CGAGCTGGGA CGTATTCCCC GGCCGTCCTG GCGACGGCGT GGGGCTGTGG GCCAGCCTGT 1860
CGAAGATCGA GGGGCTGGTC GATCCGAGCA AGACCCGCGA AGAGAACATG CAGGCGGTGT 1920
ACGACTCGTT CAAGAAGTAC GGCACCCTGG ACGGCTTCAT CGAGGCGCAG ATCCACGGCG 1980
GCGTCCTGGT CGAGGACATC AAGAAGGTCG TGTTCACGCA GCCGCCGAGC CCGATCTTCA 2040
CCGATAAACT GGACGAACTT GGAATCCCGT GGGAGGTGCA GTAATGGCGC AGATGCAGGC 2100
GACACACACA ATCGAGGGGT TCCTGGCTGT CGAGGTGGCC CCTCGGGCGT TCGTCGCAGA 2160
GAACGGCCAC GTACTGACCC GGCTGTCGGC CACGAAGTGG GGCGGTGGCG AGGGTCTCGA 2220
GATCCTCAAC TACGAGGGTC CAGGGACCGT CGAGGTCTCC GACGAGAAGC TCGCCGAAGC 2280
CCAGCGGGCC AGCGAGGTCG AGGCTGAACT TCGCCGCGAG GTCGGCAAGG AGTGAGCTGG 2340
GCCGGCTCAG GCCGGCGACA GGAACTACCA GAGGACTGGG AGCTGAATTA CCGGCTCCCG 2400
GTCCTTTCTG CTGCCAACTG GCTTTGCCAG ATCAACGGTC CCGGATGCGT AAGGGCCGCA 2460
ACCGATGTCG ACCACATCAA GCGCGGGAAC GACCACAGCC GGTCCAATCT GCAGGCAGCC 2520
TGCCATGTCT GTCACGGCAA GAAATCAGCC GCCGAGGGCG TAGCCCGACG GCGGGAACTT 2580
AGAGCCCGGA GGAAGCGACC ACCCGAACGC CATCCTGGGC GTCGATAAGC GGGCCAGGTG 2640
CCCGCTCCAC CCAGGAGGTG AACAGTGGGC ACGCGAGGCC CAATCGGAAA ACGAGATGAA 2700
GAGCGGGTTC GTCGGAACAC CCCGGACAGT CCAACCGACA CGATCCAGAT GCCCGGTCTG 2760
GTGACGATCC CCGAGATGGG CGATCTAAGC CACGACGGCC GCACGCACCA GCTCGTCAAG 2820
GACATGTACG AGTCGATCAA GCAGTCGGCA GCCGTGAAGT ACTACGAGCC GACCGACTGG 2880
CAGATGGCCC GACTCGCCCT CTACACACTT AACCAGGAAC TCATCGCAGC CGAGAACAAC 2940
GGCAAGCCCG TGGGCGCGAT GAAGCTCACT GCCATCAACC AGATGCTCTC CGCGCTGCTG 3000
CTGACCGAAG GTGACCGACG CCGCGTCCGA CTCGAAGTCG AACGAGCACC CGCTGACCCG 3060
ACAGGCGGGA AGGTCGTTGA CGTGACCGAC GTGCTCAAGC AGCGCCTCGC CAAGGCGAGC 3120
GGCGGGAGCT GATGGTCCCC CGAGGGGTTT CTAGAGCCGC TGCCGCTACC AGCCGCTCCC 3180
CCTCGGGGTA GACATCGAAA GGAACCACAT GGCCGACCTC GGCAACCCAC TCGACCTCGA 3240
GATGCTCTGC CTGGTCACAG GCCGGGACTT CCGCTGGACC ATCGATTACC CGTGGGGTCC 3300
GGGAGAGCTG TTCCTCGAAC TCGAGACCGG CGGCGAACAC AACGCGCTGC ATCAGGTCTA 3360
TGTCACCGGG GCGACCGGAG GCACGTACAC GCTGAACGTC AACGGCACCA ACACCCCGGC 3420
CATCGACTAC AACGACGTGT CGGAGAATCC GCAGGGGCTG GCAGGCGACA TCCAAGACGC 3480
TCTGGACGCA GCCGTCGGAG CCGGAAACGC TGTCGTGCAT CCGGTCTCGC TGTTCCCTGC 3540
GTGGACACTG AACTTCAACC TCAACGCCAG CAAGCCGCTC ACCGAGCAGT TGGTCAACAC 3600
GATCAACAAG GCCGCGAACG ACTTCTTCGA CACGTTCGAC CAACTACTTG GGGTCGACGT 3660
GGAGATGACG GTCACCGACA CCCTGAACTT CAAGCTCAAG GTGACCTCGC GGCGCTCGTT 3720
CGATGAGGTC GGTGTCGTCA CGTTCGCGGT CGACGTGACC AGCCAGGCAG TCATCAACTT 3780
CTTCAACTCC GTCGCCGAAC TCACCGGAGC GGTGAACACC GTCAACGTCG ACTTCTACTG 3840
GAACCGGACG TATGACATCG AGTTCACCGG ATCCCTTGGG CTGCAGCCGA TTCCGGCTAC 3900
TACAGCCGAC ATCACCAACC TGGCGGGTAC CAGCAAGGCC GTCTCAGTCA CGGTGGTCGA 3960
GCCAGGAAAG AAGAGGCTGA CCATCTGGCC GTTCACGGTC AACGGTGAAA CCGCAACCAT 4020
CAAGGTCGAG TCCGAAGAGG CCGACAAGAT CCCCAACCGC TGCCGCTGGC AGTTGGTTCA 4080
CATGCCGACC GGCGAGGCAG CCGGCGGCGA TGCAAAGCAG CTCGGCCGCG TTTACCGACA 4140
GCCGAGGTAA CACCGCACCC ATCAGAGATG GTGGGCCAGA CGGCCTTCGG GCCGTCCCCT 4200
GACGTGTAGC TCAATGGCAG AGCGCCCGAC TGTTAATCGG GTGGTTGAAG GTTCGAGTCC 4260
TTCCATGTCA GCGAGGGCTG AACCGGACCC GTGTCCGGTG TAGGCACTTT CCGCAGGCGG 4320
TTCCCCAGAG CGTGGGGAGC CCCTGCCCTG TACACGTAGC TCAATTGGTA GAGCAGCGGT 4380
CTCCAAAGCC GCCGGTTCCA GGTTCGACTC CTGGCGTGTA TGCACACACC CCTGACTCCT 4440
GCTAGCGGAG TGTTCGCCTT TCGGGCCTGG GGTCTTTTTC CCCGTTCGTC TAATCGGTAA 4500
GACACCCGGC TCTGGACCGG GCAATTGAGG TTCGAGTCCT TGGCGGGGAG CCAACTTGAC 4560
ATCCACCCGA AAGGAACAAC ATGACCTTCA CAGTCACCCG CGAGAGAGCG CAGTGGGTCC 4620
ACGACATGGC CCGCGCTCGC GACGGTCTCC CCTACGCGTA CGGCGGGGCG TTCACCAACA 4680
ACCCGAGGGT GTCGACTGAC TGCTCTGGCC TGGTGCTGCA GACCGGGGCT TGGTATGGAG 4740
GTCGCACCGA CTGGGTCGGA AACCGTTACG GCTCAACCGA ATCGTTCCGG CTCGACCACA 4800
AGATCGTCTA CGACCTAGGG TTCAAGCGGA TGCCCCGAGG CGGGCCAGCG GCCTTGCCGA 4860
TCAAGCCGGT GATGCTCGTC GGGCTCCAGC ACGGAGGCGG CGGGGTCTAC TCGCACACCG 4920
CTTGCACGTT GATGACGATG GACCACCCCG GTGGCCCGGT CAAGATGTCC GACCGAGGCG 4980
TCGACTGGGA GTCCCACGGC AACCGCAACG GCGTAGGCGT CGAACTTTAC GAGGGCGCAC 5040
GGGCATGGAA CGACCCTCTG TTCCATGACT TTTGGTACCT GGACGCAGTC CTCGAAGACG 5100
AAGGAGACGA TGACGAATTG GCTGACCCAG TTCTAGGGAA GATGATCCGC GAGATCCACG 5160
CGTGCCTGTT CAATCAGACC GCGTCGACCA GCGATCTGGC GACCCCTGGT GAAGGCGCTA 5220
TCTGGCAGCT ACACCAGAAG ATCCACTCGA TTGACGGCAT GCTCCACCCG ATCCACGCTG 5280
AGCGGCGCGC TCGCGCAGGC GATCTCGGTG AGCTGCACCG AATCGTGTTG GCCGCGAAGG 5340
GCTTGGGCGT GAAGCGCGAC GAGGTGACCA AGCGGGTCTA CCAGAGCATC CTCGCCGACA 5400
TCGAGCGGGA CAACCCCGAA GTACTTCAGC GATACATCGC AGAAAGAGGT GGCCTATGAG 5460
CCCCAAGATC CGACAGACCA TCTACCTGCT CGGCACCGCC GCCCCGGCAC TGCTGGGCAT 5520
CGTCCTGATC TGGGGCGGGC TCGACGCTGA GTCGGCGGCT GACCTCGGTG ACATCATTGC 5580
GGGCGTCGTG TCGATACTAG TCTCCGGTGC GCCGGCCGTA GCGGCAGGCA CCGTACGCAG 5640
CCAGCGCAAG GACGGCACGT TGTCCACCAG CCCGGTGGAT CAGGTCACCA AGGGCGTCGA 5700
GCAGGTGCTC GCGGCCAGGC AGAGTGCCGA GGCTGAAGTC GCGAAGGTCA AGCAGGCGCT 5760
GGAGACCGCC GTCAGCGGTT CTCTCCCCCA GCTCGGCCCG CTGGCCACGC AGATCCTCAA 5820
CGTGGCTGAC GACACCGTCT GGCGTCCATG AGCAAGCCCT GGCTGTTCAC CGTCCACGGC 5880
ACAGGCCAGC CCGACCCGCT CGGGCCTGGT CTGCCTGCCG ATACCGCACG GGACGTACTT 5940
GACATCTACC GGTGGCAGCC CATCGGCAAC TACCCGGCAG CGGCGTTCCC GATGTGGCCG 6000
TCGGTCGAAA AGGGTGTCGC TGAGCTGATC CTGCAGATCG AGCTGAAGCT GGACGCAGAT 6060
CCGTACGCGG ACTTCGCGCT GGCCGGCTAC TCGCAGGGAG CCATCGTGGT GGGCCAGGTG 6120
CTCAAGCACC ACATCATCAA CCCGAGAGGT CGACTGCACC GGTTCCTGCA CCGGCTCAGG 6180
AAGGTCATCT TCTGGGGTAA TCCGATGCGG CAGAAGGGCT TTGCCCACAC CGACGAGTGG 6240
ATTCACCAGG TCGCTGCCTC GGACACGATG GGCATCCTCG AGGACCGACT GGAGAACCTC 6300
GAGCAGTACG GCTTTGAGGT CCGCGACTAC GCGCACGACG GCGACATGTA CGCCTCCATC 6360
AAGGAGGACG ACATGCACGA GTACGAGGTG GCCATTGGCC GAATCGTGAT GAGCGCTAGG 6420
CGATTCATCG GAGGTAAGGA CTCCGTCATC GCCCAGCTCA TCGAGCTTGG ACAGCGTCCG 6480
ATCTGGGAGG GAATCGCGAT GGCCAGAGCC ATCATCGACG CCCTCACGTT CTTCGCCAAG 6540
TCGACCCAAG GCCCGAGCTG GCCGCATTTG TACAACCGCT TCCCGGCGGT CGAGTTCCTA 6600
CGACGAATCT GAGAAAGGAG GCGGGGTGAG CCTCAACAAC CACCACCCGG AGCTTGCCCC 6660
GTCTCCCCCT CACATCATCG GCCCGTCCTG GCAGAAGACG GTCGATGGTG AGTGGTATCT 6720
GCCTGAGAAG ACCCTCGGCT GGGGAGTCCT GAAGTGGCTC TCCGAGTACG TGAATACCCC 6780
TGGCGGGCAT GACGATCCGA ACCGTCTGGC GACGTTGATC GCGCTCTCCG AGGCAGGTCT 6840
TCTCGACAAC GAGAACATGT TCATCCCCAC CGACGAGCAG GTACGCCTGG TCCTCTGGTG 6900
GTACGCAGTA GATGACCAGG GCCAGTACAT CTACCGCGAG GGCGTGATCC GCCGGCTCAA 6960
GGGCTGGGGC AAGGATCCGT TCACCGCCGC GCTCTGCTTG GCGGAACTCT GTGGCCCCGT 7020
AGCCTTTTCA CACTTCGACG CCGACGGTAA CCCGGTCGGC AAGCCGCGTT CAGCCGCGTG 7080
GATCACCGTC GCGGCCGTCA GCCAGGACCA GACGAAGAAC ACGTTCTCGC TGTTCCCGGT 7140
GATGATCAGC AAGAAGCTGA AGGCCGAGTA CGGCCTGGAC GTGAACCGCT TCATCATCTA 7200
CTCCGCAGCC GGTGGCCGTA TTGAGGCAGC GACCTCGAGC CCCGCGTCGA TGGAGGGTAA 7260
CCGCCCGACG TTCGTCGTCC AGAACGAGAC GCAGTGGTGG GGCCAAGGCC CCGACGGCAA 7320
GGTCAATGAA GGCCACGCGA TGGCAGAGGT CATCGAAGGC AACATGACCA AGGTCGAGGG 7380
CTCCCGCACC CTGTCGATCT GCAACGCCCA CATCCCCGGC ACCGAGACGG TCGCCGAGAA 7440
GGCATGGGAC GAGTACCAGA AGGTCCAGGC AGGCGACTCT GTCGACACCG GGATGATGTA 7500
CGACGCGCTG GAAGCGCCGG CCGACACCCC GGTCTCCGAG ATCCCCCCGC AGAAGGAGGA 7560
TCCCGAGGGA TTCGAGAAGG GCATCGAGAA GCTCCGCGAG GGCCTGCTCA TCGCCCGAGG 7620
CGACTCCACC TGGCTGCCGA TAGACGACAT CATCAAGTCG ATTCTGTCGA CCAAGAACCC 7680
GATCACCGAG TCGCGGCGCA AGTTCCTGAA TCAGGTAAAC GCCGCTGAGG ACTCGTGGCT 7740
CTCACCGCAG GAATGGAACC GGTGCCAGGT CGACCTGGCC AAGTACCTGG ATAAGCACGG 7800
CAGGGAGTTC GCTCCGCTGC AGCGCGGTGA CCGGATCACC CTCGGGTTCG ACGGGTCGAA 7860
GTCCAACGAC TGGACCGCGC TCGTCGGCTG CCGTGTCAGC GACGGCCTGC TGTTCGTCAT 7920
CGACATCTGG GATCCCCAGA AGTACGGCGG GGAGGTTCCC CGCGAAGACG TTGACGCCAA 7980
GGTCCATTCG GCGTTCGCCC ACTACGACGT GGTGGCGTTC CGCGCCGACG TGAAGGAGTT 8040
CGAGGCGTAC GTCGACCAGT GGGGCCGGAC CTACAAGAAG AAGCTCAAGG TCAACGCCAG 8100
CCCGAACAAC CCGGTGGCGT TCGACATGCG CGGACAGCAG AAGAGGTTCG CGTTCGACTG 8160
CGAGCGACTC GAGGACGCGG TCCTTGAGGG CGAGGTCTGG CACGACGGCA ATCCCGTTCT 8220
GCGCCAACAC GTTCTGAACG CCAAACGACA CCCAACGAAC TACGACGCCA TCGCGATTCG 8280
CAAGGTCACG AAGGACTCCA GCAAGAAAAT CGACGCTGCA GTCTGCGCTG TCCTCGCGTT 8340
CGGGGCGAGA CAGGACTACC TCATGAGCAA GAAGGCCCGT AGCGGCCGGG TGGTGATGGT 8400
TCGATGACAG CACCGCTCCC CGGTATGGAG GAGATCGAAG ACCCCGCAGT CGTACGAGAA 8460
GAGATGATCT CGGCCTTCGA GGATGCTTCC AAGGATCTCG CCAGCAACAC CAGCTACTAC 8520
GACGCTGAGC GCCGGCCAGA GGCCATCGGC GTCACCGTCC CGAGAGAGAT GCAGCAACTG 8580
CTGGCTCACG TCGGATACCC CAGGCTCTAC GTCGACTCAG TCGCCGAGCG CCAGGCCGTC 8640
GAGGGTTTCC GCCTCGGCGA TGCCGACGAG GCTGACGAAG AGCTGTGGCA GTGGTGGCAG 8700
GCCAACAACC TCGACATCGA GGCACCACTG GGCTACACCG ACGCTTACGT TCACGGCCGG 8760
TCGTTCATCA CGATCAGCAA GCCAGACCCG CAGCTCGACC TGGGTTGGGA TCAGAACGTC 8820
CCGATCATCC GCGTCGAGCC GCCCACCCGA ATGCACGCCG AGATCGACCC CCGGATCAAC 8880
CGGGTGTCCA AGGCCATCCG AGTCGCATAT GACAAGGAGG GCAACGAGAT TCAGGCTGCC 8940
ACGCTGTACA CGCCGATGGA GACCATCGGC TGGTTCCGCG CTGACGGTGA GTGGGCTGAG 9000
TGGTTCAACG TCCCGCACGG TCTGGGCGTC GTTCCCGTTG TGCCGCTTCC GAACCGGACC 9060
CGGCTCTCGG ACCTGTACGG CACCAGTGAG ATCACGCCCG AGCTTCGGTC GATGACCGAC 9120
GCGGCGGCGC GCATCCTCAT GTTGATGCAG GCGACCGCCG AGCTGATGGG TGTCCCCCAG 9180
CGCCTGATCT TCGGCATCAA GCCCGAAGAG ATCGGCGTCG ACTCCGAGAC CGGCCAGACG 9240
CTGTTCGATG CGTACCTGGC CCGGATCCTG GCGTTCGAGG ACGCTGAGGG CAAGATCCAG 9300
CAGTTCTCTG CAGCCGAGCT GGCCAACTTC ACCAACGCGC TCGATCAGAT CGCCAAACAG 9360
GTCGCTGCGT ACACGGGATT GCCTCCCCAG TACCTGAGTA CCGCCGCAGA CAATCCGGCC 9420
TCCGCTGAGG CGATCAGGGC CGCTGAGAGC CGACTCATCA AGAAGGTCGA GCGGAAGAAC 9480
CTGATGTTCG GCGGCGCATG GGAAGAGGCC ATGCGGATCG CCTACCGGAT CATGAAGGGC 9540
GGCGACGTTC CCCCGGACAT GCTCCGCATG GAGACCGTCT GGCGAGACCC GAGCACTCCC 9600
ACCTACGCGG CCAAGGCCGA CGCAGCCACG AAGCTGTACG GCAACGGCCA GGGTGTCATC 9660
CCGCGTGAAC GTGCTCGCAT CGACATGGGC TACTCCGTCA AGGAGCGCGA AGAGATGCGC 9720
CGATGGGACG AGGAAGAGGC CGCAATGGGT CTCGGCCTGT TGGGCACGAT GGTCGACGCC 9780
GACCCGACGG TCCCAGGCTC CCCGAGCCCC ACGGCACCGC CGAAGCCACA GCCGGCCATC 9840
GAGTCGTCTG GTGGTGATGC GTGACCGCAG AGGAGTACGC GGCGGCTCAA GCCGCGATCA 9900
CTGCGGGTCT TGCCACATAC GTCCAGAGGT TCGCTTCGCT CTTCGTCGGT CCAGCTCTCG 9960
CTGTAGGTGA GTGGCTGCGA CTGCTGCAGG TGCTGTTCCC CGAAATCCAA CGGCGGTATG 10020
CAGATGCTGC CGCCTTGGGC AGGGACTTCT ACGACTCCCA ACGCGCACTA CACCACCCAG 10080
AGCTGCCCCG GAACGAGAGG TTCCGGGGAG AGCTTCGGTG GGAGTGGTTC GTCCAGAACA 10140
TGGAGCCCGC TCGAAAAGAG ATGTCGCAGG CCGACTCTCC GCCGAGTGCG ACCTCTAAGT 10200
TGGCTCTGGC CGCAGTTCGC GAAGTGGAGA TGGCAGCACG CCGACAGATC ATCGGCGCTG 10260
TCAAGAACGA TCCGGCCCCG CAGATCGTGC AGGGCTGGGC GAGGGTCGCC ACCGGGCGCG 10320
AAACATGCGC CTGGTGTCTG ATGCTCATCT CACGGGGTGC CGAGCTGAAT CACAAGGGCA 10380
ACTTCGCCTA CAGCTCAGCG GAAGCCGCAG GGCTCAACCT CGATGACGAG ACCGTGATCG 10440
ACCTCTGGAA CGAGTCCGGT CACGACCTTG AGAAGTTCCG CGAGGAGACC AGAGAGGACT 10500
TCGAGAAGTG GCACGCAGGG TGCGACTGTC TGGTGGTCCC GGTCTTCGAT GTGCAGAACT 10560
GGCCCGGAAG AGACGCTGCC CTACGGGCGC AGCAACTTTG GATCGAAGCC AGCGACGAAG 10620
CTGACGACCT CATTGCGTCA GGCAAGGCCC GCTCCAAGAA CAAGAACACG GAGACGCTCA 10680
ACGCGCTCCG ACGCCGCCTA GCACGCGGCG AAATCACCAT GTCCAACTAC GCCCTCGCTG 10740
CGTAGTCCCT CGAACCCCAG GTGGGTTCTC TCAACATGCC CAGGAGGCGA AAACACATGT 10800
CCGACAACCC CACTCCCGAG AGCACCCCAG AGGCCGAGAC CCCGGAGGTC GAGAAGCCGA 10860
TGGAACCGCA GGGCAAGGTC TTCGATGAAG CGTACGTTCA GTCGCTTCGC CAGGAGGCTG 10920
CAGCCGCTCG GGTGGCGAAG AAGGACGCCG TAGAAGCGGC AGAGGCTCGA GTGAAGGCCG 10980
AGTACGAGGC CAAGCTCGCT GAGCGCGACA CCGCTTACAC CGAACTGCAG AACCAGTTGG 11040
GACAGGCGTG GATTGAGCTG GAGAAGGTCT ACCTCTCTCT CGACGCCAAG GTGCCCAACG 11100
ACAAGGTTCG GGCGTTTGTC GAGATCCTCG AAGGCAACGA CAGGGACAGC ATCGCTGAGT 11160
CAGTGAAGTC CCGTCTGGAG CTGGTCGGCG GATTCGGCAA CAAGACCCCG AGTCCTGCGT 11220
TCGACCCGTC TCAGGGTCGC GGCGGTAAGC CGCCGATCCC GCTGAACGGT GACCCGATCC 11280
TCGAGGCCAT CAAGGCCGCT GTCGGGATCA AGAAGTAACC CACCCAACAG ATCTCAAGGA 11340
GAGATAAACA ATGGCAGTCA ACCCTGACCG CACCACGCCG TTCCTCGGCG TGAACGACCC 11400
CAAGGTCGCG CAGACCGGCG ACTCGATGTT CGAGGGCTAC CTCGAGCCCG AGCAGGCCCA 11460
GGACTACTTC GCCGAAGCGG AGAAGATCTC CATCGTCCAG CAGTTCGCCC AGAAGATCCC 11520
GATGGGCACG ACCGGCCAGA AGATCCCGCA CTGGACCGGC GACGTGAGTG CGTCGTGGAT 11580
CGGTGAAGGC GACATGAAGC CCATCACCAA GGGCAACATG ACCTCGCAGA CCATCGCCCC 11640
CCACAAGATC GCGACGATCT TCGTGGCCTC GGCGGAAACC GTCCGTGCGA ACCCGGCCAA 11700
CTACCTGGGC ACCATGCGGA CCAAGGTCGC GACCGCCTTC GCGATGGCGT TCGACAACGC 11760
CGCGATCAAC GGCACCGACA GCCCGTTCCC GACCTTCCTA GCGCAGACCA CCAAGGAGGT 11820
CTCGCTGGTG GACCCGGACG GCACCGGCTC CAACGCCGAC CTCACCGTCT ACGACGCGGT 11880
CGCCGTCAAC GCCCTGTCGC TGTTGGTCAA TGCCGGCAAG AAGTGGACCC ACACTCTGCT 11940
GGACGACATC ACCGAGCCGA TCCTCAACGG CGCGAAGGAC AAGAGCGGTC GCCCGCTGTT 12000
CATCGAGTCG ACCTACACCG AGGAGAACAG CCCGTTCCGC CTCGGTCGGA TTGTGGCCCG 12060
TCCGACCATC CTGAGCGACC ACGTCGCCTC GGGCACGGTC GTCGGCTACC AGGGTGACTT 12120
CCGCCAGCTC GTCTGGGGCC AGGTCGGCGG CCTGTCCTTC GACGTGACGG ATCAGGCGAC 12180
TCTGAACCTG GGCACCCCCC AGGCTCCGAA CTTCGTCTCG CTGTGGCAGC ACAACCTCGT 12240
CGCAGTCCGA GTCGAGGCCG AGTACGCCTT CCACTGCAAC GACAAGGACG CGTTCGTCAA 12300
GCTCACGAAC GTGGACGCCA CCGAAGCCTG ATCCAGGCTT GACATCCACC GGGAGGGGGC 12360
TCCTTCGGGA GCCCTCTCCT GATGTGGAGC AGGAAGGACC ACATGCGAAT CCAGTCCACC 12420
CTCAACGGCG GTTTCGCCGA GGTTTCCGAG GAGTTCGCCA AGCAGTTGAT CGCCACTGGC 12480
GGCTGGAAGG TGCCCCGGAA ACCGCGCAAC ACCAAGACCA AGACCGCTCC TGAGGAGCCC 12540
AAGAACGAGG AGTAACCCGT GGCCTACGCG ACCGCCGAAG ACGTTGTGAC GTTGTGGGCC 12600
AAGGAGCCTG AGCCCGAAGT GATGGCGCTG ATCGAGCGCC GGCTCCAGCA GATCGAGCGC 12660
ATGATCAAGC GCCGGATCCC CGACCTGGAC GTGAAAGCCG CTGCGTCGGC GACGTTCCGG 12720
GCCGATCTGA TCGACATCGA AGCTGATGCT GTTCTGCGCC TCGTGCGTAA CCCGGAGGGC 12780
TACCTCTCGG AGACCGACGG TGCGTACACC TATCAGCTCC AGGCCGACCT GTCGCAAGGC 12840
AAGCTCACCA TCCTCGATGA GGAGTGGGAG ATCCTCGGGG TCAACTCCCA GAAGCGCATG 12900
GCGGTCATCG TCCCGAACGT GGTGATGCCG ACGTGAGCGC GAGCGACCGA CACCGCGCCC 12960
CGATTGTCTA TCCGCCTGGC ACTCAGGCGG TTACGCCGGA TCGGGTCAAC GCGTTTGACT 13020
GCGATCACGA AGCTGATCCT CCGGTGTGCC GGTGCGTCCA CGACTGGCGC ATCGAGTGGG 13080
GAAACGTCAA GAAGGCCACC GCCAGATCAC GGTCGGCGGT GCTCTGATGA GCCTCCTCGA 13140
CACCGGTGCC CGGTACCAGA CCTGCATCGT CTACCCCGAA GAGATGGTCA TCGACTCCGA 13200
TGGCAACAAG CGGACCAGGC CGTCGAATAC CGGCATCCCG GCCATCGCAC GGTTCCAGGT 13260
AGCCAACCAG TCTGGTACGT CGGCACGACG TGCTGAGCAG GACAACGAGG GGTTCGAGAC 13320
CGAGAAGGTC TACCGGATGC GGTTTCCCCG CTCGTTCACC AAGGAGCACG GCATCCTCGG 13380
GGCCCAGTCC CAGATCGAGT GGCGAGACCA GCGGTGGGCG CTCTTCGGAG ACGCCACCGT 13440
CTACGACTCA TCCCCTGCGT TGGCGCGGGT CGACTACACG ATCAAGAGGT ACTGATGGCC 13500
AAGGTCTACG CGAACGCGAA CAAGGTCGCG GCCCGGTACG TCGAGACGAG GGACGCCGTC 13560
CGAGACGAGC GGAACAAGGT CACCCGTCGA GCCAAAGCCA ATCTGGCGCG GCAGAACTCG 13620
ACCACCCGCA TCACCGACGA GGGCTACTTC CCGGCCACCA TCACCGAGCA AGACGGCGAT 13680
GTCGACTTCC ACACGATCCT CAACGCGCCC AACGCGTTGG CGCTTGAGTT CGGCCACGCG 13740
CCGTCTGGCT TCTTCGCTGG CACCGACACG AAACCACCGG AGGCCACTTA CATCCTCACC 13800
CGAGCCGCCA TCGGCGGCAC CGTCTCATAA GGAGGTCACA TGGCGCGAAT GCCTCGCGTC 13860
CAGGCAGTAG CGGCCCCGAT CCTCCGGTCA GACCCCCGAC TGGAGGGAGT GACGGTCACG 13920
ACATGGGTTC CAGACGTGGA CTTCCGAGAG TTCCCGATGA TCAACCTCCG CCGCATAGGC 13980
GGGACGAGGA ACCCCAACGC ACCGACGCTG CACACGCTGC CGGTGGTCGA AATGACCGCC 14040
TACACCAGAG ACGGTCTCAT CGAGACTGAG GAGCTGTACG AGACCGCGCT AGAGGTTCTC 14100
TACGACGCGG TGGAGAACGG AACACAAACT CCCGCAGGGT ATTTGACCTC CATCTTCGAG 14160
ACGATGGGCG CCACTCAGTT CAGCTCCCTC TACCAGGACT CCTGGCGCAT CCAGGGTCTG 14220
ATCAGGCTCG GCGTCCGCAG ACCGAGAACC ACCCTCTAAC CGAAAGGTAA AGCCACATGG 14280
CTGAAAACGA CGACGCAGTG TTGACTGCGG CGGTCGGCTA CGTGTACGTC GGTGCTGCAG 14340
GCACCGCTGC TCCTACGCCG GCCTTGCTCA AGACCATCGA CCTCAGCAAG CCCGAGACCT 14400
GGACCGGTGC TACCGGTTGG ACGAGCGTCG GCCACACCAG CCGAGGCACG CTCCCTGAGT 14460
TCGGCTTCGA AGGCGGCGAG TCCGAGGTCA AGGGCTCCTG GCAGAAGAAG AAGCTCCGCG 14520
AGATCACCAC CGAGGATCCC ATCGACTACG TCACGGTCCT ACTGCACCAG TTCGATGAGC 14580
AGTCGCTGGG TCTGTACTAC GGCCCCAACG CCTCTGAGAC TCCTGGTGTG TTCGGTGTGA 14640
AGACCGGCCA GACCAACGAG AAGGCCGTGC TGGTCGTGAT CGAAGACGGC GACATGCGCC 14700
TGGGGCATCA CGCCCACAAG GCTGGAGTTC GCCGCGACGA CGCGATTGAG CTGCCCATCG 14760
ATGACCTGGC TGCGCTGCCC GTCCGGTTCA CCTACCTGGA CCACGAAGAC GAGCTGCCGT 14820
TCTCCTGGAT CAACGAAGAC CTCTTCAACG TGCCCGAGGT TCCCGAGGGC TGATCCCAAC 14880
TTGACAGCCA CCCGGCTGTC TACCCCGGAG GGGGAGGTTT CCTTGGCGGG CCTGGCCTCC 14940
CCCTCCTCCC GCCACTCACA GACCCGCCGA CACTGAAAGG TTCGCCATGA CAAACGTATT 15000
CACCATCGAC GCATTCCGCG AAGAGGTCAA GAAGAAGTAC GCTCCGGTCC TCATCGGCCT 15060
GTCCGACGAT GTGACCGTCG AGCTGAAGCC GCTGCTGAAG CTGGGCCAGA AGGCCCGCGA 15120
AGCGGTGGTC GAGGTGTTCA AGGAGTTCGC GGACATCCCC GACCTCGAAG AGGACGACGA 15180
CGACGAGTTG GTCGATGAGT ACTCGCTCCA GGTCTGCGAC ATCATCGCCA AGGCGTTCCG 15240
GCTGATCGCC ACGAAGCCCA AGAAGCTGAT CGCCGCCTTG GACGAGGAGC CGGATCCCCG 15300
TATCCGCGCA GAGCTGTATG CAGCGGTACT CAACACCTGG AAGCGAGAGA CGCAACTGGG 15360
GGAAGCCGCG CCCTCGCCGA GCTGATCGAC AAGTTCGGCG GGGCGATCCT CGCAGACCTG 15420
CTCCAGTACT ACCGGGTAGA CCTGCGCGAC CTGTTCCGCG ACGAGGATCC GCTTTCGCCG 15480
AGATTCGTTC TGTCCCTGGT GCTCTGCCTT CCCAAAGACG GCGCGTTCTA CGCAGAACGT 15540
CGTGGTGGGC AGCAGTACCG GGGCTGGACC GAGGACCGCT ACGCGCTCGC GGACATCTAC 15600
GACGCCATCC AGGCGGGCAA CCACATCCTG CTGCTGGCGA ATCGTGATCC GAAGAAGCCA 15660
AAGCCCAAGG CACCCAAGTC ATACCCGCGT CCCGACGACC TAGAGAAGAC CACACCGAAG 15720
CCGGGTTCGT TCGCCGCAAT GGTCGTGCGA GCGAAGAAGG CGGCTCGAGA GAGAAGGGAA 15780
AGGGAGGAGG AGAGTGCCGA ATAGTGCTGG CGTAGAAGTC GCCCGGATCT CGGTCAAGGT 15840
CAGCCCGAAC ACCAAGGAGT TCCGCCGGGA ACTCAAGACC GAACTCGAGA AGATCGAGCG 15900
GGAGCTTAAG GGCGATGTCG AGATCAACGG TCATCTCGAT GCGGCCCAGG CCAAGGCCGA 15960
CTTCAAGCGC ATGATGATGC AGCTCAAGAC CGAAGCTGCC AAGGGCGTTC ACGTCCCGGT 16020
CGACGTAACC GTCGACAAGA AGAGCAAGAA GGGAGGTCTC CTCGGAGGTC TCCTCGGCGG 16080
CAGCCGGGGG CTCGGAGATC TAGGCGATGA CGCCGAGAAG GCGTCGTCTC AAGTACAACA 16140
CCTTGGCAAG TCGTTCCTGG GCCTCACACG AGCCGCCTGG ATAGGCGTAG GCATCGTCGC 16200
CGTAGCAGCT CCGCTGGTCG GCATCGTGGC CGGTCTGCTG GCCGGTCTGC CGTCGCTGCT 16260
GTCTGCGTTC GGAGCCGGCG CTGGCGTAGT CGCGCTCGGC ATGGACGGCA TCAAGGCAGC 16320
CGCCTCGACG CTGGCCCCGA CGCTGGAGAC GGTCAAGGCC GCTGTCTCCT CGACGTTCCA 16380
GCAGGGACTC ACCCCGGTGT TCCAGCAGCT CGGCCCGATG CTGACCGCGA TCACCCCCAA 16440
CCTGCAGAAC GTGGCCTCGG GCCTCGTGAA CATGGCCGGG TCGATCACCG ACGTGATCAC 16500
CCAGGCTCCT GGTCTGCAGC AGATCCAGAA CATCCTCACC AAGACCGGAG AGTTCTTCAC 16560
GGGCCTCGGC CCTGTGCTCG CTACCGGCAC GCAGGCGTTC CTGACGCTGT CCAACGCCGG 16620
CGCGAACTCG TTCGGCACGC TCCTGGCTCC CCTGCAGGAG TTCACCAACG GCTTCAACGA 16680
CATGGTCAAC CGAGTCACGT CCAACGGCGT GTTCGAGGGT GCCATGCAAG GGCTTTCGCA 16740
GACGCTGGGC AGCGTCCTCA ACCTGTTCAA CCGGCTCATG GAGTCCGGTC TGCAGGCGAT 16800
GGGACAGCTC GGCGGTCCGC TGTCGACGTT CATCAACGGG TTCGGAGATC TCTTCGTCTC 16860
GCTGATGCCG GCGCTGACTT CGGTCTCTGG TCTGATCGGC AACGTCCTCG GGACGCTGGG 16920
CACACAGCTC GCTCCCATCG TCACGGCGCT CACGCCGGCC TTCCAGACGC TGGCGAGCAC 16980
GCTCGGCACG ATGCTCACCG GAGCCCTCCA AGCTCTGGGT CCGATCCTGA CTCAGGTCGC 17040
TACGTTGATC GGCACGACGC TGAACACGGC GCTGCAGGCT CTCCAGCCGA TGCTGCCGTC 17100
GCTCATGCAG AGCTTCCAGC AGATCTCCGA CGTACTGGTG ACCAGTCTGG CCCCGCACAT 17160
CCCGGCGCTG GCGACGGCCC TCGGCCAGGT CGCAGGCGCG GTGCTGCAGC TCGCTCCGAC 17220
GATCATCTCG ACGTTGGTTC CGGCGTTCGT TCAGTTGGTC CCAAAGGTCG CTGAGCTAGT 17280
TCCGACCATC GTCAACCTGG TCCAGTCGTT CGCCAACCTG ATGCCGGTGG TTCTGCCCCT 17340
GGCGCAGGCT CTGGTCAGCG TTGCTGGCGC GGTGATTCAG GTGGGTGTCT CCATCGGCGG 17400
CGCGCTCATC GGCGCGCTGG CGAACCTCAC GGAGATCATC TCCAACGTCA TCAAGAAGGT 17460
GTCCGAGTGG GTCAGCAGCT TCTCCAGCGG AGCCCAGCAG ATCGCTGCGA AGGCAGCGGA 17520
ACTGCCGGGG ATGATCCAGT CGGCTCTCGC CAACCTGATG GCCATCGGCC TGCAGGCCGG 17580
TAAGGATCTC GTCCAGGGCC TGATCAACGG CATCGGCGGG ATGGTCAGCG CAGCGGTCAA 17640
CAAGGCCAAG GAGCTGGCGT CCAGCGTGGC TGGTGCAGTG AAGGGCTTCC TGGGCATCGA 17700
GTCCCCGTCG AAGTTGTTCA CCGAGTACGG CCAGTTCACC GCCGAGGGAT TCGGCAACGG 17760
CATGGAGGCA GGGTTCAAGC CCGTCATCGA ACGGGCCAAG GATCTCGCGG CTGAGCTGTC 17820
CAGGGCGATG GAGTCGGGCA CCGACCCCTC CGGGATTCTC GCTGGGCTGG ATCAGAATGA 17880
GCTGAAGCAG ATGCTGGCGG CTCTCGAAGA GGAGCGCAAG CGACTCAAGG TCGAGAAGAA 17940
CGGTATCCCC AAGGGAGACA AGGCAGGCCG AGAGGCGCTG CAGAACCAGC TCGACCAGAT 18000
CCAGGCGCAG AAGGACATCC TGTCCTACCA GCGTGACCGC ATCAAGAACG AGTCTGAGTA 18060
CGGCGACATG GCCGGCGAAG ACCCGTTGGT GAAGGCAGCC TCCGGGCTGA TGAGCGCACC 18120
GGTCGACTTC GCGAAAGCGA CTGGCAAGCA GTTCCTTTCG GACATCGGCA TCAGCGGAGA 18180
TGGGTTCATC TCGAAGGCCA TCACCGAGGG CATCCAGTAC ATCTTCCAGA TCGGCTCTGT 18240
CGATGAGGCG CTGTCGATCA AGGACCGCGA GGAGTCGAAG AACGCGCTGT CCGTCGTTGG 18300
CCGCTGACTT GACATCCACC AGGAGGTAAG CATTGATCAC CGACACCATC GTTGAACTCG 18360
AGGGTGTCAA TGGTGAGCGT TTCAACTTGA CGACCGGTGA CCAGGGTGTG TACCTGGCCA 18420
CAGACGTGGA GGGTTGTTTC TACGACCCTC CCGTCAAGGT CGTTGTTGAA GAGCCGGGGA 18480
ACTACCCCGG CGCTCGCTAC TTGTCCCACC GAGCCCTGAA GCGAGACATC GTCTTTGGGG 18540
TCGTCATCCT CAACGACGCG AAGCAGGGGC CGCGCTCCTG GCTGTCGCGA GACTCCGAGT 18600
GGCGCAAGGC GTGGGCGTTC AACCGCACCT GCAAGCTCTA CGTCACCACC CCGGACTCCG 18660
GTACCCGCTA CCTGAAGCTG GCGCTGTTCG AGTCCCCCAC CGTCAAGATG GACACCGACC 18720
CAAGAGGTAA ACCCCTTGAG GTCACGGTGA TGTCGTGCAT CGCGTACGAC CCGTTCTGGT 18780
