HETERODUPLEX TRACKING ASSAY (HTA) FOR GENOTYPING HCV

Field ofthe Invention

This invention relates to genotyping hepatitis C viruses (HCV). In particular, this invention relates to specific primers preferably from the core and envelope region of HCV and a method to determine genotypes of HCV with a heteroduplex mobility or tracking assay which, in turn, utilizes specific primers.

Background ofthe Invention Viral hepatitis is known to be caused by five different viruses known as hepatitis

A, B, C, D, and E. HAV is an RNA virus and does not lead to long-term clinical symptoms. HBV is a DNA virus. HDV is a dependent virus that is unable to infect cells in the absence of HBV. HEV is a water-borne virus. HCV was first identified and characterized as a cause of non-A, non-B hepatitis NANBH. (Houghton et al., EPO Pub. Nos. 388,232 and 318,216). This led to the disclosure of a number of general and specific polypeptides useful as immunological reagents in identifying HCV. See, e.g., Choo et al. (1989) Science. 244:359-262; Kuo et al., (1989) Science 244:362-364 and Houghton et al. (19911 Hepatoloev 14:381-388.

HCV is a single stranded RNA virus, distantly related to the pestivirus and flavivirus and it is the causative agent of the vast majority of transfusion-associated hepatitis and of most cases of community-acquired non-A, non-B hepatitis worldwide. The HCV genome consists of 5' and 3' noncoding (NC) regions that flank a single long open reading frame (ORF). This ORF encodes for three structural proteins at the amino- terminal end and for six nonstructural (NS) proteins at the carboxyl-terminal end. The structural proteins are represented from the nucleocapsid (core; C) proteins and two glycoproteins, envelope 1 (El) and envelope 2 (E2). The nonstructural proteins are named NS2, NS3, NS4a, NS4b, NS5a, NS5b. The 5'NCR is the most highly conserved part ofthe HCV genome, whereas the sequence ofthe two envelope proteins (El and E2) is highly variable among different HCV isolates. The highest degree of variation has been observed in a region within E2, now commonly termed hypervariable region 1

(HVR1) or E2HV. A second variable region called the HVR2 also exists in a subset of isolates. Typically, the genetic heterogeneity of HCV has been classified under two headings quasispecies and genotypes. As used herein the term "quasispecies" refers to the genetic heterogeneity ofthe HCV population within an infected individual. As used herein the terms "genotype" and "subtype" refer to the genome heterogeneity observed among different HCV isolates. The analysis of nucleic acid sequence variation ofthe HCV genome, a positive stranded of approximately 9.4 kb RNA molecule, suggest that genetic variability is associated with important virological and clinical implications.

The prototype isolate of HCV was characterized in EP Publications Nos. 318,216 and 388,232. As used herein, the term "HCV" includes newly isolated N ANBH viral species. The term "HCV-1" refers to the virus described in the above-mentioned publications.

Since the initial identification of HCV, at least 6 different major viral types have been identified (full length genomes reported) and designated Type 1, 2, 3, 4, 5 and 6. Within these types are numerous subtypes. The type of virus with which a patient is infected may affect the clinical prognosis and also response to various treatments. See, Yoshioke et al., (1992) Hepatoloev 16:293-299. Considering that the most serious clinical outcome of HCV infection is heptocellular carcinoma, it would be useful to be able to determine with which type or types of HCV a patient is infected. It is thus of particular importance to develop an, accurate, reliable assay for HCV genotyping and subtypmg, that, without requinng the sequencing, could also give the genetic divergence mtra-subtype. Several classification have been proposed for HCV genotyping based on analysis of different regions, because the ideal nucleotide sequence-based system, using the complete viral genome is not practical

Summary of the Invention

The present invention includes pnmers and methods for the characterization of HCV genotyping and of variation mtra-subtype based on the heteroduplex tracking assay (HTA). The preferred probes/pπmers were single stranded derived from the carboxyl terminus of core and part of the El region of HCV.

The HTA is a hybridization based method of determining the genetic relationship between two or more viral genomes. The basis ofthe method is that related DNA products coamplified from divergent templates reanneal randomly to form heteroduplexes that migrate with reduced mobility in systems designed to separatemolecultes or the basis of size such as neutral polyacrylamide gels, HTA was originally used to genotype HIV-1 and to follow the in vivo evolution of HIV-1 in patients and populations. See, e.g., Delwart et al., (1993) Science 262:1757-1261 and Delwart et al., (1994) J. Virol. 68:6772-6883.

One aspect ofthe invention is a method for genotyping HCV comprising the steps of denaturing and reannealing partially complementary DNA or RNA strands and detecting sequence variation by noting electrophoretic mobility ofthe DNA heteroduplexes on a system designed to separate moleculte on the basis of size such as by following electrphoresis through a polyacrylamide or MDE gel.

Another aspect ofthe invention relates to the probes used in the genotyping which were selected from the core and El region of the HCV genome.

Another aspect ofthe invention relates to a method of predicting the response to drug therapy of a patient infected with a strain of HCV by determining the sensitivity of different known genotypes to drug therapy, determining the genotype ofthe HCV strain infecting the patient and comparing the genotype with its drug therapy sensitivity to predict the patient's response to the drug therapy.

Another aspect ofthe invention relates to therapeutic vaccines and predicting which therapeutic vaccine should be utilized by determining the genotype of a patient infected with a strain of HCV and administering a therapeutic vaccine ofthe same genotype. Another aspect ofthe invention relates to prophylatic vaccines and predicting which vaccine should be administered to a certain population sample by determining the prevalent genotypes in a like sample and administering a prophylatic vaccines of a genotype likely to be the prevalent genotype to the population sample.

Another aspect of the invention relates to the ability to discovering new genotypes of HCV using the method ofthe invention.

Brief Description ofthe Figures

Figures IA - IE are autoradiograms showing homoduplexes and heteroduplexes ofthe samples to be typed with the probes of known genotypes (ss probes are of genotypes la, lb, 2a, 2b, 3a in Figs. 1 A- IE respectively, lane on far left of MDE gel). The homoduplex (h) (ss probe to the double stranded RT-PCR product of known genotypetpfrom which it was derived) is shown adjacent to the probe. The heteroduplexes ofthe RT-PCR products from the 15 dialysis patients (nos. 1, 2, 3, 4, 7, 18, 20, 22, 23, 24, 26, 28, 30, 33, 35) hybridized to the ss probe is designated above the appropriate lane in each Figure.

Figures 2A - 2C are dendograms, i.e., phylogenetic trees showing the relatedness of each partial El nucleotide sequence,, formed by comparing partial El sequences obtained by sequencing of putative type 1 (nt 625-930), type 2 (nt 583-915) or type 3 (nt 558-834) isolates from the dialysis patients described hereinto published genotype sequences for type la (HCV-1) (Choo, et al, PNAS (1991) 88:2451-2455, all nucleotide, "nt", designations according to this paper), lb (HCV-J) (Kato et al, PNAS (1990) 87:9524-2528), 2a (HC-J6) (Okamoto et al Virol. (1992) 188:331-341), 2b (HC-J8) (Okamoto et al Virol. (1992) 188:331-341), 2c (Bukh, et al PNAS (1993) 90:8234-8239 ) and 3a (NZL-1) (Sakamoto, et al) J. Gen. Virol. (1994) 75:1761-1768 over the same region ofthe genome.

Figure 2D is a dendogram, phylogenetic tree, formed by comparing either partial 5'UTR sequences of isolates 23, 30 and 33 obtained by direct sequencingwith published type 1, 2 and 3 (nt -274 to -81) genotype sequences for the same region ofthe genome. Figures 3A - 3D show the nucleotide sequences for dendograms depicted in Figures 2A - 2D.