ACGAGGACGA CAAGGTCTTC TCGGCCAAGA CCAAGACCGA CACCCGGTTC GACCCGTCGT 18840
TCTGGACGCC GCCGTGGCCG TGGGAGGAAC TGCCCAAGGA GACGCTGCGG ATCAAGGTCG 18900
GCCGCGAGCA GGGTGGGCTA AACCCCACCG ACCAGTACAT CTTCCCGAAG TGGACCGTTC 18960
CCGGCTCCAC CGAGAAGGTG CCGAACTTCC CCTGGCCGTT CCCCCCGAAC GTCCCGATCC 19020
CGTGGGAGAC AGCACCGTTC ACTCAGTTCG TCATCCCGGA CTACTCGTTC GAGGATGAGG 19080
AGTTCCGCAA CCGCCGGCTC AAGACGCCGG GGTTGATCTA CGGCGAGAAC TGCGTCATCG 19140
ACACCGACCG GCGCGAGGAG CAGATCGCTT CCGAGTCGGG CTCCCCGGTG TGGGCTCGGA 19200
TGAACGGTGT CCGGTTCCGC AACTCGATCC CGCCCTACAC CGAAGAGGCT GAGTTCGTCA 19260
TAGACGCATC GGGATGCGCT CCGGGACAGG TAGTTACCCT CCGGCTCACG AGGCCGTGGT 19320
CGCGCTGCTG GGGGCTAGAG TGAGTGGTCT GACGAGCGTT CGTGAGGCCG AAGATCTCTG 19380
GCAGAAGATC CAATTGCGGC GCTGCAAGCG CGAGCAGGAA CGGCTCAAGC ATCCCGACGT 19440
AGAGCTGCGC GATGGCGACT TCCGCCTGCG CGGCCTGGTC GCTGGCGAGC GGGTGCTCGA 19500
GTGGGAGTTC ATCGAGAACG AGACTGGCAC CTGCACCTTG CAGCTCTCAC TGAGCCATTA 19560
CCTGGCGAAG TGGGTGATGG ACCACCGGGG TCGAGCAAAG CGCAACGTCA TCATCAACAT 19620
CGAGAAGCAA GGCGCTCGAT GGACCGGGAT GATGGACCAC TACCGGGTCA TCAAGACCGA 19680
CGCAGGGGAC GCCTACATCG AGATCGTGTT TTTGCACGAC TTCGAGCAGA CCAAGCATAT 19740
CCGGGTATGG TGCAACCCGT TCCTACGCCC CGAGCTGCAG TTCCCCAAGG TGTGGATCAT 19800
CTTCGGGCCG GCCAAGTGGT GTTTGCTGGT GACACTGTTC GTCAACCTGC TCAGGCTCGA 19860
GACGAGCTTG TGGACGCTGC CTGATGACCC CACGGACATC AACGAGTGGA TGGGTCCGAG 19920
CTTCAACCCA GCAAATTGGC GGAACATCGT CAAGCCGTTC CCGTTCCTGG CCGACAACTC 19980
ACCGGTCACG ATGGTGTTCA GCCGGTTCGG GACGTTCTAC GACACCGCCA AGAAGATCCT 20040
CGAAGACCAT CAGCTCACGC TGACGTGTCG TCGGTACATC AAGGACCGCG ACCCGCATCC 20100
GTTCGAAGAT CTCAAGGGGC TCTGGGGAAT TGATCCTGTC GAAGACCTGC TGCAGAAGAT 20160
CCCGCTCCGG GACGGCTGCG TGGTCTGGGA CATCGAGGAC AACTCAGGTT GGGGCACTCA 20220
GACCGCGTTC GGCGGTTCGT GGCTGACCGG GTTCGTCCGA GGGATGGTCC AACTGGCCGG 20280
CGACGGCCAG GTCGAGGGCG TCGATGTGTT CACCGGGGAC TACACGTTCC CAGGCGAGTA 20340
CTACTCCCCC TGGTTCATGG GCACCAGCCC GATAGCACCC CACGTCGTGT TCGAAGAAGG 20400
ACCGCTGACC GGGATCAAGT CGTCGGAGTT CTCGTACTAC GAGGCCACCG ACACCAGCTT 20460
CCTGGCTGGT GGACAGAGCG CACCTGGCAT CAACGAGGGC ATCTCGGCCC TGGTGAACAT 20520
CGGTGGCGAC CTGCTGACCT CGTTCATCAA CAGCCAGCTC GCCGCGCTCG GCGCGGTCGG 20580
TGGAGCGATT GACCTCCCGC CTCTGGGCGG TCTGCTCGAT GCGGTGTTGC AGCCTCTGTA 20640
CTCCGATGTG TTCGGCGCGT TCATGGAAGT TCCGACTCTG CGTGCGATGG GCATCTCGCT 20700
CCCGATCTCC GGGCTCGAGG ACATCGTCAC CGGACTGGGC GACTTCCACT ACTTCGAGAA 20760
CATGGCCGAC GGGGCGATGA AGGCGTTCAC GCTGTCAGCG TTCGCAGCCA TCGCATCGCA 20820
GATCCACAAG ACGAGGGCTC GAACGACCCA CACCCTCAAG GTGTCTGACG CCGCTCCGTA 20880
CATCTTCGCG CCAAAGCCCT ACGGGCACTG CTGGATCGGA GATCGCGTCG GCACGTCGGT 20940
CCTCGGCTAC CCGGTCGAGC ACCAGTTGTT CGTGGAGCGC ATCCGCAAGG TGAAGTACCG 21000
CATCGACAAA GACGGCATGA AGCCGTTGGA GATCGAGATC GGTTACCGCG AACCGAAGAA 21060
CCCAGCACTA CACATCCTCG AAGAGATCAA GCGCGTCAAC GGCGCTCTTG GCACTGCGGG 21120
GATTCTCTAA ACCGAAAGGC ACGCCGCATG ATTCCCTCAC AAGAGTCTCA CAATCCGAAC 21180
GACCCGCGAC AGCACGTCAT GTGGGCGCTA CGCAATCTCC CGATGATTGC TGGCGTCGGG 21240
GCGATCACGC ATCCGGGTTA CCTGGCGGAT TGGTCAGAGC ACTTGTGGAA GTGCGGCTTT 21300
CGGCACGTCG ACTGGCTCCG GGAGCTGGCT GATGAGGACG GCAACATCCA CGTCAGTCAG 21360
CTTCCTGACC AGGAGATCAA GTTTCAGCAG CCCTTCCGGG GCCAGCGAAG CGACTACAAC 21420
AACGCAGCTC GATGGGTCGG CAAAGACGAT CCTGACCCAG AGCCCGTGCG TATTCCAGAC 21480
ATTCGCAAGC TCACAGACCA GGAGAACAGA GCGATGATCG CGCAGTACGA ACGAGACGGT 21540
TGGATCAAGG ATGGATCCCC CGGCCCAGCG ATAGCCGAGG TCGTGGAGTG ACCCCGTTCA 21600
ACCCAGACTC CATAGGCGAC TACGTGACAC TGCTCGGCGT TGCGTTCCTG ACCTTCTCGG 21660
TTCCCGCATG GTTCACCGGA CGAGCACGCA AGCACAGCAG TGACATCGGC GAAATCAAGG 21720
AACAGGTATG TAACACCCAC GACACGAACC TGCGCGATGA CCTCGACAGC GTCAAGGCAG 21780
ACATCAGCGA CTTGAAAGAG ATTGTGTTGC AAGGGTTCCA CCAGGTGAAC GAGTCGATCA 21840
ACCTCGAGCG CCGTGAGCGG ATCGAAGGAG ACCGCCGAAA GGAGGTTGCG TGACCTACCC 21900
CACCAACCCA CTAGAGGCCA TCGGCGCTGA CGGCGCATTC GAGATCGGTG GGGGCGACTG 21960
GAGCTTCGGC CAGGACTACA CCGAACAGGC CATCCGGGCT CTGTTCACGA TGCCAGCGGT 22020
CACGATGGAG AACGCTCTCG GCCTGCTCGA AGAGCACCTG CTGAAGCTGC CTCTGGAGGC 22080
GCTGCAGGGC TTCAAAGACA TGATCCCGGA CTGGGTCGAA GGAGCATTCG ACACGGTCAC 22140
CGGCGCTGTG CAGGCGATCA TGAACGCGCT CCAAGACGGC CCGCTGTTCC TGAAGTTCGC 22200
CGAGTTCCAG CTCTTCCTGC AGCGTCTGCT GAACAACCCG GCCGAGGTCA TCGGCGAGAT 22260
CCCCCAGACG TTGATCGACG GCCTACAGGA CGCGCTCAAC ACCGTCAACA ACACCATCCA 22320
GACCATCGTG GACATGCTCC TGCAGGCGCT GGGCATCACC CCGGAGGGGG AGCTGATCGA 22380
CCGGATCTTC GACCTGAGCG ATGAGATGGA GTGGCTGCAG ACCGCAGCCT CGAATGCAGC 22440
TACCGGCATC CAGGACACCT GGAACAAGTT CTGGGGAGCC CTCACCGGGC GCGTCCCAGA 22500
CCAGGACCAG ACCGTCGCTG AGCCCGCCGA GCGTATCGGC GAGCTGGCCG GCACCACGTC 22560
TGCTAACTCG TCTGCCATCG CGGAGCTGCA GCGTCGACTG GACAACCAGC AGAACGCTGG 22620
CGGCGTGGCC GGCGGTGACG ACTTCGAGCG ACTGAACATA TCCGGTTGGG ACATCAGGTA 22680
TTCCAACGGA TCCAGCGGCC GAGGGTACTA CCGTGCCGAC GGCCACCAAC TGGTCTGGAT 22740
GGACGAAGGC AACCAGCAGA ACACCGCGAC GTTCGTCCGC ACCAACCCCG CAGACGAGAA 22800
GACAGCCACC GACTACCAGA AGATGACGTT GGTCGTCGGG ACTATCTCCG GTGAGGTACA 22860
GACCGTGTTC CCGCCGCAGG GAGGTTCGCA CACCCGGCTA TGGGTCCGCG TCAACGACAA 22920
CGCTCCGACC GTCGGCATCA CCGACGGCGT GTTCGTAGAG ATCGGCGGCG TATCGAAGGC 22980
CCAGATCGGC TACCGCCGCA ACGGCAATGA CACGTTCGTC GGATCTATGG TCGACTGCAC 23040
CTGGGGTGCT GGATCGATCT TCGCTCTGAC CGCCGGCACG GCCAACGGTG CTGAGAAGTT 23100
CGAGGTCTCG AAGAACGGCC CCGTGCTGGC CACATGGTCG GACGACGGCG TCGTCTCCGC 23160
GATGGGTGCG AACTACCGCC GCTGGGGCTG GGAAGGCCAG GCTCGTAACC GCAACCTCGG 23220
CCAGGGCACT CCGAACTCGG TCACCCGAGT GACGATCACC GACAACGATC CTACCGGCGC 23280
AGGCGGTGGA GCTGTCAACG TCGGAGGAGA TGTCGTAGGT GTACTCCCCA TAGAGAACGG 23340
AGGCACCGGA GCTTCGACAG CTTCGGCAGC CCGTACCGCT CTCGGAATCG ATGACCTGGT 23400
CGAAGATATG TCCGACGTAG TTCGTGGATC CGTCGAAGGA CTCCCGTTGA TACCGAAGAT 23460
CTGGGTAGGA ACAGAAGCTC AGTACACGGC TCTCGCCACC AAGGATCAGT CCACGCTATA 23520
CTTCAGGACC GCTTAATGAC TGGTATCTCG TTGGGTGTCA ACGACATCCG CAACCTCTCG 23580
ATATTCTTAG GCGTCAGCAA CAAGATATTG AAGGTCAGTC TAGGCACAGA AAAGGTCTGG 23640
CCTGCGTTCA CCCCGGTGCT GACCACGTTC GCCACGGTCG GCACGTACAC CTACAACATC 23700
CCCGACGGGG CCAAGTTCAT CGACGTCATC CTCCTCGGAG GAGGCGGCGG GGGTAAAGGC 23760
ATGGCCCTGG CTGACGGCTG GGGCAGAGGT GGAGACGCCG GAAGCTGGGC TATCGTCACT 23820
CTCGAACGCG GGGTACACAT CCCGTTGTCG ACCAAGACGA TCACCGGGCT CGTCGGAGCT 23880
GGAGGCGCAG CGGGAGCTGG CTCTGTATTC TCAGGCAAGG CCGGAGGCCC TGGAGGAAAC 23940
ACCACGGCGT CCGCTGTCGG ATGGTCAGGT TTGACCGCAA CCGGCGGTCC CGGAGGCTCT 24000
GTGATCGACA TCCTCAGCGT CGCCGGAAAG TCGCCTGGAG ATCGGACCTA CAACGACCAG 24060
CTCTACATAG GCGGCGCACA ACAGAACTCA GCTGGCGGGA ACGGCAATGC TCCTGGCGGC 24120
GGCGGGGCTG GTGCCCAGGT CTCCGCACAG AGCGGCGGTG CTGGCGCTCG CGGCCAGGCG 24180
TGGTTCTTCG CGTACTGACA AGAAACCCCC CTCTTTAGGA CTCAGTGTCC TTGGGAGGGG 24240
GGCTTTTTGC GTTTCAGGAG GTCTTGGCCA GCTTGGACAT CGCCTCAGCG ATAGCCTCGT 24300
CGCGGGCCTC AGACGCCATC TGGTACTTCA TCGCCATCCT AGGAGTCGTG TGACCGAGAC 24360
GGGCCATCAG CTCCTTGGTC GTCGCACCTG CCTGAGCGGC GAACGTAGCG CCGACAGCGC 24420
GGAGGTCGTG GATGCGGAGT TCCGGCCGAC CGATCTTGGC GTAGCCACGC TTCAGCGACT 24480
TGGTGAACGC GGACTTCGAC AGCCGGTTGC CCTGCGTCGT GGTCACCAGG AATGCCTCGG 24540
GGCCCTTGTT CATCTTCGTA CGGTCCTTCA TGTGCGCTCG GATCATCTCC GCGACGTGAG 24600
GCGGAACCGT CACAGGACGC TTCGACCGGA CGGTCTTGGC GTTGCCAACG ACGATCTTGT 24660
TCCCCACGCG GGAAGCGCCA CGGCGCACCC GGAGCTTCAT CGTCATGCCG TCGTCCACGA 24720
TGTCCTTGCG GCGAAGCTCG ATCAGCTCTC CGAACCGGAG GCTCGTCCAC GCCAGGATGT 24780
ATGCCGCGAT CCGGTAGTGC TCGAAGATCT CAGCGGCGAC GATGTCCAGC TCCTCAGGCG 24840
TCAGCGCCTC TACGTCGCGC TCATCGGCTG CCTTCTGCTC GATCCGGCAC GGGTTCTCTG 24900
CGATCAGCTT GTCCTCGACC GCTGTGTTCA TCACCGCCCG GAGGACGTTG TAGGCATGCC 24960
GGCGGGCAGT CGGGTGCTTC CTACCCATCC CGGCCCACCA CGCACGCACC AGAGCTGGCG 25020
TCATCTCTGT GACCGCCACT TCACCTAGCA CCGGGTAGAT GCGGCGCTCC GCGTGCCCGC 25080
TGTACAGATC CCTGGTGCCG TCTGCGAGGT CGCGCTCCAC GAGCCACTTC CGGGTGTACT 25140
CCTCCAGCGT GATGGCGCTG GCGGCTGCCT TCTTCGCCCG GTCCTGTGGA GGGGTCCAGG 25200
TCTCCATCTC GATGAGCCGC TTCTCGCCCG CGAGCCAGGC TTCGGCGTCC ATCTTGTTGT 25260
CGTAGGTCTG CAGCGCGTAG TACCTCACAC CGTCCTGCGG GTTGACGTAT GAGGCTTGGA 25320
TCCTCCCGCT GCGCTGAGTC TTCAGCGATC CCCATCCGCG ACGTGCCAAC TAGGTCTCCT 25380
CTCGTCGTGA ACAAGGCTAC CGGGTTGCAA CTCCTGTGCA ACTCTCAGGC TTCAACGCGC 25440
TTCTACGACC TGCAATTTCT TTCCACTTAG AGGATGCAGC CGAGAGGGGG TAAAAACCTA 25500
TCTTGACCGG CCCATATGTG GTCGGCAGAC ACCCATTCTT CCAAACTAGC TACGCGGGTT 25560
CGATTCCCGT CGCCCGCTCC GCTGGTCAGA GGGTGTTTTC GCCCTCTGGC CATTTTTCTT 25620
TCCAGGGGTC TGCAACTCTT GTGCGACTCT TCTGACCTGG GCATACGCGG TTGCAACGCA 25680
TCCCTGATCT GGCTACTTTC GATGCTGACA AACGAATAGA GCCCCCCGCC TGCGCGAACA 25740
GACGAGGGGC ATTCACACCA GATTGGAGCT GGTGCAGTGA AGAGAATAGA CCGGGACAAG 25800
GTTGCACCGG GAGTTGCAGC GGTCGGAACC CTCGCCGTCG GCGGGCTGGC GTTCGCCCTG 25860
TCGTTCACGG CTCTCAGCGA GCTGGCTGCG GCCAACGGGG TGGCCCAAGC AGAGATGGTG 25920
CCCTTGGTGG TCGACGGCCT GACGCTCGTC GCCACGGTCG CCACAGTGGC CCTCAAGCAG 25980
AACAGTTGGT ACGCGTGGTC GCTGCTGATC CTGTCCACCG TCGTATCGGT GGCCGGCAAC 26040
GTGGCACACG CCTACCCCCA CGGCATCATC GCGATGGTGA TCGCTGCGAT CCCTCCGCTC 26100
TGGCTACTGG CGTCGACCCA CCTAACCGTG ATGCTGGCGA AGCAGCACTC GGAGCACGCC 26160
GAAGTACCTG TCTCGCGGCC AGAACCCGCG CCTCGGGGCC TGGAGCCCGC TGCCGCTTGA 26220
CTGCGCCCGA CCGGGACAGA AATACATAGA GAACCTATGG ATGTAGGAGG CACAAAAAAA 26280
TACCCCCCGA GCCAGCCCGA AGGCCAGCCC AGGGGGCATG GTTCTGCTTC AGTAGACCTT 26340
GCGAGTCCGA CCCGAGTTGA TCATCGCCAT GATGACCCAG ACGGGCAACC ACATTCCGCA 26400
GGTGATGAGC GAAAGCAACA GGTGCATCGC GTGGTTCGTC CTGACAGGCA TGACAGTGGG 26460
CTGCGGCATC GGAGGAGGCG CGACCGGGTA CGGCGAGCCC GCGTACCACT GAGGTCGATC 26520
TTGTTGGGGC GGATACTGAT TGGTCATCCC GACAGCCTAC TTGCCGATGG GTCGCATCAG 26580
CTCCTCGACC GACTCGCGCT CCACGCGGAT CAGCCGGGGA CCGAGCCGAA CGGCCTTGAG 26640
CCGGCCGTCG GCGATGTAGT TGCGGACGGT CTTGGTGCTG ACACCGAGGT AGTCAGCGGT 26700
CTCCTGGATG GATGCTCTCG GGGGCATCAG CGCGGTCCTC CGTGCTTCAT CGGTTGTCTC 26760
CCGAACCCTG GATCACGCCA CGATCCTTGC GGCTCTGGAG CTTGTTGAGG TTCCTCTGGG 26820
TGACGGTGCT CAACCAGACA TCGAGCTGGT TGGCTAGCTG GGCGACGTAC CACATCACGT 26880
CTCCGAGTTC CGCCTGGAGG TCGTCTCGGT TCTCCTGGGT GATGACACCG TCTTTATCCC 26940
GGAGGATTTT CTTGACCTTG TTGGCGATCT CGCCGGCTTC GCCTACGAGA CCCATCGTCA 27000
CGTAGGAGAG ACCCTCGATG CTGTCGCAGT CGCCTGCACC GGGGTAGATC GCTGTGTCGC 27060
TCGCGGCGAT CTGGTAGATG TCGACGTGCA TCAGATCATC ACCGGGAACA ACTGGCCACC 27120
GGGCATCTGG ATGAACACCG GGACGCTGGG GGTGTAGTCC GACGAACCCG TGCCGCCCTC 27180
ACAGGCGGAC AGGCTCAGGG TGGCGGCAAG GCCGATGATG GCTGCTGCGA TGGTCTTCTT 27240
CATCTGTTGC TCCAGTAGCT AAGTTCGGAC TCCAGTTCGC GGATACGCTC CTGTAGCCCT 27300
TGGTTTTCCA GGTACGCCTC GGCGAGGTTG GCCTCGGCGC GGTCACGGGC CTCGTCCTTC 27360
GACGTGGCCT CATCGATTGC CTCGTGTAGC CGGCGGATCA GATCTGGGAT GGCACCGTGC 27420
AGACCGCATA TGAAGTCGGC GTCTGCCTCG GAGAGGTGGG ACGCCACCAG ATCCTTGTCC 27480
TGGGTCTCCT GGTTGACCGC CCAGATGACG TGATCCTCTA GCCCGTGGTC GGTCTCGCAG 27540
ATAGAAGGCG GTTCTACCTC CTCTGGCATC CAGTAAGTCT TCTCAGCCCC GGTGGACTTC 27600
GCCCACTGCT GGTAGAGGAT GTCGAAGAAC TCGTGGTCCT GTTCGTCGGC GGTAATCACA 27660
GATCGTCCTC TTCATCCCAT TCGTCGTAGT AACACGTACA GCCGCAGCAG GTGCAGCAGC 27720
CGCACTCGTA GGTGCCGTAG TCGTAGTCAT CCCAGTCGTC TTCGTCCATC TAGCTGTACT 27780
CCTTCATGAT TCGGTCGAAC GCACGCGTCT GCACGCGCAT CTCCAGGTCG ACCGTTCGCT 27840
TCAACCACGC CCATTCGCCG TCGTGGTTGA TCTCCCACTG GCTCTTGAAT GTCGCTGTCT 27900
CAACGAGGAA CTCGACAGTC AACGTGTGCA GTCCGTTGTT GCTGGGCTGG AATCCGATAC 27960
CGTCCTCAGC GATGTACCAG GGCAACTCCT GGCCGTCGAA GTAGACGGCC TTGTCGGTCA 28020
CCAGTACTTC AGGGAAGGTG TGCTCGGTCA ACGGCGTCCC AGGTATGGGA TGACGCTGGC 28080
CCGGAACTCA AGGAACACCA TGTTGTCCGG GCAGTCCTCG GGGACGTTGT CGGGGCGTTC 28140
GGCGGTGTAG ACGCCGATCT CGTTGCCCTC CAGGGTTCCA AGCTCGTTGA GCTTGTAGAT 28200
CGCCAGACCC ATCAGCTCTT CATCGAGACC GTTCGGTGCT GGCAGTACAA CTTTGGCTTG 28260
TGGCATTAGC CCTCCCTCGG AATTACGTAT GCGCTGAACT CGACGGCCGT AATGCCGTCT 28320
GGCAGTTGGA ATCCGAACCG CTCTTCGAAC TCCTCGTTGG TGATGGGGCC GTACTCGAAG 28380
GTTCCGGGCA CTACCTCGCC CTCCCCCTCG ATCAGGAGGT ACGCACCGGC GGCGTACACC 28440
TCCTCGTCGT TCGGCCATCC GACTACGGTC CCGAGGACCG TGAACTTCCT CGGCTCCATC 28500
AGGGCACGTC CACTTCGTTG ATGAGGAACC GCATCGGAGG TGGAGTGAGC ATTGCCTCGG 28560
CTATGGCGAT GAGGGCGTTC AACTGACCCT TCAGCAGCTT CTCCTCGTCG CCTGCGGGAA 28620
GGTGGCGCAC TCGGCGCTCC ATCTCCTTGG CGCGTTCCAG ATATTCGGTG GCTGTCAAGT 28680
TGTCCTCCTT AGTAATCAGC GCCGTAGAGC GAACCCCACG AACGCTTTCC GACCTCGGGG 28740
TCGGTGCCAA CCAGCACCGG ACCCATCTGT TCTTGCATCA GGTGGCCAAT GTGTGCAGCG 28800
GCTCTCTCAG CCTCTGAGGC GGGCAGAGAC GCGACGATCT CGTCGTGGAT AGGCAACCGT 28860
AGGTACGGGG TGTATCCGGC CTCGTGGAGG CGAATCAGAG CCCGACAGGT CACGTCCCGC 28920
GACGACGACT GGATCATGTA GTTCAGCGCG GAGTATGTCC GCGAGCTGTC CACCGGCAGC 28980
CGCCGGCCCA TCGCGTTGAC GATGTAGCCG TTGCGGCCAG CTTCCATCGC CAGCTTCTTG 29040
CTCAGCCGCT CCACACCGGG GTATGTCGCA GAGAACGCCT CATGAACTCG CTTGGCCACA 29100
GGGATCGAGA TCCCCACTGC CTCAGCGAGA GCCTTCGCCC CACCGCCGTA GACCTTCTGA 29160
AAGTTGGCGG TCTTCCCAAC CTTTCGCGGC ACCTGGGCTG CGTCAGCGGT CATCTGGTGG 29220
AGGTCCGCAC CGTTCTCGAA TGCCTCGATC ATGTTGCGGT CGCCCGACAG CGCCGCCAGG 29280
ACGCGAAGCT CCTGCGCCTG GTAGTCGACT GAGGCCATCA CATCGCCTGG CTCAGCGATG 29340
AAGCATCGCC GCACGATCCA GTCCGACGAC GGCAGCGTCT GCGCCGGGAT GCCGGTGATC 29400
GACATGCGCG AGGTCCGCGC CTGCAGTGGG TTGATGAACG TGTGGCAGCG GTCCTCAGAG 29460
TCCCTGGTGT CGATGAACTT CTGGACCCAG GTCTTCCGCC ACTTCCCCAG CTTCTTAGCC 29520
TCCTGAGCGA TGGCGGCAAG CTCGTTGCCA TCTTCGACCA GCTTGTCGAG CAGAGCCGCG 29580
TTGACCTGGC GCTTGCCAGT CTCGGTGCGA CCGGTGATCT TGACGCCCAT CTCCTCAAGC 29640
CCCTCGGCCA GATCCTCGGT CGAGTTGACC TTCTCCACGC CGTACTCGGT GAAAGCGATT 29700
GCCTCCCAGA CCTCCTGATC GGCCAACCAC TTCTCGGCGA GCGACCGCGA GTACTCCACA 29760
TCGAGCAGGA AGCCCTGCCT GTCGATGTAG CTGCAGATCT CACTGATCTT GTGCTCGTAC 29820
GGCACCAGCG ACCGACTCAC GTCGGGCACC AACGGTGTCA GGCTCTTGCA GACCCTCGCG 29880
GTGAAGATCG TGTCCATCCC GGCGTACAGC AGGTACTCCG GGTGGAACAG GTCGATGGTC 29940
GACCAGATCT TGGCCTTGGT CGTCTTGTGC TCGGCGGCTA GCTTGGCCAT GAGCTTCTTG 30000
ACGTTCTCGG CCTGGTCCTC GGAGATGAAC TTCGCGATCA GCTCTTCGAG CGAGTGCCCG 30060
AACCCGCCGG CCTCGAAGGG CCGGGGGTCC ACCAGCTTCG CCAGGATCTG CGTGTCAAGC 30120
ACGCGGGGCC ACAGACCCTC CATCTCGATC CCGAAGCACT GGTCGAGCAC CTGGAGGTCG 30180
AAGGAGGCGT TCTGGAGCAC CATGCGCTTG AGAGCGCCGA TGGCGATCCG CACGTCCTCG 30240
ATGAACACGT CTCCCAGCTC CACCGGCACC ACCCAGGCTT CGTCCTGAGT ACCGAACTGG 30300
ACGAGGCGGC ACTCGAAGGT GTCGCTGTAG ATGTCCAGCC CGGTGGTCTC AGTGTCGACG 30360
GCGAGGCAGT TCAGGTGAGC CCGGATGAAG TTGCGGAAGC CTTCCAGATC CTCTGGGGTT 30420
TCAACGACGT TGACGGTGAC GAGGTCTCCC TGAACCTCAT GCCGCAGCTC GATCAAAATG 30480
CTCTCCTACT GGAAGTACTG AGGCGGAATC CAGGTGGCTG AGGCCATCTC CTTGATGGCC 30540
TGCTGCATGG CCGCTTCGAA CGGACAGTCC GGGTCGATGT CCGGCTTGTA ATGGGTGACG 30600
ATGATCCGGC TGTTGCCGCC GAAGTCGTGG CTGACCAAGC CCTTTGGGGG CAGCTTCTTC 30660
AGCGCCTTGA TCAGTTCCTC AACCGTGGTC CCGGTAGGGG CCTTGCCGTC AGGCAATGCC 30720
TCCCCTCCGT ACGGCACGTC CAATGGGATC GTGTACCGCT CAACGTCTTT GATCTTCATC 30780
GAGCCTCTTC CTCTTCGACT ACCTCGTCTA CCCGGCGGAA TAACTCCGCT AGTTCTGCGG 30840
GTAGCAATAC TGGGTACTTC TCTCGGGCTT CCTGCATCGC TACCGCGATC CCAATCAGGG 30900
CAGCGAGCAG TTCATTGACG GAGTACGCCA ACAGCTCTTC GCGGATCTCT TCTCGGGTCA 30960
TTAGTGGTAG ATCCCCCGGA CGGTGCGCGA GATCGTGGCA GGGTTCACGC CGTAGTTCTC 31020
GGCGAGATCC TTCTGCTTCA TACCGCCCAG GTACGCCTGG CGGATGTCCT TGACCTCGCG 31080
CTCGGTGAGC TTCTTGCGGT TCGGCCGGCT CGGGCCGGTC TCAGGCTTGA CCTGAGCCAG 31140
CGCCTTGCCG AACAGCTCGT TCTGCGTCCG CTGCTTGATC GCGTACCGAC GGTTCGCTGC 31200
AAGCACCTCG TTGAGCCGCT GGGACAACTT GACATTGGCC TCACGCACTA CCTCGACCTC 31260
TCCGAGCAAG TTCGTGATCC GGTAGTCCTT GTCCTGGTTC TCGATGGCCA ACCGGTTGTT 31320
CTCCTCGGAA AGCATCGAGA CCTTGTATTG CGCCTCTCCC AGCGCAGCTT TCAGGTGCTT 31380
CTTCCTCATT CAGCGCCCCT CTCTCGGCGG AACTGTTCGT ACTCGTCTTC GGTCATGTAG 31440
TAGTAGTAGT CAACGACCTT GTCCCAGTTG AAGGTTCGGG ACGTGCCGTC ATCGAACGCG 31500
ATGATCAGGA CACCCTCTTG GGTGTCTAGG ATCGGCTCGC CAGCCACGAC GTGGAAGCGG 31560
TCCTCGAGGG TCACCGCAGT CGCTCTGCGT GCCATGTCAG TTCCTCTCAG TAGCTGTAGG 31620
GGACATCCGG GATGTCCTGG TAGGTGTTGG GTGCGATCTG TCGGAGCTGC CGAAGCAATT 31680
CCCCTGCCAG CTCACGGATC TCGGCATCCG CGGCCTCGTG CCAGCGGGCC TTGATGACGT 31740
ACCGCCACGC CCGATGGTTG CCCGTGACGA CCATCGGTGA GTTCGTCATG TTCGGCAGGA 31800
CAGCTCGCGC TGCCTCGCGG GCCTGCTTGC GCGGCAAGCC CCGGTCAGCC AGCCGGTTGA 31860
CGATGTGTTC GTAGACAGCG TCAATCTCAG AGCTGACGGA CTCCATGATG TGGACGAGGT 31920
CGTCTCGGTC GTCGGGGTGG AGCTTGAACA GAGCCGGGGG CAGATGGATG CCAAGGTCGG 31980
TCGGATCCAC ATATCGCTGA GACACCACCG AGAAGCTCAA GTGACGGTGA CGCTCCAGCT 32040
CGGTCAGCAC CGACCTGCTG GCCTCGATGT AGAACGTCGC CGAGGCGTGC TCGAACACGC 32100
TCTCGTGGCC CAGATCGATG ATGTGGTTGA GGTAGTCCTC GTTCTCGGCA GTTGCCGGGT 32160
TCGGTCGGTG GAACGACCGG TAGCAGTTCC GGCCCGCGAA CTCGGCCAGC TCGTCGGCAT 32220
CGAAGTCGCC GAAGTAGGGA TCTTCGTCCT TGGATTCTTC GAAGTCATCG ACCTCGAATC 32280
CGATGTCCCG CAACGCACCC GGATCGATCT CGGTGGCAGC GATCAGTTTG GCTTTCATAC 32340
TCTCCGCTCA GAGTTGGTGG AACGAGGTCA GCCAGGGGGC AGCGAAGCCC TTCTACAGCT 32400
CCCCTTGGCT CGTTACCGGC TTCTCGACCT CGGTGGATGT CAAGTAGTCG AGATGACTAC 32460
TTCTTGTCGG GCCATTGCGC GTCACACTGC TGATCGCGAG GTGCGGTGCA GGAGAACAGC 32520
GCGTACGGCT TGCCCGTCTT CTTCGAGACG CCCGACTTGT AGACCATCTC GCCGTGCTGG 32580
CAGTACCGCT TCTCGCCACC AGGCGCTTCC TGAGCTGCCT GCGGGGCGCG AGACTGCTGC 32640
TGGCCACCGC CGCCGCCGTT GGCCGGCGCG GATCCACCGG AGCCTGCGTA GTGGCCTGCG 32700
ATCTGCTGGA CCTTGTCCAT CAGCGCCTTG AACTCGGCGG TGTTGACCTT GGCCAGCACG 32760
TCGGCCGGGT CCGCACCCTT CACGACCACC CACGGGTCGC TGTACTGACC GGCGAACTTG 32820
AACGTGGCCG ACACCCCATC GGTGGAGTGC TGGACCGCCA TCGAGTCGCG CACAGCAGCC 32880
GAGGCCGTCG TCACCGTCGC CGACGGCGCG GTCTCAGGCT CAGGAGCCGG GGCCGGCTCG 32940
GGCTGGGCAG GGGCGGTGCT CCACGGATCG TCGTAGGACA ACTGGTTACC TTTCACTTAA 33000
TGGGGCATGC GCCGTTGGCG CACTCTTCAT CGACACCGTC TTCGACGGCT TTGGCCGCAG 33060
CAGATTCGTA CTGCTGCTTG GTGATTCGCT CGTACGGAGC CTGCGGGAAG CTGGACTCCG 33120
GGAAGATCGT GGAGCCCTTG ATGAGCCCCG CGAACCTCTT GAGATCGGCT GCGACATCCT 33180
CGGCCTCGTA GGCGTCTGGA TGGACGTTGG CGGTGAACGA CACCGCGTTG TCAGCCCAGC 33240
ACATCTGGTA GAGCGCCTGG AACGCCAGGA GCTGGTGGAG GGTCAACTCG TCGGCTGACT 33300
CAACGATCTC CTCGTCCCAA CCGAGTTCCT CGACAGCCTG GACCAACGTG TCCTTGGTCG 33360
GGATCGAAAC CACCTCGGTG TTCGGAGCGA AGAGATCCTT CTCGATCTCG TAACCCTCGG 33420
CTGCCAACCT CCGCAGCTCG GCCATGTCGC TGTTGAGGTT GAACCGCACA CGCCGGATGA 33480
AGTACCGCGA GAAGATCGGG TGGATCCCCT CGGAGACTCC TGGCATCTTC GCCACCGTGC 33540
CTGTGGGAGC GATGGTTCGC TTCTTCACCG GGACAGGGAT CCTCAGATCA TGGGCGAACC 33600
GTTCGGCCTC TGAGTCGACC TCAGCGGCCA TCTCCCGCAA GAACTGGGTG AACCGCTTAT 33660
CTCCGGGTGC CTCGGAGTAC CTGCTACCTG TGAGGGCCAA ATAGGAGGCA ACTCCGAGAT 33720
GACCCACGCC GATGCGACGG TTTCGGTCCA GAACCTCCCG GCTCTTCGGG TCGGCCACTT 33780
CCGAGAACGT CGCCCGGATC AGGAATCTCG TCATCAGACG ATGCGCCCGG ATCAGGTCGA 33840
GGTAGTCGGT CTTGCCGGCC GGCGTCACGA ACGCCGCCAG GTTGATGTGG CCGAGGTTGC 33900
ACGGCTCCCA CGGTTCGAGA GTGATCTCGC CGCATGGGTT GGTGCAGACC ACCCGGTTGG 33960
GCTCACCGAC GTTGGACAGT GACGAGTCCC ACATCCCCGG CTCTCCGTTG CGTACGGCTC 34020
CCTCGGAGAG TGCCTTGAGC ACTCGGTGGG CTCGCTTCTG CTTGGGCATG TCCTCGCGGG 34080
CGACCGCGAA GCTGCCGTAG CCCTCCTTGG CCAGACGCCA GAACTCGTCG TCAACCTCGA 34140
CCGAGATGTT CGTCGTCCAG TGCTCGCCCG TGCTCGCCTT GATGTTGATG AACTTGTCGA 34200
TCTGGTAGTC GTCCCAGTGC ATCATCGACA TCCGCGCCGA CCGGCGCACA CCGCCGGCCA 34260
CAACACACTG AGCGATGGCG TGGTCGACCT CCATCGCGGC GATGCCGTCG AGCGTGATCC 34320
CTGCGTACTC CGAGAAGATG TTGGCGACCT TCTGCAGCAT CACAGCGAAC GGCAGCGGGC 34380
CGCTGGCCAC TCCACCGAAC GTCTTGAGCT TGGCCCCTTG CGGCCGGATG CGGCTCACGT 34440
CGTACACCCG CTGGTAGTGG ACCGTGCCGG GTCGGTAGTG CGTGTCGATC AGATCGACCA 34500
GCGCAGCAGC CCAGCCCTCT CGTGAGTCCT CGATGGCGTA GGCACCGGCC CAGTCGTGGC 34560
TGTAGTGCTC CGACAGAATG CCTACATCCT TCATCGCCTG GTAGTCGACA TGCTCTGGAT 34620
CACAGACGAT CTCGACCCGC AGGGGGTTTA CGACCTCGGG GTAGCCTTCG AGGTAGTGGT 34680
TCGAGTAGTT CGCCCCGACT CCCCCGCCCT CCATCAGGCG CATGAACGTG AACTGGAAGT 34740
GGTCCGAGAT CTTCTCGGGC CAGCCAGCTA CCCAGCAGTT GAAGAGGTGC TGCGCGTTCT 34800
TGACCCCCGA GGCCCACAGA TGCCGACCTG CCGGCAGCAC CTTGAACTTG GTCATCAGAC 34860
GAACGAGATC TTCTCGCTCT CCTTCCAACA TATGTCGCCG GTCGACAAGA GCAAGATTGC 34920
CGTCCACGAC CCTCTCGACC GTTTCCGGCC AGGTTTCCTT CGAGCCGTCA GGCTTGGTCC 34980
TGGCGTAGGT TCGGTTGTAA ACGAGTTCAC CGGTTGGTCC CCAAGGGATT TCGTCAGTCA 35040
ACTACTTCCT CTCAGTCAGT TCGTATCGCT TGAAATAGGC GTCGGCAGAG TCGCCGCCAG 35100
AGAACGAGAC CCCGTACTCG ACCGGGCCTG CACCACGCAC CTCGCAGGTA ACGACGCCCT 35160
TCCTTCCCCG GAACATCGGC CAGGTTCCCT TGGAGGGGTG CTTGGTCTCG TCCCGCTGGA 35220
CGATGACCTT GGTGCCCTTC TTCATGCCGA CTTCCGTTCT CCGTAGCCGG GAGTGAAGCA 35280
ACCCCCGACG TACAGCTCGA GATCTTCTTG CGACCAGTTC TCCAGTCGCA TCGGCGGCTG 35340
GTGCGGGAAC AGCTCCGGGA ACACCTCGGC CCGGTACAGC TCCGAACCGG GCATCCCGTT 35400
GAACGTCGGA TCAAGAATGT TGTGCATGGC ACCTCCCTCC CAAGAACTCG GAGATCGGCG 35460
GCTCGTAGAG GTAGCCATCG CGCAGCTCGG GGTTCTCGAT GAGCATGATC GCGATGTTCG 35520
CTGTGGGGTC AGAGTGCCCA TCCCCCTGCG ACTTTCGGAT GTCTGGGAAG ATAGCGTGCT 35580 TGCTGCCCGG ACCATCCTTG ACGATGACCT TGCCCTTGTC GTCCTTCTCC ACGCCAGCCG 35640
TGATCGCGAT GATGTTGACG TGCTCGGTCA GCGACTTGTG AGCGCGGAAC AACCGGTTCT 35700
GCCCGCTCTT ATCCTTCGGG GAGATCCCGT CGGTGTAGCG GCTCCTGATC GCCTCTGCAT 35760
AGCCCCCGTT CTGAGCGTCC AGAGCCTTCA TCGCCAGCGG GAGGATGTCG ACCAGGTACC 35820
GATTGGTCGA CTCCCCCTGC AGAGCCTCTT TGACGTTCTC GGACGAGTAG TGGCTGCGCT 35880
CCTGGAACAA GTCGCGGGCC TTGGCCGCTC CCGACAGGAT GTTGCGAACC TGATTGCGTA 35940
CGTAGTGAAC TGCCTCACCA CGGTGCAAGC TCTCCAGCGT CTTCTGGATG TACGGGCTCT 36000
CGAGGTACCA GACCCACAGC TCTTGGATGA TCTCCTCGGC TGTCAGGTTG GTCTCCCAAC 36060
CGATCAGCGC CTTCCGGGTG GCCCTGCTGA ACAGCTTGCT GATGTCGTCG GTCAAGGCAT 36120
CACCTTTCGT AGGTACTCCT CCCGGTCCAA TCGGCGGTCG AGGTGTCGAG TGACCTCCTC 36180
CGCGAAGACC TCGCGGACTT CGCTGGAGGT GATCTGGCGC GAACGTGCGT TCTTGTGCAG 36240
GTACGGCAGC TTGGTGGCTG TCAAGTTCTA GACCTCCCAG ACTCGGCCGT CGACCGAGAA 36300
CCGGCCTCCG ACAATCGGAA CAAGCTCAGG CTTGACGTGC TGGCCGTCGA CCGTCAGCAG 36360
AGCAAAACCA CTCTGCCAGT TGGCTGTTGC ACCCTTGAGG TACTGAGCTA GCTTCATGTT 36420
CATCAGGTTG CCGACCTCCA TCGACCACAG CACCTTCTGG TTGCCGCCGT AGCCCAGCGT 36480
GTGTGGCTTG ATGCCCTGGC GGTGGGTGTG TCCGATGATC ACCGACGTGC CGAACCGCAT 36540
CATCGCGTTG TACGCGGTGT CAGCGGACTT CTGCGTCACC CGGACCCCAC CACGGTGGCC 36600
GTGGGTGGAG ATCCAGCCTG GAGCGATCTT GTAGAACTCA GGCAGCACGT CAACACCGAA 36660
CCCGTCGAAG TCCAGCAGGT TCTGGAACTG GAACGAGCTG ACGTACTCGA CCAGCGCCGG 36720
GGCGAACTGG TGCAGGTAGT CGACTGGCCG GCGGTCGTGG TTGCCCTCGT GGACACCAAC 36780
CGGGCCGTCG TAGACCTGGC GCAGCGGCTC CAGGAACCGC CGCTTGCACT GCTCGGAGTC 36840
GGGCTTGATC CGCTGAGCGA ACTCTTCCTT GGTGCCCTTG GTCCACCGAG ACGGGCTCGG 36900
GTAGTCCATC AGGTCACCGA TGTGGACGAC CTCGTCAGGC TGGGTGTCCC CGATGTAGCC 36960
GATGACCGCC TTCAACTGCT TGCGATCATC GAACGGAATC TGGGTGTCCG AGATGACGAC 37020
GATGCGCTTG CTCACTCAGC GACCTCGGTG AAGGGGCCCC GCATACGTTC CTCGTGGGAG 37080
CTGGCGTTGC CTCCTGACCA GCGTCGCTTG CCCACCTTGG TGTGGTGCAA CCCGTTGGGG 37140
TAGTAGATCC ACTTCACTCC TGTGGCGTTG GTGACGGTCT TCACATCGGC AGGAACGTCC 37200
AGCAAGGTGT CCCACTGGCG AGGCCCCTTG GGATACCGCT CGTCCTCGGG GAGCTGCATC 37260
TTCTCCAGAA CGCCTGCGTA ACCGGCGATG TCGACCACCG TGTCCTGGTG GTAGCCGTTC 37320
TCCATGAACC GGGCGATCTT CAGCAGGATC ATCATGACGG CCACGTCCTC CGGGGTGAAC 37380
TCGACGCCGC GCTTGTACGC GCCCCACAGG GTCGCGATGC GTTCGTGGTT CTCCTTGGCG 37440
TCCCCGTAGT CCTGGGCTCG CTGTCCGTTG ATGATCTCTT CGGCGGTGGT CAGAATGCTC 37500
ACAGTCCAGT CTCCGATGCG GTGTAGTAGT CGATCAGCTC ATCGAGCTGG TCCGGTTGAT 37560
AGCCGAGGAT CGGCTTGTGG GTGTCAGTGA CGACGACGGG AACCGACATC GCGTTGAGCA 37620
CCTTGGTGAC GTAGTCGTAC GCCTCCGAGT TGGCCGTGAC ATCGACTGCG TCGAAGTCGA 37680
TCCCGGCAGC CGTCAGCTTG TCTTTGACTC GCTCGCATGG CTTGCAGCCG GGACGGGTGT 37740
ACACCGTGAC CGGCGCGAAC AGCGTTCTCA CGTGAGCACC ATCCCAGTCG ATGTATCGGT 37800
CTCCATACAT CAGATCCTTT CCAGCAGAGC AGCTTTGCCC TGCGATGTGA CTAGTGAGTT 37860
GACATCCTCG CCTTCTGGCA TCGGGATGAT TCGGGCGTTC GGCAGCGTCT TCGCCACCGA 37920
CCGGGCGAAC TCCATACCGG CGTCGTCGCC GTCGGCCAGG ATGTTCACGT TGCGGTAGCC 37980
CAGGAACAGC TCTCGGAAGT ACGGCTTCCA CTTCTGGGCT CCGCTGAGCC CCACCGTCGG 38040
CAGCCCACAC AGCTCGGCGG TGATCGTGTC GAGTTCTCCC TCGCAGATCG CCATGTCCTT 38100
GCTGTATTTG GTCAGCGCGT AGGTGTTGTA GAGCCGGTCC TTCTCCCCTG GCATCGACAG 38160
GTACTTCGGT GTGCCACCGT CGATTCGGCG ATACCGGATC GCAGCTACCG TCCAGTGACG 38220
CCAGGGCGAC CACCGCATAT ACGGAATCGC CAGGCAGCCC CGGTACATCT CATGTCCAGG 38280
GAGTGGGTCG TCCACGAATC CCAGACCGAA CCGGCTTAGT TCCGCTCGGC CGGCCAGCCC 38340
GCGACTCGCC AAATACTCGT CGGCTGGGCT TCCGGGCAGG CTTTCTCTGT ACCGGGACGT 38400
TGCCTCCCAC AGATAGGTTC TCTGCGATTC GCTTAGCCTC TGCAAATGTC ACCTCCTCTT 38460
CGTGACGAAT GATCGAGATC ACGTCTCCAC GGACCCCGCA GGCCATGCAG TTGTAGCCCT 38520
GTAGGTCGTA ACTGACTGCG GCAGACGGCG TTTCGTCGCC GTGGAAGGGG CACAGGCACT 38580
TGTTCCACTC GTGGTGGTCA GGTGGTGGTT CCCAATCCGG GTGGTAGCGA AGAATCGCCC 38640
TCGCGATGGG CGAGTCGTTC ATTCGTCCTC GTCAAGCTCC TCGGGAGAGA GCCCTTCGAA 38700
GATCCCGTTC AGGACGGCGG CGAAGCCCTC GCCGGTCTCC GCTGCGTCGA GCATCTCTGC 38760
AATCGTCTTT GCCATGTTTC CTCCTGGTGG ATGTCAAGTT CGAGACAGCT TGTCAGCCTC 38820
GACTGGAGCG ATGCGCTCCC CGATGACTTG GACGGCCGGC GGGTTCAGCA GGTACTCGAT 38880
GGCCCGTTTG AAGAACTCGA TGCAGTCCCT CGCCCAGCCC AGCGTGTACT TGTTGCACAT 38940
CGTGCAGAGC AACCCTCGGA CGATGCCTGT CTTGTGATCG TGGTCGACCG ACAGGCGCTT 39000
CTTCTTACCG TTGGCTCGCT GGCAGATGTA GCACCGACCA CCTTGGAACT CGTAGATCTG 39060
CCAATACTCA TCGCCGGTGA TGCCGTAGGT GGCCAGGATC CGGGTCTCCC AGCTCGTAGA 39120
GCTGCGAGCC GTCCTGAACT CTCGGTGATG AGTAGCGCAT CGTGGCCCTG GATACTTGGC 39180
GTCTCGCGTG AGCGGGAGCC CCTGTGCGAC ACAGTCTTTG CAAGGCTTCC GCTTGTGCTT 39240
ACGGTTCTGC ACCCGGTACC CCGGAGACCT CTTCGCCGCC CTCGGCACGC GCGTCCTCCT 39300
CCCGGTTCTC CATCACCATG CAGAACCACG ACAGCAGCCC TGCCAGGGAG ATGTAGAAGG 39360
CCACCAGAAC TTGGCCGCTC ACTTCACCAT TCCTCGAACC CACCAGCGAG ACAGCGCCTT 39420
ACGCCCTTTG TCGAGCGGGG TCAGCTCGCG CTCATCGTCC TCACCGAAGT CGAACTCGAT 39480
GCTGGCGATC TCGTAGCCGA GGATCTTGAA CGACACGTTC ATAGGCGGTC TCCGAAGTTG 39540
ATGACGGGAA TGCCGGCCCT TTCGGCCTCT CGCATGCAGT GCCGGGTGCC GACTGAGTTG 39600
CCGAGGGGGA ACGCCAGACA GATGTCCGCA CCGGCCCTGA CCATCTCGAT GTTGCGGAGG 39660
ATGCCAGCCC GCTTGCCGTA GCGTTCCCAG TCGGCTCGGT GCAGCTCGGG GAGCACGTCC 39720
CATCCCTCCT GCTTCATCCC CCAGGCCCAG CGGTCTGCGA TGTCGTCAGC GCCGCGAGCG 39780
CCGCCGTGGA CGACCGTGAG ACCGGAGAAG GACCGGTGGT ACTCAGTGGC CAACGCTTCC 39840
CAGACCGTGG TGCGGTCCTT CCAGATCCGA GATCCGGTGA TCAGTACTCG CCGCATCAGA 39900
TCGCCTCCCA CTGCAGGCCG TCGTGCGACG TGACCAGCTC CGCTTCGTAG ACGCCGTAGC 39960
GGGTGGCCAG GAACTGGATC ATCTGCGCCT GCTTGTACCC GAAGGGACAT TCGTGGACGC 40020
CGCTGATCGG GTATCTGACT CCGTATTTCA CTTGATCCAC CGCTTCGCGA TTCGGTCGAC 40080
GTTCTCCTCG GAGACGTTGC GGGCGAGGCC GGTGAACTCC TGGCCGTGGA CCTTGGTCTC 40140
GATCACGCGA GGCTTGCGGG GATCCGGGCT CTCCGGGTCG ATCCGCTTGT GGGTCCAGAC 40200
GGTCGGCTTC GTCTTGATCA GAGCGCCCAG CACCTGCTGG CGCAGTGGGT TGGTCTTGCG 40260
GGGCATAGCG TTTGGAGTGG TCATCTGGAT CCTTTCCTCG GTGGCTGTCA AGTCGGTGTG 40320
CGTAGTGAAG CCCCCCCAGG CATGCGCGCC CCGCCTGGGG AGAGTTGATC AGCGCAGTTC 40380
GATGTCGGGC AGGATCGCCT GCGGCTTGAA GTTGACCTGG TAGAAGTCGG TCGAGACGTT 40440
TGCGCCATCG ACCTGCTCCA TGAAGTAGGA GACGTTGTCC GACAGGCCCA GGAAGTGCTT 40500
CTTGATCCCG TCCTTGGTCT TGCAGGTCAC GTCGAGCTTC TTCGACGCGG TGTCCGCGTT 40560
GATTGAGCAC CGGCCCTGGA TCTCGAGCAG GTACTTGTCC GTGATCCCGT TGAAGAACAC 40620
GATCCGGCGA TTGATCTCGA AGTTGTCAGC GGCCTTGCTG ACGTTCTCCG ATGCGACGTC 40680
GGCGTCGGAG GTACACGCGG AGAGGCCCAG GATCGCCGAT CCGGCGATGA GTGCGGTGGC 40740
GATGATCTTC TTCATGTTCG CTACTTTCTG TTTGGTGGAT GTCAAGTTAG TGACCGAAGT 40800
CGTTGATCTG CATAGTGTCT CCGACGAACT CCAAGGAAGC GAAGTCTTGT CCCGACGGGT 40860
CCGACTTCCC CCCTCGGTTC TTGACCGTGG AGACGTTGAG CATGTCCGGG CCGAACCCGT 40920
CCGATACTCG GTGGAGAGTG AGGATCATCT CAGGAACACG CCCGATCTGA CCTTTGATGC 40980
CCGACAACGG GATCGGCTTG TCGCCGTCGT TGTGCGGGCC GGTGACGTGG TGGAGCCCGA 41040
CGACGCATGA GCCTGTCTCA CGGCCCATCT CGTGTAGGTA GTCCATCAGC GACTCCAGAC 41100
CCGAGAACGG GTCGTCTCCC TCGCTTGAAT CGGTGCGGAC GTTGGTGATG TTGTCCACGA. 41160
CGATCAACGC TGGGAAGTCC TCGTACAGCG CGTCATACGC GGCCAGAGCG TTCTCGATCT 41220
CGTCCAACGA CGGTGATGCC TTGTAGTTGA ACCGGATCGG GATCTCGTCT AGTGAGTCAG 41280
CTACCGCGTC CTCGATGTTC TGCTCGCGAA CAGCCCGCGT AGCTCGTTCG AGCGACCATC 41340
CGCTGAGGAT GGACACCGAA CGGGAGAGCT GGGTGAACGC ATCAGAGTCG GCCGAGAAGT 41400
ACAACGTCGG CACCTTCGAC TTGAGCGCGT AGGCGAGGAC GAACGCCGAC TTCCCGGTGC 41460
CGGGGCCGGC GCAGACCAGG ACTAGCTGGC CTCGTCGGAG ATGTGTACCT TTCTGGTCAA 41520
GCGCGGCCCA GACCGGGGGT AGCGGATCCC CCGCCGACCC TCGGATGTAG AGCGATTGTC 41580 TAGGTGTGTA CACCTTCCTC CTCGTGGATG TGATTGACCA GGTCATAGAT CTCGTCGCGA 41640
GAGACCAGCC GGCCCCAGGC GTCGATCCCC ACGTGGATCT GTCTCCGGTG GATGTGTCGG 41700
GACAGGATCA TCGGCGAATG CGTGTGCCCG TGGATCAGGA TCTTGCCATC GTCACGGAGC 41760
CTCCACTGGG TGTGTCGGTC CTCGCTGGTG TGGTCCCCGA CGTATGGGAA GTGGCTCAGC 41820
AGAACATCTG TGTGCCCGCC AGCGTCCCCG TACAGCGGCA CCCGGATACG AGCTGCCGTC 41880
GACACATGCT CGAACACCAT CCAGTACGCA CCAACCAGCT TGTGAGCATC GCGGTTCATC 41940
GGGTGGGGCC CATCGTGGTT GCCCAGGATC AGCCGTTTGC GGCCTGGCCG ATCCGAGATC 42000
CACCCGAGGG CATGTATCTG CCCCTTGGTG GAGCCAGAGG AGATGTCACC TAGGATCCAG 42060
ACCGTGTCGT CCTTGCCGAC GACCGAGTCC CACGCCTTCG CCAGGGTGGC GTCGTGCTCT 42120
TCGACATCAT CCGCCAGGTT GCGGATCTCC ATCAGCCGCT TGTGTCCGAT GTGTAGATCG 42180
GACGTGAACC AGGTGTTGCT CATGGCTTCC TTTCAGAACG GCGGGCCGTA CAGCTCGATC 42240
ACCAGCGCGT GCAGCTCCTC TGCCGCGTCG TCACGCTCGA ATCCGCAGCA GGAATCGTGC 42300
CGGTCGAGGA TTGCGACGAT CTGGTCGTAG AGGCTGGGCC TCACTTCACC TTCTTCGGAT 42360
CGATCAAGGC GTCGTGAATC GGCCGACCGG CGCGAGCCGC GTGCGTCTCG GCGTCCAAGG 42420
CTCGCTGCAT CTGGTTCATC AGCCGGGTGC CGCGCAGCTT GAGGATCTTC ATGGTCGCCC 42480
GACCCTTGTA TCCAGCGCGG TGCATCCGTA GGACGCAGGC TGTCTCGTGC GGGGCTATAG 42540
GTGACCTCAG CGACGGGTGG TTTGGATCCC AGTTCGTCAT GTCTTCCTCT CGGTGGCTGT 42600
CAAGTTGGTC ACAGACCGAA CTCTTCCTGG TACTGCGGGA TGAAGTGGCC GGCCGTTCAT 42660
GTTCGGCTCG ATACCTCTCG CGTCACGAAC TCCTGCCCGT TCCATCTCCG ACCGTCCTCG 42720
AACTCGATCA CGATCTCTCG TCCGGGATGA CGCACGGCCT CCGCTTGGGC AAACCTGCGT 42780
GCAGCCTCTG GGGTCGGGAA CGGAAACTTC TGCGAGGCGT ACAGCTCCTG GTGCCACTTC 42840
GGCTTGTCAG GAATCGGCCC CATTTCCACG TACGTGTAAC CCGCGTCGGG GTCGAGTTCG 42900
AGCGTTTTCT TGTATTCCTT CGTGCCTGCC TTAGAGGGAA GGTGAGTATC GGTGGCTGTC 42960
AAGGTGACCT CACTTAAAAA CAGGGCAGCT GTAATTCACA TCACAGAAGC CGCATTTGTC 43020
AGGTTCAGGC AGAGGCTCGA AGTCACCAGC CTGGATCCGA GCCTCGACCT CATGGAACCT 43080
CTCGGTGATC CGCTCCCGCG TCCAATCGGT CAGGTCGTAG GGCGCAGTGG GCTTCGCCTT 43140
GATGCCCTTC TTCCCCGCCA TGAAGTAGTC GCCCGTCTTC GGAGCCTCCA CGTCATAGGT 43200
CATCGCGACC GCGAGCGCGT ACACGCCGAG CTGGAAGTCG TCACCCGGCG AGTTGCCGGT 43260
CTTGTAGTCC CGGACTCGAA GCTCACCGTT GACCACGACG ACCGCGTCGA TGAACCCTCG 43320
GACGCGGATG CCGTCCAGCT CGATGTTGAA CGGAAGCTCG ATGGCCGGCT TGGGCTGTTC 43380
ACACTCCTTG CAGTTGGTGT CTTTCCACGC CTCCGTAGAG CAGATCCCTC GCCCAGGGGT 43440
AGTCCAGATC TGCTGGCCCT TGTCCTTCCG CCACGCGATG AACTTCTCTA CCTGCTCCAG 43500
TCCAAGGTGG AACCGGCGCT CGATGTCACG CTCACCGTTG TACGGCCCGG ACCAAAACCA 43560
CCACTCGAAG TTCGGGGTTT CGTCGCACAG TGCTCCGATG TCCTTGGCGT ACTCCTCGCG 43620
GAAGATCTCT TGTGCCCGTT CGAGGCTCAT CTCGCGGCCC TCGGCCAGAG CCTTCTCGTA 43680
GACCTCAGCG ACGGTGTGAA ACGCGGTGCC CTGCGGCAAC CACGCCGCAG GACGAGCCCA 43740
TACCTTGTCG ATGCGAGCCA GCTTGTACGC CTGCGGGCAA CGTGTGTATT GGTTCAACTG 43800
GCTGACGCTT CGCAGCGGCA GCAATGTCTT GGTGTCTGTC ACGCAGCGGC CATCCTTCCC 43860
TTGCCTATCG TCTCGTTCAG CGCCCCGTCG ACAGCGACAC TGAGCAGTTT TGCGACCTCC 43920
GACATGTCAA TCGGATCCTT GGGGAATTGG TCAGCCTGAG TCATCCTGAG CACCATCCAC 43980
TCGGTGCCCT TGTCGCAGTG GATCATGGTC GGATCAAAGC GAGTTCCCCG TGCTACGTAC 44040
TCGACTTTGT TCGCGGAAAG AATCAAATTC GACACAGGCC GATAAAGTCG TGAGGTGTCT 44100
TTTACACGAG GACTGCGGTA GACGAGCAGA ACTGAGACTG GGTCTTCGTC CAGTTGGCCC 44160
TTCCACCACG CCTCACACCT CTGCGCGAAC AGCCACCCTG GATGATCGGC GATGACTTGC 44220
GGTGAGGTGT GGACGAGGTT GTCTGCGAAC AGCTTTGCGA GCCGAGTGAG GGGCACGGGG 44280
TTTCCTTTCG TTGCGCGGCC TGGGTTGGCT CACACAACCG GTCGTGACTT TTAGGGCTCC 44340
GAGAGAAGCT CCTCGATGTC GTCTGGCCAC GACCAGAGGA GTTCACCCTC GGCGGTGAGG 44400
TTGGTGTGCT CGTTCACCCG GATCAGGAGA TCGTCATCCT CGATGCCTCG GGGGACGTAC 44460
CTGAACCCGC CGCCGGCCAT ACCTTCGTAG GGCTCGATGG ATGGGTCGAA CTCGAGCACT 44520
AAGTCGTCGT CGCGGAGCAT CTTCCACCAC GACAATAGGC GCTTCTTCTT GTCTTCGGAC 44580
ATCGTGCGGA AGCTACCCAC TCGCATGTAC TCGCCGTGAT CCCGGAGCCT CTGAAAAGCC 44640
TTCGACTTAT CGTGAGGTTT CCGCGTGTCC CACGGCCAGT TCTGCTGGAC GATCTGCCTG 44700
GTGGTCAACC GTCCTCCGTA GGTCTTCTTG TGCCACGACA CCGCTTGTCG AGTCACGCCA 44760
TACAGCTCTG CGATTTCGGT CTGATTAAAC CCCTTCCTGC GAAGATCTTC GATCTCGCTG 44820
AGAGTGAGTG GTATTCGGCT AGGGGCCGGA ACCACTGCTT TGTGTTGGAT TTTGCCGCTC 44880
ATGTTTCCCT CCATGAGAAA GGTGCGTGCG TCTCCGCCGA TTACGGAGAC ATGTTGGTGC 44940
CTGTCAAGGA TACCCCTAAT TTAGTTGCGT CTGCGGAACC ATATTCAGTT GTGTTCCCCG 45000
ACGCCGTGGC CGTCTCCCAC TGGGCGTGGG ATCGACTGGC GTTACGCGGT CGTAAATGTA 45060
GCGGCCTGCC CCACTCGGTA GCAAACCTTG TGACAGGTAT CACTTAGGTC GCCTTCTGTT 45120
ACACGTTGAC CTCGGGTTTC ATCGTCACGA CTCTCCTTTC TTAGACAGCC TCAAGATCGT 45180
TACACCGGCT TGCGAAGATG TACCTTCGCC TTGAATCCGG CCCTTGCCAG CTCGAACTCG 45240
ACCACCTGGC GGGCGGTCTC CTTCAGGTCG GACTTCGCCG ACAGCGGCCC GACGAACCCG 45300
TAGCTCTTGA TGTACTCCTC GAGGTCGATG TCGACGTACA GCGTGACAGG GACCACCGAC 45360
AAGTCACACC TCCAATTCGT GGGGCTTGAT CTCGTTGGTC ACGTCGTAGT CGTTCAGCAG 45420
CGACTGGAAG TCGGAGTCTG TCAAGTCGTC CAACTCATCC TGCTCGAACG GCGCGGGCTC 45480
GTCATGCCAC GTCTTCCACT GGTCGTGGTC GGCGCGGAAC CACTTCCGCA GATCCTTGAT 45540
GGCCTCGTCC TCGGTGGCGA AGACGTAGGT CTCGAGCACG TCCTCGTACT CGACGGTCAG 45600
CGACCAGACG GTGATCTTCA CTCCCCGTTC ACCTCCGCTT TGTAGTTCAT CTCGGCGGTC 45660
TCCTCCTAGT TGGGTAGCAG TCGGTTGTAC TCGTCGTGGC TGATCTCGCC AACGATGAAC 45720
TGGCGCATCA GATTTGCGAC CGAAGCCGCG TCCATCCCTT CGGGAATGGG CTTGGCGTGG 45780
CCGAACTGCC AGTCTCGTGA GCGCCAGCGG AACCAGAGTT GGACCTTGTC CAGTGAGGTC 45840
AGGTGCAGGC ACTGAAACGT CATGCCTCCG AACGGGAACT CCATCACACC TCCTGTTTGA 45900
CCTTGACGGT GTGGCCTGTC ATTACTTCGT GGATTCGGAT GCTGGTGCCG AACGTCTTTC 45960
GCGTCTCGGC CTTGAACTCG GTGGAGCACC CCGAGCACTT CGCTTTGAAT CGCACTAGCA 46020
GTACCAACGC TTTCTGCAGA ATCGGGACTT GCCGCCGTCC CGGTTGTCGT TGTCCCGGCG 46080
GGCTTCGCCC TTCGGTGATT CGTCACATGA CGGAAGCTCG CCATGCTTGA TGTGCCATGC 46140
GTCGTCGGCG ACTTTTCCGC CGTGCTCGGC GATGTGCGCT GCGCTCCGGT ACTCACAGAG 46200
CGGGGAAGCC GATGCCTCGG CGATGATCCC AGGCAGGTTG CCTAGAACCA CCGCCAAGCA 46260
CATCAGCAGA ACGACGTGCC ACGCCTTCAT CAGCCCGCCA GCGCGTGGTT CATCGCCGCG 46320
TTGCGGCCGT CGCGCTGACC GTGGGCATAG CCGCTGAGGT CGTACCGGGT CCGAGGCTTG 46380
ACGTTCTTGG TGCGAGGATG CGCCTGGCGC AGAGCCAGCG CAGCTCGTTC CTTGTCGCCT 46440
CGGTAGAGCA CCAACGCTCC CCCGCCGGCC GATTCCACGG CCTTGTTCTC CTCGGCGGTC 46500
AGGCGTTCCT TGACGGCCTG GGCGAAGCCT GCGATCCACG ACCGGCGGTA GCTCTTGAGC 46560
TGGCCAGCGG TGCTCTTCGG CTTGTACTCC CCGGTGTTGT AGTCGTACTT GTACCGAGGC 46620
TCGAAAGCCT GCTCCGGGCG GACATTCTCA ACCAGGCGCA TCATCTGCGG CTGCATGATC 46680
GACCAGAGGA ATTGGAGCCT CTCGATGTGG CGGGGCACGC CGTAGACGTA GATCCGCTGA 46740
CCGCCCGTGA GGCTGGCGTA CACCGTCTTG CAGTGCAGGG CCTGAGCCAT GCCGTGCAGC 46800
AACAACGCTT GTGCGGCAAC GTACTTGCCG GTGACGTAGG TGACCCACTG GATGGCGTCG 46860
GGCAGGTCGG TGGTGTCCAA CCCTTGCTTG CTCGCCTCGA CCTGGGCCAT CTCCAGCCCG 46920
TACTTGGCCA TCAGCTCGAA CGCTTTCGCC TGGAACACAG CCTCTTCCGG CGTACCGGCC 46980
ACGTCTTCGG CCTGGCGCAG CAGCTTGGCG ACCTTGTCCT GCATCTTCTT CGTCTTGCCG 47040
TCGATCATGG TCAGTACTCC TTCTTCCAGT TGTTCCGGTT GCCCTTGCCG GGGCGCTTCA 47100
TCTCTCGCTT GCGGTTACGG TGCGGCTGCG CCGCGTTGGA GAGACGCAAC TCGAGCCGTG 47160
CCTTGAGCTG GTCGCTCATC TTCTTCACCT CTTCTGGTTC AGCGGATCTG GTCGACGTGG 47220
ATGCAGCCGA CGCGGTCTGG CCCGAACTCG GGAGCGAAGC CCAAGACTTC GTCCTCCTCG 47280
CATGGGAACG CTCGCTGGTC GAACGTGATT GGGTCGGCCG AAGCCTCGTA TGGATCGGCC 47340
AAGGCCATCG CTCCGACCGC TGTAGCGAAT GCAACGACGA CGGTGATCAG GTGCTTCTTC 47400
ACTCTTCTTC CCTCCACTTT TGGTCTGCGA GAAGCCTTCT GGCGATCTCG ATAGGTTCGA 47460
TCTCAGGAGT CACTCATCGC CCTCCAAGAT CTTCAGGTTG GCCAGCAGTG CATTGGCCAC 47520
AGCTCCGATG TGGCCACCGC CCTTACCTCC ACGGCGGGAG TACTCGCGGT TCGCGGCCTG 47580
CATGAAGTGG AACCTCGGTG AGCCGTCCTC GTGAACCCAC GAGGCTTTCT CGGCGGGCAG 47640
AGCCCGGTTC ATCTCCACCG ACATCGTGAC GATGATGTGG TCCCTCTGGA GCCGAGCCTC 47700
GGTCTCGGCG TAGTGGGCAG CTTGGATTAC TGCGCCTCGT GTGGTCATGT CTTCTCCTTC 47760
GGTAGATGTC AAGCTGTCGT CACCACTCTT CGACCGGTAT CGGTTTGTCA CAGCCAGCAA 47820
GGATCGCGGC GTTGCTGCGG TGATGCCCGT CCCACAGCGT CTTTCGGTCC CTCGAAACCT 47880
CGAGGGGTTC GAACGGCCAC TCGTTCGATG AGTTGAGGAT GTCCACGACT TCGTGGACCT 47940
TGGCCCAGAA CTTGCCGGTC ACGCCTCCCT GGTAGTTGTA GCGGGGCGTG GTCTGGTAGA 48000
ACTCTTCGAG CACTGGTCCG CTGTCGGCGA CGGTGCAGTC GACACCAGCG CAGGACATGC 48060
AGTCGCTGGC GCGGAGCTGG GCAACTTCAT CGGTGGTCAT GAACGCCGTG GTCACATCGA 48120
GCCTTTCAGG TGTATGTCAA GCGGCGCGGA CGCCGGAATC GGAGAGGTAG ACGCGGTCAG 48180
CTCCCAGGAA CGGAGCCTGT GTGTTGGCGT GGACGAACGT GTCGTTCTCG TAGGGGTTGT 48240
AGGCGATCTT CGATCCCACG AAGTCTTGCG GGAGAAGCGA GATCAGCTCG CCTACGATGC 48300
CAGCGTGGAC CACCTTGCGG CGCTCGCGCC GTACCTTGTC GCGGCCGGCC GGCCGAACCA 48360
CACCCTTGGC GTGGGCCAGC AGGACGTGGC CGCTGCGGTG GATGACTCGA CCCTTGAAGT 48420
CTCCCTCCAA GGCTTGCACC GAGTACCACG GCTTGCCCTC GCGGTGCGTG CGGTGCAGGT 48480
TCTTGTAGAC GAAGACTCGG ATCGGCTTGG GAGTCATGAG ACCTCCAGTG TGCGAACGGC 48540
CTTGTAGGCA CTGATGAGTG ACGCCCCCGA CAGCTCGTTA CCGTGCAGGT GATACCTGTA 48600
TTTCAGATAC ACGGCTTGGT CGACCGGCTT GTACTCGACC GAAGTGACCT CGACAACCAT 48660
CCCGTCGATG ATCGCGAAGT CTCCAGCGCG GAGATGGGTG GGGAATTTGA TCTCGGTGTT 48720
GACTACGGTC ACAGCTTCGA AACCTCCCAG GTACCAACGA ACTTGCCGTT GCGCTTGATG 48780
TATCCGCTCT CACCGGGCTC GTACCAATCG ACCTCGAACC CGTAGCGGGC GGCGCAAGCC 48840
TCGAGGTGGT CGAGCAGGAC GCGGCGACCG GACGCGGTAG CTTCTCCGGT CAGCCCGCTG 48900
TCGTTCTTGC GGACGATGAG CTTGAACACT TGGTGCCTAC CCTTCTGCGA TGTCTCGGGA 48960
GATCTCGGCG AAGACTTTCT TTGCCCACGC CACGCCGTCC CAGGTGATGT CGAACAGTGC 49020
CTCGTAGAAC TGGTCTCGCA AGGCTTCGTT GCCGTCGGCC AGCGTTGTGA CGAGCCGGTC 49080
GATGCGGTCC TCGTGGAACT TGTAGACCGA GTGGTTGTAC GGCTCAGCCA TATTGGCGTT 49140
GGCTCGTTTC ACGTTCTCAA CCACGATGGC TTCGAATAGG TGGTTAACCA GCTCCTCGGT 49200
CATGTTCTAT CTCTCCTCAG TAGTCGCTGT GCTGGGTCTC GAAGCCTTCG AGGTCACCGA 49260
CCTCGTCGTC GTACGCGCTC GGGTTGCCGC GCCAGTCGTC GCGGAGCCTT TGACCGCTGG 49320
CGTTGTAGCA GGCACCACAG TTCGGGCAGT CCACATCGCT CTGGCCGTAG TAGCGGCAAA 49380
CCTCGCCGCC GCAGCGTTGG CAGTCCCACG CGCTGTAACC AGGGATCAGG AAACCTTGGT 49440
CGTCGGTCTG ATCAGGGATG CGTCGGAAGT TCTTGGCAGG CATAGCTACT CCTCATAGAA 49500
ACTCGTGGTT GATGGCTCGG TGGGCAGCCT CGCGGAAGGT CAGCCCGTCG TCGTACGCGT 49560
CCCGGTACGT CCAGTCCGCG ATGTCTTGGT AACCAAGACC AAAGGTCTCG GTCATGTAGC 49620
CGTCCAGCGC GGCCATCCAG GTCTCGAAGC TCATGTCTTC CCTCACTTCT TTGTGGTCGA 49680
GAACAGCACG TTCCTGCGGC CGTTGACGCA CAGACCGCAA CGGGCACAAG CCGATCCCTT 49740
GTCGTTGATC AGGTCGATGG CTTTGTTGTT CTCCGGGCAG CGCACCGCCG TCGGAAACTC 49800
GGCCTTGCCT TTGGCGAACG TGGTGTCGAC GTAGGCGATG TTGATGCCCT TGTCTTCCAA 49860
GAAGCGCGCC ACGTCGATGT TGTCCGGGTC TGCGCTGAAG TACAGCGCCA GGTTGTCGAG 49920
CCTCTGCGAG TGCAGGTAGA CAGCCGCCGT CTGAACCCTT GTGTAGGCCC AGAACTGGAC 49980
ATCCGGGTTG TCGCGGATGA CTCGACCCCA AGCGGCCACA TAGGTGGGGC TGAAGAAGTC 50040
TCCATCCCAG TGGATGCGGA ACAGCTTCGG AGCCTTGCGA CGGTCGCAAT CCTTGACGAA 50100
CTCGGCGACC ATCTCGGACA GCAGCGTCAC GGTGTCTGTC AAGTCAGCGT CACGCAACAG 50160
TTCCCAGTTG TGCAGCAGGA CCGAGCTGAC AGCCTTGCGA ACTTTCTCCA GCTTGCCGGC 50220
GTAGCACACC TTGGCACAGA AGGCCGTCGC GTCCGGGCAG GAGAAGCCTT GACCGGAGGG 50280
CAGGCCGATG CTGTTGGCGA TACCTACGGT GGCGTTGCCG CCCTTGGTGA CGTGGACGTA 50340
GTTGGTGACC TTGCGGTCGT TCGAACGCTT CAGCTTGGCC ATACCTAGCC TTCCTTCGGT 50400
GGCTGTCAAG TTGTTGGATA CAAAGCGCCC CGAGAGGGAG TCGAACCCTC ACACCGCGAA 50460
CCGTCGCGGG GCCACCGTGC CTAGTCGATA GAGGTCACTC GACTCTCGTG GACGTAGACC 50520
ACGGTGTTGC CTACGTTCAC CGCGTAGTAC AGGCCATCGG CACCTCGTAG CTTGTGCCGA 50580
ACCGTGCCCG ACGTGGCCGT CATGTCTTCG CCCCAGTCGG CGTTAGGTGC CCAGGTGACT 50640
CGCATGGTGA TCCCTTCAGT AGTCGGTGGC TGTCAAGTCA GCGGATACGG ACGTACCCGT 50700
TGCCTCGAGC GACGTAGATC TTGCCGTCGA TGTAAACGCG CTGCTGCTGG TTCATAATCC 50760
TATTCCTTTC GGTGGCTGTC AAGTCTCAGG CCCAGCGACG AGTCGTCGGC CGGGGGCGGC 50820
GCACCTTGGG CGCGTTGGCT CGCGGTGCCT TACGGATGGC GGTGCCTACC GTGATCTCTT 50880
CCAACTGGCG TTCAGCCAGG CCGACAGGCC GGGCGTCACC GGGCAGTTCG ATCTTGTAAT 50940
CGAAGTCAGT CCACCCCTTC AGACCCTTCT CCAGCTCGCG ATCCAACAGA CGCGGAGCCG 51000
ACAGCTCAGG CGCAACAAAC GGTGTCTTGA CGCTCTCGCG GGCAGTAACC CGAACCTCAC 51060
GGTGCTCAGC GAAGACTGGC ATAGTTCACC CCTTTGGTGG ATGTCAAGCC TGAGCACCAA 51120
AGCTCAGGCG TAGTGGGTAG TCGGGAATCG AACCCGATAG CTTCATAGCC ACGTTCTACG 51180
GCTCAGCCAT AGCTCAGCGA TCATTCCATC GCGCCAAGAG CTACCCTCCC GAATGCCGAA 51240
CCAAAGCTCA GCATTCGTAA GTGTGTATTC TCCCCGTGGC TCAGACAGTA TCTATCAGAA 51300
CCTAACCACA GGTCTACATT TAGTTATCCG CAGTGCTCGC ACTTTAACGG CATCGAGCTT 51360
CCGCCGACCC TCAGTCCTCT GGCAGCGAAC TAAAGGTTTG AGTCGGGCTG CGGCCCTTCT 51420
CGGTCTTGCG TGATTCTCAC TCTACCGGAT GTTTCGGTGG CTGTCAAGCG GGCCGTTTTG 51480
GTGTTGCAAC GATGCCCTCG TTTAGCGCCG CTGGCGTAAT GCGCTACCCG CCTGATCTCA 51540
CCGGTCCAAG TTGGTGATGC TTGCAGCTTA CCCGATAACC GGGTGGCTGT CAAACCGGAG 51600
AATCTTGCCG CCGGATTTTC ACCGGCACCG GCACGATCCT CTCGGATCCG CCTACCGCCT 51660
TGCTGCTGCG GTGACACAAG AATGCACTAC TGGCCGGGTG GCTGTCAAGC CCTAATCGCA 51720
AATTGGTGCC CTAGCTGCAG ATATGGCGCG TTCTCGGTGG CTGTAAAGGG CACTACGTGC 51780
CGCTATCCGC TGGTCACGCT GGACAGTCCC GGCAGCCCGT GCCGCGCATA GGCTGCTCAC 51840
TACGTGCCCG GTATCGGCGT TGTCGTGCCG CTGTCGTGGT CGTCGCCCCG TCGCTGTCGC 51900
TGGTCTCGGT GGCATCGCTT GACAGTCGCC CCGCTATCCC CCGTTGCCGC TGGTCAGACG 51960
CTAATCCGCT TATTTCGCAT AGGCTGCTCA CTATCGCATC GGTATGCGTA TGCGCTGGTC 52020
ACATATGCGT GTGGTGGTGG TGTGGTGTGC GTGTGTTTGC GCTGGTCAGC CGTGTGCGTA 52080
CCGTATCCGC ACACTGTGCT TGTGCGTTTG CTGTGTGTCG AGGCCGGCTC TCGCATCGTC 52140 GCATGTCAGC GCGGGTATGG GCGTGTATCG CACGCTTTGC TAGCCGCGTG CCGCGGCGCT 52200 CTCGCATCGC ATCGAGTGTT TGCTGTGTCT CTCATCGTCG CAGGTCAGAA GGGGTAGGGG 52260 GGTTCCCCCT AGGGGTCGGT CCTTGACCGG TCGGTTA 52297
It is known that during the establishment of lysogeny, the L5 genome becomes integrated into the mycobacterial chromosome via the phage attachment site (attP).