Description ofthe Invention

The practice ofthe present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, polypeptide and nucleic acid synthesis, and immunology, which are within the skill of

the art. Such techniques are explained fully in the literature. See e.g., Sambrook, et al., MOLECULAR CLONrNG; A LABORATORY MANUAL, SECOND EDITION (1989); DNA CLONING, VOLUMES I AND II (D.N Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (MJ. Gait ed, 1984); NUCLEIC ACID HYBRIDIZATION (B.D. Hames & S.J. Higgins eds. 1984); TRANSCRIPTION AND TRANSLATION (B.D. Hames & SJ. Higgins eds. 1984); ANIMAL CELL CULTURE (R.I. Freshney ed. 1986); IMMOBILIZED CELLS AND ENZYMES (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press, Inc.); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J.H. Miller and M.P. Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in Enzymology Vol. 154 and' Vol. 155 (Wu and Grossman, and Wu, eds., respectively), Mayer and Walker, eds. (1987), IMMUNOCHEMICAL METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London), Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE, Second Edition (Springer-Verlag, N.Y.),and HANDBOOK OF

EXPERIMENTAL IMMUNOLOGY, VOLUMES I-IV (D.M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in this specification. All publications, patents, and patent applications cited herein are incorporated by reference.

The term "recombinant polynucleotide" as used herein intends a polynucleotide of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation:

(1) is not associated with all or a portion of a polynucleotide with which it is associated in nature, (2) is linked to a polynucleotide other than that to which it is linked in nature, or(3) does not occur in nature.

The term "polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure ofthe molecule. Thus, this term includes double- and single-stranded DNA and RNA. It also includes known types of modifications, for

example, labels which are known in the art, methylation, "caps", substitution of one or more ofthe naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties, such as, for example proteins (including for e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric nucleic acids, etc.), as well as unmodified forms ofthe polynucleotide.

By "PCR" is meant herein the polymerase chain reaction (PCR) technique, disclosed by Mullis in U.S. Pat. Nos. 4,683,195 (Mullis et al) and 4,683,202, incorporated herein by reference. In the PCR technique, short oligonucleotide primers are prepared which match opposite ends of a desired sequence. The sequence between the primers need not be known. A sample of DNA (or RNA) is extracted and denatured (preferably by heat). Then, oligonucleotide primers are added in molar excess, along with dNTPs and a polymerase (preferably Taq polymerase, which is stable to heat). The DNA is replicated, then again denatured. This results in two "long products," which begin with the respective primers, and the two original strands (per duplex DNA molecule). The reaction mixture is then returned to polymerizing conditions (e.g., by lowering the temperature, inactivating a denaturing agent, or adding more polymerase), and a second cycle initiated. The second cycle provides the two original strands, the two long products from cycle 1 , two new long products (replicated from the original strands), and two "short products" replicated from the long products. The short products have the sequence ofthe target sequence (sense or antisense) with a primer at each end. On each additional cycle, an additional two long products are produced, and a number of short products equal to the number of long and short products remaining at the end ofthe previous cycle. Thus, the number of short products grows exponentially with each cycle. This amplification of a specific analyte sequence allows the detection of extremely small quantities of DNA.

λ

The term "3SR" as used herein refers to a method of target nucleic acid amplification also known as the "self-sustained sequence replication" system as described in European Patent Publication No. 373,960 (published June 20, 1990). The term "LCR" as used herein refers to a method of target nucleic acid amplification also known as the "ligase chain reaction" as described by Barany, Proc. Natl. Acad. Sci. (USA) (1991) 88:189-193.

An "open reading frame" (ORF) is a region of a polynucleotide sequence which encodes a polypeptide; this region may represent a portion of a coding sequence or a total coding sequence. A "coding sequence" is a polynucleotide sequence which is translated into a polypeptide, usually via mRNA, when placed under the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, cDNA, and recombinant polynucleotide sequences. As used herein, the term "polypeptide" refers to a polymer of amino acids and does not refer to a specific length of the product; thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not refer to or exclude post expression modifications of the polypeptide, for example, glycosylations, acety lations, phosphorylationsand the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.

A polypeptide or amino acid sequence "derived from" a designated nucleic acid sequence refers to a polypeptide having an amino acid sequence identical to that of a polypeptide encoded in the sequence, or a portion thereof wherein the portion consists of at least 3-5 amino acids, and more preferably at least 8-10 amino acids, and even more preferably at least 11-15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence. This terminology also includes a polypeptide expressed from a designated nucleic acid sequence.

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The protein may be used for producing antibodies, either monoclonal or polyclonal, specific to the protein. The methods for producing these antibodies are known in the art.

"Recombinant host cells", "host cells," "cells," "cell cultures," and other such terms denote, for example, rmcroorganisms, insect cells, and mammalian cells, that can be, or have been, used as recipients for recombinant vector or other transfer DNA, and include the progeny ofthe original cell which has been transformed. It is understood that the progeny of a single parental cell may not necessarily be completely identical in moφhology or in genomic or total DNA complement as the original parent, due to natural, accidental, or deliberate mutation. Examples for mammalian host cells include Chinese hamster ovary (CHO) and monkey kidney (COS) cells.

By "cDNA" is meant a complimentary mRNA sequence thafhybridizesto a complimentary strand of mRNA.

By "purified" and "isolated" is meant, when referring to a polypeptide or nucleotide sequence, that the indicated molecule is present in the substantial absence of other biological macromolecules of the same type. The term "purified" as used herein preferably means at least 75% by weight, more preferably at least 85% by weight, more preferably still at least 95% by weight, and most preferably at least 98% by weight, of biological macromolecules of the same type present (but water, buffers, and other small molecules, especially molecules having a molecular weight of less than 1000, can be present). By "pharmaceutical acceptable carrier," is meant any pharmaceutical carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules such as proteins, polysaccharides, poly lactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers; and inactive virus particles. Such carriers are well known to those of ordinary skill in the art.

The therapeutic compositions typically will contain pharmaceutically acceptable vehicles, such as water, saline, glycerol, ethanol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.

Typically, the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared. The preparation also may be emulsified or encapsulated in liposomes for enhanced adjuvant effect Evidence indicates that different HCV geneotypes may have different pathogenicities as well as distinct geographical distributions and may elicit partly different serological profiles in infected patients. See Cammarota, et al. J. Clin. Microb. (1995) 33:2781-2784. The invention includes methods for detecting HCV and identifying infection by different types of HCV. The invention includes genotyping HCV, the potential to discover a new genotype of HCV, and assessing viral populations for ability to predict response to drug therapy. The invention also includes probes for use in the genotyping of HCV.

The methods for genotyping HCV include but are not limited to a heteroduplex tracking or mobility assay utilizing probes/primers from the core/El region ofthe HCV genome. The documented antigenic differences between HCV genotypes would have usefulness not only in blood donor screening and in predicting response to IFN treatment, but also for the designated composition of candidate vaccines for HCV in different countries, choice of therapeutic vaccines, as well as in the identificaiton of new genotypes. Other methods have been proposed to identify the main genotypes infecting populations, based on analysis of different regions of the genome, such as RFLP. See Davidson et al., J. Gen Virol. (1995) 76: 1 197-1204 for discussion of genotyping HCV using RFCP of sequences camplified form the 5' non-coding region (NCR).

The known nucleic acid based methods of genotyping require a sub-type specific RT-PCR(reverse transcriptase-PCR) primers (see Okamoto (1992) J. Gen Virol 73:673- 679) U.S. Patent 5,427.909; (2) specific probes (G. Marteen, et al.. Line probe assay); (3) restriction site polymoφhism (a function ofthe nucleotide sequence (nt)) or (4) direct sequences to determine genotype. The analysis of the 5' NC sequence with RFLP is easy to perform, but does not accurately predict all HCV genotypes, and. some subtypes may be misclassified. For example, the change in sequence between la and lb recognized by the restriction enzyme is not absolute and sequences other than la and lb, and 2a and 2b

are misclassified. For example, type lc would appear as type la, type 2c as either type

2a or 2b. See Cammarota et al.. J. Clin. Microb.. f !995 ι 33:2781-2784. For this reason,

RFLP is not able to detect "escape" species, new divergent species, or epidemiological trends. It is likely that a typing method like RFLP will have to be continuously modified to accommodate the rapidly increasing information collected on HCV sequence heterogeneity.