Integration-proficient plasmid vectors have been constructed which efficiently transform both fast-growing and slow-growing mycobacteria through stable integration of the plasmid sequences into the bacterial chromosomal attachment site (attB).
Because the L5 sequence is now known, and because L5 has been previously characterized, the use of transcriptional promoters with this mycobacteriophage may be evaluated efficiently, and host synthesis inhibition may also be evaluated efficiently.
Figure 1 represents the genome organization of the entire L5 genome. DNA analysis has indicated that the L5 genome is organized into a right and left arm with the attachment site at the center of the genome. The integration functions have been successfully employed to construct integration-proficient vectors for mycobacteria.
Part of the L5 genome is not essential for mycobacteriophage growth. By way of example, gene 71-70-69 may be deleted without affecting the lytic cycle of the L5 phage. Therefore, it may be a suitable region in the L5 mycobacteriophage for the insertion of reporter genes. As a general role, it is critical that reporter genes be inserted into non-essential regions of the mycobacteriophage. Otherwise, the mycobacteriophage will be unable to survive and replicate.
For example, the L5 mycobacteriophage may have introduced therein promoter gene 71 fused to reporter gene lacZ, and this reporter mycobacteriophage would be capable of rapid diagnosis of mycobacterial infection and accurate assessment of mycobacterial strain drug susceptibilities.
Another mycobacteriophage which may be successfully used to produce the reporter mycobacteriophages is the mycobacteriophage TM4. TM4 has been used to construct a first generation reporter mycobacteriophage, and has the ability to discriminate between M. tuberculosis and BCG. A shuttle plasmid may be employed with TM4, and may be useful in the construction of recombinant and other mycobacteriophages. Unlike L5, which is a broad host-range mycobacteriophage, TM4 is a species-specific mycobacteriophage. However, TM4 is not as well characterized as the L5 mycobacteriophage, and therefore it is more difficult to analyze its functions.
DS6A is a mycobacteriophage that has been found to be specific for the M. tuberculosis complex of mycobacteria. It has been shown to infect both M. tuberculosis and BCG. It has been demonstrated that DS6A can infect over 3,000 different types of M. tuberculosis strains. Current efforts are under way to develop DS6A shuttle phasmids containing Firefly luciferase genes as the reporter molecule.
Different mycobacteriophages have varying host specificities. For example, DS6A mycobacteriophage is specific for only M. tuberculosis strains. In contrast, L5 and TM4 mycobacteriophages are specific for several mycobacteria, including M. tuberculosis and M. smegmatis. In order to diagnose tuberculosis according to the invention, it is necessary to use mycobacteriophages which are specific for M. tuberculosis strains only. Because DS6A mycobacteriophage is specific for M. tuberculosis strains only, it can be used to narrow the host specificity of L5 and TM4 mycobacteriophages so that L5 and TM4 mycobacteriophages can be used to accurately diagnose tuberculosis. For example, a clinical sample (control) can be infected with L5 reporter mycobacteriophages. Another clinical sample from the same source (experimental) can be co-infected with both L5 reporter mycobacteriophages and DS6A reporter mycobacteriophages. If the photon signal generated by the experimental sample is lower than the signal generated by the control sample, a diagnosis of tuberculosis is confirmed. If, however, there is no decrease in photon signal, then the diagnosis for tuberculosis is negative. Hence, DS6A mycobacteriophages can be used to confer specificity for M. tuberculosis onto L5 mycobacteriophages. Figure 31 represents an outline of a method which can be used to diagnose tuberculosis and determine drug susceptibility using reporter mycobacteriophage DS6A.
In anticipation of the need for a diverse set of mycobacteriophages that can effect a broad or limited range of mycobacterial cells, a total of more than 50 unique mycobacteriophages have been collected and isolated by the inventors. 21 new mycobacteriophages have been isolated from soil samples from India, France, England, Israel, Tunisia, Carville, LA and New York. In addition, another 30 mycobacteriophages from both the Centers for Disease Control in Atlanta and the World Health Organization Phage Reference Laboratory in Amsterdam were collected. The characterization of the nucleic acid content of the phage particles of 30 of these mycobacteriophages have revealed that all of the mycobacteriophages contain double stranded DNA whose genome sizes range from 45 to 100kb as sized on pulsed field gels. Restriction analysis has shown that all of these mycobacteriophages are different, except that one of the mycobacteriophages from France had a considerable similarity to the L5 mycobacteriophage, which was originally isolated in Japan. The host range of the mycobacteriophages varies greatly, some being able to infect only M. smegmatis and others being able to infect M. smegmatis, BCG and M. tuberculosis, but not M. avium. These mycobacteriophages may be developed into reporter mycobacteriophages and cosmid cloning systems, and may provide a source of useful transcriptional translation initiating sequences, transcriptional terminators, or host-range specificity genes.
In addition, the choice of reporter gene and its method of expression are critical. It is necessary to choose a reporter gene whose product would not normally be found in clinical samples, but whose product is also easily detectable.
Luciferase reporter genes have been used in many diversified biological systems, including E. coli, cyanobacteria, phytopathogenic bacteria and Bacillus. The presence of luciferase reporter genes can be detected by the emission of photons in the presence of a substrate, such as luciferin or decanal. Luciferin and decanal can permeate mycobacteria, and thereby allow for the detection of gene products, such as photons. Since one molecule of the luciferase gene product can yield 0.85 photons of light, it is the most sensitive biological reporter molecule known. The preferred reporter genes of this invention are luciferase reporter genes, such as the Firefly lux gene (FFlux), the Vibrio fischeri lux genes and the Xenorhabdus luminescens lux genes, as well as the E. coli β-galactosidase (lacZ) genes. Luciferase genes, especially the Firefly lux gene, generate a high amount of luminescence activity. They generate photons. the detection of which is simple and sensitive, using commercially available luminometers that can detect 100-1000 molecules of luciferase with a linear relationship to enzyme concentration. In addition, it is unlikely that clinical samples will contain significant levels of endogenous luciferase activity.
In choosing transcriptional promoters to be introduced into the mycobacteriophages, it is desirable to use strong promoters since this will increase the sensitivity of the system. In addition, it is important that the promoter be active following mycobacteriophage infection. Promoter candidates currently available are the BCG hsp60 promoter and the L5 gene 71 promoter, which are of comparable strength. The hspβo promoter gives good levels of luciferase expression from plasmid recombinants, but lower levels of luciferase expression where the mycobacteriophage is TM4. It is possible that the reason for this is that the hsp60 promoter is shut off by the TM4 enzymes following infection, thus producing only a modest level of luciferase. The gene 71 promoter may behave in a similar manner with the TM4 phage since the gene 71 product is a good candidate for the L5 repressor and is expressed at high levels in the absence of other mycobacteriophage functions. Knowing the sequence of the mycobacteriophage used will help in identifying, characterizing and cloning the appropriate promoter to be used in the reporter mycobacteriophages of this invention. There are several methods which can be utilized to introduce the reporter genes and transcriptional promoters into mycobacterial species-specific mycobacteriophages. One method is the utilization of shuttle phasmids. When utilizing shuttle phasmid technology, it is necessary to know the sequence of the mycobacteriophage so that the reporter genes are inserted into non-essential regions of the mycobacteriophage. Insertion of reporter genes into non-essential regions permits the mycobacteriophage to survive and replicate. In order to use the shuttle phasmid methodology, it is necessary to first generate a cosmid library of large double-stranded recombinant DNA fragments of mycobacteriophage. This can be done using cosmid cloning in E. coli. Next, the cosmid library is introduced into the mycobacteria of interest to select for cosmids which have been inserted into non-essential regions of the mycobacteriophage. The shuttle phasmids, which consist of the E. coli cosmid, the reporter genes and mycobacteriophage promoters, may then be characterized. Shuttle phasmids can be propagated in E. coli as plasmids, and propagated in mycobacteria as mycobacteriophages.
A second method of introducing the reporter genes and transcriptional promoters into mycobacteriophages is by homologous recombination. First, non-essential regions of a mycobacteriophage must be determined. Again, in order to do this, it is necessary to know the sequence of the mycobacteriophage. Consequently, L5 is an ideal phage to use with this method as its genome has already been sequenced and characterized by the inventors. Next, plasmids are constructed wherein reporter genes hooked to transcriptional promoters are flanked by mycobacteriophage non-essential region sequences in mycobacterial plasmids. Then, homologous recombination systems may be utilized in M. smegmatis or E. coli to perform gene replacement whereby the plasmid constructs containing the reporter genes are put into mycobacteriophages.
A third method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by use of transposons. For example, transposon IS1096 may be utilized. In order to use this methodology, reporter genes and transcriptional promoters are put into transposons, and the transposons containing the reporter genes and transcriptional promoters are delivered on plasmids in mycobacteria. Next, it is necessary to grow up the mycobacteriophages on a strain such as M. smegmatis, which strain contains the transposons. At certain frequencies, the transposons will hop into non-essential regions of the mycobacteriophages, thereby introducing themselves therein. The mycobacteriophages are still viable, and contain the reporter genes and transcriptional promoters.
A fourth method of introducing reporter genes and transcriptional promoters into mycobacteriophages is by debilitated phages packaged into phage heads and tails (phage particles). To utilize this methodology, it is necessary to develop helper phage systems which allow for pieces of DNA containing pac sites to be packaged. These helper phages allow for the synthesis of head and tail genes at will in mycobacteria, prevent themselves from being packaged into phage heads and tails, and facilitate packaging of pacmids into phage heads and tails. Helper phage systems may be generated from the L5 mycobacteriophage. The genome of the helper phage is put into the mycobacterial chromosome, at which time the mycobacteria are grown up. Next, pacmids which comprise phages which have pac sites, reporter genes, transcriptional promoters and mycobacterial replicons are transformed onto the mycobacterial strain. The production of head and tail proteins may be induced, for example, through an increase in temperature, and the pacmids are then packaged into phage heads and tails. The L5 genome has cohesive (cos) termini. This suggests the possibility of constructing L5 cosmid vectors, which could be packaged through the cos sites into L5 particles either in vivo or in vitro. Then, a large number of genes could be easily and efficiently delivered to mycobacteria.
Packaging into phage heads and tails may also be utilized in a fifth methodology wherein the pacmid is a plasmid. The methodology is similar to the methodology wherein a debilitated phage is used, however, instead of using phage pacmids, the pacmids comprise plasmids which have pac sites, reporter genes, transcriptional promoters, and plasmid replicons.
Finally, direct cloning using recombinant DNA techniques in vitro may be used to introduce reporter genes and transcriptional promoters into mycobacteriophages. This methodology consists of ligating a mycobacteriophage, identifying or introducing unique restriction enzyme sites in non-essential regions of the mycobacteriophage, cleaving the mycobacteriophage with the restriction enzyme sites, and cleaving DNA which encodes the promoter and the reporter gene so that it has the unique sites flanking it on either side. Next, ligation is set up in vitro between the cleaved mycobacteriophage with the unique restriction enzyme sites and the reporter gene cassette. The result is a circular DNA molecule which consists of the mycobacteriophage, the reporter genes and the transcriptional promoters. The circular DNA may then be electroporated directly into mycobacteria.
EXAMPLES
Expression of Reporter Gene
lacZ and FFlux in Mycobacteria
A promoter probe vector was constructed which incorporated a truncated E. coli β-galactosidase (lacZ) gene as a reporter probe into a shuttle plasmid vector that replicated in either mycobacteria or E. coli. Random DNA fragments from the three mycobacteriophages Ll, TM4 and Bxbl were cloned into a unique BamHI site immediately upstream of the lacZ gene and screened for their ability to produce β-galactosidase. This established that lacZ could be used as a reporter gene in the mycobacteria, and identified the DNA sequences which could effectively express foreign genes in both M. smegmatis and M. tuberculosis. β-galactosidase activity could be detected from lysed cells using OMPG, or from unlysed cells using either X-gal or a fluorescent methylumbelliferyl β-galactosidase derivative. The promoter hsp60 gene highly expressed the lacZ gene in both M. smegmatis and BCG.
The FFlux gene was cloned into pMV261 downstream from the hsp60 promoter in plasmid pYUB180 (see Figure 2), which plasmid was shown to express the FFlux gene in M. smegmatis, BCG and M. tuberculosis H37Ra. The expression of the FFlux gene was detected by observing luminescence of mycobacterial clones containing the cloned gene in the dark room, and verified use in photographic film. This demonstrated that the luciferase was expressed in the mycobacteria, and that luciferin, the substrate used, was able to penetrate mycobacterial cell walls and yield photons expressed by the mycobacteria.
Detection of Photons In
Mycobacterial Cells Expressing FFlux
The expression of FFlux from the plasmid pYUB180 in
M. smegmatis provided a model with which to determine a minimal number of individual cells detectable with the luciferase assay. M. smegmatis containing pYUB180 were grown in the presence of kanamycin to ensure that every cell contained the plasmid. The cells were diluted 10-fold serially and the amount of luciferase activity was determined using a luminometer. Figure 3 shows that the amount of luciferase activity from 5 X 10 7 cells approached 108 luciferase units, though at this level of activity the luminometer was unable to yield an accurate measurement. However, the activity decreased in a linear manner down to 1200 units for 500 cells. Hence, 5000 cells expressing the FFlux gene can be clearly discerned above the background measurement, which approaches the number of cells that one would expect to observe in clinical samples.
Demonstration of Luciferin Uptake by Mycobacteria
In order to ascertain whether the substrate luciferin could be transported across the intact mycobacterial cell wall, the firefly luciferase (FFlux) gene was cloned downstream of the hsp60 promoter in a mycobacterial extrachromosomal plasmid, and was also cloned downstream of the gene 71 promoter of the mycobacteriophage L5 in a mycobacterial integrating vector. Figure 12 shows a schematic diagram of the extrachromosomal plasmid pYUB180 and the integration plasmid pGS16. Both of the luciferase constructs were electroporated into the M. smegmatis strain mc2155. Kanr transformants were grown to a density of approximately 5 × 108 cells/ml and 10-fold serial dilutions were prepared. 100 μl samples were mixed with 250 μl of 0.1 M Na citrate, pH5 in a 13 × 75 mM polystyrene tube. This mixture was placed in the monolight 2010 luminometer (Analytical Luminescence Laboratory, San Diego, CA) and 100 μl of 1 mM luciferin (Sigma, St. Louis, MO) was injected into the tube and the luciferase activity was measured as relative light units. As shown in Figure 13, upon the addition of luciferin, luciferase activity was readily measured from intact mycobacterial cells infected with both the extrachromosomal and the integrating vectors. Serial dilutions indicated that it was possible to detect as few as 500 to 5,000 M. smegmatis cells expressing firefly luciferase, thereby establishing that the luciferase-luciferin system could be developed as a sensitive reporter system for ATP in mycobacteria. Distinguishing Drug-Resistant Mycobacteria From
Drug-Sensitive Mycobacteria Using Luciferase Activity
Since Firefly luciferase activity requires ATP, and
ATP is produced only by living cells which are metabolically active, luciferase is a powerful indicator of the metabolic abilities of a bacterial cell. Since anti-tuberculosis drugs are likely to significantly decrease the metabolic activity of a cell, the measurement of luciferase activity should provide a sensitive means of distinguishing drug-resistant mycobacteria from drug-sensitive mycobacteria.
First, the kinetics of the production of luciferase activity of M. smegmatis containing pYUB180 following the addition of streptomycin, isoniazid, ethambutol, rifampicin, ciprofloxacin, novobiocin or cyanide, added at levels that inhibit the growth of M. smegmatis in plate assays, was measured.
As shown in Figure 4A, the levels of luciferase production were 100 to 1000 times less at eight hours after the addition of the drugs compared to the untreated control.
Next, this approach was used to distinguish drug-resistant from drug-sensitive mycobacteria. The pYUB180 deposit was transformed into streptomycin-resistant or novobiocin-resistant M. smegmatis mutants. Photon production by the drug-sensitive parent was compared to the streptomycin-resistant or novobiocin-resistant mutants. The drug-resistant mutants continued to produce luciferase activity levels comparable to the untreated patent in the presence of the appropriate antibiotic. In addition, the drug-resistant mutants produced 100 to 1000 times more luciferase activity than the drug-sensitive parent (see Figures 4B and 4C). Hence, a luciferase-based assay may be used to determine mycobacterial drug susceptibility. Construction of TM4 Reporter Mycobacteriophages
(phAE39, phAE37 and phAE40) and
Detection of Photons Following TM4 : :lux Infection
The first vectors developed to introduce recombinant DNA into mycobacteria were shuttle phasmid phage vectors.
Shuttle phasmids have the ability to replicate in E. coli as cosmids and then replicate in mycobacteria as phages. Shuttle phasmids of TM4 which contained the FFlux and lacZ genes transcribed from hsp60 and L1 promoters, respectively, were constructed (see Figure 5).
A deposit of the shuttle phasmid (reporter mycobacteriophage) phAE39 which contains mycobacteriophage TM4, cosmid pYUB216, reporter gene FFlux and promoter hsp60, was made with the American Type Culture Collection on January 15, 1992 and catalogued as ATCC No. 75183. When the TM4 ::lux shuttle phasmid phAE39 was mixed with M. smegmatis cells, luciferase activity could be detected within 15 minutes of incubation, and continued to increase slightly over the next 4 hours (see Figure 6). These results show that the TM4::lux mycobacteriophage is capable of introducing the FFlux gene into mycobacterial cells, and that the FFlux gene can be expressed in mycobacteriophage-infected cells. Figure 7 represents a flow chart for cloning different promoters into the TM4::lux shuttle phasmid phAE39.
A deposit of the shuttle phasmid (reporter mycobacteriophage) phAE37 which contains mycobacteriophage TM4, cosmid pYUB216, reporter gene lacZ and promoter L1, was made with the American Type Culture Collection on February 10, 1992 and catalogued as ATCC No. 75204. The TM4::lacZ mycobacteriophage formed bright blue plaques when plated on media containing X-gal.
A mutant of the shuttle phasmid phAE39, designated phAE40, was isolated. As discussed hereinabove, in order to produce shuttle phasmid phAE39, E. coli cosmid pYUB216 was inserted into a non-essential region of the mycobacteriophage TM4. The pYUB216 cosmid contained FFlux in a transcriptional fusion with the hsp60 promoter of BCG, a ColEl origin and an ampicillin-resistant gene (AP) for replication and selection in E. coli, and a bacteriophage lambda cos sequence as well as a unique Bc/l site. The phAE39 shuttle phasmid was constructed with Bc/l-digested pYUB216 being ligated to Sau3A-partially digested TM4 DNA. As shown in Figure 8, the shuttle phasmid phAE39 readily forms plaques of M. tuberculosis, but does not efficiently plaque on BCG. A spontaneous host range mutant of phAE39 was isolated at a frequency of 10 to 10 , and designated phAE40. Mutant shuttle phasmid phAE40 was found to be capable of infecting BCG vaccine strains, in addition to being capable of infecting M. smegmatis and M. tuberculosis strains. The shuttle phasmid phAE40 was deposited with the American Type Culture Collection on April 29, 1993 and catalogued as ATCC No. 75457. In order to test whether the phAE39 and phAE40 reporter mycobacteriophages were capable of eliciting the production of light following infection of mycobacteria, the reporter mycobacteriophages were mixed with M. smegmatis cells and then exposed at different times to luciferin. In order to perform this, high titers of phAE40 were prepared as described above for TM4 phages. Both M. smegmatis, mc2155 cells and
BCG-Pasteur cells were grown in roller bottles to approximately 5 × 107 cells per ml in M-ADC-TW broth at 37ºC. Either the M. smegmatis or the BCG cells were harvested by centrifugation and washed two times in M-ADC broth, containing no tween. The resulting pellet was resuspended in the original volume of M-ADC broth. The cells were then diluted into fresh M-ADC broth and allowed to incubate overnight standing at 37ºC. Tween-80 appeared to remove the receptors, and it was determined that the optimal activities were achieved if the cells were given a chance to grow in the absence of tween.
This may have allowed the regeneration of phage receptors. Next, 1 ml of washed cells (approximately 5 × 107 cells) was mixed with 0.1 ml phAE40 particles (5 × 108 pfu/ml) that had been concentrated on CsCl gradients to achieve a multiplicity of infection of 10. The cells phage mixture was incubated at 37ºC. Beginning at the time of the addition of the phAE40, 0.1 ml samples were removed. Luciferase activity was measured as described in Figure 13. Light signals were detected within minutes following infection using a luminometer and increased 1,000 fold within 2 hours. The rapid kinetics of light production allowed for the testing of the simple hypothesis that one reason slow-growing mycobacteria, such as BCG and M. tuberculosis, have generation times 10-fold longer than other mycobacteria is the consequence of a generalized slow rate of transcription or translation. The observation that the kinetics of light production following infection of BCG with the reporter mycobacteriophages is almost identical to that of M. smegmatis, thereby suggesting that the slow growth of slow growing mycobacteria is unlikely to be attributable to slower rates of metabolic processes, but rather is the result of a highly regulated event, such as the initiation of chromosome replication or cell division.
Since it was determined that the phAE39 and phAE40 reporter mycobacteriophages were able to elicit the production of light following infection of mycobacteria, they were used to distinguish between drug-resistant and drug-sensitive organisms. In order to perform this, mutants of BCG were selected that were resistant to streptomycin, isoniazid and rifampicin. Spontaneous mutants of BCG-Pasteur were isolated on Middlebrook 7H10 agar containing either 50 μg/ml rifampicin, 250 μg/ml streptomycin or 50 μg/ml isoniazid.
The rifampicin-, streptomycin-, or isoniazid-resistant mutants were purified and designated mc2768, mc2767 and mc2765, respectively. All three mutants and the BCG parent were grown to midlog phase, harvested and washed. As shown in Figure 10C, the mc2768 cells and the BCG cells were incubated standing at
37°C in the presence or absence of rifampicin (50 μg/ml) for 24 hours. A 0.5 ml sample (approximately 5 × 107 viable cells) was mixed with 0.1 ml (5 × 107 pfu) of phAE40 particles and luciferase activity was determined. The samples were removed and luciferase activity was measured. As shown in
Figure 10B, the mc2767 cells and the BCG cells were incubated standing at 37°C in the presence or absence of streptomycin
(250 μg/ml) for 24 hours. A reporter assay was performed as described above. As shown in Figure 10C, the mc2765 cells and the BCG cells were incubated standing at 37°C in the presence or absence of isoniazid (50 μg/ml) for 24 hours. The reporter assay was performed as described above. As shown in Figure 10, when wild-type BCG and the mutants were cultured for 24 hours with the antibiotics, the parental strain (wild-type BCG) failed to produce any signal, whereas light was produced by the drug-resistant mutants.
Next, the luciferase reporter phage assay was tested on clinically-derived M. tuberculosis strains, which were both singly and multiply drug-resistant. In order to perform this, the following M. tuberculosis strains were grown in a biological safety level 3 containment facility: (i) the virulent drug-sensitive M. tuberculosis Erdman strain; (ii) strain 92-2025, a singly isoniazid-resistant strain; and (iii) an MDR strain of tuberculosis that has been shown to be resistant to rifampicin, streptomycin, isoniazid, ethambutol and ethionamide and the cause of several nosocomial outbreaks in New York City. The Erdman strain was subcultured from the starter culture by inoculation of 0.4 ml into 20 ml of Middlebrook 7H9 broth containing OADC enrichment (Difco Laboratories, Detroit, MI) plus 0.5 Tween-80 (M-OADC-TW broth). The 92-2025 and the MDR strains, which grow more slowly than the Erdman strain, were subcultured by inoculation of 2 ml into 20 ml M-OADC-TW broth.
All three cultures were grown standing at 37°C for 7 to 8 days. The cells were washed and resuspended in 0.5 × the original volume. Washed cells (0.2 ml) were inoculated into 0.7 ml of M-OADC broth and incubated in 13 × 100 mm polypropylene tubes in a heating block in a Biohazard hood for 48 hours. Rifampicin, streptomycin, or isoniazid were added to final concentrations of 2 μg/ml, 0 μg/ml, and 1 μg/ml, respectively. After 48 hours of incubation, 0.1 ml of phAE40 particles (1 × 10 particles) were added to attain a multiplicity of infection of 1000. Samples of 100 μl were removed at 1, 3 and 5 hours after addition of the phage and were mixed with 250 μl of 0.1 M sodium citrate (pH 5) in a Lumacuvette (Lumac, BV, Netherlands). One hundred microliters of 1 mM luciferin were added, and the Lumacuvette was plugged with cotton. The tube was placed in a Lumac Biocounter (M1500P), and readings were recorded as described above. (The Lumac Biocounter had dimensions that permit it to fit in a standard biohazard hood.) The light production followed kinetics similar to the BCG experiments, and the readings at 3 and 5 hours differed by no more than twofold. The results at 3 hours are shown for the Erdman (Figure 11A), 92-2025 (Figure 11B), and the MDR (Figure 11C) M. tuberculosis strains.
A repeated experiment gave similar results, with the samples cultured in the absence of drug exhibiting an 80-fold greater luminescence than the cells cultured with rifampicin or streptomycin and greater than 10-fold luminescence relative to those cultured with isoniazid at 3 and 5 hours. Open bars represent cells alone; filled bars represent cells plus LRP; diagonal lines represent cells plus rifampicin plus LRP; cross-hatching represents cells plus streptomycin plus LRP; squares represents cells plus isoniazid. As shown in Figures 11A-C, the luciferase reporter phages were capable of rapidly revealing the patterns of drug-susceptibility or resistance of M. tuberculosis strains.
Construction of L5 Reporter
Mycobacteriophages (phGS1 and phGS5)
In order to construct L5::FFlux phages, a plasmid (pGS12) was constructed in which a DNA segment of the L5 genome was inserted into the E. coli-mycobacterial shuttle plasmid pMD31. pMD31 is described by Donnelly-Wu et al. in "Superinfection Immunity of Mycobacteriophage L5: Applications for Genetic Transformation of Mycobacteria", Molecular Microbiology, Vol. 7, No. 3, pages 407-417 (1993). This DNA segment contained the tRNA gene cluster from L5 as described by Hatfull et al. in "DNA Sequence, Structure and Gene Expression of Mycobacteriophage L5: A Phage System for Mycobacterial Genetics", Molecular Microbiology, Vol. 7, No. 3, pages 395-405 (1993). Next, this plasmid was further manipulated by insertion of a segment of DNA containing the FFlux gene between the second and third tRNA, to produce pGS24. The resulting plasmid DNA was introduced into M. smegmatis by electroporation, and an L5 lysate was prepared by growth of L5 phage on this plasmid-containing strain.
Individual phages were screened by hybridization using an FFlux probe and filters containing 106-107 plaques.
Several positive plaques were identified and two were purified and characterized. These two phages were named phGS1 and phGS5.
Construction of plasmids pGS11, pGS12, pGS22 and pGS24
Plasmids pGS11, pGS12 and pGS22 were constructed as described below and then used to construct plasmid pGS24. L5 DNA was cleaved with Xba I and Bcl I and the 3,993bp fragment was purified. This DNA fragment represents the L5 segment defined by the coordinates 3,150-7,143. Figure 14 is a segment of L5 DNA used for FFlux insertion which shows the left arm of the L5 genome with genes 1-33 indicated. The segment of L5 taken to make FFlux inserts is between the Xba I and Bcl I sites indicated. The Nhe I site that defines the position of insertion of FFlux is shown. This DNA fragment was inserted into plasmid pMV2611acZ (see Stover et al., Nature, Vol. 351, pp. 456-460, 1991) cleaved with Xba I and Bam HI to produce plasmid pGS1l (see Figure 15). Figure 15 is a map of plasmid pGS11 which contains the Xba I - Bcl I segment of L5 inserted into pMV2611acZ. The Bcl I end was inserted into the Bam HI site of the vector and both the Bcl I and Bam HI sites were destroyed. The Hind III and Xba I sites that were used to construct pGS12 are indicated.
Plasmid pGS1l DNA was cleaved with Xba I and Hind III and the 4,013bp fragment was purified and inserted into plasmid pMD31 (Donnelly-Wu et al., 1993) cleaved with Xba I and Hind III. This plasmid was named pGS12. Figure 16 is a map of plasmid pGS12 showing the location of the Xba I and Hind III sites used to insert the Xba I - Hind III piece from pGS11 into pMD31. The unique Nhe I site used for the insertion of FFlux is also shown. Plasmid pGS12 contains a unique Nhe I restriction site which corresponds to the Nhe I site at position 4,441 in the L5 genome which is located between the tRNA-trp and tRNA-gln genes (genes 8 and 9).
Plasmid pYUB216 was cut with Hind III, the sticky ends converted to blunt ends by Klenow enzyme and dNTP's and the DNA religated. The resulting plasmid was named pGS22. Figure 17 is a map of plasmid pGS22 which shows the two Nhe I sites that flank the FFlux gene. This procedure was followed to generate an additional Nhe I site upstream of the FFlux gene in pYUB216.
pGS12 was digested with Nhe I. pGS22 was also digested with Nhe I which produces a fragment of approximately 2.4kb. The DNA's were mixed, ligated and a recombinant recovered in which the Nhe I fragment derived from pGS22 was inserted into the Nhe I site of pGS12. This plasmid was named pGS24. Figure 18 is a map of plasmid pGS24 which contains the Nhe I FFlux DNA fragment inserted into the unique Nhe I site of pGS12. The two Nhe I sites are indicated. The orientation of the inserted DNA was determined by restriction enzyme digestion and found to be in the appropriate orientation for FFlux to be expressed from the same DNA strand as the L5 tRNA's. pGS24 is thus a E. coli-mycobacterial shuttle plasmid that contains the FFlux gene flanked upstream by approximately 1,291bp of L5 DNA and downstream by approximately 2,702bp L5 DNA.
Construction of phGS1 and phGS5
Having constructed a plasmid containing FFlux flanked by L5 DNA, FFlux was inserted onto the L5 genome by a double crossover event between plasmid pGS24 and L5. This was achieved by growth of an L5 lysate on M. smegmatis carrying plasmid pGS24 and searching among the progeny for FFlux-containing phage by hybridization. Figure 19 shows the strategy for recombination between pGS24 and L5. Specifically, Figures 19A and 19B show the left arm of L5 and the position of genes 1-33. Figure 19A shows the segment of L5 DNA present in pGS24 and the location of FFlux inserted between the tRNA-trp and tRNA-gln genes. It was hoped that by growth of L5 phage in cells containing plasmid pGS24 that progeny could be recovered in which the FFlux gene had been inserted into the L5 genome by homologous recombination within the common sequences to the left and right of FFlux in pGS24 and those in L5.
Plasmid pGS24 DNA was introduced into M. smegmatis mc2-155 by electroporation, and transformants recovered by selection with kanamycin. A lysate of phage L5 was prepared by infection of approximately 0.5 ml late-log phage M. smegmatis cells containing plasmid pGS24 with approximately 10 L5ts11 particles and incubation on solid media at 37°C. [L5ts11 is a poorly characterized temperature-sensitive mutant of L5]. The phages were harvested and shown to have a titer of approximately 10 plaque forming particles/ml (pfu/ml).
Approximately 10 6-107 phage articles were added to
M. smegmatis mc2-155 cells and plated onto large agar plates. After incubation, plaques were transferred to nitrocellulose filters and probed with radioactively labeled pYUB216 DNA. About 15 positive plaques were identified.
Several positive plaques were recovered from the agar plates purified through several rounds of plaque purification, checking with positive hybridization to the pYUB216 DNA probe at each stage. At the end of this procedure, two of the phages were chosen for further characterization. These phages were named phGS1 and phGS5.
Characterization of phGS1 and phGS5 DNA's
Phage DNA's were prepared from high titer stocks of phGS1 and phGS5 using standard methods.
phGS1 and phGS5 DNA's were digested with several different restriction enzymes (including Bam HI, Nhe I, Bst E II, Asp718, Cla I, Bgl II) and the patterns observed compared with those obtained from wild-type L5, using agarose gel electrophoresis. Several differences were observed between phGS1 and phGS5 as compared to L5 DNA. Some of these changes were consistent with a double crossover recombination event inserting FFlux onto the L5 genome as anticipated. Other differences were consistent with deletion of some of the L5 DNA close to the right end of the genome.
Confirmation of the structures of phGS1 and phGS5 was obtained by hybridization of Southern blots of the DNA's using a variety of DNA probes. phGS1, phGS5 and L5 DNA's were digested with either Bam HI, BamHI and EcoRI, Asp718 or Asp718 and ClaI. DNA fragments were separated by agarose gel electrophoresis and transferred to a nitrocellulose filter. This filter was probed with radiolabelled pGS12 DNA and the hybridizing bands detected by autoradiography. The results are shown in Figures 20A and 20B. Figure 20A shows a Southern blot of the insertion of FFlux into L5. DNA purified from L5, phGS1 and phGS5 particles was cleaved with restriction enzymes as indicated and the fragments separated by agarose gel electrophoresis. The DNA fragments were transferred to a nitrocellulose filter and probed with radiolabelled pGS12 DNA. Following autoradiography, the filter was stripped and probed with radiolabelled pYUB216 DNA (Figure 20B). These data conclusively demonstrate that FFlux is inserted into the L5 genome in a corresponding location to that in pGS24 as would be expected from a pair of homologous recombination events in the common flanking sequences. A map of the expected DNA fragments is shown in Figure 21.
Figures 21A-1 and 21A-2 maps show the expected restriction products from FFlux insertion - Bam HI. The location of the L5 probe (from pGS12) used for hybridization is shown (labeled 'probe'). This probe is expected to hybridize to two comigrating Bam HI fragments (3.010bp and 3,104bp) in wild-type L5 DNA (shown as 'labeled BAM HI fragments' in the top part of the figure). Figure 21A-2 shows the anticipated structure of the FFlux insertion and the expected fragments resulting from digestion with either Bam HI or BAM HI + EcoRI that hybridize with the probe. These are 3,010bp and 4,937bp fragments from Bam HI digestion and 3,010bp, l,183bp and 3,754bp fragments from Bam HI and EcoRI digestion. The location of the FFlux probe derived from pYUB216 is indicated in Figure 21A-2. it is expected to hybridize to the 4,937bp Bam HI fragment, and the l,183bp and 3,754bp Bam HI/EcoRI fragments in the recombinants, but not to L5 at all. The data shown in Figures 21A-1 and 21A-2 agree well with these predictions.
Figures 21B-1 and 21B-2 maps show the expected restriction products from FFlux insertion - Asp718/Cla I. The pGS12 probe is anticipated to hybridize to 2,690bp, 1,148bp and 8,078bp fragments resulting from Asp718 digestion of L5 and 2,690bp, 2,981bp and 8,078bp from the FFlux recombinants. This probe is also expected to hybridize to 645bp, 1,148bp and 8,078bp L5 fragments from Asp718 + Cla I digestion and 645bp, 1,078bp and 8,078bp fragments from this digestion of the FFlux recombinants. Note that the l,078bp fragment migrates as a fragment of approximately 1.5kb which reflects the difference between the DNA strider generated maps and empirically-determined maps. The FFlux probe in pYUB216 hybridizes to the 2,981bp Asp718 fragments and the 1,903bp and 1,078bp fragments from Asp718 - Cla I digestion's of the recombinant phage.
Digestion of phGS1 and phGS5 DNA with Bam HI indicated that neither contained the largest Bam HI fragment (7.711bp). The coordinates of the Bam HI sites that yield this fragment in wild-type L5 are 43,933 and 51,644. phGS1 and phGS5 thus appear to have lost a segment of L5 DNA close to the right end of the genome. Since the adjacent Bam HI fragments appear to be intact, it seemed probable that both Bam HI sites were present in phGS1 and phGS5 and that segments within the 7.7llbp Bam HI fragment were deleted. It was also not clear whether phGS1 and phGS5 were identical in this respect.
Hybridization of a specific L5 DNA probe derived from the 7.7llbp Bam HI fragment to a Southern blot of digested DNA's showed that phGS1 and phGS5 contain deletions of different sizes. phGS1 contained a new hybridizing fragment of approximately 3.4kb indicating that 4.3kb of DNA internal to the 7.7kb Bam HI fragment had been deleted. phGS5 contained a new hybridizing fragment of 5.3kb indicating that 2.4kb of DNA internal to the 7.7kb Bam HI fragment had been deleted.
The exact end points of the deletions in phGS1 and phGS5 have not yet been determined. However, approximate end points were determined through a combination of restriction enzyme mapping and Southern blot hybridization as shown in Figure 22. Figure 22 map shows deleted regions in phGS1 and phGS5. phGS1 and phGS5 DNA's were found to contain deletions of L5 DNA in the right arm close to the right end of the genome. The location of these deletions was determined by a series of restriction enzyme digestions and Southern blot hybridizations and the approximate locations are shown here. The dark box represents the deleted portions and the limits of the positions are defined by the vertical lines. phGS1 contains a deletion of approximately 4.3kb DNA and phGS5 contains a deletion of approximately 2.4kb DNA.
For phGS1, the left end point appears to be to the right of the Bgl II site located at position 44,803 (wild-type L5 coordinates); the right end point is to the right of the BstE II site at 49,588 and to the left of the Bgl II site at 50,716. For phGS5, the left end point is to the right of the Sca I site at 47,559 and to the left of the Asp718 site at 48,750; the right end point is to the right of the Bgl II site at 50,716 and to the left of the Mse I site at 51,344.
Plasmids phGS1 and phGS5 were deposited on April 27, 1993 with the American Type Culture Collection, Rockville, Maryland, and catalogued as ATCC Nos. 75454 and 75453, respectively.
Construction of other phGS1 and phGS5 Derivatives
In order to fully evaluate the behaviors of the L5::FFlux recombinants, several additional derivatives were isolated. Isolation of phGS1ts+ and phGS5ts+:
It should be noted phGS1 and phGS5 are both derivatives of L5ts11. L5ts11 was chosen because some preliminary data indicated the temperature-sensitive mutation may lie within the region of DNA represented in pGS12. However, both phGS1 and phGS5 are still temperature-sensitive and fail to grow at 42°C. Temperature-resistant derivatives were isolated from phGS1 and phGS5 by plating approximately 10 particles at 42°C and recovering a derivative that was now competent to grow normally at 42°C. These were named phGS1ts+ and phGS5ts+, respectively. It is likely that these are simply derivatives of phGS1 and phGS5 that have resulted from the initial temperature-sensitive mutation in
L5tsll. Phages phGS1ts+ and phGS5ts+ behave similarly to their direct parents in all respects except that they are competent to grow at high temperatures.
Isolation of phGS5ts+cpml, phGS5ts+cpm2, phGS5ts+cpm3, phGS5ts+cpm4, and phGS5ts+cpm5.
Several clear plaque mutants of phGS5ts+ were isolated that are unable to form lysogens. These were isolated by plating various numbers of phage particles on M. smegmatis cells at 42ºC and looking for clear plague versions. We have shown previously that these mutants arise at a frequency of 10-3 - 10-4 (Donnelly-Wu et al., 1993). Five separate mutants were isolated and named phGS5ts+cpml, phGS5ts+cpm2, phGS5ts+cpm3, phGS5ts+cpm4, and phGS5ts+cpm5.
Isolation of phGS1 and phGS5 lysoqens of M. smegmatis
phGS1 and phGS5 lysogens of M. smegmatis mc2-155 were generated using standard methods. The phage lysates were used to infect M. smegmatis mc2-155. Cells were then recovered from the infected area and purified by plating for isolated colonies. One lysogenic isolate was prepared from each phage and shown to confer immunity to superinfection by
L5, a known property of L5 lysogens (Donnelly-Wu et al., 1993). These were named M. smegmatis mc2-155(phGS1) and
M. smegmatis mc2-155(phGS5). Luciferase activity of plasmids and lysogens
It was anticipated that plasmid pGS24 would have little or no luciferase activity in M. smegmatis mc2-155.
Likewise, it was not anticipated that M. smegmatis mc2-155(phGS1) and M. smegmatis mc2-155(phGS5) lysogens would have much luciferase activity. This view was arrived at via the assumption that transcriptional promoters for expression of the L5 genes 1-32 probably resided between genes 88 and 1. These promoters would thus have been removed from plasmid pGS24 and were expected to be inactive in the lysogenic state. Determination of the luciferase activity of pGS24 and the lysogens indicated that these assumptions were incorrect.
Figure 23 shows luciferase activity of pGS24 and phGS1 and phGS5 lysogens. Cultures of either M. smegmatis mc2-155 lysogens of phGS1 or phGS5 or M. smegmatis mc2-155 carrying pGS24 were grown to early log phase and the optical density (O.D.) determined at A600. A portion (10-20 μl) was removed and FFlux activity determined in a Luminometer (Analytical Luminescence Monolight 2010) using luciferin as a substrate. The activities shown are normalized for 1.0 O.D. unit for 1 ml culture.
As shown in Figure 23, lysogens of phGS1 and phGS5 and pGS24 all have considerable amounts of luciferase activity. There is a small difference between the activities of phGS1 and phGS5 which is probably not significant.
Luciferase activity following infection of M. smegmatis mc2-155
Luciferase activity was determined following liquid infection of M. smegmatis mc2-155 with phGS1 and phGS5.
Figure 24 shows luciferase activity following infection of
M. smegmatis with phGS1 and phGS5. phGS1 or phGS5
(approximately 4 × 107 pfu) were added to an early log phase culture of M. smegmatis mc2-155 (O.D. A600=0.1) incubated at 30°C and 50 μl samples removed for FFlux activity determination at various times. The absolute relative light units (RLU) obtained are shown at each time point. As shown in Figure 24, activity increased sharply for four hours and then increased less rapidly for as long as the experiment was pursued, up to about 50 hours. phGS1 consistently produced less activity than phGS5 in this assay. These phages are extremely active in FFlux activity, and phGS5 produced almost 107 relative light units (RLU) after 50 hours. The background in this assay (for example if phages are omitted) is routinely between 180 and 200 RLU's.
Efficient light production reguires the formation of lysoqens
Since light production increases over a long period of time after phGS1 and phGS5 infection, it was reasoned that this could result from formation and growth of stable lysogens. It was shown above phGS1 and phGS5 strongly express FFlux in the lysogenic state. This hypothesis was tested by comparing the activity of phGS5 with clear plaque mutant derivatives that are not competent to form lysogens. The data are shown in Figure 25. It was apparent that where the activity of phGS5 continued with time, the clear plaque mutant derivatives rose to a maximum activity after about three hours and then declined to a background level. The difference in activity of phGS5 and the clear plaque mutants was greater than 104-fold at 20 hours after infection. Comparison of L5::FFlux and TM4::FFlux phages
The activity of phages phAE40 and the L5::FFlux phages were compared following infection of M. smegmatis mc2-155 (see Figure 26). The activity of phAE40 had characteristics similar to that of the clear plaque mutants of phGS5 although the maximum activity was greater. However, at all points after 4 hours, the phGS5 and phGS1 phage had substantially greater activity. Sensitivity of the phGS5 phage
It was apparent that phGS5 has the greatest potential of all of the luciferase reporter phages constructed to detect small numbers of mycobacterial cells. To evaluate its sensitivity, serial dilutions of a culture of M. Smegmatis mc2-155 was prepared, infected with phGS5 and then FFlux activity 20 hours after infection was determined. Two different concentrations of phGS5 phage were used. A culture of M. smegmatis mc2-155 (O.D. A600=0.1) was diluted by serial 10-fold dilution. 100 μl portions were infected with either 4 × 10 7 pfu or 4 × 105 pfu phGS5 as indicated.
After 20 hours incubation at 30°C, 50 μl samples were removed for measuring FFlux activity. Assuming that a culture of
M. smegmatis mc2-155 with an O.D. A600=0.1 contains approximately 10 bacteria/ml, this experiment demonstrates that approximately 5,000 cells of M. smegmatis can be readily detected in this assay. The results are shown in Figure 27. These data demonstrate that a 50 μl culture with an O.D. ~0.0001 (equivalent to approximately 5,000 bacterial cells) infected with 4 × 105 pfu phGS5 produced a signal (4,000 RLU) more than 10-fold greater than in a culture containing no mycobacteria (180-200 RLU). A culture containing approximately
500 cells produced a signal (approximately 600 RLU) infected with a similar titer of phage gave a signal 2-fold greater than background. It was concluded that phGS5 offers exquisite sensitivity for the detection of small numbers of M. smegmatis cells.
Further Evaluation of Sensitivity of L5::FFlux Phages
An experiment similar to that described in Figure 27 was performed to measure for light production (RLU) at both 20 hours and 40 hours after the addition of phage phGS18. Aliquots were plated onto agar for viable colony counts from several samples either before or after phage infection. Infections were done in duplicate and average numbers are shown in Figure 28A.
10 μl aliquots were removed from samples either immediately prior to addition of phage (T=0), or 20 hours (T=20) or 40 hours (T=40) following addition of phage. Each row shows viable colony forming units present in a 50 μl sample for a given size of phage (either 5 × 10 or 5 × 107pfu) and starting cell innoculum. Figure 28B shows the light produced (RLU) for each sample and the calculated light per colony forming unit (RLA/Cell) at both 20 hours and
40 hours. These numbers correlate to Figure 28A such that the sample shown as 1.5 × 10e-5 in Figure 28A contained a starting innoculum of an estimated 69 colony forming units and when infected with 5 × 10 phage yielded a signal of 2,229 RLU after 40 hours at 30°C. This illustrates the exquisite sensitivity of these reporter phages and the use of lysogeny to amplify the signal.
Infection of an L5 Lysogen
To evaluate the influence of lysogeny of the host, light production following infection of an M. smegmatis L5 lysogen [155(L5)] with a non-lysogen (155) was compared. Figure 29 shows the data from a similar experiment using phAE40 which was expected to be unaffected by L5 lysogeny. Interestingly, it was observed that infection of an L5 lysogen [155(L5)] with phGS18 produced at least as much light (RLU) as from infection of a non-lysogen and is actually consistently 2-5 fold higher. Given this observation, it was predicted that the clear plaque mutant phGS26 would give extended light production after infection of an L5 lysogen, in contrast to the characteristic pattern observed in a non-lysogen (see also figure 25). Figure 29 shows this to be exactly what was observed. In addition, Figure 29 shows that L5 lysogeny has no effect on phAE40 infection. It was concluded that the potential problem of naturally occurring lysogens of M. tuberculosis does not present a significant one in this assay.
Each L5::FFlux phage constructed to date is listed in Figure 30 along with any alternative or previous designation, whether or not it has been characterized further, a brief description and the date of isolation. Note that of the initial phage derivatives, only phGS1 and phGS5 have been characterized further with respect to the point of insertion of FFlux and approximate location of deletions in the right arm of the phage genome. The ts derivatives and clear plaque mutant derivatives have not been fully characterized with respect to their specific differences from their phGS1 or phGS5 precursors.
Other Methods of Constructing
L5 Reporter Mycobacteriophages
The use of the shuttle phasmid approach starting with L5 deletion derivatives, in which the size of the genome has been reduced, should be further explored in determining strategies for the construction of recombinant L5 mycobacteriophages. Initially, the largest gene 71 deletion available could be used, or deletions of the gene 72-88 region similar to those described for phGS1 and phGS5 as described in Figure 22 could be used. Another approach would be to attempt to introduce genes by homologous recombination with plasmids. Still another approach would be to transpose lux genes onto L5 using either the mini-Mu in vitro transposition system or a mycobacterial transposon such as IS1096.
Recombining reporter genes from additional recombinant plasmids onto L5 using a double recombination event may be performed. This involves first constructing a recombinant plasmid that carries a reporter gene (lacZ may be more suitable) inserted into gene 71 such that both the upstream and downstream parts of gene 71 are present. Advantages of this approach are that lacZ can be easily detected in agar media, that gene 71 is not an essential gene, and that lacZ is efficiently expressed from a promoter immediately upstream of gene 71. An L5 mycobacteriophage lysate may be prepared by growth of the plasmid-containing strain and recombinant mycobacteriophage progeny identified by plating the lysate on wild-type M. smegmatis for individual plaques on agar containing the indicator X-gal. Alternatively, recombinant phage derivatives could be identified by hybridization.
This recombination approach may be expanded to introduce other gene or DNA segments of the L5 genome. For example, it should be possible to add luciferase genes from FFlux in an identical manner, provided that packaging limits are not exceeded. In addition, inclusion of polylinker containing restriction enzyme sites unique for L5 would open the way for construction of L5 recombinants in vitro. Similar genetic strategies may be used to systematically reduce the size of the L5 genome by deletion of non-essential sequences.
Transposition offers an alternative method for the construction of reporter mycobacteriophages. A transposition system which is available is the mini-Mu in vitro transposition system. This is a defined biochemical reaction in which a mini-Mu transposon carrying the desired gene is transposed onto the phage genome using purified MuA and MuB proteins. Similar transposition experiments have been tried with L5, but few L5 mini-Mu derivatives have been isolated. It is possible that this is due to the relatively large size of the transposon used. It is necessary to first construct a small Mu transposon which contains the reporter gene, a promoter and the two Mu in order for these experiments to be successful.
Development of L5 in vivo
and in vitro Packaging Systems
λ cosmids and packaging systems provide the efficiency of mycobacteriophage infection with the ability to inject large segments of non-mycobacteriophage DNA. Analogous mycobacterial systems would overcome packaging constraints encountered with recombinant mycobacteriophage genomes and allow the introduction of multiple copies or types of reporter genes into mycobacteria, potentially enhancing the sensitivity of the assay. In addition, they would help overcome any problems with host synthesis inhibition.
The development of L5 cosmids and packaging systems is dependent on the finding that the L5 genome contains cohesive termini. The λ paradigm suggests that a relatively small region of DNA (approximately 500bp) around the cos site (in the ligated form) is necessary to promote packaging. The first series of experiments with L5 would therefore be to identify the segment of the genome required for packaging by constructing a series of plasmids containing the L5 cos site and surrounding sequences. Cos activity may be determined by preparation of an L5 lysate on plasmid-containing M. smegmatis strains, followed by the identification of antibiotic-resistant transductants in the lysate, by transduction of M. smegmatis. This assay assumes that plasmid multimers of a total size of approximately 50kb are present in the cell and will be packaged. Although the presence of such multimers has not been demonstrated directly, they are likely to be generated by the homologous recombination system of M. smegmatis. If this assay should fail, cosmid vectors which contain both L5 λ cos sites may be constructed. Insertion of 40-45kb of DNA (as in the construction of cosmid libraries) followed by λ packaging in vitro and infection with E. coli will generate 50kb sized molecules containing L5 cos site. These should be isolated from E. coli and introduced by electroporation into M. smegmatis. Assuming that one of these approaches is successful, it would then be possible to define a small segment of L5 DNA required for packaging.
The construction of in vivo cosmid packaging systems is a particularly attractive idea since it has proven very useful in E. coli. Thermoinducible lysogens of L5 may be suitable for in vivo packaging of L5 cosmids without further modification, since prophage excision may be a temperature-sensitive event. Efficient packaging of extrachromosomal cosmids present in the lysogen may be achieved by simple induction and growth at 42°C.
It is possible that some process other than excision is temperature-sensitive in lysogen induction. If so, it will be necessary to further debilitate the prophage in order to prevent DNA packaging of the prophage. There are a variety of ways to accomplish this. For example, the excise gene itself could be deleted (using a recombination strategy similar to that described above) such as to prevent excision. Another approach is to damage the cohesive termini (by exonucleolytic digestion) of an L5 thermoinducible derivative and construct a defective lysogen. A combination of approaches may be desirable, since even if prophage excision is a temperature-sensitive process, the destruction of cos might effectively reduce the background of spontaneous mycobacteriophage release. Construction of in vitro packaging systems will follow similar lines. Extracts may be prepared from thermoinducible strains with non-packagable prophages and assessed for their ability to package exogenously added L5 cosmid or mycobacteriophage DNA. Optimization of conditions should follow both empirical biochemical approaches and the well-established λ systems. For example, it may be necessary to supplement the extracts with purified mycobacteriophage products such as the terminase or the tape-measure analogues (genes A/Nu and H of λ respectively), neither of which have yet been identified.
Construction of Novel Shuttle
Phasmids From Any Mycobacteriophage
Although mycobacteriophages L5 and TM4 can be used in the development of diagnostic luciferase and β-galactosidase shuttle phasmids, there may be other mycobacteriophages, such as the mycobacteriophage DS6A which only infects BCG and
M. tuberculosis strains, that might prove to have a more useful host range for clinical isolates. Diagnostic luciferase mycobacteriophages from these other mycobacteriophages may be developed by using the shuttle phasmid methodology described herein that has been proven successful for constructing mycobacteriophage vectors from both TM4 and phage L1. Isolate Mycobacteriophage L5 and TM4 Mutants to
Infect the Maximum Number of Clinical Isolates
For the diagnostic luciferase mycobacteriophage system to have maximal use in the clinical laboratory, it will be essential that to develop a set of diagnostic mycobacteriophages that can efficiently infect any clinical isolate and possibly distinguish M. tuberculosis from M. avium and BCG. Both mycobacteriophages TM4 and L5 appear to have the ability to infect a large number of M. tuberculosis isolates. TM4 is very closely related to phage 33D, a mycobacteriophage that has been found not to infect every M. tuberculosis isolate used to define the mycobacteriophage typing schemes for M. tuberculosis isolates. However, this mycobacteriophage does not infect BCG. TM4 has been found to be almost identical by DNA hybridization and restriction analysis to 33D, and it shares the host-specificity with 33D in that it infects M. tuberculosis, but fails to infect BCG. Mycobacteriophage L5 appears to share the same receptor as mycobacteriophage D29 which receptor has been previously shown to infect a very large number of M. tuberculosis isolates. L5, unlike 33D or TM4, infects all three morphotypes of M. avium including a wide range of serovariants.
If L5 or TM4 are found not to infect certain M. tuberculosis isolates, it may be possible to isolate mutants of these mycobacteriophages which plaque on the particular isolate. The inability to plaque on a particular isolate could result from the lack of a mycobacteriophage receptor or be the result of lysogenization of the isolate with a homoimmune phage. Phage mutants with altered host range specificities or mutants which no longer bind a repressor (equivalent to virulent mutant of λ) have been isolated in other systems.
Variants of TM4 which can efficiently infect BCG have been isolated at frequencies of 107. Previous work has demonstrated that 33D, similarly to TM4, cannot adsorb to BCG cells. Host-range variants of TM4 which not only plaque BCG, but also still plaque M. tuberculosis have been isolated.
Similar strategies for M. tuberculosis isolates which are uninfected by L5 or TM4 may be used.
Detecting the Presence of
M. tuberculosis in Clinical Samples
The combined sensitivities of luciferase and mycobacteriophage infections should permit the detection of previously undetectable levels of M. tuberculosis cells in sputum, blood samples, or cerebral spinal fluid. A number of preliminary studies to optimize the detection of M. tuberculosis cells in a variety of body samples will be performed.
Detecting M. tuberculosis Grown In Primary Human Macrophages and Macrophage Cell Lines
As a model system for optimizing detection of M. tuberculosis in infected monocytes and macrophages, primary human monocytes which have been purified by adherence for 1 hour or primary macrophages which have been cultured for 6 days in microwells will be infected with M. tuberculosis H37Ra at varying multiplicities. The number of cells initially infected will be determined microscopically, and then at various periods of time from 2 hours to 30 days, the cells will by lysed by non-ionic detergent NP40 which has no effect on viability of mycobacteria, concentrated by centrifugation, plated for viable organisms and infected with the luciferase plasmids. Quantitative studies at different moi's and with varying numbers of infected cells will indicate how few bacilli/cell and bacilli/specimen can be detected.
The inability of M. tuberculosis cells isolated from macrophages to be infected with diagnostic shuttle phasmids could result from either the absence of the expression of the mycobacteriophage-receptor or the masking of the receptor with a membrane from a phagosome of the macrophage. The level of expression of phage receptors may be regulated by the environment in which the host cell is grown. For example, the λ repressor of E. coli is induced by maltose and repressed by glucose. Studies to identify the receptors for mycobacteriophage L5 have been initiated. Similar studies for mycobacteriophage TM4 will also be performed. By identifying the genes encoding the receptor, it is possible to assay gene repression of the mycobacteriophage receptor of M. tuberculosis
cells when grown in macrophages by hybridization for the mRNA synthesis. If the receptor is not expressed in macrophages, it may be necessary to use a mycobacteriophage which recognizes a receptor that is constitutively expressed.
If the receptor is masked by a membrane of the macrophage, the cells isolated from macrophages may be treated with a variety of different detergents to find a treatment that would allow infection of the M. tuberculosis cells with the mycobacteriophages. Again, it may be necessary to cultivate the detergent-treated macrophages in broth for a few generations to gain expression of the receptors. The assays to determine the infectability of macrophages from mycobacteria include not only the luciferase assay for the TM4 ::lux mycobacteriophages, but also infectious centers assays in which free mycobacteriophages are removed and mycobacteriophage-producing cells are scored by a mixed plating on a lawn of M. smegmatis. This assay would be useful since infectability can be scored even if there are insufficient M. tuberculosis cells to form a bacterial lawn. It is important to re-evaluate the host range specificities of all of the mycobacteriophages in this assay. Free mycobacteriophages can simply be removed through the use of specific anti-mycobacteriophage antibodies. Detecting M. tuberculosis in Sputum Samples
Sputum from a patient infected with M. tuberculosis contains a mixture of mucopolysaccharide, free M. tuberculosis cells, macrophages containing M. tuberculosis cells and a variety of cellular debris. Sputum samples from patients thought to have pulmonary tuberculosis may be used for a study in which various numbers of M. tuberculosis cells are added to sputum samples found to have no or few organisms by acid-fast staining. A variety of methods can be used to treat sputum samples so as to liquify the mucous and decontaminate the specimen under conditions in which all bacteria other than mycobacteria are killed. Because of the specificity of the phasmids, decontamination may not be as important as preserving the mycobacteriophage receptors. Nonetheless, the sputum samples may be treated initially with 2% w/v NaOH for 30 minutes at 37ºC or with 0.5% N-acetyl cysteine + 1% NaOH. Alternatively, the sample may be treated with a variety of hydrolytic enzymes, such as collagenase, to help dissolve the sputum sample. If mycobacteriophage receptors are carbohydrates possibly sensitive to these conditions, other conditions may be utilized or the cells will be cultured 3-16 hours to allow recovery of infectivity before mycobacteriophage infection.
Detecting Mycobacteria In Blood Samples
Tuberculosis has been known to have a bacteremia. If the sensitivity necessary to detect 100 to 200 M. tuberculosis cells in a ml of sample can be obtained, levels of bacteremia in tuberculosis patients which were not previously observable may be observed. White cells should be purified over
Ficoil-hypaque and lysed with 2% NP40, 1% SDS or freeze-thawing in the presence of DNAse to liberate intracellular mycobacteria. The pellet should then be infected with the diagnostic luciferase mycobacteriophage, or if only few organisms are present they can be concentrated by filtration onto filters, and filter areas cut out and infected.
Assuring Specificity On a Variety of Clinical Isolates and Species;
Assessment of False Positives and Negatives