As above-mentioned, when using the nucleic acid based methods of genotyping, one obtains a result of either a type or subtype or a negative that is "untypeable" result. See, e.g. Cammarota, et al., J. Clin. Microb. (1995) 33:2781-2784, isolates that remained untyped by genotype-specific PCR were classified subtype 2c on the basis of sequence analysis of PCR amplications obtained from the core and NS5 genes. This problem is avoided by using the presently claimed invention to determine HCV genotypes by choosing RT-PCR primers in the C-terminus or core/mid 2/3 of El . In addition, the subtype ofthe isolate can be accurately determined using the present invention of HCV genotyping and isolates can be detected, even those less than in approximately 30% divergent, enabling the characterization of new sub-types without sequencing.

Heteroduplex Tracking or Mobility Assay

The method of determining the genotype of HCV in the present invention utilizes minor variants in complex quasispecies. One such technique is the heteroduplex tracking assay (HTA). HTA, well known in the art for use with HIV, (see e.g., Delwart,et al., J. Virol. (1994) 68:6672-6683; Delwart, et al., Science (1993) 262:1257-1261 ; Delwart, et al., PCR Methods and Applications 4:S202-S216 (19950 Cold Springs Harbor; and Delwart, et al., Heteroduplex Mobility Analysis HIV-1 env Subtyping Kit Protocol Version 3, each of which is incoφorated herein by reference in its entirety), grew out of the observation that when sequences were amplified by nested PCR from peripheral blood mononuclear celis of infected individuals, related DNA products coamplified from divergent templates could randomly reanneal to form heteroduplexes that migrate with reduced mobility in neutral polyacrylamide gels. Using these techniques, one can establish genetic relationships between multiple viral DNA template molecules.

l l HTA in particular utilizes a first PCR product as a labeled probe, it may be radioactive, which is mixed with an excess (driver) of an unlabeled PCR product from a different source, i.e., the source for which typing is desired. The probe sequences are then driven completely into heteroduplexes with the driver, and are separated on the basis of size. An autoradiogram for example ofthe resulting polyacrylamide gel reveals only these heteroduplexes and provides a visual display ofthe relationship between the two virus populations under study. The fact that heteroduplexes migrate with distinct mobilities indicates that the strand-specific composition of mismatched and unpaired nucleotides affects their mobility. An exponential equation described in Delwart et al., is then used to describe a curve fitting the experimental data from pairwise analysis of genes'of known sequence. In the present invention, the equation is used to estimate the genetic distance between the known genotypes ofthe probes and the unknown genotypes ofthe patient samples.

Primers for Use in the HTA

It was determined that the El or core region could be the best region in to study the HCV heterogeneity, thus the El region became the choice for primers in the present invention. The use of the partial El sequence, the most heterogeneous region ofthe genome for the present invention, as well as a longer fragment, i.e. 400nt, althought it could have been as long as 1 OOOnt, enabled the design of probes which do not cross hybridize among sub-types/types and thus allow accurate geneotyping. By flanking the heterogeneous region, conserved nt sequences for sense and antisense primers were identified. Preferably, a combination of universal sense and type specific antisense primers for the first PCR round and a universal antisense and type specific sense primers for the second round were utilized. The PCR need not be two rounds and the primers are not limited to the above-described combination. The preferred combination, however, enabled the preparation of single stranded probes and minimized the number of PCR primer combinations.

Preferred probes are sequences in the core and El regions of which the sequences for a wide range of genotypes are published and grouped into at least 12

IZ distinct genotypes and subtype: I/la, H/lb, III/2a, IV/2b, 2c, 3a, 4a, 4b, 4c, 4d,5a, 6a.

The nucleotide sequence identities of the El gene among HCV isolates ofthe same genotype ranges from 88.0% to 99.1%, whereas those of HCV isolates of different genotypes ranges from 53.5 to 78.6%. The degree of variation for good discrimination of heteroduplex in neutral polyacrylamide gels is comfortably within the range of 3-20%, so that is likely that divergent templates reanneal to form a heteroduplex if they are ofthe same subtype. For this reason, a single stranded 32p labelled DNA probe was used so that if the formation ofthe heteroduplex is impossible, the ss-DNA probe could likely not reanneal and form a homoduplex band. Without direct sequencing, the present invention can rapidly give not only a certainly identification ofthe subtypes, but also the genetic relations inside the same subtypes. For example, the genotypes analyzed, i.e., (la, lb, 2a, 2b, 3a) showed no overlapping between different subtypes.

Further since isolates approximately 30% divergent can be visualized on the gel- new subtypes can be visualized and the distribution of isolates in a population could be characterized and populations or individual isolates can be followed in population or in individuals in epidemiological studies.

HCV Genotyping Kits

A kit for determining the genotype of HCV is within the scope of this invention. As described for HIV in Delwart et al, Heteroduplex Mobility Analysis HIV-1 env Subtyping Kit Protocol Version 3, such a kit would include the specific primers. Preferred primers are from the core and El region of the HCV genome. If two stages of PCR are desired, the first round primers could include for example a universal sense probe, preferably located in the core/El region ofthe HCV genome. One such universal primer is located from nucleotide 508 to 529 of HCV-1 and is shown in Table 1. Coupled with the universal primer could be a type specific antisense primer also preferably located in the core/El region of the HCV genome. Examples of these primers are from nucleotides 1032 to 1012 for type 1, type 2a, type 2b and type 3a of the HCV genomes and are also shown in Table 1.

If a second round of PCR is desired, the second round primers would likewise be from the core/El region ofthe HCV genome. Preferred second round primers could include a universal antisense primer from nucleotides 978 to 958 ofthe HCV-1 genome, this primer is shown in Table 1. In addition the second round primers could include a type specific sense primer from the core/El region. Preferred second round type specific sense primers are from nucleotides 536 to 557 of HCV genomes type 1, type 2 or type 3, and are shown in Table 1.

The first or second round of primers may be sufficient to amplify the viral RNA without using a second round of PCR if the concentration ofthe virus is sufficiently high, ie., nested PCR is not necessarily required, what is required is PCR products in lOOx excess of probe.

An HCV genotyping kit ofthe present invention would also include subtype references which may change as new subtypes are discovered and evaluated for use in the kit. Use of more than one reference from a given subtype is recommended because comparison to a single reference does not always provide an unambiguous result.

The foregoing discussion and following examples only illustrate the invention, persons of ordinary skill in the art will appreciate that the invention can be implemented in other ways, and the invention is defined solely by reference to the claims.

Example 1

Patient samples 35 hemodialyzed patients undergoing regular hemodialysis were studied: 20 men (57%) and 15 women (43%) with a mean age of 64.8 + 13 years. Serum samples were collected in August 1995, divided into aliquots and stored at -80 degrees Celsius. 26 patients were anti-HCV ELISA positive and 9 anti-HCV ELISA negative. 25 of the 26 ELISA positive were also RIBA III positive, while 1 was indeterminate. The 9 ELISA negative were all RIBA III negative. 15 patients were HCV-RNA 5 ¹ UTR and El PCR positive. By direct sequencing of 15 5' NCR products, 5 patients resulted type 1 ; 3 patients type 2; and 7 patients type 3.

Example 2 cDNA and PCR HCV -RNA was extracted at least two different times using a Stratagene reagent from a Strategene RNA Isolation Kit (Chomezynsky and Sacchi method). RNA extracted from 20ul of plasma that was reverse transcribed in a 25 ul of cDNA mixture (BRL cDNA synthesis kit, 8085SB) using 100 pmol of PCR primers. The cDNA mixture was boiled for 5 minutes, quick-cooled on ice and added to the PCR cDNA reagents with final concentrations according to the Perkin PCR kit (N801-0055) specification. 40 PCR cycles (94 degrees Celsius for 10 seconds, 55 degrees Celsius for 30 seconds and 72 degrees Celsius for 30 seconds were performed. Ten ul ofthe first PCR reaction mixture was added to a second PCR reaction mixture" containing nested PCR primers and was amplified for 40 cycles as indicated above.