The luciferase assay may be optimized such that positive correlations of M. tuberculosis infections as indicated in the clinical lab may be obtained. The recombinant mycobacteriophages may be tested to ascertain the range of specificity that they have for other mycobacteria, and for the closely related genera Norcardia, Corynebacterium, and Actinomycetes strains. These strains may be obtained from the ATCC. A number of blinded tests including negative controls, M. tuberculosis-infected patients, samples from patients infected with M. avium, and samples infected with other
non-mycobacterial pathogens may be performed to ascertain the range of specificity.
The ability to rapidly assess the susceptibilities of M. tuberculosis isolates to isoniazid, ethambutol, rifampicin, pyrazinamide and other antibiotics will have a major impact on the treatment of tuberculosis patients. After the isolation of M. tuberculosis cells from a sputum sample, which may take several weeks, the assessment of drug-susceptibilities may take an additional 2 to 9 weeks. Diagnostic reporter mycobacteriophages may allow for evaluations of drug-susceptibilities at the time a sputum sample is collected. Alternatively, this approach would shorten the time necessary to assess drug-susceptibilities of purified M. tuberculosis colonies grown up from clinical samples. Luciferase Assays for M. tuberculosis
Cells in the Presence of Drugs
The results of the experiments suggest that by using luciferase as an indicator for the metabolic ability of the cell, it may be possible to define conditions which will enable us to distinguish drug-resistant mycobacteria from drug-sensitive mycobacteria. To test this hypothesis, isolated mutants of M. tuberculosis H37Ra which are resistant to isoniazid, rifampicin, ethambutol, or pyrazinamide would be used to generate a set of cogenic mutants. These independent mutants and the parent strains would be transformed with pYUB180. Luciferase activity will be assessed in the presence and absence of drugs in order to determine the optimal conditions for distinguishing between drug-resistant and drug-sensitive cells. It is quite possible that the window of time to observe differences for different drugs could vary and require different incubation times for each drug.
The choice of the promoter for expressing luciferase may provide a needed parameter to more readily assess drug action. For example, in the case of E. coli, gyrase promoters are greatly stimulated in the presence of gyrase inhibitors.
Clinical isolates of M. tuberculosis may be transformed with PYUB180 and tested for luciferase activity in the presence and absence of drugs. The luciferase assays with mycobacteriophage infections with lux mycobacteriophages on in vitro-grown M. tuberculosis cells will first be optimized, and then extended to M. tuberculosis cells grown in macrophages or isolated from sputum samples.
Critical Assessment of Drug-Susceptibility Testing
As for the detection of M. tuberculosis from clinical samples, the luciferase assay may be optimized so that the drug-susceptibility patterns for any clinical isolate may be obtained. It may be possible to add diagnostic mycobacteriophages to a single clinical specimen, aliquot the mixture into various tubes and add antibiotic drugs. Thus
every experiment would have an internal control and each drug-treated sample could be compared to an untreated control. The critical parameter to conclude drug-resistance or sensitivity lies in the comparison.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: William R. Jacobs, Jr.
Barry R. Bloom
Graham F. Hatfull
(ii) TITLE OF INVENTION: MYCOBACTERIAL SPECIES-SPECIFIC
REPORTER MYCOBACTERIOPHAGES (iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amster, Rothstein & Ebenstein
(B) STREET: 90 Park Avenue
(C) CITY: New York
(D) STATE : New York
(E) COUNTRY: U.S.A.
(F) ZIP: 10016
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch 1.44 Mb storage diskette
(B) COMPUTER: IBM PC Compatible
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: Word Processor (ASCII)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Not yet assigned
(B) FILING DATE: Not yet assigned
(C) CLASSIFICATION: Not yet assigned
(vii) PRIOR APPLICATION DATA: Continuation-In-Part
(A) APPLICATION NUMBER: 07/833,431
(B) FILING DATE: February 7, 1992
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Pasqualini, Patricia A.
(B) REGISTRATION NUMBER: 34,894
(C) REFERENCE/DOCKET NUMBER: 96700/238
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 697-5995
(B) TELEFAX: (212) 286-0854 or 286-0082
(C) TELEX: TWX 710-581-4766
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 52297
(B) TYPE: nucleotide
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(A) DESCRIPTION: phage genome sequence
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: no
(v) FRAGMENT TYPE: not applicable.
(vi) ORIGINAL SOURCE:
(A) ORGANISM: mycobacteriophage L5
(B) STRAIN: not applicable
(C) INDIVIDUAL ISOLATE: L5
(D) DEVELOPMENTAL STAGE: not applicable
(E) HAPLOTYPE: not applicable
(F) TISSUE TYPE: not applicable
(G) CELL TYPE: not applicable
(H) CELL LINE: not applicable
(I) ORGANELLE: not applicable
(vii) IMMEDIATE SOURCE: mycobacteriophage L5 particles
(viii) POSITION IN GENOME: entire genome
( ix) FEATURE :
(A) NAME/KEY:
(B) LOCATION:
( C) IDENTIFICATION METHOD :
(D) OTHER INFORMATION :
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Hatfull and Sarkis
(B) TITLE: DNA Sequence, Structure and Gene
Expression of Mycobacteriophage L5: A Phage System for Mycobacterial Genetics
( C) JOURNAL : Molecular Microbiology
( D) VOLUME : 7
(F) PAGES: 395-405
(G) DATE: 1993
( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 1 :
GGTCGGTTAT GCGGCCGAGC CATCCTGTAC GGGTTTCCAA GTCGATCAGA GGTAGGGGCC 60
GGCACAGAAA CCACTCACAT CAGGGCTGTG CGCCTCCAGG GCGCGTGAAC TCCCACACCC 120
CGGTGTAGTT ACATCCCGGA ATTGTCTCAG CGCCTCTCAG GGCGCTTCTC ATAAACAGTG 180
ATCTACGCCA CTCCTGACGG GTGGCTGTCA AGGATACTCA CCTTCCCTAC TAATGAGGGG 240
CTAAGAGCCC CTCTCTATAG AGCGCCGCAC AGGCGGCGCG ATAAGAGCGC CACCAGGCGC 300
TCATCTAAAG ACCGGCCTTG AAGGGCCGGT CATAGAGATC TATTCGATCC GGCAACCGCC 360
GGATCTCAAG GCCGCGCCAG TGCGCGGCCC TATAGAGGGG TGACTCAACT GTGCATGGCA 420
CTCGCTCGAG TGCCCACTGG AGCACTCAAC CGGGGAAGTT CGACGTTCTC AACCTGCGAA 480
TGACGTTTGA ATCGTCATCC GCGTACGAAA TCCCCGATCT GCGGCCGACC GACTTCGTGC 540
CGGCCTATCT CGCGGCCTGG AATATGCCGC GTCACCGCGA TTACGCCGCC AAGAACGGCG 600
GCGCGCTGCA CTTCTTCCTT GACGATTACC GGTTTGAGAC CGCGTGGTCG TCCCCCGAGC 660
GCCTTCTCGA CCGCGTAAAG CAGGTCGGCG CTGCACTCAC GCCGGATTTC AGCCTCTGGA 720
CGAACATGCC GAAGGCGGCG CAGCTATGGA ACGTCTACCG CTCCCGCTGG TGTGGCGCGT 780
ATTGGCAGTC GGAAGGAATC GAGGTGATTC CGACGGCGTG TTGGGCGACT CCCGACACGT 840
TCGATTTCTG TTTCGACGGG ATCCCGATGG GATCGACCGT CGCAATTTCT TCGATGGGCA 900
TTCGCTCTTC AAAAGTCGAC CAGGAGCTTT TCCGGTACGG ACTACGCGAA CTCATCGATC 960
GCACTCAACC GCAACTGCTT TTGGCATATG GCCAGCTTCG GCATTGCGAC GACATGGATT 1020
TACCAGAGGT CCGCGAATAC CCGACCTACT GGGACAGACG ACGAAAGTGG GTAACTGCCG 1080
ATGGGAGGCC GGGGAAGTAA AGGCGGCCCC GGTCCCGGAA CCGGAGCACG CAACCGCAGA 1140
GGCGCTGGAG CCCCCGGATC GGGCGGCGTA GGCGGCGTCG GAGGCGGGGG TGGAGCTGCA 1200
GGGAGCAGCG GAGGCGGCAA GGGAACGGCA GCGCCGGTAC CGGAGGCGTC ACCGGTGGCG 1260
GCGGAAGTGG AGCCGGCGGC GGTGGCAGCA GCCCCAACAC CCCGGTGCCC CCCACCGAGC 1320
TGGAGAAGAA GCGCGGCGAA TACAACCAGA TCGCCATCGA CGCCCAGAAA CAGCACGCGC 1380
CCACCGATGA GAAGCGCGAG GCCAAGCGCA AGCAACTGAT GGATCGAGTC GGAGGAGACT 1440
GGCAGGCTTT GGACCCGGAT CACCACGACG CCATCAAGGT GGCGATGGAT GACGCCATGC 1500
GGAAGATCCT CTCCGAGGAG GAGATCGTCC ACCGCACCAA GCACTTCGGC GACCTACTCG 1560
ACTCCGGTCG ACTCAAGTCG CTGTTCGAGG TCGGCTTCTC AGCCGGTGGC GACACCCCGA 1620
CCGAACGCGC CCTCCTCGAG GACGCCTGGT TCGGCGCAGG CAAGGTTCCC CCGATCTACT 1680
CGGCAATCGA GTTCAACGGC GCTCCGACAG CCGGCCTCGG CATGTACGGC GGCACCAAGC 1740
TCTACATGAA GGACTCGGTC AAGGACCGCG TCACCGTGAC CATCGGCGAC TCGCTGATGT 1800
CGAGCTGGGA CGTATTCCCC GGCCGTCCTG GCGACGGCGT GGGGCTGTGG GCCAGCCTGT 1860
CGAAGATCGA GGGGCTGGTC GATCCGAGCA AGACCCGCGA AGAGAACATG CAGGCGGTGT 1920
ACGACTCGTT CAAGAAGTAC GGCACCCTGG ACGGCTTCAT CGAGGCGCAG ATCCACGGCG 1980
GCGTCCTGGT CGAGGACATC AAGAAGGTCG TGTTCACGCA GCCGCCGAGC CCGATCTTCA 2040
CCGATAAACT GGACGAACTT GGAATCCCGT GGGAGGTGCA GTAATGGCGC AGATGCAGGC 2100
GACACACACA ATCGAGGGGT TCCTGGCTGT CGAGGTGGCC CCTCGGGCGT TCGTCGCAGA 2160
GAACGGCCAC GTACTGACCC GGCTGTCGGC CACGAAGTGG GGCGGTGGCG AGGGTCTCGA 2220
GATCCTCAAC TACGAGGGTC CAGGGACCGT CGAGGTCTCC GACGAGAAGC TCGCCGAAGC 2280
CCAGCGGGCC AGCGAGGTCG AGGCTGAACT TCGCCGCGAG GTCGGCAAGG AGTGAGCTGG 2340
GCCGGCTCAG GCCGGCGACA GGAACTACCA GAGGACTGGG AGCTGAATTA CCGGCTCCCG 2400
GTCCTTTCTG CTGCCAACTG GCTTTGCCAG ATCAACGGTC CCGGATGCGT AAGGGCCGCA 2460
ACCGATGTCG ACCACATCAA GCGCGGGAAC GACCACAGCC GGTCCAATCT GCAGGCAGCC 2520
TGCCATGTCT GTCACGGCAA GAAATCAGCC GCCGAGGGCG TAGCCCGACG GCGGGAACTT 2580
AGAGCCCGGA GGAAGCGACC ACCCGAACGC CATCCTGGGC GTCGATAAGC GGGCCAGGTG 2640
CCCGCTCCAC CCAGGAGGTG AACAGTGGGC ACGCGAGGCC CAATCGGAAA ACGAGATGAA 2700
GAGCGGGTTC GTCGGAACAC CCCGGACAGT CCAACCGACA CGATCCAGAT GCCCGGTCTG 2760
GTGACGATCC CCGAGATGGG CGATCTAAGC CACGACGGCC GCACGCACCA GCTCGTCAAG 2820
GACATGTACG AGTCGATCAA GCAGTCGGCA GCCGTGAAGT ACTACGAGCC GACCGACTGG 2880
CAGATGGCCC GACTCGCCCT CTACACACTT AACCAGGAAC TCATCGCAGC CGAGAACAAC 2940
GGCAAGCCCG TGGGCGCGAT GAAGCTCACT GCCATCAACC AGATGCTCTC CGCGCTGCTG 3000
CTGACCGAAG GTGACCGACG CCGCGTCCGA CTCGAAGTCG AACGAGCACC CGCTGACCCG 3060
ACAGGCGGGA AGGTCGTTGA CGTGACCGAC GTGCTCAAGC AGCGCCTCGC CAAGGCGAGC 3120
GGCGGGAGCT GATGGTCCCC CGAGGGGTTT CTAGAGCCGC TGCCGCTACC AGCCGCTCCC 3180
CCTCGGGGTA GACATCGAAA GGAACCACAT GGCCGACCTC GGCAACCCAC TCGACCTCGA 3240
GATGCTCTGC CTGGTCACAG GCCGGGACTT CCGCTGGACC ATCGATTACC CGTGGGGTCC 3300
GGGAGAGCTG TTCCTCGAAC TCGAGACCGG CGGCGAACAC AACGCGCTGC ATCAGGTCTA 3360
TGTCACCGGG GCGACCGGAG GCACGTACAC GCTGAACGTC AACGGCACCA ACACCCCGGC 3420
CATCGACTAC AACGACGTGT CGGAGAATCC GCAGGGGCTG GCAGGCGACA TCCAAGACGC 3480
TCTGGACGCA GCCGTCGGAG CCGGAAACGC TGTCGTGCAT CCGGTCTCGC TGTTCCCTGC 3540
GTGGACACTG AACTTCAACC TCAACGCCAG CAAGCCGCTC ACCGAGCAGT TGGTCAACAC 3600
GATCAACAAG GCCGCGAACG ACTTCTTCGA CACGTTCGAC CAACTACTTG GGGTCGACGT 3660
GGAGATGACG GTCACCGACA CCCTGAACTT CAAGCTCAAG GTGACCTCGC GGCGCTCGTT 3720
CGATGAGGTC GGTGTCGTCA CGTTCGCGGT CGACGTGACC AGCCAGGCAG TCATCAACTT 3780
CTTCAACTCC GTCGCCGAAC TCACCGGAGC GGTGAACACC GTCAACGTCG ACTTCTACTG 3840
GAACCGGACG TATGACATCG AGTTCACCGG ATCCCTTGGG CTGCAGCCGA TTCCGGCTAC 3900
TACAGCCGAC ATCACCAACC TGGCGGGTAC CAGCAAGGCC GTCTCAGTCA CGGTGGTCGA 3960
GCCAGGAAAG AAGAGGCTGA CCATCTGGCC GTTCACGGTC AACGGTGAAA CCGCAACCAT 4020
CAAGGTCGAG TCCGAAGAGG CCGACAAGAT CCCCAACCGC TGCCGCTGGC AGTTGGTTCA 4080
CATGCCGACC GGCGAGGCAG CCGGCGGCGA TGCAAAGCAG CTCGGCCGCG TTTACCGACA 4140
GCCGAGGTAA CACCGCACCC ATCAGAGATG GTGGGCCAGA CGGCCTTCGG GCCGTCCCCT 4200
GACGTGTAGC TCAATGGCAG AGCGCCCGAC TGTTAATCGG GTGGTTGAAG GTTCGAGTCC 4260
TTCCATGTCA GCGAGGGCTG AACCGGACCC GTGTCCGGTG TAGGCACTTT CCGCAGGCGG 4320
TTCCCCAGAG CGTGGGGAGC CCCTGCCCTG TACACGTAGC TCAATTGGTA GAGCAGCGGT 4380
CTCCAAAGCC GCCGGTTCCA GGTTCGACTC CTGGCGTGTA TGCACACACC CCTGACTCCT 4440
GCTAGCGGAG TGTTCGCCTT TCGGGCCTGG GGTCTTTTTC CCCGTTCGTC TAATCGGTAA 4500
GACACCCGGC TCTGGACCGG GCAATTGAGG TTCGAGTCCT TGGCGGGGAG CCAACTTGAC 4560
ATCCACCCGA AAGGAACAAC ATGACCTTCA CAGTCACCCG CGAGAGAGCG CAGTGGGTCC 4620
ACGACATGGC CCGCGCTCGC GACGGTCTCC CCTACGCGTA CGGCGGGGCG TTCACCAACA 4680
ACCCGAGGGT GTCGACTGAC TGCTCTGGCC TGGTGCTGCA GACCGGGGCT TGGTATGGAG 4740
GTCGCACCGA CTGGGTCGGA AACCGTTACG GCTCAACCGA ATCGTTCCGG CTCGACCACA 4800
AGATCGTCTA CGACCTAGGG TTCAAGCGGA TGCCCCGAGG CGGGCCAGCG GCCTTGCCGA 4860
TCAAGCCGGT GATGCTCGTC GGGCTCCAGC ACGGAGGCGG CGGGGTCTAC TCGCACACCG 4920
CTTGCACGTT GATGACGATG GACCACCCCG GTGGCCCGGT CAAGATGTCC GACCGAGGCG 4980
TCGACTGGGA GTCCCACGGC AACCGCAACG GCGTAGGCGT CGAACTTTAC GAGGGCGCAC 5040
GGGCATGGAA CGACCCTCTG TTCCATGACT TTTGGTACCT GGACGCAGTC CTCGAAGACG 5100
AAGGAGACGA TGACGAATTG GCTGACCCAG TTCTAGGGAA GATGATCCGC GAGATCCACG 5160
CGTGCCTGTT CAATCAGACC GCGTCGACCA GCGATCTGGC GACCCCTGGT GAAGGCGCTA 5220
TCTGGCAGCT ACACCAGAAG ATCCACTCGA TTGACGGCAT GCTCCACCCG ATCCACGCTG 5280
AGCGGCGCGC TCGCGCAGGC GATCTCGGTG AGCTGCACCG AATCGTGTTG GCCGCGAAGG 5340
GCTTGGGCGT GAAGCGCGAC GAGGTGACCA AGCGGGTCTA CCAGAGCATC CTCGCCGACA 5400
TCGAGCGGGA CAACCCCGAA GTACTTCAGC GATACATCGC AGAAAGAGGT GGCCTATGAG 5460
CCCCAAGATC CGACAGACCA TCTACCTGCT CGGCACCGCC GCCCCGGCAC TGCTGGGCAT 5520
CGTCCTGATC TGGGGCGGGC TCGACGCTGA GTCGGCGGCT GACCTCGGTG ACATCATTGC 5580
GGGCGTCGTG TCGATACTAG TCTCCGGTGC GCCGGCCGTA GCGGCAGGCA CCGTACGCAG 5640
CCAGCGCAAG GACGGCACGT TGTCCACCAG CCCGGTGGAT CAGGTCACCA AGGGCGTCGA 5700
GCAGGTGCTC GCGGCCAGGC AGAGTGCCGA GGCTGAAGTC GCGAAGGTCA AGCAGGCGCT 5760
GGAGACCGCC GTCAGCGGTT CTCTCCCCCA GCTCGGCCCG CTGGCCACGC AGATCCTCAA 5820
CGTGGCTGAC GACACCGTCT GGCGTCCATG AGCAAGCCCT GGCTGTTCAC CGTCCACGGC 5880
ACAGGCCAGC CCGACCCGCT CGGGCCTGGT CTGCCTGCCG ATACCGCACG GGACGTACTT 5940
GACATCTACC GGTGGCAGCC CATCGGCAAC TACCCGGCAG CGGCGTTCCC GATGTGGCCG 6000
TCGGTCGAAA AGGGTGTCGC TGAGCTGATC CTGCAGATCG AGCTGAAGCT GGACGCAGAT 6060
CCGTACGCGG ACTTCGCGCT GGCCGGCTAC TCGCAGGGAG CCATCGTGGT GGGCCAGGTG 6120
CTCAAGCACC ACATCATCAA CCCGAGAGGT CGACTGCACC GGTTCCTGCA CCGGCTCAGG 6180
AAGGTCATCT TCTGGGGTAA TCCGATGCGG CAGAAGGGCT TTGCCCACAC CGACGAGTGG 6240
ATTCACCAGG TCGCTGCCTC GGACACGATG GGCATCCTCG AGGACCGACT GGAGAACCTC 6300
GAGCAGTACG GCTTTGAGGT CCGCGACTAC GCGCACGACG GCGACATGTA CGCCTCCATC 6360
AAGGAGGACG ACATGCACGA GTACGAGGTG GCCATTGGCC GAATCGTGAT GAGCGCTAGG 6420
CGATTCATCG GAGGTAAGGA CTCCGTCATC GCCCAGCTCA TCGAGCTTGG ACAGCGTCCG 6480
ATCTGGGAGG GAATCGCGAT GGCCAGAGCC ATCATCGACG CCCTCACGTT CTTCGCCAAG 6540
TCGACCCAAG GCCCGAGCTG GCCGCATTTG TACAACCGCT TCCCGGCGGT CGAGTTCCTA 6600
CGACGAATCT GAGAAAGGAG GCGGGGTGAG CCTCAACAAC CACCACCCGG AGCTTGCCCC 6660
GTCTCCCCCT CACATCATCG GCCCGTCCTG GCAGAAGACG GTCGATGGTG AGTGGTATCT 6720
GCCTGAGAAG ACCCTCGGCT GGGGAGTCCT GAAGTGGCTC TCCGAGTACG TGAATACCCC 6780
TGGCGGGCAT GACGATCCGA ACCGTCTGGC GACGTTGATC GCGCTCTCCG AGGCAGGTCT 6840
TCTCGACAAC GAGAACATGT TCATCCCCAC CGACGAGCAG GTACGCCTGG TCCTCTGGTG 6900
GTACGCAGTA GATGACCAGG GCCAGTACAT CTACCGCGAG GGCGTGATCC GCCGGCTCAA 6960
GGGCTGGGGC AAGGATCCGT TCACCGCCGC GCTCTGCTTG GCGGAACTCT GTGGCCCCGT 7020
AGCCTTTTCA CACTTCGACG CCGACGGTAA CCCGGTCGGC AAGCCGCGTT CAGCCGCGTG 7080
GATCACCGTC GCGGCCGTCA GCCAGGACCA GACGAAGAAC ACGTTCTCGC TGTTCCCGGT 7140
GATGATCAGC AAGAAGCTGA AGGCCGAGTA CGGCCTGGAC GTGAACCGCT TCATCATCTA 7200
CTCCGCAGCC GGTGGCCGTA TTGAGGCAGC GACCTCGAGC CCCGCGTCGA TGGAGGGTAA 7260
CCGCCCGACG TTCGTCGTCC AGAACGAGAC GCAGTGGTGG GGCCAAGGCC CCGACGGCAA 7320
GGTCAATGAA GGCCACGCGA TGGCAGAGGT CATCGAAGGC AACATGACCA AGGTCGAGGG 7380
CTCCCGCACC CTGTCGATCT GCAACGCCCA CATCCCCGGC ACCGAGACGG TCGCCGAGAA 7440
GGCATGGGAC GAGTACCAGA AGGTCCAGGC AGGCGACTCT GTCGACACCG GGATGATGTA 7500
CGACGCGCTG GAAGCGCCGG CCGACACCCC GGTCTCCGAG ATCCCCCCGC AGAAGGAGGA 7560
TCCCGAGGGA TTCGAGAAGG GCATCGAGAA GCTCCGCGAG GGCCTGCTCA TCGCCCGAGG 7620
CGACTCCACC TGGCTGCCGA TAGACGACAT CATCAAGTCG ATTCTGTCGA CCAAGAACCC 7680
GATCACCGAG TCGCGGCGCA AGTTCCTGAA TCAGGTAAAC GCCGCTGAGG ACTCGTGGCT 7740
CTCACCGCAG GAATGGAACC GGTGCCAGGT CGACCTGGCC AAGTACCTGG ATAAGCACGG 7800
CAGGGAGTTC GCTCCGCTGC AGCGCGGTGA CCGGATCACC CTCGGGTTCG ACGGGTCGAA 7860
GTCCAACGAC TGGACCGCGC TCGTCGGCTG CCGTGTCAGC GACGGCCTGC TGTTCGTCAT 7920
CGACATCTGG GATCCCCAGA AGTACGGCGG GGAGGTTCCC CGCGAAGACG TTGACGCCAA 7980
GGTCCATTCG GCGTTCGCCC ACTACGACGT GGTGGCGTTC CGCGCCGACG TGAAGGAGTT 8040
CGAGGCGTAC GTCGACCAGT GGGGCCGGAC CTACAAGAAG AAGCTCAAGG TCAACGCCAG 8100
CCCGAACAAC CCGGTGGCGT TCGACATGCG CGGACAGCAG AAGAGGTTCG CGTTCGACTG 8160
CGAGCGACTC GAGGACGCGG TCCTTGAGGG CGAGGTCTGG CACGACGGCA ATCCCGTTCT 8220
GCGCCAACAC GTTCTGAACG CCAAACGACA CCCAACGAAC TACGACGCCA TCGCGATTCG 8280
CAAGGTCACG AAGGACTCCA GCAAGAAAAT CGACGCTGCA GTCTGCGCTG TCCTCGCGTT 8340
CGGGGCGAGA CAGGACTACC TCATGAGCAA GAAGGCCCGT AGCGGCCGGG TGGTGATGGT 8400
TCGATGACAG CACCGCTCCC CGGTATGGAG GAGATCGAAG ACCCCGCAGT CGTACGAGAA 8460
GAGATGATCT CGGCCTTCGA GGATGCTTCC AAGGATCTCG CCAGCAACAC CAGCTACTAC 8520
GACGCTGAGC GCCGGCCAGA GGCCATCGGC GTCACCGTCC CGAGAGAGAT GCAGCAACTG 8580
CTGGCTCACG TCGGATACCC CAGGCTCTAC GTCGACTCAG TCGCCGAGCG CCAGGCCGTC 8640
GAGGGTTTCC GCCTCGGCGA TGCCGACGAG GCTGACGAAG AGCTGTGGCA GTGGTGGCAG 8700
GCCAACAACC TCGACATCGA GGCACCACTG GGCTACACCG ACGCTTACGT TCACGGCCGG 8760
TCGTTCATCA CGATCAGCAA GCCAGACCCG CAGCTCGACC TGGGTTGGGA TCAGAACGTC 8820
CCGATCATCC GCGTCGAGCC GCCCACCCGA ATGCACGCCG AGATCGACCC CCGGATCAAC 8880
CGGGTGTCCA AGGCCATCCG AGTCGCATAT GACAAGGAGG GCAACGAGAT TCAGGCTGCC 8940
ACGCTGTACA CGCCGATGGA GACCATCGGC TGGTTCCGCG CTGACGGTGA GTGGGCTGAG 9000
TGGTTCAACG TCCCGCACGG TCTGGGCGTC GTTCCCGTTG TGCCGCTTCC GAACCGGACC 9060
CGGCTCTCGG ACCTGTACGG CACCAGTGAG ATCACGCCCG AGCTTCGGTC GATGACCGAC 9120
GCGGCGGCGC GCATCCTCAT GTTGATGCAG GCGACCGCCG AGCTGATGGG TGTCCCCCAG 9180
CGCCTGATCT TCGGCATCAA GCCCGAAGAG ATCGGCGTCG ACTCCGAGAC CGGCCAGACG 9240
CTGTTCGATG CGTACCTGGC CCGGATCCTG GCGTTCGAGG ACGCTGAGGG CAAGATCCAG 9300
CAGTTCTCTG CAGCCGAGCT GGCCAACTTC ACCAACGCGC TCGATCAGAT CGCCAAACAG 9360
GTCGCTGCGT ACACGGGATT GCCTCCCCAG TACCTGAGTA CCGCCGCAGA CAATCCGGCC 9420
TCCGCTGAGG CGATCAGGGC CGCTGAGAGC CGACTCATCA AGAAGGTCGA GCGGAAGAAC 9480
CTGATGTTCG GCGGCGCATG GGAAGAGGCC ATGCGGATCG CCTACCGGAT CATGAAGGGC 9540
GGCGACGTTC CCCCGGACAT GCTCCGCATG GAGACCGTCT GGCGAGACCC GAGCACTCCC 9600
ACCTACGCGG CCAAGGCCGA CGCAGCCACG AAGCTGTACG GCAACGGCCA GGGTGTCATC 9660
CCGCGTGAAC GTGCTCGCAT CGACATGGGC TACTCCGTCA AGGAGCGCGA AGAGATGCGC 9720
CGATGGGACG AGGAAGAGGC CGCAATGGGT CTCGGCCTGT TGGGCACGAT GGTCGACGCC 9780
GACCCGACGG TCCCAGGCTC CCCGAGCCCC ACGGCACCGC CGAAGCCACA GCCGGCCATC 9840
GAGTCGTCTG GTGGTGATGC GTGACCGCAG AGGAGTACGC GGCGGCTCAA GCCGCGATCA 9900
CTGCGGGTCT TGCCACATAC GTCCAGAGGT TCGCTTCGCT CTTCGTCGGT CCAGCTCTCG 9960
CTGTAGGTGA GTGGCTGCGA CTGCTGCAGG TGCTGTTCCC CGAAATCCAA CGGCGGTATG 10020
CAGATGCTGC CGCCTTGGGC AGGGACTTCT ACGACTCCCA ACGCGCACTA CACCACCCAG 10080
AGCTGCCCCG GAACGAGAGG TTCCGGGGAG AGCTTCGGTG GGAGTGGTTC GTCCAGAACA 10140
TGGAGCCCGC TCGAAAAGAG ATGTCGCAGG CCGACTCTCC GCCGAGTGCG ACCTCTAAGT 10200
TGGCTCTGGC CGCAGTTCGC GAAGTGGAGA TGGCAGCACG CCGACAGATC ATCGGCGCTG 10260
TCAAGAACGA TCCGGCCCCG CAGATCGTGC AGGGCTGGGC GAGGGTCGCC ACCGGGCGCG 10320
AAACATGCGC CTGGTGTCTG ATGCTCATCT CACGGGGTGC CGAGCTGAAT CACAAGGGCA 10380
ACTTCGCCTA CAGCTCAGCG GAAGCCGCAG GGCTCAACCT CGATGACGAG ACCGTGATCG 10440
ACCTCTGGAA CGAGTCCGGT CACGACCTTG AGAAGTTCCG CGAGGAGACC AGAGAGGACT 10500
TCGAGAAGTG GCACGCAGGG TGCGACTGTC TGGTGGTCCC GGTCTTCGAT GTGCAGAACT 10560
GGCCCGGAAG AGACGCTGCC CTACGGGCGC AGCAACTTTG GATCGAAGCC AGCGACGAAG 10620
CTGACGACCT CATTGCGTCA GGCAAGGCCC GCTCCAAGAA CAAGAACACG GAGACGCTCA 10680
ACGCGCTCCG ACGCCGCCTA GCACGCGGCG AAATCACCAT GTCCAACTAC GCCCTCGCTG 10740
CGTAGTCCCT CGAACCCCAG GTGGGTTCTC TCAACATGCC CAGGAGGCGA AAACACATGT 10800
CCGACAACCC CACTCCCGAG AGCACCCCAG AGGCCGAGAC CCCGGAGGTC GAGAAGCCGA 10860
TGGAACCGCA GGGCAAGGTC TTCGATGAAG CGTACGTTCA GTCGCTTCGC CAGGAGGCTG 10920
CAGCCGCTCG GGTGGCGAAG AAGGACGCCG TAGAAGCGGC AGAGGCTCGA GTGAAGGCCG 10980
AGTACGAGGC CAAGCTCGCT GAGCGCGACA CCGCTTACAC CGAACTGCAG AACCAGTTGG 11040
GACAGGCGTG GATTGAGCTG GAGAAGGTCT ACCTCTCTCT CGACGCCAAG GTGCCCAACG 11100
ACAAGGTTCG GGCGTTTGTC GAGATCCTCG AAGGCAACGA CAGGGACAGC ATCGCTGAGT 11160
CAGTGAAGTC CCGTCTGGAG CTGGTCGGCG GATTCGGCAA CAAGACCCCG AGTCCTGCGT 11220
TCGACCCGTC TCAGGGTCGC GGCGGTAAGC CGCCGATCCC GCTGAACGGT GACCCGATCC 11280
TCGAGGCCAT CAAGGCCGCT GTCGGGATCA AGAAGTAACC CACCCAACAG ATCTCAAGGA 11340
GAGATAAACA ATGGCAGTCA ACCCTGACCG CACCACGCCG TTCCTCGGCG TGAACGACCC 11400
CAAGGTCGCG CAGACCGGCG ACTCGATGTT CGAGGGCTAC CTCGAGCCCG AGCAGGCCCA 11460
GGACTACTTC GCCGAAGCGG AGAAGATCTC CATCGTCCAG CAGTTCGCCC AGAAGATCCC 11520
GATGGGCACG ACCGGCCAGA AGATCCCGCA CTGGACCGGC GACGTGAGTG CGTCGTGGAT 11580
CGGTGAAGGC GACATGAAGC CCATCACCAA GGGCAACATG ACCTCGCAGA CCATCGCCCC 11640
CCACAAGATC GCGACGATCT TCGTGGCCTC GGCGGAAACC GTCCGTGCGA ACCCGGCCAA 11700
CTACCTGGGC ACCATGCGGA CCAAGGTCGC GACCGCCTTC GCGATGGCGT TCGACAACGC 11760
CGCGATCAAC GGCACCGACA GCCCGTTCCC GACCTTCCTA GCGCAGACCA CCAAGGAGGT 11820
CTCGCTGGTG GACCCGGACG GCACCGGCTC CAACGCCGAC CTCACCGTCT ACGACGCGGT 11880
CGCCGTCAAC GCCCTGTCGC TGTTGGTCAA TGCCGGCAAG AAGTGGACCC ACACTCTGCT 11940
GGACGACATC ACCGAGCCGA TCCTCAACGG CGCGAAGGAC AAGAGCGGTC GCCCGCTGTT 12000
CATCGAGTCG ACCTACACCG AGGAGAACAG CCCGTTCCGC CTCGGTCGGA TTGTGGCCCG 12060
TCCGACCATC CTGAGCGACC ACGTCGCCTC GGGCACGGTC GTCGGCTACC AGGGTGACTT 12120
CCGCCAGCTC GTCTGGGGCC AGGTCGGCGG CCTGTCCTTC GACGTGACGG ATCAGGCGAC 12180
TCTGAACCTG GGCACCCCCC AGGCTCCGAA CTTCGTCTCG CTGTGGCAGC ACAACCTCGT 12240
CGCAGTCCGA GTCGAGGCCG AGTACGCCTT CCACTGCAAC GACAAGGACG CGTTCGTCAA 12300
GCTCACGAAC GTGGACGCCA CCGAAGCCTG ATCCAGGCTT GACATCCACC GGGAGGGGGC 12360
TCCTTCGGGA GCCCTCTCCT GATGTGGAGC AGGAAGGACC ACATGCGAAT CCAGTCCACC 12420
CTCAACGGCG GTTTCGCCGA GGTTTCCGAG GAGTTCGCCA AGCAGTTGAT CGCCACTGGC 12480
GGCTGGAAGG TGCCCCGGAA ACCGCGCAAC ACCAAGACCA AGACCGCTCC TGAGGAGCCC 12540
AAGAACGAGG AGTAACCCGT GGCCTACGCG ACCGCCGAAG ACGTTGTGAC GTTGTGGGCC 12600
AAGGAGCCTG AGCCCGAAGT GATGGCGCTG ATCGAGCGCC GGCTCCAGCA GATCGAGCGC 12660
ATGATCAAGC GCCGGATCCC CGACCTGGAC GTGAAAGCCG CTGCGTCGGC GACGTTCCGG 12720
GCCGATCTGA TCGACATCGA AGCTGATGCT GTTCTGCGCC TCGTGCGTAA CCCGGAGGGC 12780
TACCTCTCGG AGACCGACGG TGCGTACACC TATCAGCTCC AGGCCGACCT GTCGCAAGGC 12840
AAGCTCACCA TCCTCGATGA GGAGTGGGAG ATCCTCGGGG TCAACTCCCA GAAGCGCATG 12900
GCGGTCATCG TCCCGAACGT GGTGATGCCG ACGTGAGCGC GAGCGACCGA CACCGCGCCC 12960
CGATTGTCTA TCCGCCTGGC ACTCAGGCGG TTACGCCGGA TCGGGTCAAC GCGTTTGACT 13020
GCGATCACGA AGCTGATCCT CCGGTGTGCC GGTGCGTCCA CGACTGGCGC ATCGAGTGGG 13080
GAAACGTCAA GAAGGCCACC GCCAGATCAC GGTCGGCGGT GCTCTGATGA GCCTCCTCGA 13140
CACCGGTGCC CGGTACCAGA CCTGCATCGT CTACCCCGAA GAGATGGTCA TCGACTCCGA 13200
TGGCAACAAG CGGACCAGGC CGTCGAATAC CGGCATCCCG GCCATCGCAC GGTTCCAGGT 13260
AGCCAACCAG TCTGGTACGT CGGCACGACG TGCTGAGCAG GACAACGAGG GGTTCGAGAC 13320
CGAGAAGGTC TACCGGATGC GGTTTCCCCG CTCGTTCACC AAGGAGCACG GCATCCTCGG 13380
GGCCCAGTCC CAGATCGAGT GGCGAGACCA GCGGTGGGCG CTCTTCGGAG ACGCCACCGT 13440
CTACGACTCA TCCCCTGCGT TGGCGCGGGT CGACTACACG ATCAAGAGGT ACTGATGGCC 13500
AAGGTCTACG CGAACGCGAA CAAGGTCGCG GCCCGGTACG TCGAGACGAG GGACGCCGTC 13560
CGAGACGAGC GGAACAAGGT CACCCGTCGA GCCAAAGCCA ATCTGGCGCG GCAGAACTCG 13620
ACCACCCGCA TCACCGACGA GGGCTACTTC CCGGCCACCA TCACCGAGCA AGACGGCGAT 13680
GTCGACTTCC ACACGATCCT CAACGCGCCC AACGCGTTGG CGCTTGAGTT CGGCCACGCG 13740
CCGTCTGGCT TCTTCGCTGG CACCGACACG AAACCACCGG AGGCCACTTA CATCCTCACC 13800
CGAGCCGCCA TCGGCGGCAC CGTCTCATAA GGAGGTCACA TGGCGCGAAT GCCTCGCGTC 13860
CAGGCAGTAG CGGCCCCGAT CCTCCGGTCA GACCCCCGAC TGGAGGGAGT GACGGTCACG 13920
ACATGGGTTC CAGACGTGGA CTTCCGAGAG TTCCCGATGA TCAACCTCCG CCGCATAGGC 13980
GGGACGAGGA ACCCCAACGC ACCGACGCTG CACACGCTGC CGGTGGTCGA AATGACCGCC 14040
TACACCAGAG ACGGTCTCAT CGAGACTGAG GAGCTGTACG AGACCGCGCT AGAGGTTCTC 14100
TACGACGCGG TGGAGAACGG AACACAAACT CCCGCAGGGT ATTTGACCTC CATCTTCGAG 14160
ACGATGGGCG CCACTCAGTT CAGCTCCCTC TACCAGGACT CCTGGCGCAT CCAGGGTCTG 14220
ATCAGGCTCG GCGTCCGCAG ACCGAGAACC ACCCTCTAAC CGAAAGGTAA AGCCACATGG 14280
CTGAAAACGA CGACGCAGTG TTGACTGCGG CGGTCGGCTA CGTGTACGTC GGTGCTGCAG 14340
GCACCGCTGC TCCTACGCCG GCCTTGCTCA AGACCATCGA CCTCAGCAAG CCCGAGACCT 14400
GGACCGGTGC TACCGGTTGG ACGAGCGTCG GCCACACCAG CCGAGGCACG CTCCCTGAGT 14460
TCGGCTTCGA AGGCGGCGAG TCCGAGGTCA AGGGCTCCTG GCAGAAGAAG AAGCTCCGCG 14520
AGATCACCAC CGAGGATCCC ATCGACTACG TCACGGTCCT ACTGCACCAG TTCGATGAGC 14580
AGTCGCTGGG TCTGTACTAC GGCCCCAACG CCTCTGAGAC TCCTGGTGTG TTCGGTGTGA 14640
AGACCGGCCA GACCAACGAG AAGGCCGTGC TGGTCGTGAT CGAAGACGGC GACATGCGCC 14700
TGGGGCATCA CGCCCACAAG GCTGGAGTTC GCCGCGACGA CGCGATTGAG CTGCCCATCG 14760
ATGACCTGGC TGCGCTGCCC GTCCGGTTCA CCTACCTGGA CCACGAAGAC GAGCTGCCGT 14820
TCTCCTGGAT CAACGAAGAC CTCTTCAACG TGCCCGAGGT TCCCGAGGGC TGATCCCAAC 14880
TTGACAGCCA CCCGGCTGTC TACCCCGGAG GGGGAGGTTT CCTTGGCGGG CCTGGCCTCC 14940
CCCTCCTCCC GCCACTCACA GACCCGCCGA CACTGAAAGG TTCGCCATGA CAAACGTATT 15000
CACCATCGAC GCATTCCGCG AAGAGGTCAA GAAGAAGTAC GCTCCGGTCC TCATCGGCCT 15060
GTCCGACGAT GTGACCGTCG AGCTGAAGCC GCTGCTGAAG CTGGGCCAGA AGGCCCGCGA 15120
AGCGGTGGTC GAGGTGTTCA AGGAGTTCGC GGACATCCCC GACCTCGAAG AGGACGACGA 15180
CGACGAGTTG GTCGATGAGT ACTCGCTCCA GGTCTGCGAC ATCATCGCCA AGGCGTTCCG 15240
GCTGATCGCC ACGAAGCCCA AGAAGCTGAT CGCCGCCTTG GACGAGGAGC CGGATCCCCG 15300
TATCCGCGCA GAGCTGTATG CAGCGGTACT CAACACCTGG AAGCGAGAGA CGCAACTGGG 15360
GGAAGCCGCG CCCTCGCCGA GCTGATCGAC AAGTTCGGCG GGGCGATCCT CGCAGACCTG 15420
CTCCAGTACT ACCGGGTAGA CCTGCGCGAC CTGTTCCGCG ACGAGGATCC GCTTTCGCCG 15480
AGATTCGTTC TGTCCCTGGT GCTCTGCCTT CCCAAAGACG GCGCGTTCTA CGCAGAACGT 15540
CGTGGTGGGC AGCAGTACCG GGGCTGGACC GAGGACCGCT ACGCGCTCGC GGACATCTAC 15600
GACGCCATCC AGGCGGGCAA CCACATCCTG CTGCTGGCGA ATCGTGATCC GAAGAAGCCA 15660
AAGCCCAAGG CACCCAAGTC ATACCCGCGT CCCGACGACC TAGAGAAGAC CACACCGAAG 15720
CCGGGTTCGT TCGCCGCAAT GGTCGTGCGA GCGAAGAAGG CGGCTCGAGA GAGAAGGGAA 15780
AGGGAGGAGG AGAGTGCCGA ATAGTGCTGG CGTAGAAGTC GCCCGGATCT CGGTCAAGGT 15840
CAGCCCGAAC ACCAAGGAGT TCCGCCGGGA ACTCAAGACC GAACTCGAGA AGATCGAGCG 15900
GGAGCTTAAG GGCGATGTCG AGATCAACGG TCATCTCGAT GCGGCCCAGG CCAAGGCCGA 15960
CTTCAAGCGC ATGATGATGC AGCTCAAGAC CGAAGCTGCC AAGGGCGTTC ACGTCCCGGT 16020
CGACGTAACC GTCGACAAGA AGAGCAAGAA GGGAGGTCTC CTCGGAGGTC TCCTCGGCGG 16080
CAGCCGGGGG CTCGGAGATC TAGGCGATGA CGCCGAGAAG GCGTCGTCTC AAGTACAACA 16140
CCTTGGCAAG TCGTTCCTGG GCCTCACACG AGCCGCCTGG ATAGGCGTAG GCATCGTCGC 16200
CGTAGCAGCT CCGCTGGTCG GCATCGTGGC CGGTCTGCTG GCCGGTCTGC CGTCGCTGCT 16260
GTCTGCGTTC GGAGCCGGCG CTGGCGTAGT CGCGCTCGGC ATGGACGGCA TCAAGGCAGC 16320
CGCCTCGACG CTGGCCCCGA CGCTGGAGAC GGTCAAGGCC GCTGTCTCCT CGACGTTCCA 16380