The first extraction was used for the nested-PCR reaction with primers specific for the 5' NCR as previously described in Shimizu et al, PNAS (1992) 5477-5481 and this product was directly sequenced and used for the RFLP. RNA from the same extraction was used for HTA using core/El primers. A second RNA extraction was performed for RFLP and/or HTA to confirm the results. The primers used for the HTA are listed in Table 1. The nested pairs of PCR primers used to obtain these El products were different for the types 1 , 2a, 2b, and 3a. The universal sense probe for the first round of amplification corresponds to 5'-3' nt 508-529, amino acids 170-176, of Choo, et al., PNAS, 1991 , while the universal antisense primer for the second round of amplification corresponds to nt 978-958. amino acids 320-326 of Choo, et al., PNAS, 1991.

When the ssDN A DNA probes were prepared for use in the HTA, one of the primers for the nested PCR was biotinylated. See e.g. SEQ ID NO:6 in Table 1.

IS

Table 1

HCV-1 ^■ 3' nt ^■ 5' nt Ammo Acid Primer Type

(SEQ ID NO: 1 ) 508-529 529-508 170-176 Universal sense probe PCR I Purified, C170S

(SEQ ID NO:2) 1032-1012 1012-1032 338-344 Type 1 antisense PCR I Purified, E338A1

(SEQ ID N0:3) 1032-1012 1012-1032 338-344 Type 2a antisense PCR 1 Purified, E338A2a

(SEQ ID N0:4) 1032-1012 1012-1032 338-344 Type 2b antisense PCR I Purified, E338A2b

(SEQ ID N0:5) 1032-1012 1012-1032 338-344 Type 3 a antisense PCR 1 Purified, E338A3a

(SEQ ID N0:6) 978-958 958-978 320-326 'Universal antisense PCR II Purified, E320A

(SEQ ID N0:7) 536-557 958-978 179-186 Type 1 sense PCR II Purified, C179S1

(SEQ ID N0:8) 536-557 958-978 179-186 Type 2 sense PCR II Purified, C179S2

(SEQ ID NO:9) 536-557 958-978 179-186 Type 3 sense PCR II Purified, C179S3

Example 3 HTA

The single stranded probes were prepared by RT-PCR of HCV ELISA and RIBA positive sera of known genotypes with the same PCR primers described, as above, except that one ofthe primers 320A was biotinylated. ssDNA probes were generated with the Dynabeads M-280 Streptavidin following the protocol of Heng Pan and Eric Delwart. The non-biotinyl single strand was eluted was from the magnetic bead/streptavidin column. Probes were generated from 20 ng of ssDNA ofthe different genotypes and end labeled using T4 polynucleotide kinase (Gibco BRL) and 100 microCi of 32P ATP and then column purified. The kinase probe was separated from 32P ATP using a Pharmacia Bio Sepharose column. The 32P-labeled single strand probes were mixed with a 100- fold excess driver, and the PCR products were generated from the patient samples or the control serum/plasma. Hybridization was in 2 x SSC. The mixtures were put on a 94 degree Celsius heat block for 3 minutes. They were then transferred to a 55 degree

I U Celsius heat block for at least 2 hours. The entire reaction volume was loaded on 1mm thick, 6% polyacrylamide MDE gel (Baker) and electrophoresed for 16 h at 500V. The gel was vacuum dried at 80 degrees Celsius on filter paper and exposed to X-ray film.

The genotypes of each ofthe samples were determined based on the Delwart method. Table 2 depicts the genotype results determined by using HTA.

Figures 1 A- IE are antoradiograms showing each ofthe single strand probes in

Table 1, that is the probes specific known for genotypes la, lb, 2a, 2b, 3a in Figures 1 A-

E respectively, see the lane on the far left ofthe MDE gel. The homoduplex(h) (ss probe to the double stranded RT-PCR product form which it was derived) is shown adjacent to the probe. RT-PCR products from the 15 dialysis patients (nos. 1 , 2, 3, 4, 7, 18, 20, 22,

23, 24, 26, 28, 30, 33, 35) hybridized to the probe is designated ais© as the appropriate lane in each Figure.

As can be seen in Figures 1 A- IE, Type 1 ss subtypes probes were specific for each type 1 sub-type and did not cross hybridize with other subtypes lb, 2a, 2b, 3a (2a, 2b not shown). Type 3a ss sub-type specific probe was also specific for subtype 3a and did not cross hybridized with la, 2c, or 2a, 2s isolates (data not shown), ss Sub-type 2 probes do not cross hybridize with each other (data not shown) but did cross-hybridized with subtype 2c isolates; however, the distance between the homoduplex and the 2c isolates indicates a high degree of divergence suggesting that patients 23, 30 and 33 had different sub-types. The virus in sera 23, 30 and 33 was confirmed by sequencing the partial El to be most closely related to sub-type 2c (see figure 2b) but was ambiguous by

51 UTR sequencing, See Figure 2D.

Isolates 23, 30 and 33 hybridized with the 2a probe, while only 30 and 33 hybridized to the 2b probe. The gels also indicate that isolate 30 is more closely related to 2a than to 2b. Therefore, while all three sera are clearly type 2 non-a, non-b subtype, they are not all equally divergent from types 2a and 2b. As seen in Figures IB and ID, patient 4 appears to be co-infected with types lb and a non-a, non-b type subtype.

The lb probe was derived from a patient (JK 16) and appeared to have two viral genomes which is reflected in the homoduplex lane (h) and therefore each lb patient has two bands.

The ss probe 3a was derived from a plasmid clone of one RT-PLR product from a type 3a individual (JK3a), see Fig. IE, lane h, therefore, multiple bands in lane 22 most likely reflect two closely related viruses in this patient.

It appeared that most often patients had unique viral isolates. It is possible that patents 3 and 18 had identical or highly related virus isolates. Similarly, patients 20 and 26 had the same type 3a viral isolate and patients 2 and 4 has the same type lb isolate based on the co-migration ofthe bands on MDE gels.

Figures 2a-2c depict phylogenetic trees, dendrograms, showing the genetic relatedness of each ofthe partial El nucleotide sequences. These denrograms were constructed by pairwise progressive alignment ofthe nucleotide sequences to one another by using the computer software program Gene Works Unweighted Pair Group Methods with Arithmetic mean, as described in Weiner, et al., J. Virol. 67: pg. 4365-4368 (1993). The dendrograms, in Figures 2a-2c were formed by comparing partial El sequences of putative type 1 (nt 625-93), type 2 (nt 583-915) or type 3 (nt 558-834) isolates from the dialysis patients, as determined by sequence analsyis to published genotype sequences for type la (HCV-1) (Choo, et al. PNAS 1991); lb (HCV-J) (Kato et al).; 2a (HC- J6)(Okamoto, et al (1992); 2b (HC-J8)(Okamoto, et al, 1992); 2c (Bukh, et al.PNAS 1993) and 3a (NZL-1) (Sakamoto, et al. 1994) over the same region ofthe genome. Figure 2D is a dendrogram formed as above-described by comparing either partial 5' UTR sequences of isolates 23, 30 and 33 with published type 1, 2 and 3 (nt-274 to -81) genotype sequences for the same region ofthe genome.

The results ofthe RFLP and HTA were compared and are presented in Table 2.