GCAGGGACTC ACCCCGGTGT TCCAGCAGCT CGGCCCGATG CTGACCGCGA TCACCCCCAA 16440
CCTGCAGAAC GTGGCCTCGG GCCTCGTGAA CATGGCCGGG TCGATCACCG ACGTGATCAC 16500
CCAGGCTCCT GGTCTGCAGC AGATCCAGAA CATCCTCACC AAGACCGGAG AGTTCTTCAC 16560
GGGCCTCGGC CCTGTGCTCG CTACCGGCAC GCAGGCGTTC CTGACGCTGT CCAACGCCGG 16620
CGCGAACTCG TTCGGCACGC TCCTGGCTCC CCTGCAGGAG TTCACCAACG GCTTCAACGA 16680
CATGGTCAAC CGAGTCACGT CCAACGGCGT GTTCGAGGGT GCCATGCAAG GGCTTTCGCA 16740
GACGCTGGGC AGCGTCCTCA ACCTGTTCAA CCGGCTCATG GAGTCCGGTC TGCAGGCGAT 16800
GGGACAGCTC GGCGGTCCGC TGTCGACGTT CATCAACGGG TTCGGAGATC TCTTCGTCTC 16860
GCTGATGCCG GCGCTGACTT CGGTCTCTGG TCTGATCGGC AACGTCCTCG GGACGCTGGG 16920
CACACAGCTC GCTCCCATCG TCACGGCGCT CACGCCGGCC TTCCAGACGC TGGCGAGCAC 16980
GCTCGGCACG ATGCTCACCG GAGCCCTCCA AGCTCTGGGT CCGATCCTGA CTCAGGTCGC 17040
TACGTTGATC GGCACGACGC TGAACACGGC GCTGCAGGCT CTCCAGCCGA TGCTGCCGTC 17100
GCTCATGCAG AGCTTCCAGC AGATCTCCGA CGTACTGGTG ACCAGTCTGG CCCCGCACAT 17160
CCCGGCGCTG GCGACGGCCC TCGGCCAGGT CGCAGGCGCG GTGCTGCAGC TCGCTCCGAC 17220
GATCATCTCG ACGTTGGTTC CGGCGTTCGT TCAGTTGGTC CCAAAGGTCG CTGAGCTAGT 17280
TCCGACCATC GTCAACCTGG TCCAGTCGTT CGCCAACCTG ATGCCGGTGG TTCTGCCCCT 17340
GGCGCAGGCT CTGGTCAGCG TTGCTGGCGC GGTGATTCAG GTGGGTGTCT CCATCGGCGG 17400
CGCGCTCATC GGCGCGCTGG CGAACCTCAC GGAGATCATC TCCAACGTCA TCAAGAAGGT 17460
GTCCGAGTGG GTCAGCAGCT TCTCCAGCGG AGCCCAGCAG ATCGCTGCGA AGGCAGCGGA 17520
ACTGCCGGGG ATGATCCAGT CGGCTCTCGC CAACCTGATG GCCATCGGCC TGCAGGCCGG 17580
TAAGGATCTC GTCCAGGGCC TGATCAACGG CATCGGCGGG ATGGTCAGCG CAGCGGTCAA 17640
CAAGGCCAAG GAGCTGGCGT CCAGCGTGGC TGGTGCAGTG AAGGGCTTCC TGGGCATCGA 17700
GTCCCCGTCG AAGTTGTTCA CCGAGTACGG CCAGTTCACC GCCGAGGGAT TCGGCAACGG 17760
CATGGAGGCA GGGTTCAAGC CCGTCATCGA ACGGGCCAAG GATCTCGCGG CTGAGCTGTC 17820
CAGGGCGATG GAGTCGGGCA CCGACCCCTC CGGGATTCTC GCTGGGCTGG ATCAGAATGA 17880
GCTGAAGCAG ATGCTGGCGG CTCTCGAAGA GGAGCGCAAG CGACTCAAGG TCGAGAAGAA 17940
CGGTATCCCC AAGGGAGACA AGGCAGGCCG AGAGGCGCTG CAGAACCAGC TCGACCAGAT 18000
CCAGGCGCAG AAGGACATCC TGTCCTACCA GCGTGACCGC ATCAAGAACG AGTCTGAGTA 18060
CGGCGACATG GCCGGCGAAG ACCCGTTGGT GAAGGCAGCC TCCGGGCTGA TGAGCGCACC 18120
GGTCGACTTC GCGAAAGCGA CTGGCAAGCA GTTCCTTTCG GACATCGGCA TCAGCGGAGA 18180
TGGGTTCATC TCGAAGGCCA TCACCGAGGG CATCCAGTAC ATCTTCCAGA TCGGCTCTGT 18240
CGATGAGGCG CTGTCGATCA AGGACCGCGA GGAGTCGAAG AACGCGCTGT CCGTCGTTGG 18300
CCGCTGACTT GACATCCACC AGGAGGTAAG CATTGATCAC CGACACCATC GTTGAACTCG 18360
AGGGTGTCAA TGGTGAGCGT TTCAACTTGA CGACCGGTGA CCAGGGTGTG TACCTGGCCA 18420
CAGACGTGGA GGGTTGTTTC TACGACCCTC CCGTCAAGGT CGTTGTTGAA GAGCCGGGGA 18480
ACTACCCCGG CGCTCGCTAC TTGTCCCACC GAGCCCTGAA GCGAGACATC GTCTTTGGGG 18540
TCGTCATCCT CAACGACGCG AAGCAGGGGC CGCGCTCCTG GCTGTCGCGA GACTCCGAGT 18600
GGCGCAAGGC GTGGGCGTTC AACCGCACCT GCAAGCTCTA CGTCACCACC CCGGACTCCG 18660
GTACCCGCTA CCTGAAGCTG GCGCTGTTCG AGTCCCCCAC CGTCAAGATG GACACCGACC 18720
CAAGAGGTAA ACCCCTTGAG GTCACGGTGA TGTCGTGCAT CGCGTACGAC CCGTTCTGGT 18780
ACGAGGACGA CAAGGTCTTC TCGGCCAAGA CCAAGACCGA CACCCGGTTC GACCCGTCGT 18840
TCTGGACGCC GCCGTGGCCG TGGGAGGAAC TGCCCAAGGA GACGCTGCGG ATCAAGGTCG 18900
GCCGCGAGCA GGGTGGGCTA AACCCCACCG ACCAGTACAT CTTCCCGAAG TGGACCGTTC 18960
CCGGCTCCAC CGAGAAGGTG CCGAACTTCC CCTGGCCGTT CCCCCCGAAC GTCCCGATCC 19020
CGTGGGAGAC AGCACCGTTC ACTCAGTTCG TCATCCCGGA CTACTCGTTC GAGGATGAGG 19080
AGTTCCGCAA CCGCCGGCTC AAGACGCCGG GGTTGATCTA CGGCGAGAAC TGCGTCATCG 19140
ACACCGACCG GCGCGAGGAG CAGATCGCTT CCGAGTCGGG CTCCCCGGTG TGGGCTCGGA 19200
TGAACGGTGT CCGGTTCCGC AACTCGATCC CGCCCTACAC CGAAGAGGCT GAGTTCGTCA 19260
TAGACGCATC GGGATGCGCT CCGGGACAGG TAGTTACCCT CCGGCTCACG AGGCCGTGGT 19320
CGCGCTGCTG GGGGCTAGAG TGAGTGGTCT GACGAGCGTT CGTGAGGCCG AAGATCTCTG 19380
GCAGAAGATC CAATTGCGGC GCTGCAAGCG CGAGCAGGAA CGGCTCAAGC ATCCCGACGT 19440
AGAGCTGCGC GATGGCGACT TCCGCCTGCG CGGCCTGGTC GCTGGCGAGC GGGTGCTCGA 19500
GTGGGAGTTC ATCGAGAACG AGACTGGCAC CTGCACCTTG CAGCTCTCAC TGAGCCATTA 19560
CCTGGCGAAG TGGGTGATGG ACCACCGGGG TCGAGCAAAG CGCAACGTCA TCATCAACAT 19620
CGAGAAGCAA GGCGCTCGAT GGACCGGGAT GATGGACCAC TACCGGGTCA TCAAGACCGA 19680
CGCAGGGGAC GCCTACATCG AGATCGTGTT TTTGCACGAC TTCGAGCAGA CCAAGCATAT 19740
CCGGGTATGG TGCAACCCGT TCCTACGCCC CGAGCTGCAG TTCCCCAAGG TGTGGATCAT 19800
CTTCGGGCCG GCCAAGTGGT GTTTGCTGGT GACACTGTTC GTCAACCTGC TCAGGCTCGA 19860
GACGAGCTTG TGGACGCTGC CTGATGACCC CACGGACATC AACGAGTGGA TGGGTCCGAG 19920
CTTCAACCCA GCAAATTGGC GGAACATCGT CAAGCCGTTC CCGTTCCTGG CCGACAACTC 19980
ACCGGTCACG ATGGTGTTCA GCCGGTTCGG GACGTTCTAC GACACCGCCA AGAAGATCCT 20040
CGAAGACCAT CAGCTCACGC TGACGTGTCG TCGGTACATC AAGGACCGCG ACCCGCATCC 20100
GTTCGAAGAT CTCAAGGGGC TCTGGGGAAT TGATCCTGTC GAAGACCTGC TGCAGAAGAT 20160
CCCGCTCCGG GACGGCTGCG TGGTCTGGGA CATCGAGGAC AACTCAGGTT GGGGCACTCA 20220
GACCGCGTTC GGCGGTTCGT GGCTGACCGG GTTCGTCCGA GGGATGGTCC AACTGGCCGG 20280
CGACGGCCAG GTCGAGGGCG TCGATGTGTT CACCGGGGAC TACACGTTCC CAGGCGAGTA 20340
CTACTCCCCC TGGTTCATGG GCACCAGCCC GATAGCACCC CACGTCGTGT TCGAAGAAGG 20400
ACCGCTGACC GGGATCAAGT CGTCGGAGTT CTCGTACTAC GAGGCCACCG ACACCAGCTT 20460
CCTGGCTGGT GGACAGAGCG CACCTGGCAT CAACGAGGGC ATCTCGGCCC TGGTGAACAT 20520
CGGTGGCGAC CTGCTGACCT CGTTCATCAA CAGCCAGCTC GCCGCGCTCG GCGCGGTCGG 20580
TGGAGCGATT GACCTCCCGC CTCTGGGCGG TCTGCTCGAT GCGGTGTTGC AGCCTCTGTA 20640
CTCCGATGTG TTCGGCGCGT TCATGGAAGT TCCGACTCTG CGTGCGATGG GCATCTCGCT 20700
CCCGATCTCC GGGCTCGAGG ACATCGTCAC CGGACTGGGC GACTTCCACT ACTTCGAGAA 20760
CATGGCCGAC GGGGCGATGA AGGCGTTCAC GCTGTCAGCG TTCGCAGCCA TCGCATCGCA 20820
GATCCACAAG ACGAGGGCTC GAACGACCCA CACCCTCAAG GTGTCTGACG CCGCTCCGTA 20880
CATCTTCGCG CCAAAGCCCT ACGGGCACTG CTGGATCGGA GATCGCGTCG GCACGTCGGT 20940
CCTCGGCTAC CCGGTCGAGC ACCAGTTGTT CGTGGAGCGC ATCCGCAAGG TGAAGTACCG 21000
CATCGACAAA GACGGCATGA AGCCGTTGGA GATCGAGATC GGTTACCGCG AACCGAAGAA 21060
CCCAGCACTA CACATCCTCG AAGAGATCAA GCGCGTCAAC GGCGCTCTTG GCACTGCGGG 21120
GATTCTCTAA ACCGAAAGGC ACGCCGCATG ATTCCCTCAC AAGAGTCTCA CAATCCGAAC 21180
GACCCGCGAC AGCACGTCAT GTGGGCGCTA CGCAATCTCC CGATGATTGC TGGCGTCGGG 21240
GCGATCACGC ATCCGGGTTA CCTGGCGGAT TGGTCAGAGC ACTTGTGGAA GTGCGGCTTT 21300
CGGCACGTCG ACTGGCTCCG GGAGCTGGCT GATGAGGACG GCAACATCCA CGTCAGTCAG 21360
CTTCCTGACC AGGAGATCAA GTTTCAGCAG CCCTTCCGGG GCCAGCGAAG CGACTACAAC 21420
AACGCAGCTC GATGGGTCGG CAAAGACGAT CCTGACCCAG AGCCCGTGCG TATTCCAGAC 21480
ATTCGCAAGC TCACAGACCA GGAGAACAGA GCGATGATCG CGCAGTACGA ACGAGACGGT 21540
TGGATCAAGG ATGGATCCCC CGGCCCAGCG ATAGCCGAGG TCGTGGAGTG ACCCCGTTCA 21600
ACCCAGACTC CATAGGCGAC TACGTGACAC TGCTCGGCGT TGCGTTCCTG ACCTTCTCGG 21660
TTCCCGCATG GTTCACCGGA CGAGCACGCA AGCACAGCAG TGACATCGGC GAAATCAAGG 21720
AACAGGTATG TAACACCCAC GACACGAACC TGCGCGATGA CCTCGACAGC GTCAAGGCAG 21780
ACATCAGCGA CTTGAAAGAG ATTGTGTTGC AAGGGTTCCA CCAGGTGAAC GAGTCGATCA 21840
ACCTCGAGCG CCGTGAGCGG ATCGAAGGAG ACCGCCGAAA GGAGGTTGCG TGACCTACCC 21900
CACCAACCCA CTAGAGGCCA TCGGCGCTGA CGGCGCATTC GAGATCGGTG GGGGCGACTG 21960
GAGCTTCGGC CAGGACTACA CCGAACAGGC CATCCGGGCT CTGTTCACGA TGCCAGCGGT 22020
CACGATGGAG AACGCTCTCG GCCTGCTCGA AGAGCACCTG CTGAAGCTGC CTCTGGAGGC 22080
GCTGCAGGGC TTCAAAGACA TGATCCCGGA CTGGGTCGAA GGAGCATTCG ACACGGTCAC 22140
CGGCGCTGTG CAGGCGATCA TGAACGCGCT CCAAGACGGC CCGCTGTTCC TGAAGTTCGC 22200
CGAGTTCCAG CTCTTCCTGC AGCGTCTGCT GAACAACCCG GCCGAGGTCA TCGGCGAGAT 22260
CCCCCAGACG TTGATCGACG GCCTACAGGA CGCGCTCAAC ACCGTCAACA ACACCATCCA 22320
GACCATCGTG GACATGCTCC TGCAGGCGCT GGGCATCACC CCGGAGGGGG AGCTGATCGA 22380
CCGGATCTTC GACCTGAGCG ATGAGATGGA GTGGCTGCAG ACCGCAGCCT CGAATGCAGC 22440
TACCGGCATC CAGGACACCT GGAACAAGTT CTGGGGAGCC CTCACCGGGC GCGTCCCAGA 22500
CCAGGACCAG ACCGTCGCTG AGCCCGCCGA GCGTATCGGC GAGCTGGCCG GCACCACGTC 22560
TGCTAACTCG TCTGCCATCG CGGAGCTGCA GCGTCGACTG GACAACCAGC AGAACGCTGG 22620
CGGCGTGGCC GGCGGTGACG ACTTCGAGCG ACTGAACATA TCCGGTTGGG ACATCAGGTA 22680
TTCCAACGGA TCCAGCGGCC GAGGGTACTA CCGTGCCGAC GGCCACCAAC TGGTCTGGAT 22740
GGACGAAGGC AACCAGCAGA ACACCGCGAC GTTCGTCCGC ACCAACCCCG CAGACGAGAA 22800
GACAGCCACC GACTACCAGA AGATGACGTT GGTCGTCGGG ACTATCTCCG GTGAGGTACA 22860
GACCGTGTTC CCGCCGCAGG GAGGTTCGCA CACCCGGCTA TGGGTCCGCG TCAACGACAA 22920
CGCTCCGACC GTCGGCATCA CCGACGGCGT GTTCGTAGAG ATCGGCGGCG TATCGAAGGC 22980
CCAGATCGGC TACCGCCGCA ACGGCAATGA CACGTTCGTC GGATCTATGG TCGACTGCAC 23040
CTGGGGTGCT GGATCGATCT TCGCTCTGAC CGCCGGCACG GCCAACGGTG CTGAGAAGTT 23100
CGAGGTCTCG AAGAACGGCC CCGTGCTGGC CACATGGTCG GACGACGGCG TCGTCTCCGC 23160
GATGGGTGCG AACTACCGCC GCTGGGGCTG GGAAGGCCAG GCTCGTAACC GCAACCTCGG 23220
CCAGGGCACT CCGAACTCGG TCACCCGAGT GACGATCACC GACAACGATC CTACCGGCGC 23280
AGGCGGTGGA GCTGTCAACG TCGGAGGAGA TGTCGTAGGT GTACTCCCCA TAGAGAACGG 23340
AGGCACCGGA GCTTCGACAG CTTCGGCAGC CCGTACCGCT CTCGGAATCG ATGACCTGGT 23400
CGAAGATATG TCCGACGTAG TTCGTGGATC CGTCGAAGGA CTCCCGTTGA TACCGAAGAT 23460
CTGGGTAGGA ACAGAAGCTC AGTACACGGC TCTCGCCACC AAGGATCAGT CCACGCTATA 23520
CTTCAGGACC GCTTAATGAC TGGTATCTCG TTGGGTGTCA ACGACATCCG CAACCTCTCG 23580
ATATTCTTAG GCGTCAGCAA CAAGATATTG AAGGTCAGTC TAGGCACAGA AAAGGTCTGG 23640
CCTGCGTTCA CCCCGGTGCT GACCACGTTC GCCACGGTCG GCACGTACAC CTACAACATC 23700
CCCGACGGGG CCAAGTTCAT CGACGTCATC CTCCTCGGAG GAGGCGGCGG GGGTAAAGGC 23760
ATGGCCCTGG CTGACGGCTG GGGCAGAGGT GGAGACGCCG GAAGCTGGGC TATCGTCACT 23820
CTCGAACGCG GGGTACACAT CCCGTTGTCG ACCAAGACGA TCACCGGGCT CGTCGGAGCT 23880
GGAGGCGCAG CGGGAGCTGG CTCTGTATTC TCAGGCAAGG CCGGAGGCCC TGGAGGAAAC 23940
ACCACGGCGT CCGCTGTCGG ATGGTCAGGT TTGACCGCAA CCGGCGGTCC CGGAGGCTCT 24000
GTGATCGACA TCCTCAGCGT CGCCGGAAAG TCGCCTGGAG ATCGGACCTA CAACGACCAG 24060
CTCTACATAG GCGGCGCACA ACAGAACTCA GCTGGCGGGA ACGGCAATGC TCCTGGCGGC 24120
GGCGGGGCTG GTGCCCAGGT CTCCGCACAG AGCGGCGGTG CTGGCGCTCG CGGCCAGGCG 24180
TGGTTCTTCG CGTACTGACA AGAAACCCCC CTCTTTAGGA CTCAGTGTCC TTGGGAGGGG 24240
GGCTTTTTGC GTTTCAGGAG GTCTTGGCCA GCTTGGACAT CGCCTCAGCG ATAGCCTCGT 24300
CGCGGGCCTC AGACGCCATC TGGTACTTCA TCGCCATCCT AGGAGTCGTG TGACCGAGAC 24360
GGGCCATCAG CTCCTTGGTC GTCGCACCTG CCTGAGCGGC GAACGTAGCG CCGACAGCGC 24420
GGAGGTCGTG GATGCGGAGT TCCGGCCGAC CGATCTTGGC GTAGCCACGC TTCAGCGACT 24480
TGGTGAACGC GGACTTCGAC AGCCGGTTGC CCTGCGTCGT GGTCACCAGG AATGCCTCGG 24540
GGCCCTTGTT CATCTTCGTA CGGTCCTTCA TGTGCGCTCG GATCATCTCC GCGACGTGAG 24600
GCGGAACCGT CACAGGACGC TTCGACCGGA CGGTCTTGGC GTTGCCAACG ACGATCTTGT 24660
TCCCCACGCG GGAAGCGCCA CGGCGCACCC GGAGCTTCAT CGTCATGCCG TCGTCCACGA 24720
TGTCCTTGCG GCGAAGCTCG ATCAGCTCTC CGAACCGGAG GCTCGTCCAC GCCAGGATGT 24780
ATGCCGCGAT CCGGTAGTGC TCGAAGATCT CAGCGGCGAC GATGTCCAGC TCCTCAGGCG 24840
TCAGCGCCTC TACGTCGCGC TCATCGGCTG CCTTCTGCTC GATCCGGCAC GGGTTCTCTG 24900
CGATCAGCTT GTCCTCGACC GCTGTGTTCA TCACCGCCCG GAGGACGTTG TAGGCATGCC 24960
GGCGGGCAGT CGGGTGCTTC CTACCCATCC CGGCCCACCA CGCACGCACC AGAGCTGGCG 25020
TCATCTCTGT GACCGCCACT TCACCTAGCA CCGGGTAGAT GCGGCGCTCC GCGTGCCCGC 25080
TGTACAGATC CCTGGTGCCG TCTGCGAGGT CGCGCTCCAC GAGCCACTTC CGGGTGTACT 25140
CCTCCAGCGT GATGGCGCTG GCGGCTGCCT TCTTCGCCCG GTCCTGTGGA GGGGTCCAGG 25200
TCTCCATCTC GATGAGCCGC TTCTCGCCCG CGAGCCAGGC TTCGGCGTCC ATCTTGTTGT 25260
CGTAGGTCTG CAGCGCGTAG TACCTCACAC CGTCCTGCGG GTTGACGTAT GAGGCTTGGA 25320
TCCTCCCGCT GCGCTGAGTC TTCAGCGATC CCCATCCGCG ACGTGCCAAC TAGGTCTCCT 25380 CTCGTCGTGA ACAAGGCTAC CGGGTTGCAA CTCCTGTGCA ACTCTCAGGC TTCAACGCGC 25440
TTCTACGACC TGCAATTTCT TTCCACTTAG AGGATGCAGC CGAGAGGGGG TAAAAACCTA 25500
TCTTGACCGG CCCATATGTG GTCGGCAGAC ACCCATTCTT CCAAACTAGC TACGCGGGTT 25560
CGATTCCCGT CGCCCGCTCC GCTGGTCAGA GGGTGTTTTC GCCCTCTGGC CATTTTTCTT 25620
TCCAGGGGTC TGCAACTCTT GTGCGACTCT TCTGACCTGG GCATACGCGG TTGCAACGCA 25680
TCCCTGATCT GGCTACTTTC GATGCTGACA AACGAATAGA GCCCCCCGCC TGCGCGAACA 25740
GACGAGGGGC ATTCACACCA GATTGGAGCT GGTGCAGTGA AGAGAATAGA CCGGGACAAG 25800
GTTGCACCGG GAGTTGCAGC GGTCGGAACC CTCGCCGTCG GCGGGCTGGC GTTCGCCCTG 25860
TCGTTCACGG CTCTCAGCGA GCTGGCTGCG GCCAACGGGG TGGCCCAAGC AGAGATGGTG 25920
CCCTTGGTGG TCGACGGCCT GACGCTCGTC GCCACGGTCG CCACAGTGGC CCTCAAGCAG 25980
AACAGTTGGT ACGCGTGGTC GCTGCTGATC CTGTCCACCG TCGTATCGGT GGCCGGCAAC 26040
GTGGCACACG CCTACCCCCA CGGCATCATC GCGATGGTGA TCGCTGCGAT CCCTCCGCTC 26100
TGGCTACTGG CGTCGACCCA CCTAACCGTG ATGCTGGCGA AGCAGCACTC GGAGCACGCC 26160
GAAGTACCTG TCTCGCGGCC AGAACCCGCG CCTCGGGGCC TGGAGCCCGC TGCCGCTTGA 26220
CTGCGCCCGA CCGGGACAGA AATACATAGA GAACCTATGG ATGTAGGAGG CACAAAAAAA 26280
TACCCCCCGA GCCAGCCCGA AGGCCAGCCC AGGGGGCATG GTTCTGCTTC AGTAGACCTT 26340
GCGAGTCCGA CCCGAGTTGA TCATCGCCAT GATGACCCAG ACGGGCAACC ACATTCCGCA 26400
GGTGATGAGC GAAAGCAACA GGTGCATCGC GTGGTTCGTC CTGACAGGCA TGACAGTGGG 26460
CTGCGGCATC GGAGGAGGCG CGACCGGGTA CGGCGAGCCC GCGTACCACT GAGGTCGATC 26520
TTGTTGGGGC GGATACTGAT TGGTCATCCC GACAGCCTAC TTGCCGATGG GTCGCATCAG 26580
CTCCTCGACC GACTCGCGCT CCACGCGGAT CAGCCGGGGA CCGAGCCGAA CGGCCTTGAG 26640
CCGGCCGTCG GCGATGTAGT TGCGGACGGT CTTGGTGCTG ACACCGAGGT AGTCAGCGGT 26700
CTCCTGGATG GATGCTCTCG GGGGCATCAG CGCGGTCCTC CGTGCTTCAT CGGTTGTCTC 26760
CCGAACCCTG GATCACGCCA CGATCCTTGC GGCTCTGGAG CTTGTTGAGG TTCCTCTGGG 26820
TGACGGTGCT CAACCAGACA TCGAGCTGGT TGGCTAGCTG GGCGACGTAC CACATCACGT 26880
CTCCGAGTTC CGCCTGGAGG TCGTCTCGGT TCTCCTGGGT GATGACACCG TCTTTATCCC 26940
GGAGGATTTT CTTGACCTTG TTGGCGATCT CGCCGGCTTC GCCTACGAGA CCCATCGTCA 27000
CGTAGGAGAG ACCCTCGATG CTGTCGCAGT CGCCTGCACC GGGGTAGATC GCTGTGTCGC 27060
TCGCGGCGAT CTGGTAGATG TCGACGTGCA TCAGATCATC ACCGGGAACA ACTGGCCACC 27120
GGGCATCTGG ATGAACACCG GGACGCTGGG GGTGTAGTCC GACGAACCCG TGCCGCCCTC 27180
ACAGGCGGAC AGGCTCAGGG TGGCGGCAAG GCCGATGATG GCTGCTGCGA TGGTCTTCTT 27240
CATCTGTTGC TCCAGTAGCT AAGTTCGGAC TCCAGTTCGC GGATACGCTC CTGTAGCCCT 27300
TGGTTTTCCA GGTACGCCTC GGCGAGGTTG GCCTCGGCGC GGTCACGGGC CTCGTCCTTC 27360
GACGTGGCCT CATCGATTGC CTCGTGTAGC CGGCGGATCA GATCTGGGAT GGCACCGTGC 27420
AGACCGCATA TGAAGTCGGC GTCTGCCTCG GAGAGGTGGG ACGCCACCAG ATCCTTGTCC 27480
TGGGTCTCCT GGTTGACCGC CCAGATGACG TGATCCTCTA GCCCGTGGTC GGTCTCGCAG 27540
ATAGAAGGCG GTTCTACCTC CTCTGGCATC CAGTAAGTCT TCTCAGCCCC GGTGGACTTC 27600
GCCCACTGCT GGTAGAGGAT GTCGAAGAAC TCGTGGTCCT GTTCGTCGGC GGTAATCACA 27660
GATCGTCCTC TTCATCCCAT TCGTCGTAGT AACACGTACA GCCGCAGCAG GTGCAGCAGC 27720
CGCACTCGTA GGTGCCGTAG TCGTAGTCAT CCCAGTCGTC TTCGTCCATC TAGCTGTACT 27780
CCTTCATGAT TCGGTCGAAC GCACGCGTCT GCACGCGCAT CTCCAGGTCG ACCGTTCGCT 27840
TCAACCACGC CCATTCGCCG TCGTGGTTGA TCTCCCACTG GCTCTTGAAT GTCGCTGTCT 27900
CAACGAGGAA CTCGACAGTC AACGTGTGCA GTCCGTTGTT GCTGGGCTGG AATCCGATAC 27960
CGTCCTCAGC GATGTACCAG GGCAACTCCT GGCCGTCGAA GTAGACGGCC TTGTCGGTCA 28020
CCAGTACTTC AGGGAAGGTG TGCTCGGTCA ACGGCGTCCC AGGTATGGGA TGACGCTGGC 28080
CCGGAACTCA AGGAACACCA TGTTGTCCGG GCAGTCCTCG GGGACGTTGT CGGGGCGTTC 28140
GGCGGTGTAG ACGCCGATCT CGTTGCCCTC CAGGGTTCCA AGCTCGTTGA GCTTGTAGAT 28200
CGCCAGACCC ATCAGCTCTT CATCGAGACC GTTCGGTGCT GGCAGTACAA CTTTGGCTTG 28260
TGGCATTAGC CCTCCCTCGG AATTACGTAT GCGCTGAACT CGACGGCCGT AATGCCGTCT 28320
GGCAGTTGGA ATCCGAACCG CTCTTCGAAC TCCTCGTTGG TGATGGGGCC GTACTCGAAG 28380
GTTCCGGGCA CTACCTCGCC CTCCCCCTCG ATCAGGAGGT ACGCACCGGC GGCGTACACC 28440
TCCTCGTCGT TCGGCCATCC GACTACGGTC CCGAGGACCG TGAACTTCCT CGGCTCCATC 28500
AGGGCACGTC CACTTCGTTG ATGAGGAACC GCATCGGAGG TGGAGTGAGC ATTGCCTCGG 28560
CTATGGCGAT GAGGGCGTTC AACTGACCCT TCAGCAGCTT CTCCTCGTCG CCTGCGGGAA 28620
GGTGGCGCAC TCGGCGCTCC ATCTCCTTGG CGCGTTCCAG ATATTCGGTG GCTGTCAAGT 28680
TGTCCTCCTT AGTAATCAGC GCCGTAGAGC GAACCCCACG AACGCTTTCC GACCTCGGGG 28740
TCGGTGCCAA CCAGCACCGG ACCCATCTGT TCTTGCATCA GGTGGCCAAT GTGTGCAGCG 28800
GCTCTCTCAG CCTCTGAGGC GGGCAGAGAC GCGACGATCT CGTCGTGGAT AGGCAACCGT 28860
AGGTACGGGG TGTATCCGGC CTCGTGGAGG CGAATCAGAG CCCGACAGGT CACGTCCCGC 28920
GACGACGACT GGATCATGTA GTTCAGCGCG GAGTATGTCC GCGAGCTGTC CACCGGCAGC 28980
CGCCGGCCCA TCGCGTTGAC GATGTAGCCG TTGCGGCCAG CTTCCATCGC CAGCTTCTTG 29040
CTCAGCCGCT CCACACCGGG GTATGTCGCA GAGAACGCCT CATGAACTCG CTTGGCCACA 29100
GGGATCGAGA TCCCCACTGC CTCAGCGAGA GCCTTCGCCC CACCGCCGTA GACCTTCTGA 29160
AAGTTGGCGG TCTTCCCAAC CTTTCGCGGC ACCTGGGCTG CGTCAGCGGT CATCTGGTGG 29220
AGGTCCGCAC CGTTCTCGAA TGCCTCGATC ATGTTGCGGT CGCCCGACAG CGCCGCCAGG 29280
ACGCGAAGCT CCTGCGCCTG GTAGTCGACT GAGGCCATCA CATCGCCTGG CTCAGCGATG 29340
AAGCATCGCC GCACGATCCA GTCCGACGAC GGCAGCGTCT GCGCCGGGAT GCCGGTGATC 29400
GACATGCGCG AGGTCCGCGC CTGCAGTGGG TTGATGAACG TGTGGCAGCG GTCCTCAGAG 29460
TCCCTGGTGT CGATGAACTT CTGGACCCAG GTCTTCCGCC ACTTCCCCAG CTTCTTAGCC 29520
TCCTGAGCGA TGGCGGCAAG CTCGTTGCCA TCTTCGACCA GCTTGTCGAG CAGAGCCGCG 29580
TTGACCTGGC GCTTGCCAGT CTCGGTGCGA CCGGTGATCT TGACGCCCAT CTCCTCAAGC 29640
CCCTCGGCCA GATCCTCGGT CGAGTTGACC TTCTCCACGC CGTACTCGGT GAAAGCGATT 29700
GCCTCCCAGA CCTCCTGATC GGCCAACCAC TTCTCGGCGA GCGACCGCGA GTACTCCACA 29760
TCGAGCAGGA AGCCCTGCCT GTCGATGTAG CTGCAGATCT CACTGATCTT GTGCTCGTAC 29820
GGCACCAGCG ACCGACTCAC GTCGGGCACC AACGGTGTCA GGCTCTTGCA GACCCTCGCG 29880
GTGAAGATCG TGTCCATCCC GGCGTACAGC AGGTACTCCG GGTGGAACAG GTCGATGGTC 29940
GACCAGATCT TGGCCTTGGT CGTCTTGTGC TCGGCGGCTA GCTTGGCCAT GAGCTTCTTG 30000
ACGTTCTCGG CCTGGTCCTC GGAGATGAAC TTCGCGATCA GCTCTTCGAG CGAGTGCCCG 30060
AACCCGCCGG CCTCGAAGGG CCGGGGGTCC ACCAGCTTCG CCAGGATCTG CGTGTCAAGC 30120
ACGCGGGGCC ACAGACCCTC CATCTCGATC CCGAAGCACT GGTCGAGCAC CTGGAGGTCG 30180
AAGGAGGCGT TCTGGAGCAC CATGCGCTTG AGAGCGCCGA TGGCGATCCG CACGTCCTCG 30240
ATGAACACGT CTCCCAGCTC CACCGGCACC ACCCAGGCTT CGTCCTGAGT ACCGAACTGG 30300
ACGAGGCGGC ACTCGAAGGT GTCGCTGTAG ATGTCCAGCC CGGTGGTCTC AGTGTCGACG 30360
GCGAGGCAGT TCAGGTGAGC CCGGATGAAG TTGCGGAAGC CTTCCAGATC CTCTGGGGTT 30420
TCAACGACGT TGACGGTGAC GAGGTCTCCC TGAACCTCAT GCCGCAGCTC GATCAAAATG 30480
CTCTCCTACT GGAAGTACTG AGGCGGAATC CAGGTGGCTG AGGCCATCTC CTTGATGGCC 30540
TGCTGCATGG CCGCTTCGAA CGGACAGTCC GGGTCGATGT CCGGCTTGTA ATGGGTGACG 30600
ATGATCCGGC TGTTGCCGCC GAAGTCGTGG CTGACCAAGC CCTTTGGGGG CAGCTTCTTC 30660
AGCGCCTTGA TCAGTTCCTC AACCGTGGTC CCGGTAGGGG CCTTGCCGTC AGGCAATGCC 30720
TCCCCTCCGT ACGGCACGTC CAATGGGATC GTGTACCGCT CAACGTCTTT GATCTTCATC 30780
GAGCCTCTTC CTCTTCGACT ACCTCGTCTA CCCGGCGGAA TAACTCCGCT AGTTCTGCGG 30840
GTAGCAATAC TGGGTACTTC TCTCGGGCTT CCTGCATCGC TACCGCGATC CCAATCAGGG 30900
CAGCGAGCAG TTCATTGACG GAGTACGCCA ACAGCTCTTC GCGGATCTCT TCTCGGGTCA 30960
TTAGTGGTAG ATCCCCCGGA CGGTGCGCGA GATCGTGGCA GGGTTCACGC CGTAGTTCTC 31020
GGCGAGATCC TTCTGCTTCA TACCGCCCAG GTACGCCTGG CGGATGTCCT TGACCTCGCG 31080
CTCGGTGAGC TTCTTGCGGT TCGGCCGGCT CGGGCCGGTC TCAGGCTTGA CCTGAGCCAG 31140
CGCCTTGCCG AACAGCTCGT TCTGCGTCCG CTGCTTGATC GCGTACCGAC GGTTCGCTGC 31200
AAGCACCTCG TTGAGCCGCT GGGACAACTT GACATTGGCC TCACGCACTA CCTCGACCTC 31260
TCCGAGCAAG TTCGTGATCC GGTAGTCCTT GTCCTGGTTC TCGATGGCCA ACCGGTTGTT 31320
CTCCTCGGAA AGCATCGAGA CCTTGTATTG CGCCTCTCCC AGCGCAGCTT TCAGGTGCTT 31380
CTTCCTCATT CAGCGCCCCT CTCTCGGCGG AACTGTTCGT ACTCGTCTTC GGTCATGTAG 31440
TAGTAGTAGT CAACGACCTT GTCCCAGTTG AAGGTTCGGG ACGTGCCGTC ATCGAACGCG 31500
ATGATCAGGA CACCCTCTTG GGTGTCTAGG ATCGGCTCGC CAGCCACGAC GTGGAAGCGG 31560
TCCTCGAGGG TCACCGCAGT CGCTCTGCGT GCCATGTCAG TTCCTCTCAG TAGCTGTAGG 31620
GGACATCCGG GATGTCCTGG TAGGTGTTGG GTGCGATCTG TCGGAGCTGC CGAAGCAATT 31680
CCCCTGCCAG CTCACGGATC TCGGCATCCG CGGCCTCGTG CCAGCGGGCC TTGATGACGT 31740
ACCGCCACGC CCGATGGTTG CCCGTGACGA CCATCGGTGA GTTCGTCATG TTCGGCAGGA 31800
CAGCTCGCGC TGCCTCGCGG GCCTGCTTGC GCGGCAAGCC CCGGTCAGCC AGCCGGTTGA 31860
CGATGTGTTC GTAGACAGCG TCAATCTCAG AGCTGACGGA CTCCATGATG TGGACGAGGT 31920
CGTCTCGGTC GTCGGGGTGG AGCTTGAACA GAGCCGGGGG CAGATGGATG CCAAGGTCGG 31980
TCGGATCCAC ATATCGCTGA GACACCACCG AGAAGCTCAA GTGACGGTGA CGCTCCAGCT 32040
CGGTCAGCAC CGACCTGCTG GCCTCGATGT AGAACGTCGC CGAGGCGTGC TCGAACACGC 32100
TCTCGTGGCC CAGATCGATG ATGTGGTTGA GGTAGTCCTC GTTCTCGGCA GTTGCCGGGT 32160
TCGGTCGGTG GAACGACCGG TAGCAGTTCC GGCCCGCGAA CTCGGCCAGC TCGTCGGCAT 32220
CGAAGTCGCC GAAGTAGGGA TCTTCGTCCT TGGATTCTTC GAAGTCATCG ACCTCGAATC 32280
CGATGTCCCG CAACGCACCC GGATCGATCT CGGTGGCAGC GATCAGTTTG GCTTTCATAC 32340
TCTCCGCTCA GAGTTGGTGG AACGAGGTCA GCCAGGGGGC AGCGAAGCCC TTCTACAGCT 32400
CCCCTTGGCT CGTTACCGGC TTCTCGACCT CGGTGGATGT CAAGTAGTCG AGATGACTAC 32460
TTCTTGTCGG GCCATTGCGC GTCACACTGC TGATCGCGAG GTGCGGTGCA GGAGAACAGC 32520
GCGTACGGCT TGCCCGTCTT CTTCGAGACG CCCGACTTGT AGACCATCTC GCCGTGCTGG 32580
CAGTACCGCT TCTCGCCACC AGGCGCTTCC TGAGCTGCCT GCGGGGCGCG AGACTGCTGC 32640
TGGCCACCGC CGCCGCCGTT GGCCGGCGCG GATCCACCGG AGCCTGCGTA GTGGCCTGCG 32700
ATCTGCTGGA CCTTGTCCAT CAGCGCCTTG AACTCGGCGG TGTTGACCTT GGCCAGCACG 32760
TCGGCCGGGT CCGCACCCTT CACGACCACC CACGGGTCGC TGTACTGACC GGCGAACTTG 32820
AACGTGGCCG ACACCCCATC GGTGGAGTGC TGGACCGCCA TCGAGTCGCG CACAGCAGCC 32880
GAGGCCGTCG TCACCGTCGC CGACGGCGCG GTCTCAGGCT CAGGAGCCGG GGCCGGCTCG 32940
GGCTGGGCAG GGGCGGTGCT CCACGGATCG TCGTAGGACA ACTGGTTACC TTTCACTTAA 33000
TGGGGCATGC GCCGTTGGCG CACTCTTCAT CGACACCGTC TTCGACGGCT TTGGCCGCAG 33060
CAGATTCGTA CTGCTGCTTG GTGATTCGCT CGTACGGAGC CTGCGGGAAG CTGGACTCCG 33120
GGAAGATCGT GGAGCCCTTG ATGAGCCCCG CGAACCTCTT GAGATCGGCT GCGACATCCT 33180
CGGCCTCGTA GGCGTCTGGA TGGACGTTGG CGGTGAACGA CACCGCGTTG TCAGCCCAGC 33240
ACATCTGGTA GAGCGCCTGG AACGCCAGGA GCTGGTGGAG GGTCAACTCG TCGGCTGACT 33300
CAACGATCTC CTCGTCCCAA CCGAGTTCCT CGACAGCCTG GACCAACGTG TCCTTGGTCG 33360
GGATCGAAAC CACCTCGGTG TTCGGAGCGA AGAGATCCTT CTCGATCTCG TAACCCTCGG 33420
CTGCCAACCT CCGCAGCTCG GCCATGTCGC TGTTGAGGTT GAACCGCACA CGCCGGATGA 33480
AGTACCGCGA GAAGATCGGG TGGATCCCCT CGGAGACTCC TGGCATCTTC GCCACCGTGC 33540
CTGTGGGAGC GATGGTTCGC TTCTTCACCG GGACAGGGAT CCTCAGATCA TGGGCGAACC 33600
GTTCGGCCTC TGAGTCGACC TCAGCGGCCA TCTCCCGCAA GAACTGGGTG AACCGCTTAT 33660
CTCCGGGTGC CTCGGAGTAC CTGCTACCTG TGAGGGCCAA ATAGGAGGCA ACTCCGAGAT 33720
GACCCACGCC GATGCGACGG TTTCGGTCCA GAACCTCCCG GCTCTTCGGG TCGGCCACTT 33780
CCGAGAACGT CGCCCGGATC AGGAATCTCG TCATCAGACG ATGCGCCCGG ATCAGGTCGA 33840
GGTAGTCGGT CTTGCCGGCC GGCGTCACGA ACGCCGCCAG GTTGATGTGG CCGAGGTTGC 33900
ACGGCTCCCA CGGTTCGAGA GTGATCTCGC CGCATGGGTT GGTGCAGACC ACCCGGTTGG 33960
GCTCACCGAC GTTGGACAGT GACGAGTCCC ACATCCCCGG CTCTCCGTTG CGTACGGCTC 34020
CCTCGGAGAG TGCCTTGAGC ACTCGGTGGG CTCGCTTCTG CTTGGGCATG TCCTCGCGGG 34080
CGACCGCGAA GCTGCCGTAG CCCTCCTTGG CCAGACGCCA GAACTCGTCG TCAACCTCGA 34140
CCGAGATGTT CGTCGTCCAG TGCTCGCCCG TGCTCGCCTT GATGTTGATG AACTTGTCGA 34200
TCTGGTAGTC GTCCCAGTGC ATCATCGACA TCCGCGCCGA CCGGCGCACA CCGCCGGCCA 34260
CAACACACTG AGCGATGGCG TGGTCGACCT CCATCGCGGC GATGCCGTCG AGCGTGATCC 34320
CTGCGTACTC CGAGAAGATG TTGGCGACCT TCTGCAGCAT CACAGCGAAC GGCAGCGGGC 34380
CGCTGGCCAC TCCACCGAAC GTCTTGAGCT TGGCCCCTTG CGGCCGGATG CGGCTCACGT 34440
CGTACACCCG CTGGTAGTGG ACCGTGCCGG GTCGGTAGTG CGTGTCGATC AGATCGACCA 34500
GCGCAGCAGC CCAGCCCTCT CGTGAGTCCT CGATGGCGTA GGCACCGGCC CAGTCGTGGC 34560
TGTAGTGCTC CGACAGAATG CCTACATCCT TCATCGCCTG GTAGTCGACA TGCTCTGGAT 34620
CACAGACGAT CTCGACCCGC AGGGGGTTTA CGACCTCGGG GTAGCCTTCG AGGTAGTGGT 34680
TCGAGTAGTT CGCCCCGACT CCCCCGCCCT CCATCAGGCG CATGAACGTG AACTGGAAGT 34740
GGTCCGAGAT CTTCTCGGGC CAGCCAGCTA CCCAGCAGTT GAAGAGGTGC TGCGCGTTCT 34800
TGACCCCCGA GGCCCACAGA TGCCGACCTG CCGGCAGCAC CTTGAACTTG GTCATCAGAC 34860
GAACGAGATC TTCTCGCTCT CCTTCCAACA TATGTCGCCG GTCGACAAGA GCAAGATTGC 34920
CGTCCACGAC CCTCTCGACC GTTTCCGGCC AGGTTTCCTT CGAGCCGTCA GGCTTGGTCC 34980
TGGCGTAGGT TCGGTTGTAA ACGAGTTCAC CGGTTGGTCC CCAAGGGATT TCGTCAGTCA 35040
ACTACTTCCT CTCAGTCAGT TCGTATCGCT TGAAATAGGC GTCGGCAGAG TCGCCGCCAG 35100
AGAACGAGAC CCCGTACTCG ACCGGGCCTG CACCACGCAC CTCGCAGGTA ACGACGCCCT 35160
TCCTTCCCCG GAACATCGGC CAGGTTCCCT TGGAGGGGTG CTTGGTCTCG TCCCGCTGGA 35220
CGATGACCTT GGTGCCCTTC TTCATGCCGA CTTCCGTTCT CCGTAGCCGG GAGTGAAGCA 35280
ACCCCCGACG TACAGCTCGA GATCTTCTTG CGACCAGTTC TCCAGTCGCA TCGGCGGCTG 35340
GTGCGGGAAC AGCTCCGGGA ACACCTCGGC CCGGTACAGC TCCGAACCGG GCATCCCGTT 35400
GAACGTCGGA TCAAGAATGT TGTGCATGGC ACCTCCCTCC CAAGAACTCG GAGATCGGCG 35460
GCTCGTAGAG GTAGCCATCG CGCAGCTCGG GGTTCTCGAT GAGCATGATC GCGATGTTCG 35520
CTGTGGGGTC AGAGTGCCCA TCCCCCTGCG ACTTTCGGAT GTCTGGGAAG ATAGCGTGCT 35580
TGCTGCCCGG ACCATCCTTG ACGATGACCT TGCCCTTGTC GTCCTTCTCC ACGCCAGCCG 35640
TGATCGCGAT GATGTTGACG TGCTCGGTCA GCGACTTGTG AGCGCGGAAC AACCGGTTCT 35700
GCCCGCTCTT ATCCTTCGGG GAGATCCCGT CGGTGTAGCG GCTCCTGATC GCCTCTGCAT 35760
AGCCCCCGTT CTGAGCGTCC AGAGCCTTCA TCGCCAGCGG GAGGATGTCG ACCAGGTACC 35820
GATTGGTCGA CTCCCCCTGC AGAGCCTCTT TGACGTTCTC GGACGAGTAG TGGCTGCGCT 35880
CCTGGAACAA GTCGCGGGCC TTGGCCGCTC CCGACAGGAT GTTGCGAACC TGATTGCGTA 35940
CGTAGTGAAC TGCCTCACCA CGGTGCAAGC TCTCCAGCGT CTTCTGGATG TACGGGCTCT 36000
CGAGGTACCA GACCCACAGC TCTTGGATGA TCTCCTCGGC TGTCAGGTTG GTCTCCCAAC 36060
CGATCAGCGC CTTCCGGGTG GCCCTGCTGA ACAGCTTGCT GATGTCGTCG GTCAAGGCAT 36120
CACCTTTCGT AGGTACTCCT CCCGGTCCAA TCGGCGGTCG AGGTGTCGAG TGACCTCCTC 36180
CGCGAAGACC TCGCGGACTT CGCTGGAGGT GATCTGGCGC GAACGTGCGT TCTTGTGCAG 36240
GTACGGCAGC TTGGTGGCTG TCAAGTTCTA GACCTCCCAG ACTCGGCCGT CGACCGAGAA 36300
CCGGCCTCCG ACAATCGGAA CAAGCTCAGG CTTGACGTGC TGGCCGTCGA CCGTCAGCAG 36360
AGCAAAACCA CTCTGCCAGT TGGCTGTTGC ACCCTTGAGG TACTGAGCTA GCTTCATGTT 36420
CATCAGGTTG CCGACCTCCA TCGACCACAG CACCTTCTGG TTGCCGCCGT AGCCCAGCGT 36480
GTGTGGCTTG ATGCCCTGGC GGTGGGTGTG TCCGATGATC ACCGACGTGC CGAACCGCAT 36540
CATCGCGTTG TACGCGGTGT CAGCGGACTT CTGCGTCACC CGGACCCCAC CACGGTGGCC 36600
GTGGGTGGAG ATCCAGCCTG GAGCGATCTT GTAGAACTCA GGCAGCACGT CAACACCGAA 36660
CCCGTCGAAG TCCAGCAGGT TCTGGAACTG GAACGAGCTG ACGTACTCGA CCAGCGCCGG 36720
GGCGAACTGG TGCAGGTAGT CGACTGGCCG GCGGTCGTGG TTGCCCTCGT GGACACCAAC 36780
CGGGCCGTCG TAGACCTGGC GCAGCGGCTC CAGGAACCGC CGCTTGCACT GCTCGGAGTC 36840
GGGCTTGATC CGCTGAGCGA ACTCTTCCTT GGTGCCCTTG GTCCACCGAG ACGGGCTCGG 36900
GTAGTCCATC AGGTCACCGA TGTGGACGAC CTCGTCAGGC TGGGTGTCCC CGATGTAGCC 36960
GATGACCGCC TTCAACTGCT TGCGATCATC GAACGGAATC TGGGTGTCCG AGATGACGAC 37020
GATGCGCTTG CTCACTCAGC GACCTCGGTG AAGGGGCCCC GCATACGTTC CTCGTGGGAG 37080
CTGGCGTTGC CTCCTGACCA GCGTCGCTTG CCCACCTTGG TGTGGTGCAA CCCGTTGGGG 37140
TAGTAGATCC ACTTCACTCC TGTGGCGTTG GTGACGGTCT TCACATCGGC AGGAACGTCC 37200
AGCAAGGTGT CCCACTGGCG AGGCCCCTTG GGATACCGCT CGTCCTCGGG GAGCTGCATC 37260
TTCTCCAGAA CGCCTGCGTA ACCGGCGATG TCGACCACCG TGTCCTGGTG GTAGCCGTTC 37320
TCCATGAACC GGGCGATCTT CAGCAGGATC ATCATGACGG CCACGTCCTC CGGGGTGAAC 37380