7 6062

I S Table 2

Comparison of Partial El HTA and RFLP Genotyping Results

Patient HTA RFLP

1 lb lb

2 lb lb

3 3a 3a

4 lb lb

7 3a 3a

18 3a 3a

20 3a 3a

22 3a 3a

23 2?* 2a

24 lb lb

26 3a 3a

28 lb lb

30 2?* 2a

33 2?* 2a

35 3a 3a sample is neither 2a nor 2b

The partial El sequences depicted in Figures 3a-3d confirm the HTA sub-type designations given in Table 2 and definitively show that patients 23, 30 and 33 are most closely related to 2c with 33 being the most distantly related to 2c. (18.6% divergent). The RFLP results using ScrFI (see Davidson, et al., J. Gen. Virol. (1995)

76:1197-1204) wrongly designated 23, 30 and 33 as type 2a. This wrong designation is reflected in Figure 2D which shows that based on the 5' UTR nt sequence, the computer did not accurately sub-type HCV 2c due to insufficient nt divergence in this region ofthe genome. The present invention of HTA utilizing primers for the core and envelope region allowed for 3 levels of characterization of HCV genomes. The first was type specificity in the choice of RT-PCR primers. The second was sub-type specificity, based on choosing primers in the core/El region, and from a region greater than 400 nt, which

/ <? resulted in a lack of cross-hybridization between sub-type probes, e.g. 1 and 3, 2a, 2b; and a high degree of heterogenity to maximize differences between genotypes (lack of cross-hybridization). Finally, isolate specificity was determined by the distance from the homoduplex as exemplified in Figures I.E - 1-E. Other genotyping methods do not have the ability to analyze isolate differences

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(l) APPLICANT: CHIROil CORPORATION

(ii) TITLE OF INVENTION: HETERODUPLEX TRACKING ASSAY (HTA) FOR GENOTYPING HCV

(iii) NUMBER OF SEQUENCES: 52

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Chiron Corporation

(B) STREET: 4560 Horton Street - R440

(C) CITY: Emeryville

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 9460B-2916

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: Unassigned

(B) FILING DATE: Even date herewith

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Harbin, Alisa A.

(B) REGISTRATION NUMBER: 33,895

(C) REFERENCE/DOCKET NUMBER: 1226.100

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (510) 923-3274

(B) TELEFAX: (510) 655-3542

(C) TELEX: N/A

(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

CCTGGTTGCT CTTTCTCTAT CT 22

(2) INFORMATION FOR SEQ ID NO:2:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs

£ /

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: GATGGCTTGT GGGATCCGGA G 21

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GATGACCTCG GGGACGCGCA T 21

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4 : GACCAGTTCT GGAACACGAG C 21

(2) INFORMATION FOR SEQ ID NO: 5:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 CAAGGTCTGG GGTAAACGCA G 21

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

Z? (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6: CCAGTTCATC ATCATATCCC A ₂ 1

(2) INFORMATION FOR SEQ ID NO:7:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TGGCCCTGCT CTCTTGCTTG AC 22

(2) INFORMATION FOR SEQ ID NO: 8:

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : TTGCTCTTCT GTCGTGCGTC AC 22

(2) INFORMATION FOR SEQ ID NO: 9:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 9 TTGCTCTGTT CTCTTGCTTA AT 22

(2) INFORMATION FOR SEQ ID NO: 10-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:10:

AACTCAAGCA TTGTGTATGA AGCGGCGGAC ATGATCATGC ACACCCCCGG GTGCGTGCCA 60

TGCGTCCGGG AGGGCAATCT CTCCCGCTGC TGGGTAGCGC TCACTCCCAC GCTCGCGGCC 120

AGAAACAGCA GCGTTCCTAC TACGACAATA CGACGCCATG TCGACTTGCT AGTAGGAGCG 180

GCTGCTTTTT GCTCCGCCAT GTACGTGGGG GACCTCTGCG GATCTATTTT CCTCGTCTCC 240

CAACTGTTCA CCTTCTCGCC CCGCCGGCAT CATACAGTAC AGGACTGCAA TTGCTCGATC 300

TATCCC 306 (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:11:

AACTCAAGCA TCGTGTATGA GGCAGCGGAA GTGATCATGC ACATTCCCGG GTGCGTGCCC 60

TGCGTTCGGG AGAGCAATCT CTCCCGCTGC TGGGTAGCGC TCACCCCCAC ACTCGCGGCC 120

AGGAACAGCA GCGTCCCCAC CACGACAATA CGACGCCACG TCGACTTGCT CGTTGGGGCG 180

GCTGCCTTCT GCTCCGCTAT GTATGTGGGG GATCTCTGCG GATCTGTTTT CCTTGTCTCC 240

CAACTGTTCA CCTTTTCGCC TCGCCGGCAT GAGACAGTAC AGGACTGCAA TTGTTCAATC 300

TATCCC 306 (2) INFORMATION FOR SEQ ID NO:12:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:12

AACTCAAGCA TAGTATATGA GGCAGCGGAC ATAATCATGC ATACCCCCGG GTGCGTGCCC 60

TGTGTTCGGG AGGTCAACTC CTCCCGCTGC TGGGCAGCGC TCACCCCTAC GCTCGCGGCC 120

AGGAACTCCA GCGTGCCCAC TACGACAATA CGACGCCACG TCGACTTGCT CGTTGGGGCG 180

£1

GCTGCTTTCT GCTCCGCTAT GTACGTGGGG GATCTATGCG GATCTGTTCT ACTTGTCTCT 240

CAGCTGTTCA CCTTCTCACC TCGCCGGCAC GAGACAGTGC AGGACTGCAA TTGTTCAATC 300

TATCCC 306 (2) INFORMATION FOR SEQ ID NO:13:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:13:

AACACGAGCA TTGTGTATGA GGCAGCGGAC TTGATCATGC ACGTCCCCGG GTGCGTGCCC 60

TGCGTTCGGG AGGGCAACTC CTCCCGATGC TGGGTAGCGC TCACTCCCAC GATCGCGGCC 120

AGGAACAGCA GTGTCCCCGT TACGACCATA CGACGCCACG TCGATTTGCT CGTTGGGGCG 180

GCTGCTCTTT GCTCCGCCAT GTACGTGGGG GATCTCTGCG GATCTGTCTT CCTCGCTTCC 240

CAGTTGTTCA CTTTCTCGCC TCGCCAGCAT CAGACGGTAC AGGACTGCAA CTGCTCAATC 300

TATCCC 306 (2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION- SEQ ID NO 14

AACTCAAGCA TCGTGTATGA GGCGGCGGAA GTGATCATGC ACATTCCTGG GTGCGTGCCC 60

TGCGTTCGGG AGGGCGACTT CTCCCGCTGC TGGGTAGCGC TCACCCCCAC ACTCGCGGCC 120

AGGAATAACA GCGTCCCCAC TACGACAATA CGACGCCACG TCGACTTGCT CGTTGGGGCG 180

GCTGCCTTCT GCTCCGCTAT GTACGTGGGG GATCTCTGCG GATCTGTTTT CCTTGTCTCC 240

CAACTGTTCA CCTTTTCGCC TCGCCGGCAT GCGACAGTAC AGGACTGCAA TTGTTCAATC 300

TATCCC 306

(2) INFORMATION FOR SEQ ID NO: 15.

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 306 base pairs

&S

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:IS: AACTCGAGTA TTGTGTACGA GGCGGCCGAT GCCATCCTGC ACACTCCGGG GTGCGTCCCT 60

TGCGTTCGTG AGGGCAACGC CTCGAGGTGT TGGGTGGCGA TGACCCCTAC GGTGGCCACC 120

AGGGATGGCA AACTCCCCGC GACGCAGCTT CGACGTCACA TCGATCTGCT TGTCGGGAGC 180

GCCACCCTCT GTTCGGCCCT CTACGTGGGG GACCTATGCG GGTCTGTCTT TCTTGTCGGC 240

CAACTGTTCA CCTTCTCTCC CAGGCGCCAC TGGACGACGC AAGGTTGCAA TTGCTCTATC 300

TATCCC 306 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

AACTCAAGTA TTGTGTATGA GGCAGCGGAC ATGATCATGC ACACCCCCGG GTGCGTGCCC 60

TGCGTCCGGG AGAGTAATTT CTCCCGTTGC TGGGTAGCGC TCACTCCCAC GCTCGCGGCC 120

AGGAACAGCA GCATCCCCAC CACGACAATA CGACGCCACG TCGATTTGCT CGTTGGGGCG 180

GCTGCTCTCT GTTCCGCTAT GTACGTTGGG GATCTCTGCG GATCCGTTTT TCTCGTCTCC 240

CAGCTGTTCA CCTTCTCACC TCGCCGGTAT GAGACGGTAC AAGATTGCAA TTGCTCAATC 300

TATCCC 306 (2) INFORMATION FOR SEQ ID NO:17:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(ll) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: AATGATAGCA TTACCTGGCA ACTCCAGGCT GCTGTCCTCC ACGTCCCCGG GTGCGTCCCG 60

A-fc>

TGCGAGAAAG TGGGGAATAC ATCTCGGTGC TGGATACCGG TCTCACCGAA TGTGGCCGTG 120

CAGCAGCCCG GCGCCCTCAC GCAGGGCTTA CGGACGCACA TTGACATGGT TGTGATGTCC 180

GCCACGCTCT GCTCCGCTCT TTACGTGGGG GACCTCTGCG GTGGGGTGAT GCTTGCAGCC 240

CAGATGTTCA TTGTCTCGCC ACAGCACCAC TGGTTTGTGC AAGACTGCAA TTGCTCCATC 300

TACCCT 306 (2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

AACAACAGCA TCACCTGGCA GCTCACTGAC GCAGTTCTCC ATCTTCCTGG ATGCGTCCCA 60

TGTGAGAATG ATAATGGCAC CTTGCATTGC TGGATACAAG TAACACCCAA CGTGGCTGTG 120

AAACACCGCG GTGCGCTCAC TCGTAGCCTG CGAACACACG TCGACATGAT CGTAATGGCA 180

GCTACGGCCT GCTCGGCCTT GTATGTGGGA GATGTGTGCG GGGCCGTGAT GATTCTATCG 240

CAGGCTTTCA TGGTATCACC ACAACGCCAC AACTTCACCC AAGAGTGCAA CTGTTCCATC 300

TACCAA 306 (2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:

AATAGCAGTA TTGTGTATGA GGCCGATGAT GTCATTCTGC ACACACCCGG CTGTGTACCT 60

TGTGTCCAGG ACGGCAATAC ATCTACGTGC TGGACCCCAG TGACACCTAC AGTGGCAGTC 120

AGGTACGTCG GAGCAACTAC TGCTTCGATA CGCAGTCATG TGGACCTATT AGTAGGCGCG 180

GCCACGATGT GCTCTGCGCT CTACGTGGGT GATATGTGTG GGGCTGTCTT TCTCGTGGGA 240

CAAGCCTTCA CGTTCAGACC TCGACGCCAT CAAACGGTCC AGACCTGTAA CTGCTCGCTG 300

TACCCA 306 (2) INFORMATION FOR SEQ ID NO: 20:

*7

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH. 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:20.

CGCAACTCCA CGGGGCTTTA CCACGTCACC AATGATTGCC CTAACTCGAG TATTGTGTAC 60

GAGACGGCCG ATGCCATCCT GCACACTCCG GGGTGCGTCC CTTGTGTTCG CGAGGGCAAC 120

GCCTCGAGGT GTTGGGTGGC GATGACCCCT ACGGTGGCCA CCAGGGATGG CAAACTCCCC 180

GCGACGCAGC TTCGACGTCA CATCGATCTG CTTGTCGGGA GCGCCACCCT CTGTTCGGCC 240

CTCTACGTGG GGGATCTGTG CGGGTCTGTC TTTCTTGTCG GCCAACTGTT TACCTTCTCT 300

CCCAGGCGCC ACTGGACGAC GCAAGGTTGC AAT 333 (2) INFORMATION FOR SEQ ID NO: 21

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH. 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO- 21.

AAGAACACCA GCGACAGCTA CATGGTGACC AATGACTGCC AAAATGACAG CATCACCTGG 60

CAGCTTGAGG CTGCGGTCCT CCACGTCCCC GGGTGCGTCC CGTGCGAGAG AGTGGGAAAT 120

ACATCTCGGT GCTGGATACC GGTCTCACCA AACGTGGCTG TGCGGCAGCC CGGCGCCCTC 180

ACGCAGGGCT TGCGGACGCA CATCGACATG ATTGTGATGT CCGCCACGCT CTGCTCCGCT 240

CTCTACGTGG GGGACCTCTG TGGCGGGATG ATGCTCGCAG CCCAGATGTT CATCGTTTCG 300

CCGCAGAACC ACTGGTTCGT GCAGGAATGC AAT 333 (2) INFORMATION FOR SEQ ID NO.22

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH. 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO 22

AGGAACATCA GTTCTAGCTA CTACGCCACT AATGACTGCT CGAACAACAG CATCACCTGG 60

CAGCTCACCA ACGCAGTTCT CCACCTTCCC GGATGCGTCC CATGTGAGAA TAATAATGGC 120

ACCTTGCATT GCTGGATACA AGTAACACCT AATGTGGCCG TAAAACATCG CGGCGCACTC 180

ACTCACAACC TGCGGACACA TGTCGACATG ATCGTAATGG CAGCTACGGT CTGTTCGGCC 240

TTGTACGTAG GAGACGTGTG TGGGGCTGTG ATGATTGTGT CTCAGGCCCT TATAATATCA 300

CCAGAACACC ATAACTTCAC CCAAGAGTGC AAC 333 (2 ) INFORMATION FOR SEQ ID NO : 23 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 333 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

( ii ) MOLECULE TYPE : DNA (genomic )

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 23 :

AAGGACACCG GCGACTCCTA CATGCCGACC AACGATTGCT CCAACTCTAG TATCGTTTGG 60

CAGCTTGAAG GAGCAGTGCT TCATACTCCT GGATGCGTCC CTTGTGAGCG TACCGCCAAC 120

GTCTCTCGAT GTTGGGTGCC GGTTGCCCCC AATCTCGCCA TAAGTCAACC TGGCGCTCTC 180

ACTAAGGGCC TGCGAGCACA CATCGATATC ATCGTGATGT CTGCTACGGT CTGTTCTGCC 240

CTTTATGTGG GGGACGTGTG TGGCGCGCTG ATGCTGGCCG CTCAGGTCGT CGTCGTGTCG 300

CCACAACACC ATACGTTTGT CCAGGAATGC AAC 333 ( 2 ) INFORMATION FOR SEQ ID NO : 24 : d ) SEQUENCE CHARACTERISTICS .

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

( n ) MOLECULE TYPE : DNA (genomic )

(Xl ) SEQUENCE DESCRIPTION : SEQ ID NO : 24

AAAAACACCA GCATCTCCTA TATGGCGACC AACGACTGCT CCAATTCCAG CATCGCTTGG 60

CAGTTTGACG GCGCAGTGCT CCATACTCCT GGATGTGTCC CTTGCGAACG GACCGGCAAC 120

GCGTCCCGGT GTTGGGTGCC GGTTGCCCCC AATGTGGCTA TAAGACAACC CGGCGCCCTC 180

ACTAAGGGCA TACGAACGCA CATTGATGTC ATCGTAATGT CTGCTACGCT CTGTTCTGCC 240

CTTTACGTGG GGGACGTGTG TGGTGCGCTG ATGATTGCCG CTCAGGTCGT CATTGTGTCT 300

13

CCGCAGCATC ACCACTTTGT CCAGGACTGC AAT 333

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

AAGAACACCA GCGACTCCTA CATGGCGACT AACGACTGCT CTAACTCCAG CATCGTTTGG 60

CAGCTTGAGG ACGCAGTGCT CCATGTCCCT GGATGTGTCC CTTGTGAGAA GACTGGCAAT 120

ACGTCTCGGT GCTGGGTGCC GGTTACCCCC AATGTGGCTA CAAGTCAACC CGGCGCTCTC 180

ACCAGGGGCT TGCGGACGCA CATCGATGTC ATCGTGATGT CAGCCACGCT CTGCTCCGCT 240

CTCTATGTGG GGGACGTGTG TGGCGCGTTG ACGATAGCCG CTCAGGTTGT CATCGTATCG 300

CCACGGCACC ACCACTTTGT CCAGGACTGC AAT 333 (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

( i i ) MOLECULE TYPE : DNA (genomic )

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 26 :