TCGACGCCGC GCTTGTACGC GCCCCACAGG GTCGCGATGC GTTCGTGGTT CTCCTTGGCG 37440
TCCCCGTAGT CCTGGGCTCG CTGTCCGTTG ATGATCTCTT CGGCGGTGGT CAGAATGCTC 37500
ACAGTCCAGT CTCCGATGCG GTGTAGTAGT CGATCAGCTC ATCGAGCTGG TCCGGTTGAT 37560
AGCCGAGGAT CGGCTTGTGG GTGTCAGTGA CGACGACGGG AACCGACATC GCGTTGAGCA 37620
CCTTGGTGAC GTAGTCGTAC GCCTCCGAGT TGGCCGTGAC ATCGACTGCG TCGAAGTCGA 37680
TCCCGGCAGC CGTCAGCTTG TCTTTGACTC GCTCGCATGG CTTGCAGCCG GGACGGGTGT 37740
ACACCGTGAC CGGCGCGAAC AGCGTTCTCA CGTGAGCACC ATCCCAGTCG ATGTATCGGT 37800
CTCCATACAT CAGATCCTTT CCAGCAGAGC AGCTTTGCCC TGCGATGTGA CTAGTGAGTT 37860
GACATCCTCG CCTTCTGGCA TCGGGATGAT TCGGGCGTTC GGCAGCGTCT TCGCCACCGA 37920
CCGGGCGAAC TCCATACCGG CGTCGTCGCC GTCGGCCAGG ATGTTCACGT TGCGGTAGCC 37980
CAGGAACAGC TCTCGGAAGT ACGGCTTCCA CTTCTGGGCT CCGCTGAGCC CCACCGTCGG 38040
CAGCCCACAC AGCTCGGCGG TGATCGTGTC GAGTTCTCCC TCGCAGATCG CCATGTCCTT 38100
GCTGTATTTG GTCAGCGCGT AGGTGTTGTA GAGCCGGTCC TTCTCCCCTG GCATCGACAG 38160
GTACTTCGGT GTGCCACCGT CGATTCGGCG ATACCGGATC GCAGCTACCG TCCAGTGACG 38220
CCAGGGCGAC CACCGCATAT ACGGAATCGC CAGGCAGCCC CGGTACATCT CATGTCCAGG 38280
GAGTGGGTCG TCCACGAATC CCAGACCGAA CCGGCTTAGT TCCGCTCGGC CGGCCAGCCC 38340
GCGACTCGCC AAATACTCGT CGGCTGGGCT TCCGGGCAGG CTTTCTCTGT ACCGGGACGT 38400
TGCCTCCCAC AGATAGGTTC TCTGCGATTC GCTTAGCCTC TGCAAATGTC ACCTCCTCTT 38460
CGTGACGAAT GATCGAGATC ACGTCTCCAC GGACCCCGCA GGCCATGCAG TTGTAGCCCT 38520
GTAGGTCGTA ACTGACTGCG GCAGACGGCG TTTCGTCGCC GTGGAAGGGG CACAGGCACT 38580
TGTTCCACTC GTGGTGGTCA GGTGGTGGTT CCCAATCCGG GTGGTAGCGA AGAATCGCCC 38640
TCGCGATGGG CGAGTCGTTC ATTCGTCCTC GTCAAGCTCC TCGGGAGAGA GCCCTTCGAA 38700
GATCCCGTTC AGGACGGCGG CGAAGCCCTC GCCGGTCTCC GCTGCGTCGA GCATCTCTGC 38760
AATCGTCTTT GCCATGTTTC CTCCTGGTGG ATGTCAAGTT CGAGACAGCT TGTCAGCCTC 38820
GACTGGAGCG ATGCGCTCCC CGATGACTTG GACGGCCGGC GGGTTCAGCA GGTACTCGAT 38880
GGCCCGTTTG AAGAACTCGA TGCAGTCCCT CGCCCAGCCC AGCGTGTACT TGTTGCACAT 38940
CGTGCAGAGC AACCCTCGGA CGATGCCTGT CTTGTGATCG TGGTCGACCG ACAGGCGCTT 39000
CTTCTTACCG TTGGCTCGCT GGCAGATGTA GCACCGACCA CCTTGGAACT CGTAGATCTG 39060
CCAATACTCA TCGCCGGTGA TGCCGTAGGT GGCCAGGATC CGGGTCTCCC AGCTCGTAGA 39120
GCTGCGAGCC GTCCTGAACT CTCGGTGATG AGTAGCGCAT CGTGGCCCTG GATACTTGGC 39180
GTCTCGCGTG AGCGGGAGCC CCTGTGCGAC ACAGTCTTTG CAAGGCTTCC GCTTGTGCTT 39240
ACGGTTCTGC ACCCGGTACC CCGGAGACCT CTTCGCCGCC CTCGGCACGC GCGTCCTCCT 39300
CCCGGTTCTC CATCACCATG CAGAACCACG ACAGCAGCCC TGCCAGGGAG ATGTAGAAGG 39360
CCACCAGAAC TTGGCCGCTC ACTTCACCAT TCCTCGAACC CACCAGCGAG ACAGCGCCTT 39420
ACGCCCTTTG TCGAGCGGGG TCAGCTCGCG CTCATCGTCC TCACCGAAGT CGAACTCGAT 39480
GCTGGCGATC TCGTAGCCGA GGATCTTGAA CGACACGTTC ATAGGCGGTC TCCGAAGTTG 39540
ATGACGGGAA TGCCGGCCCT TTCGGCCTCT CGCATGCAGT GCCGGGTGCC GACTGAGTTG 39600
CCGAGGGGGA ACGCCAGACA GATGTCCGCA CCGGCCCTGA CCATCTCGAT GTTGCGGAGG 39660
ATGCCAGCCC GCTTGCCGTA GCGTTCCCAG TCGGCTCGGT GCAGCTCGGG GAGCACGTCC 39720
CATCCCTCCT GCTTCATCCC CCAGGCCCAG CGGTCTGCGA TGTCGTCAGC GCCGCGAGCG 39780
CCGCCGTGGA CGACCGTGAG ACCGGAGAAG GACCGGTGGT ACTCAGTGGC CAACGCTTCC 39840
CAGACCGTGG TGCGGTCCTT CCAGATCCGA GATCCGGTGA TCAGTACTCG CCGCATCAGA 39900
TCGCCTCCCA CTGCAGGCCG TCGTGCGACG TGACCAGCTC CGCTTCGTAG ACGCCGTAGC 39960
GGGTGGCCAG GAACTGGATC ATCTGCGCCT GCTTGTACCC GAAGGGACAT TCGTGGACGC 40020
CGCTGATCGG GTATCTGACT CCGTATTTCA CTTGATCCAC CGCTTCGCGA TTCGGTCGAC 40080
GTTCTCCTCG GAGACGTTGC GGGCGAGGCC GGTGAACTCC TGGCCGTGGA CCTTGGTCTC 40140
GATCACGCGA GGCTTGCGGG GATCCGGGCT CTCCGGGTCG ATCCGCTTGT GGGTCCAGAC 40200
GGTCGGCTTC GTCTTGATCA GAGCGCCCAG CACCTGCTGG CGCAGTGGGT TGGTCTTGCG 40260
GGGCATAGCG TTTGGAGTGG TCATCTGGAT CCTTTCCTCG GTGGCTGTCA AGTCGGTGTG 40320
CGTAGTGAAG CCCCCCCAGG CATGCGCGCC CCGCCTGGGG AGAGTTGATC AGCGCAGTTC 40380
GATGTCGGGC AGGATCGCCT GCGGCTTGAA GTTGACCTGG TAGAAGTCGG TCGAGACGTT 40440
TGCGCCATCG ACCTGCTCCA TGAAGTAGGA GACGTTGTCC GACAGGCCCA GGAAGTGCTT 40500
CTTGATCCCG TCCTTGGTCT TGCAGGTCAC GTCGAGCTTC TTCGACGCGG TGTCCGCGTT 40560
GATTGAGCAC CGGCCCTGGA TCTCGAGCAG GTACTTGTCC GTGATCCCGT TGAAGAACAC 40620
GATCCGGCGA TTGATCTCGA AGTTGTCAGC GGCCTTGCTG ACGTTCTCCG ATGCGACGTC 40680
GGCGTCGGAG GTACACGCGG AGAGGCCCAG GATCGCCGAT CCGGCGATGA GTGCGGTGGC 40740
GATGATCTTC TTCATGTTCG CTACTTTCTG TTTGGTGGAT GTCAAGTTAG TGACCGAAGT 40800
CGTTGATCTG CATAGTGTCT CCGACGAACT CCAAGGAAGC GAAGTCTTGT CCCGACGGGT 40860
CCGACTTCCC CCCTCGGTTC TTGACCGTGG AGACGTTGAG CATGTCCGGG CCGAACCCGT 40920
CCGATACTCG GTGGAGAGTG AGGATCATCT CAGGAACACG CCCGATCTGA CCTTTGATGC 40980
CCGACAACGG GATCGGCTTG TCGCCGTCGT TGTGCGGGCC GGTGACGTGG TGGAGCCCGA 41040
CGACGCATGA GCCTGTCTCA CGGCCCATCT CGTGTAGGTA GTCCATCAGC GACTCCAGAC 41100
CCGAGAACGG GTCGTCTCCC TCGCTTGAAT CGGTGCGGAC GTTGGTGATG TTGTCCACGA 41160
CGATCAACGC TGGGAAGTCC TCGTACAGCG CGTCATACGC GGCCAGAGCG TTCTCGATCT 41220
CGTCCAACGA CGGTGATGCC TTGTAGTTGA ACCGGATCGG GATCTCGTCT AGTGAGTCAG 41280
CTACCGCGTC CTCGATGTTC TGCTCGCGAA CAGCCCGCGT AGCTCGTTCG AGCGACCATC 41340
CGCTGAGGAT GGACACCGAA CGGGAGAGCT GGGTGAACGC ATCAGAGTCG GCCGAGAAGT 41400
ACAACGTCGG CACCTTCGAC TTGAGCGCGT AGGCGAGGAC GAACGCCGAC TTCCCGGTGC 41460
CGGGGCCGGC GCAGACCAGG ACTAGCTGGC CTCGTCGGAG ATGTGTACCT TTCTGGTCAA 41520
GCGCGGCCCA GACCGGGGGT AGCGGATCCC CCGCCGACCC TCGGATGTAG AGCGATTGTC 41580
TAGGTGTGTA CACCTTCCTC CTCGTGGATG TGATTGACCA GGTCATAGAT CTCGTCGCGA 41640
GAGACCAGCC GGCCCCAGGC GTCGATCCCC ACGTGGATCT GTCTCCGGTG GATGTGTCGG 41700
GACAGGATCA TCGGCGAATG CGTGTGCCCG TGGATCAGGA TCTTGCCATC GTCACGGAGC 41760
CTCCACTGGG TGTGTCGGTC CTCGCTGGTG TGGTCCCCGA CGTATGGGAA GTGGCTCAGC 41820
AGAACATCTG TGTGCCCGCC AGCGTCCCCG TACAGCGGCA CCCGGATACG AGCTGCCGTC 41880
GACACATGCT CGAACACCAT CCAGTACGCA CCAACCAGCT TGTGAGCATC GCGGTTCATC 41940
GGGTGGGGCC CATCGTGGTT GCCCAGGATC AGCCGTTTGC GGCCTGGCCG ATCCGAGATC 42000
CACCCGAGGG CATGTATCTG CCCCTTGGTG GAGCCAGAGG AGATGTCACC TAGGATCCAG 42060
ACCGTGTCGT CCTTGCCGAC GACCGAGTCC CACGCCTTCG CCAGGGTGGC GTCGTGCTCT 42120
TCGACATCAT CCGCCAGGTT GCGGATCTCC ATCAGCCGCT TGTGTCCGAT GTGTAGATCG 42180
GACGTGAACC AGGTGTTGCT CATGGCTTCC TTTCAGAACG GCGGGCCGTA CAGCTCGATC 42240
ACCAGCGCGT GCAGCTCCTC TGCCGCGTCG TCACGCTCGA ATCCGCAGCA GGAATCGTGC 42300
CGGTCGAGGA TTGCGACGAT CTGGTCGTAG AGGCTGGGCC TCACTTCACC TTCTTCGGAT 42360
CGATCAAGGC GTCGTGAATC GGCCGACCGG CGCGAGCCGC GTGCGTCTCG GCGTCCAAGG 42420
CTCGCTGCAT CTGGTTCATC AGCCGGGTGC CGCGCAGCTT GAGGATCTTC ATGGTCGCCC 42480
GACCCTTGTA TCCAGCGCGG TGCATCCGTA GGACGCAGGC TGTCTCGTGC GGGGCTATAG 42540
GTGACCTCAG CGACGGGTGG TTTGGATCCC AGTTCGTCAT GTCTTCCTCT CGGTGGCTGT 42600
CAAGTTGGTC ACAGACCGAA CTCTTCCTGG TACTGCGGGA TGAAGTGGCC GGCCGTTCAT 42660
GTTCGGCTCG ATACCTCTCG CGTCACGAAC TCCTGCCCGT TCCATCTCCG ACCGTCCTCG 42720
AACTCGATCA CGATCTCTCG TCCGGGATGA CGCACGGCCT CCGCTTGGGC AAACCTGCGT 42780
GCAGCCTCTG GGGTCGGGAA CGGAAACTTC TGCGAGGCGT ACAGCTCCTG GTGCCACTTC 42840
GGCTTGTCAG GAATCGGCCC CATTTCCACG TACGTGTAAC CCGCGTCGGG GTCGAGTTCG 42900
AGCGTTTTCT TGTATTCCTT CGTGCCTGCC TTAGAGGGAA GGTGAGTATC GGTGGCTGTC 42960
AAGGTGACCT CACTTAAAAA CAGGGCAGCT GTAATTCACA TCACAGAAGC CGCATTTGTC 43020
AGGTTCAGGC AGAGGCTCGA AGTCACCAGC CTGGATCCGA GCCTCGACCT CATGGAACCT 43080
CTCGGTGATC CGCTCCCGCG TCCAATCGGT CAGGTCGTAG GGCGCAGTGG GCTTCGCCTT 43140
GATGCCCTTC TTCCCCGCCA TGAAGTAGTC GCCCGTCTTC GGAGCCTCCA CGTCATAGGT 43200
CATCGCGACC GCGAGCGCGT ACACGCCGAG CTGGAAGTCG TCACCCGGCG AGTTGCCGGT 43260
CTTGTAGTCC CGGACTCGAA GCTCACCGTT GACCACGACG ACCGCGTCGA TGAACCCTCG 43320
GACGCGGATG CCGTCCAGCT CGATGTTGAA CGGAAGCTCG ATGGCCGGCT TGGGCTGTTC 43380
ACACTCCTTG CAGTTGGTGT CTTTCCACGC CTCCGTAGAG CAGATCCCTC GCCCAGGGGT 43440
AGTCCAGATC TGCTGGCCCT TGTCCTTCCG CCACGCGATG AACTTCTCTA CCTGCTCCAG 43500
TCCAAGGTGG AACCGGCGCT CGATGTCACG CTCACCGTTG TACGGCCCGG ACCAAAACCA 43560
CCACTCGAAG TTCGGGGTTT CGTCGCACAG TGCTCCGATG TCCTTGGCGT ACTCCTCGCG 43620
GAAGATCTCT TGTGCCCGTT CGAGGCTCAT CTCGCGGCCC TCGGCCAGAG CCTTCTCGTA 43680
GACCTCAGCG ACGGTGTGAA ACGCGGTGCC CTGCGGCAAC CACGCCGCAG GACGAGCCCA 43740
TACCTTGTCG ATGCGAGCCA GCTTGTACGC CTGCGGGCAA CGTGTGTATT GGTTCAACTG 43800
GCTGACGCTT CGCAGCGGCA GCAATGTCTT GGTGTCTGTC ACGCAGCGGC CATCCTTCCC 43860
TTGCCTATCG TCTCGTTCAG CGCCCCGTCG ACAGCGACAC TGAGCAGTTT TGCGACCTCC 43920
GACATGTCAA TCGGATCCTT GGGGAATTGG TCAGCCTGAG TCATCCTGAG CACCATCCAC 43980
TCGGTGCCCT TGTCGCAGTG GATCATGGTC GGATCAAAGC GAGTTCCCCG TGCTACGTAC 44040
TCGACTTTGT TCGCGGAAAG AATCAAATTC GACACAGGCC GATAAAGTCG TGAGGTGTCT 44100
TTTACACGAG GACTGCGGTA GACGAGCAGA ACTGAGACTG GGTCTTCGTC CAGTTGGCCC 44160
TTCCACCACG CCTCACACCT CTGCGCGAAC AGCCACCCTG GATGATCGGC GATGACTTGC 44220
GGTGAGGTGT GGACGAGGTT GTCTGCGAAC AGCTTTGCGA GCCGAGTGAG GGGCACGGGG 44280
TTTCCTTTCG TTGCGCGGCC TGGGTTGGCT CACACAACCG GTCGTGACTT TTAGGGCTCC 44340
GAGAGAAGCT CCTCGATGTC GTCTGGCCAC GACCAGAGGA GTTCACCCTC GGCGGTGAGG 44400
TTGGTGTGCT CGTTCACCCG GATCAGGAGA TCGTCATCCT CGATGCCTCG GGGGACGTAC 44460
CTGAACCCGC CGCCGGCCAT ACCTTCGTAG GGCTCGATGG ATGGGTCGAA CTCGAGCACT 44520
AAGTCGTCGT CGCGGAGCAT CTTCCACCAC GACAATAGGC GCTTCTTCTT GTCTTCGGAC 44580
ATCGTGCGGA AGCTACCCAC TCGCATGTAC TCGCCGTGAT CCCGGAGCCT CTGAAAAGCC 44640
TTCGACTTAT CGTGAGGTTT CCGCGTGTCC CACGGCCAGT TCTGCTGGAC GATCTGCCTG 44700
GTGGTCAACC GTCCTCCGTA GGTCTTCTTG TGCCACGACA CCGCTTGTCG AGTCACGCCA 44760
TACAGCTCTG CGATTTCGGT CTGATTAAAC CCCTTCCTGC GAAGATCTTC GATCTCGCTG 44820
AGAGTGAGTG GTATTCGGCT AGGGGCCGGA ACCACTGCTT TGTGTTGGAT TTTGCCGCTC 44880
ATGTTTCCCT CCATGAGAAA GGTGCGTGCG TCTCCGCCGA TTACGGAGAC ATGTTGGTGC 44940
CTGTCAAGGA TACCCCTAAT TTAGTTGCGT CTGCGGAACC ATATTCAGTT GTGTTCCCCG 45000
ACGCCGTGGC CGTCTCCCAC TGGGCGTGGG ATCGACTGGC GTTACGCGGT CGTAAATGTA 45060
GCGGCCTGCC CCACTCGGTA GCAAACCTTG TGACAGGTAT CACTTAGGTC GCCTTCTGTT 45120
ACACGTTGAC CTCGGGTTTC ATCGTCACGA CTCTCCTTTC TTAGACAGCC TCAAGATCGT 45180
TACACCGGCT TGCGAAGATG TACCTTCGCC TTGAATCCGG CCCTTGCCAG CTCGAACTCG 45240
ACCACCTGGC GGGCGGTCTC CTTCAGGTCG GACTTCGCCG ACAGCGGCCC GACGAACCCG 45300
TAGCTCTTGA TGTACTCCTC GAGGTCGATG TCGACGTACA GCGTGACAGG GACCACCGAC 45360
AAGTCACACC TCCAATTCGT GGGGCTTGAT CTCGTTGGTC ACGTCGTAGT CGTTCAGCAG 45420
CGACTGGAAG TCGGAGTCTG TCAAGTCGTC CAACTCATCC TGCTCGAACG GCGCGGGCTC 45480
GTCATGCCAC GTCTTCCACT GGTCGTGGTC GGCGCGGAAC CACTTCCGCA GATCCTTGAT 45540
GGCCTCGTCC TCGGTGGCGA AGACGTAGGT CTCGAGCACG TCCTCGTACT CGACGGTCAG 45600
CGACCAGACG GTGATCTTCA CTCCCCGTTC ACCTCCGCTT TGTAGTTCAT CTCGGCGGTC 45660
TCCTCCTAGT TGGGTAGCAG TCGGTTGTAC TCGTCGTGGC TGATCTCGCC AACGATGAAC 45720
TGGCGCATCA GATTTGCGAC CGAAGCCGCG TCCATCCCTT CGGGAATGGG CTTGGCGTGG 45780
CCGAACTGCC AGTCTCGTGA GCGCCAGCGG AACCAGAGTT GGACCTTGTC CAGTGAGGTC 45840
AGGTGCAGGC ACTGAAACGT CATGCCTCCG AACGGGAACT CCATCACACC TCCTGTTTGA 45900
CCTTGACGGT GTGGCCTGTC ATTACTTCGT GGATTCGGAT GCTGGTGCCG AACGTCTTTC 45960
GCGTCTCGGC CTTGAACTCG GTGGAGCACC CCGAGCACTT CGCTTTGAAT CGCACTAGCA 46020
GTACCAACGC TTTCTGCAGA ATCGGGACTT GCCGCCGTCC CGGTTGTCGT TGTCCCGGCG 46080
GGCTTCGCCC TTCGGTGATT CGTCACATGA CGGAAGCTCG CCATGCTTGA TGTGCCATGC 46140
GTCGTCGGCG ACTTTTCCGC CGTGCTCGGC GATGTGCGCT GCGCTCCGGT ACTCACAGAG 46200
CGGGGAAGCC GATGCCTCGG CGATGATCCC AGGCAGGTTG CCTAGAACCA CCGCCAAGCA 46260
CATCAGCAGA ACGACGTGCC ACGCCTTCAT CAGCCCGCCA GCGCGTGGTT CATCGCCGCG 46320
TTGCGGCCGT CGCGCTGACC GTGGGCATAG CCGCTGAGGT CGTACCGGGT CCGAGGCTTG 46380
ACGTTCTTGG TGCGAGGATG CGCCTGGCGC AGAGCCAGCG CAGCTCGTTC CTTGTCGCCT 46440
CGGTAGAGCA CCAACGCTCC CCCGCCGGCC GATTCCACGG CCTTGTTCTC CTCGGCGGTC 46500
AGGCGTTCCT TGACGGCCTG GGCGAAGCCT GCGATCCACG ACCGGCGGTA GCTCTTGAGC 46560
TGGCCAGCGG TGCTCTTCGG CTTGTACTCC CCGGTGTTGT AGTCGTACTT GTACCGAGGC 46620
TCGAAAGCCT GCTCCGGGCG GACATTCTCA ACCAGGCGCA TCATCTGCGG CTGCATGATC 46680
GACCAGAGGA ATTGGAGCCT CTCGATGTGG CGGGGCACGC CGTAGACGTA GATCCGCTGA 46740
CCGCCCGTGA GGCTGGCGTA CACCGTCTTG CAGTGCAGGG CCTGAGCCAT GCCGTGCAGC 46800
AACAACGCTT GTGCGGCAAC GTACTTGCCG GTGACGTAGG TGACCCACTG GATGGCGTCG 46860
GGCAGGTCGG TGGTGTCCAA CCCTTGCTTG CTCGCCTCGA CCTGGGCCAT CTCCAGCCCG 46920
TACTTGGCCA TCAGCTCGAA CGCTTTCGCC TGGAACACAG CCTCTTCCGG CGTACCGGCC 46980
ACGTCTTCGG CCTGGCGCAG CAGCTTGGCG ACCTTGTCCT GCATCTTCTT CGTCTTGCCG 47040
TCGATCATGG TCAGTACTCC TTCTTCCAGT TGTTCCGGTT GCCCTTGCCG GGGCGCTTCA 47100
TCTCTCGCTT GCGGTTACGG TGCGGCTGCG CCGCGTTGGA GAGACGCAAC TCGAGCCGTG 47160
CCTTGAGCTG GTCGCTCATC TTCTTCACCT CTTCTGGTTC AGCGGATCTG GTCGACGTGG 47220
ATGCAGCCGA CGCGGTCTGG CCCGAACTCG GGAGCGAAGC CCAAGACTTC GTCCTCCTCG 47280
CATGGGAACG CTCGCTGGTC GAACGTGATT GGGTCGGCCG AAGCCTCGTA TGGATCGGCC 47340
AAGGCCATCG CTCCGACCGC TGTAGCGAAT GCAACGACGA CGGTGATCAG GTGCTTCTTC 47400
ACTCTTCTTC CCTCCACTTT TGGTCTGCGA GAAGCCTTCT GGCGATCTCG ATAGGTTCGA 47460
TCTCAGGAGT CACTCATCGC CCTCCAAGAT CTTCAGGTTG GCCAGCAGTG CATTGGCCAC 47520
AGCTCCGATG TGGCCACCGC CCTTACCTCC ACGGCGGGAG TACTCGCGGT TCGCGGCCTG 47580
CATGAAGTGG AACCTCGGTG AGCCGTCCTC GTGAACCCAC GAGGCTTTCT CGGCGGGCAG 47640
AGCCCGGTTC ATCTCCACCG ACATCGTGAC GATGATGTGG TCCCTCTGGA GCCGAGCCTC 47700
GGTCTCGGCG TAGTGGGCAG CTTGGATTAC TGCGCCTCGT GTGGTCATGT CTTCTCCTTC 47760
GGTAGATGTC AAGCTGTCGT CACCACTCTT CGACCGGTAT CGGTTTGTCA CAGCCAGCAA 47820
GGATCGCGGC GTTGCTGCGG TGATGCCCGT CCCACAGCGT CTTTCGGTCC CTCGAAACCT 47880
CGAGGGGTTC GAACGGCCAC TCGTTCGATG AGTTGAGGAT GTCCACGACT TCGTGGACCT 47940
TGGCCCAGAA CTTGCCGGTC ACGCCTCCCT GGTAGTTGTA GCGGGGCGTG GTCTGGTAGA 48000
ACTCTTCGAG CACTGGTCCG CTGTCGGCGA CGGTGCAGTC GACACCAGCG CAGGACATGC 48060
AGTCGCTGGC GCGGAGCTGG GCAACTTCAT CGGTGGTCAT GAACGCCGTG GTCACATCGA 48120
GCCTTTCAGG TGTATGTCAA GCGGCGCGGA CGCCGGAATC GGAGAGGTAG ACGCGGTCAG 48180
CTCCCAGGAA CGGAGCCTGT GTGTTGGCGT GGACGAACGT GTCGTTCTCG TAGGGGTTGT 48240
AGGCGATCTT CGATCCCACG AAGTCTTGCG GGAGAAGCGA GATCAGCTCG CCTACGATGC 48300
CAGCGTGGAC CACCTTGCGG CGCTCGCGCC GTACCTTGTC GCGGCCGGCC GGCCGAACCA 48360
CACCCTTGGC GTGGGCCAGC AGGACGTGGC CGCTGCGGTG GATGACTCGA CCCTTGAAGT 48420
CTCCCTCCAA GGCTTGCACC GAGTACCACG GCTTGCCCTC GCGGTGCGTG CGGTGCAGGT 48480
TCTTGTAGAC GAAGACTCGG ATCGGCTTGG GAGTCATGAG ACCTCCAGTG TGCGAACGGC 48540
CTTGTAGGCA CTGATGAGTG ACGCCCCCGA CAGCTCGTTA CCGTGCAGGT GATACCTGTA 48600
TTTCAGATAC ACGGCTTGGT CGACCGGCTT GTACTCGACC GAAGTGACCT CGACAACCAT 48660
CCCGTCGATG ATCGCGAAGT CTCCAGCGCG GAGATGGGTG GGGAATTTGA TCTCGGTGTT 48720
GACTACGGTC ACAGCTTCGA AACCTCCCAG GTACCAACGA ACTTGCCGTT GCGCTTGATG 48780
TATCCGCTCT CACCGGGCTC GTACCAATCG ACCTCGAACC CGTAGCGGGC GGCGCAAGCC 48840
TCGAGGTGGT CGAGCAGGAC GCGGCGACCG GACGCGGTAG CTTCTCCGGT CAGCCCGCTG 48900
TCGTTCTTGC GGACGATGAG CTTGAACACT TGGTGCCTAC CCTTCTGCGA TGTCTCGGGA 48960
GATCTCGGCG AAGACTTTCT TTGCCCACGC CACGCCGTCC CAGGTGATGT CGAACAGTGC 49020
CTCGTAGAAC TGGTCTCGCA AGGCTTCGTT GCCGTCGGCC AGCGTTGTGA CGAGCCGGTC 49080
GATGCGGTCC TCGTGGAACT TGTAGACCGA GTGGTTGTAC GGCTCAGCCA TATTGGCGTT 49140
GGCTCGTTTC ACGTTCTCAA CCACGATGGC TTCGAATAGG TGGTTAACCA GCTCCTCGGT 49200
CATGTTCTAT CTCTCCTCAG TAGTCGCTGT GCTGGGTCTC GAAGCCTTCG AGGTCACCGA 49260
CCTCGTCGTC GTACGCGCTC GGGTTGCCGC GCCAGTCGTC GCGGAGCCTT TGACCGCTGG 49320
CGTTGTAGCA GGCACCACAG TTCGGGCAGT CCACATCGCT CTGGCCGTAG TAGCGGCAAA 49380
CCTCGCCGCC GCAGCGTTGG CAGTCCCACG CGCTGTAACC AGGGATCAGG AAACCTTGGT 49440
CGTCGGTCTG ATCAGGGATG CGTCGGAAGT TCTTGGCAGG CATAGCTACT CCTCATAGAA 49500
ACTCGTGGTT GATGGCTCGG TGGGCAGCCT CGCGGAAGGT CAGCCCGTCG TCGTACGCGT 49560
CCCGGTACGT CCAGTCCGCG ATGTCTTGGT AACCAAGACC AAAGGTCTCG GTCATGTAGC 49620
CGTCCAGCGC GGCCATCCAG GTCTCGAAGC TCATGTCTTC CCTCACTTCT TTGTGGTCGA 49680
GAACAGCACG TTCCTGCGGC CGTTGACGCA CAGACCGCAA CGGGCACAAG CCGATCCCTT 49740
GTCGTTGATC AGGTCGATGG CTTTGTTGTT CTCCGGGCAG CGCACCGCCG TCGGAAACTC 49800
GGCCTTGCCT TTGGCGAACG TGGTGTCGAC GTAGGCGATG TTGATGCCCT TGTCTTCCAA 49860
GAAGCGCGCC ACGTCGATGT TGTCCGGGTC TGCGCTGAAG TACAGCGCCA GGTTGTCGAG 49920
CCTCTGCGAG TGCAGGTAGA CAGCCGCCGT CTGAACCCTT GTGTAGGCCC AGAACTGGAC 49980
ATCCGGGTTG TCGCGGATGA CTCGACCCCA AGCGGCCACA TAGGTGGGGC TGAAGAAGTC 50040
TCCATCCCAG TGGATGCGGA ACAGCTTCGG AGCCTTGCGA CGGTCGCAAT CCTTGACGAA 50100
CTCGGCGACC ATCTCGGACA GCAGCGTCAC GGTGTCTGTC AAGTCAGCGT CACGCAACAG 50160
TTCCCAGTTG TGCAGCAGGA CCGAGCTGAC AGCCTTGCGA ACTTTCTCCA GCTTGCCGGC 50220
GTAGCACACC TTGGCACAGA AGGCCGTCGC GTCCGGGCAG GAGAAGCCTT GACCGGAGGG 50280
CAGGCCGATG CTGTTGGCGA TACCTACGGT GGCGTTGCCG CCCTTGGTGA CGTGGACGTA 50340
GTTGGTGACC TTGCGGTCGT TCGAACGCTT CAGCTTGGCC ATACCTAGCC TTCCTTCGGT 50400
GGCTGTCAAG TTGTTGGATA CAAAGCGCCC CGAGAGGGAG TCGAACCCTC ACACCGCGAA 50460
CCGTCGCGGG GCCACCGTGC CTAGTCGATA GAGGTCACTC GACTCTCGTG GACGTAGACC 50520
ACGGTGTTGC CTACGTTCAC CGCGTAGTAC AGGCCATCGG CACCTCGTAG CTTGTGCCGA 50580
ACCGTGCCCG ACGTGGCCGT CATGTCTTCG CCCCAGTCGG CGTTAGGTGC CCAGGTGACT 50640
CGCATGGTGA TCCCTTCAGT AGTCGGTGGC TGTCAAGTCA GCGGATACGG ACGTACCCGT 50700
TGCCTCGAGC GACGTAGATC TTGCCGTCGA TGTAAACGCG CTGCTGCTGG TTCATAATCC 50760
TATTCCTTTC GGTGGCTGTC AAGTCTCAGG CCCAGCGACG AGTCGTCGGC CGGGGGCGGC 50820
GCACCTTGGG CGCGTTGGCT CGCGGTGCCT TACGGATGGC GGTGCCTACC GTGATCTCTT 50880
CCAACTGGCG TTCAGCCAGG CCGACAGGCC GGGCGTCACC GGGCAGTTCG ATCTTGTAAT 50940
CGAAGTCAGT CCACCCCTTC AGACCCTTCT CCAGCTCGCG ATCCAACAGA CGCGGAGCCG 51000
ACAGCTCAGG CGCAACAAAC GGTGTCTTGA CGCTCTCGCG GGCAGTAACC CGAACCTCAC 51060
GGTGCTCAGC GAAGACTGGC ATAGTTCACC CCTTTGGTGG ATGTCAAGCC TGAGCACCAA 51120
AGCTCAGGCG TAGTGGGTAG TCGGGAATCG AACCCGATAG CTTCATAGCC ACGTTCTACG 51180
GCTCAGCCAT AGCTCAGCGA TCATTCCATC GCGCCAAGAG CTACCCTCCC GAATGCCGAA 51240
CCAAAGCTCA GCATTCGTAA GTGTGTATTC TCCCCGTGGC TCAGACAGTA TCTATCAGAA 51300
CCTAACCACA GGTCTACATT TAGTTATCCG CAGTGCTCGC ACTTTAACGG CATCGAGCTT 51360
CCGCCGACCC TCAGTCCTCT GGCAGCGAAC TAAAGGTTTG AGTCGGGCTG CGGCCCTTCT 51420
CGGTCTTGCG TGATTCTCAC TCTACCGGAT GTTTCGGTGG CTGTCAAGCG GGCCGTTTTG 51480
GTGTTGCAAC GATGCCCTCG TTTAGCGCCG CTGGCGTAAT GCGCTACCCG CCTGATCTCA 51540
CCGGTCCAAG TTGGTGATGC TTGCAGCTTA CCCGATAACC GGGTGGCTGT CAAACCGGAG 51600
AATCTTGCCG CCGGATTTTC ACCGGCACCG GCACGATCCT CTCGGATCCG CCTACCGCCT 51660
TGCTGCTGCG GTGACACAAG AATGCACTAC TGGCCGGGTG GCTGTCAAGC CCTAATCGCA 51720
AATTGGTGCC CTAGCTGCAG ATATGGCGCG TTCTCGGTGG CTGTAAAGGG CACTACGTGC 51780
CGCTATCCGC TGGTCACGCT GGACAGTCCC GGCAGCCCGT GCCGCGCATA GGCTGCTCAC 51840
TACGTGCCCG GTATCGGCGT TGTCGTGCCG CTGTCGTGGT CGTCGCCCCG TCGCTGTCGC 51900
TGGTCTCGGT GGCATCGCTT GACAGTCGCC CCGCTATCCC CCGTTGCCGC TGGTCAGACG 51960
CTAATCCGCT TATTTCGCAT AGGCTGCTCA CTATCGCATC GGTATGCGTA TGCGCTGGTC 52020
ACATATGCGT GTGGTGGTGG TGTGGTGTGC GTGTGTTTGC GCTGGTCAGC CGTGTGCGTA 52080
CCGTATCCGC ACACTGTGCT TGTGCGTTTG CTGTGTGTCG AGGCCGGCTC TCGCATCGTC 52140
GCATGTCAGC GCGGGTATGG GCGTGTATCG CACGCTTTGC TAGCCGCGTG CCGCGGCGCT 52200
CTCGCATCGC ATCGAGTGTT TGCTGTGTCT CTCATCGTCG CAGGTCAGAA GGGGTAGGGG 52260
GGTTCCCCCT AGGGGTCGGT CCTTGACCGG TCGGTTA 52297
Figure imgf000140_0001
Figure imgf000141_0001
Figure imgf000142_0001

Claims

WHAT IS CLAIMED IS:
1. The reporter mycobacteriophage designated phAE40 deposited on April 29, 1993 with the American Type Culture Collection and catalogued as ATCC No. 75457.
2. The reporter mycobacteriophage designated phGS1 deposited on April 27, 1993 with the American Type Culture Collection and catalogued as ATCC No. 75454.
3. The reporter mycobacteriophage designated phGS5 deposited on April 27, 1993 with the American Type Culture Collection and catalogued as ATCC No. 75453.
4. A method of diagnosing a mycobacterial disease which comprises incubating a sample which may contain mycobacteria with mycobacterial species-specific mycobacteriophages which contain reporter genes and transcriptional promoters in their genomes, wherein the reporter genes produce a gene product upon incubation with the mycobacteria for which the mycobacteriophage is specific, and wherein the gene product is detectable.
5. The method according to Claim 4 wherein the mycobacterial disease is tuberculosis.
6. The method according to Claim 4 wherein the mycobacteria is M. tuberculosis.
7. The method according to Claim 4 wherein the mycobacterial species-specific mycobacteriophage is L5, TM4 or DS6A.
8. The method according to Claim 7 wherein the mycobacterial species-specific mycobacteriophage is temperate.
9. The method according to Claim 4 wherein the reporter genes are luciferase genes or the β-galactosidase gene.
10. The method according to Claim 9 wherein the luciferase genes are selected from the group consisting of Firefly lux gene. Vibrio fischeri lux genes, Xenorhabdus luminescens lux genes and lacZ genes.
11. The method according to Claim 4 wherein the transcriptional promoter is hsp60 or the L5 gene 71 promoter.
12. The method according to Claim 4 wherein the gene product is photons.
13. The method according to Claim 4 wherein the gene product is made detectable by contacting said gene product with a substrate.
14. The method according to Claim 13 wherein the substrate is luciferin or decanal.
15. The method according to Claim 4 wherein the sample is blood or sputum.
16. A method of assessing drug resistance of a mycobacterial strain which comprises:
(a) incubating a sample which contains a mycobacterial strain with mycobacterial species-specific mycobacteriophages which contain in their genomes transcriptional promoters and reporter genes which produce gene products; (b) adding an anti-mycobacterial drug to the incubation; and
(c) detecting whether the gene product is present in the sample, such presence indicating drug resistance of the mycobacterial strain.
17. The method according to Claim 16 wherein the mycobacterial strain is a strain of M. tuberculosis.
18. The method according to Claim 16 wherein the mycobacterial species-specific mycobacteriophage is L5, or TM4 or DS6A.
19. The method according to Claim 18 wherein the mycobacterial species-specific mycobacteriophage is temperate.
20. The method according to Claim 16 wherein the reporter genes are luciferase genes or the β-galactosidase.
21. The method according to Claim 20 wherein the luciferase genes are selected from the group consisting of Firefly lux, gene. Vibrio fischeri lux genes, Xenorhabdus luminescens lux genes and lacZ genes.
22. The method according to Claim 16 wherein the gene product is photons.
23. The method according to Claim 16 wherein the transcriptional promoter is hsp60 or the L5 gene 71 promoter.
24. The method according to Claim 16 wherein the anti-mycobacterial drug is selected from the group consisting of streptomycin, isoniazid, ethambutol, rifampicin, ciprofloxacin, novobiocin and cyanide.
25. The method according to Claim 16 wherein the gene product is made detectable by contacting said gene product with a substrate.
26. The method according to Claim 25 wherein the substrate is luciferin or decanal.
27. The method according to Claim 16 wherein the sample is blood or sputum.
PCT/US1994/004788 1993-04-29 1994-04-29 Mycobacterial species-specific reporter mycobacteriophages WO1994025572A1 (en)

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WO2006075996A2 (en) * 2004-04-07 2006-07-20 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Reporter plasmid phage packaging system for detection of bacteria
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CN114164183A (en) * 2021-12-17 2022-03-11 中国科学院大学 Nocardia south Africa bacteriophage P69 and application thereof

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US5922282A (en) * 1995-06-07 1999-07-13 Ledley; Robert S. Super fast tuberculosis diagnosis and sensitivity testing method
WO2006075996A2 (en) * 2004-04-07 2006-07-20 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Reporter plasmid phage packaging system for detection of bacteria
WO2006075996A3 (en) * 2004-04-07 2006-10-26 Us Gov Health & Human Serv Reporter plasmid phage packaging system for detection of bacteria
EP2142667A1 (en) * 2007-04-05 2010-01-13 Sequella, Inc. Improved methods and compositions for determining the pathogenic status of infectious agents
JP2010531636A (en) * 2007-04-05 2010-09-30 セケラ インコーポレイテッド Improved methods and compositions for determining the pathogenic status of infectious pathogens
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US8501400B2 (en) 2007-04-05 2013-08-06 Sequella, Inc. Methods and compositions for determining the pathogenic status of infectious agents
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US9481903B2 (en) 2013-03-13 2016-11-01 Roche Molecular Systems, Inc. Systems and methods for detection of cells using engineered transduction particles
US10240212B2 (en) 2013-03-13 2019-03-26 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
US9133497B2 (en) 2013-03-13 2015-09-15 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
EP3699592A1 (en) * 2013-03-13 2020-08-26 Geneweave Biosciences Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US9546391B2 (en) 2013-03-13 2017-01-17 GeneWeave Biosciences, Inc. Systems and methods for detection of cells using engineered transduction particles
US9752200B2 (en) 2013-03-13 2017-09-05 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US9771622B2 (en) 2013-03-13 2017-09-26 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US9388453B2 (en) 2013-03-13 2016-07-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US10227663B2 (en) 2013-03-13 2019-03-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US10227662B2 (en) 2013-03-13 2019-03-12 GeneWeave Biosciences, Inc. Non-replicative transduction particles and transduction particle-based reporter systems
US10125386B2 (en) 2013-10-29 2018-11-13 GeneWeave Biosciences, Inc. Reagent cartridge and methods for detection of cells
US9540675B2 (en) 2013-10-29 2017-01-10 GeneWeave Biosciences, Inc. Reagent cartridge and methods for detection of cells
US10351893B2 (en) 2015-10-05 2019-07-16 GeneWeave Biosciences, Inc. Reagent cartridge for detection of cells
WO2021220302A1 (en) * 2020-04-29 2021-11-04 Indian Council Of Medical Research A paper disc based method for determining the drug susceptibility of mycobacterium tuberculosis
CN114164183A (en) * 2021-12-17 2022-03-11 中国科学院大学 Nocardia south Africa bacteriophage P69 and application thereof

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