AAGAACACCA GCACCTCCTA CATGGTGACT AACGATTGCT CCAACTCCAG CATCGTTTGG 60

CAACTTGAAG GCGCAGTGCT CCATGTTCCT GGATGTGTCC CTTGTGAGCA GATCGGCAAC 120

GTGTCTCAGT GTTGGGTGCC GGTTACCCCC AATATGGCCA TAAGTACACC CGGCGCTCTC 180

ACTAAGGGCT TGCGAACGCA CATCGACGGC ATCGTGATGT CCGCTACGCT CTGTTCTGCC 240

CTTTATGTGG GGGACGTGTG TGGCGCGTTG ATGATAGCCG CCCAGGTCGT CATCGTATCG 300

CCACAGCACC ACCACTTTGT CCACGACTGC AAC 333 ( 2 ) INFORMATION FOR SEQ ID NO : 27 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 333 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : l inear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:27:

CGCAACTCCA CGGGGCTTTA CCACGTCACC AATGATTGCC CTAACTCGAG TATTGTGTAC 60

GAGGCGGCCG ATGCCATCCT GCACACTCCG GGGTGCGTCC CTTGCGTTCG TGAGGGCAAC 120

GCCTCGAGGT GTTGGGTGGC GATGACCCCT ACGGTGGCCA CCAGGGATGG CAAACTCCCC 180

GCGACGCAGC TTCGACGTCA CATCGATCTG CTTGTCGGGA GCGCCACCCT CTGTTCGGCC 240

CTCTACGTGG GGGACCTATG CGGGTCTGTC TTTCTTGTCG GCCAACTGTT CACCTTCTCT 300

CCCAGGCGCC ACTGGACGAC GCAAGGTTGC AAT 333 (2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:28.

CGCAACGTGT CCGGGATATA CCATGTCACG AACGACTGCT CCAACTCAAG TATTGTGTAT 60

GAGGCAGCGG ACATGATCAT GCACACCCCC GGGTGCGTGC CCTGCGTCCG GGAGAGTAAT 120

TTCTCCCGTT GCTGGGTAGC GCTCACTCCC ACGCTCGCGG CCAGGAACAG CAGCATCCCC 180

ACCACGACAA TACGACGCCA CGTCGATTTG CTCGTTGGGG CGGCTGCTCT CTGTTCCGCT 240

ATGTACGTTG GGGATCTCTG CGGATCCGTT TTTCTCGTCT CCCAGCTGTT CACCTTCTCA 300

CCTCGCCGGT ATGAGACGGT ACAAGATTGC AAT 333 (2) INFORMATION FOR SEQ ID NO:29:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:

AGGAACATTA GTTCTAGCTA CTACGCCACT AATGATTGCT CAAACAACAG CATCACCTGG 60

CAGCTCACTG ACGCAGTTCT CCATCTTCCT GGATGCGTCC CATGTGAGAA TGATAATGGC 120

ACCTTGCATT GCTGGATACA AGTAACACCC AACGTGGCTG TGAAACACCG CGGTGCGCTC 180

ACTCGTAGCC TGCGAACACA CGTCGACATG ATCGTAATGG CAGCTACGGC CTGCTCGGCC 240

3/

TTGTATGTGG GAGATGTGTG CGGGGCCGTG ATGATTCTAT CGCAGGCTTT CATGGTATCA 300 CCACAACGCC ACAACTTCAC CCAAGAGTGC AAC 333

(2) INFORMATION FOR SEQ ID NO:30:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:

CGGAATACGT CTGGCCTCTA CGTCCTTACC AACGACTGTT CCAATAGCAG TATTGTGTAT 60

GAGGCCGATG ATGTCATTCT GCACACACCC GGCTGTGTAC CTTGTGTCCA GGACGGCAAT 120

ACATCTACGT GCTGGACCCC AGTGACACCT ACAGTGGCAG TCAGGTACGT CGGAGCAACT 180

ACTGCTTCGA TACGCAGTCA TGTGGACCTA TTAGTAGGCG CGGCCACGAT GTGCTCTGCG 240

CTCTACGTGG GTGATATGTG TGGGGCTGTC TTTCTCGTGG GACAAGCCTT CACGTTCAGA 300

CCTCGACGCC ATCAAACGGT CCAGACCTGT AAC 333 (2) INFORMATION FOR SEQ ID NO:31:

(l) SEQUENCE CHARACTERISTICS .

(A) LENGTH: 333 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31.

AAGAACATCA GTACCGGCTA CATGGTGACC AACGACTGCA CCAATGATAG CATTACCTGG 60

CAACTCCAGG CTGCTGTCCT CCACGTCCCC GGGTGCGTCC CGTGCGAGAA AGTGGGGAAT 120

ACATCTCGGT GCTGGATACC GGTCTCACCG AATGTGGCCG TGCAGCAGCC CGGCGCCCTC 1B0

ACGCAGGGCT TACGGACGCA CATTGACATG GTTGTGATGT CCGCCACGCT CTGCTCCGCT 240

CTTTACGTGG GGGACCTCTG CGGTGGGGTG ATGCTTGCAG CCCAGATGTT CATTGTCTCG 300

CCACAGCACC ACTGGTTTGT GCAAGACTGC AAT 333 (2) INFORMATION FOR SEQ ID NO.32.

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TTACCAACGA 60

CTGTTCCAAT AACATTATTG TGTATGAGGC CGATGACGTC ATCCTGCACA CGCCCGGCTG 120

TGTACCTTGT GTTCAGGACG GTAATACATC CAAGTGCTGG ACCCCAGTGA CACCTACAGT 180

GGCAGTCAGG TACGTCGGAG CAACCACCGC TTCAATACGC AGCCACGTGG ACCTATTATT 240

GGGCGCGGCC ACGATGTGCT CTGCGCTCTA CGTGGGT 277 (2) INFORMATION FOR SEQ ID NO: 33:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO-.33:

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TTACCAACGA 60

CTGTTCCAAT AACATCATTG TGTATGAGGC CGATGACGTC ATCCTGCACG CACCCGGCTG 120

TGTACCTTGT GTTCAGGACG GCAATACATC CACGTGCTGG ACCCCAGTGA CACCTACAGT 180

GGCAGTCAGG TACGTCGGAG CAACCACCGC TTCAATACGC AGCCATGTGG ACCTATTAGT 240

GGGCGCGGCC ACGATGTGCT CTGCGCTCTA CGTGGGT 277 (2) INFORMATION FOR SEQ ID NO: 34-

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:34.

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TTACCAACGA 60

CTGTTCCAAT AATATTATTG TGTATGAGGC CGACGACGTC ATCCTGCACG CCCCCGGCTG 120

TGTACCTTGT GTTCAGGACG GCAATACATC CACGTGCTGG ATCCCAGTGA CACCTACAGT 180

GGCAGTCAGG TACGCCGGAG CAACCACCGC TTCAATACGC AGCCATGTGG ACCTGTTAGT 240

35

GGGCGCGGCC ACGATGTGCT CTGCGCTCTA CGTGGGT 277

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TTACCAACGA 60

CTGTTCCAAT AACATTATTG TGTATGAGGC CGATGACGTC ATCCTGCACA CACCCGGCTG 120

TGTACCTTGT GTTCAGGACG GCAATACATC CACGTGCTGG ACCCCAGTGA CACCTACAGT 180

ATCAGTCAGG TACGTCGGAG CAACCACCGC TTCAATACGC AGCCATGTGG ACCTACTATT 240

GGGCGCGGCC ACGATGTGCT CCGCGCTCTA CGTGGGT 277 (2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TTACCAACGA 60

CTGTTCCAAT AACAGTATTG TGTATGAGGC CGATCACGTC ATCCTGCACA CACCCGGCTG 120

TGTACCTTGT GTTCAAGCCA ACAATAAATC CAAATGCTGG ACCCCAGTGA CACCTACAGT 180

ATCAGTCGAG TACGTCGGAG CAACCACCGC TTCAATACGC AGCCATGTGG ACCTACTATT 240

GGGCGCGGCC ACGATGTGCT CTGCGCTCTA CGTGGGT 277 (2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TTACCAACGA 60

CTGTTCTAAT AACATTATTG TGTATGAGGC CGATGACGTC ATCCTGCACA CACCCGGCTG 120

TGTACCTTGT GTTCAGGACG GCAATGCATC CACGTGCTGG ACCCCAGTAA CACCTACAGT 1B0

ATCAGTCAGG TACGTCGGAG CAACCACCGC TTCAGTACGC AGCCATGTGG ACCTACTATT 240

GGGCGCGGCC ACGATGTGCT CTGCGCTCTA TGTGGGT 277 (2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl ) SEQUENCE DESCRIPTION: SEQ ID NO: 38:

TCATCCAACA TCTAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTATGTCC TCACCAACGA 60

CTGTTCCAAC AACATTATTG TGTATGAGGC CGATGACGTC ATTCTGCACA CGCCCGGCTG 120

CGTACCTTGT GTACAGGACG GCAATACATC CACGTGCTGG ACCCCAGTGA CACCTACAGT 180

GGCAGTCAGG TACGTCGGAG CAACTACCGC TTCAATACGC AGCCATGTGG ACCTACTATT 240

GGGCGCGGCC ACGATGTGCT CTGCGCTCTA CGTGGGT 277 (2) INFORMATION FOR SEQ ID NO:39-

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(XI ) SEQUENCE DESCRIPTION: SEQ ID NO: 39:

TCATCCAGCA GCCAGTCTAG AGTGGCGGAA TACGTCTGGC CTCTACGTCC TTACCAACGA 60

CTGTTCCAAT AGCAGTATTG TGTATGAGGC CGATGATGTC ATTCTGCACA CACCCGGCTG 120

TGTACCTTGT GTCCAGGACG GCAATACATC TACGTGCTGG ACCCCAGTGA CACCTACAGT 180

GGCAGTCAGG TACGTCGGAG CAACTACTGC TTCGATACGC AGTCATGTGG ACCTATTAGT 240

AGGCGCGGCC ACGATGTGCT CTGCGCTCTA CGTGGGT 277 (2) INFORMATION FOR SEQ ID NO: 40

(l) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

-\t>

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:

TGTGCCCGCT TCGGCCTACC AAGTGCGCAA CTCCACGGGG CTTTACCACG TCACCAATGA 60

TTGCCCTAAC TCGAGTATTG TGTACGAGGC GGCCGATGCC ATCCTGCACA CTCCGGGGTG 120

CGTCCCTTGC GTTCGTGAGG GCAACGCCTC GAGGTGTTGG GTGGCGATGA CCCCTACGGT 180

GGCCACCAGG GATGGCAAAC TCCCCGCGAC GCAGCTTCGA CGTCACATCG ATCTGCTTGT 240

CGGGAGCGCC ACCCTCTGTT CGGCCCTCTA CGTGGGG 277 (2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:

CATCCCAGCT TCCGCTTACG AGGTGCGCAA CGTGTCCGGG ATATACCATG TCACGAACGA 60

CTGCTCCAAC TCAAGTATTG TGTATGAGGC AGCGGACATG ATCATGCACA CCCCCGGGTG 120

CGTGCCCTGC GTCCGGGAGA GTAATTTCTC CCGTTGCTGG GTAGCGCTCA CTCCCACGCT 180

CGCGGCCAGG AACAGCAGCA TCCCCACCAC GACAATACGA CGCCACGTCG ATTTGCTCGT 240

TGGGGCGGCT GCTCTCTGTT CCGCTATGTA CGTTGGG 277 (2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:

CACCCCGGTC TCCGCTGCCG AAGTGAAGAA CATCAGTACC GGCTACATGG TGACCAACGA 60

CTGCACCAAT GATAGCATTA CCTGGCAACT CCAGGCTGCT GTCCTCCACG TCCCCGGGTG 120

CGTCCCGTGC GAGAAAGTGG GGAATACATC TCGGTGCTGG ATACCGGTCT CACCGAATGT 180

GGCCGTGCAG CAGCCCGGCG CCCTCACGCA GGGCTTACGG ACGCACATTG ACATGGTTGT 240

/

36

GATGTCCGCC ACGCTCTGCT CCGCTCTTTA CGTGGGG 277

(2) INFORMATION FOR SEQ ID NO-43

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH: 277 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION- SEQ ID NO:43:

AGTGCCAGTG TCTGCAGTGG AAGTCAGGAA CATTAGTTCT AGCTACTACG CCACTAATGA 60

TTGCTCAAAC AACAGCATCA CCTGGCAGCT CACTGACGCA GTTCTCCATC TTCCTGGATG 120

CGTCCCATGT GAGAATGATA ATGGCACCTT GCATTGCTGG ATACAAGTAA CACCCAACGT 180

GGCTGTGAAA CACCGCGGTG CGCTCACTCG TAGCCTGCGA ACACACGTCG ACATGATCGT 240

AATGGCAGCT ACGGCCTGCT CGGCCTTGTA TGTGGGA 277 (2) INFORMATION FOR SEQ ID NO:44

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH. 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY- linear

(ii) MOLECULE TYPE- DNA (genomic)

(xi) SEQUENCE DESCRIPTION- SEQ ID NO-44:

GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTA CAGCCTCCAG GCCCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CGGGAAGACT 120

GGGTCCTTTC TTGGATAAAC CCACTCTATG CCCGGTCATT TGGGCGTGCC CCCGCAAGAC 180

TGCTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO 45 d) SEQUENCE CHARACTERISTICS

(A) LENGTH 194 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(li) MOLECULE TYPE DNA (genomic)

(Xl) SEQUENCE DESCRIPTION SEQ ID NO 45 GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTA CAGCCTCCAG GCCCCCCCCT 60

3/

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTAC CGGAAAGACT 120

GGGTCCTTTC TTGGATAAAC CCACTCTATG TCCGGTCATT TGGGCACGCC CCCGCAAGAC 180

TGCTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTG CAGCCTCCAG GACCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CAGGACGACC 120

GGGTCCTTTC TTGGATCAAC CCGCTCAATG CCTGGAGATT TGGGCGTGCC CCCGCGAGAC 180

TGCTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:

GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTG CAGCCTCCAG GACCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CAGGACGACC 120

GGGTCCTTTC TTGGATCAAC CCGCTCAATG CCTGGAGATT TGGGCGTGCC CCCGCAAGAC 180

TGCTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO:48:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION. SEQ ID NO: 48:

it

GCGGAAAGCG CCTAGCCATG GCGTTAGTAC GAGTGTCGTG CAGCCTCCAG GACCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATCGC TGGGGTGACC 120

GGGTCCTTTC TTGGAGCAAC CCGCTCAATA CCCAGAAATT TGGGCGTGCC CCCGCGAGAT 180

CACTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTA CAGCCTCCAG GCCCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CGGGAAGACT 120

GGGTCCTTTC TTGGATAAAC CCACTCTATG CCCGGCCATT TGGGCGTGCC CCCGCAAGAC 180

TGCTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 50: GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTA CAGCCTCCAG GCCCCCCCCT 60 CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CGGGAAGACT 120 GGGTCCTTTC TTGGATAAAC CCACTCTATG CCCGGCCATT TGGGCGTGCC CCCGCAAGAC 180 TGCTAGCCGA GTAG 194

(2) INFORMATION FOR SEQ ID NO: 51:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTA CAGCCTCCAG GCCCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CAGGAAGACT 120

GGGTCCTTTC TTGGATAAAC CCACTCTATG CCTGGCCATT TGGGCGTGCC CCCGCAAGAC 180

TGCTAGCCGA GTAG 194 (2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 194 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

GCAGAAAGCG TCTAGCCATG GCGTTAGTAT GAGTGTCGTA CAGCCTCCAG GTCCCCCCCT 60

CCCGGGAGAG CCATAGTGGT CTGCGGAACC GGTGAGTACA CCGGAATTGC CGGGAAGACT 120

GGGTCCTTTC TTGGATAAAC CCACTCTATG CCCGGCCATT TGGGCGTGCC CCCGCAAGAC 180

TGCTAGCCGA GTAG 194