AU740741B2 - Determination of genetic sex in equine species by analysis of Y-chromosomal DNA sequences - Google Patents

Description

WO 99/02672 PCT/AU98/00533 1 Determination of genetic sex in equine species by analysis of Ychromosomal DNA sequences Field of the Invention The present invention relates to polynucleotide sequences associated with the equine Y chromosome and to methods of identifying such polynucleotide sequences. The present invention also relates to methods of determining the primary genetic) sex of individuals and of samples of cells removed from individuals, and is particularly concerned with equine sex determination.

Background of the Invention Many sectors of the various horse industries prefer a preponderance of animals of one sex. This may be for reasons of reproductive potential, heritability of particular traits, tractability, performance, stature and physique, appearance or other reasons.

The ability to determine the sex of a fetus is advantageous since it allows optimal management and valuation of pregnancies.

Where methods of assisted reproduction are available, by embryo transfer (with or without induced multiple ovulation) or by recovery and return into the donor or by in vitro fertilisation, the ability to determine the sex of an embryo is advantageous since it allows the sex of potential progeny to be predetermined. If combined with artificial twinning by means of embryo bisection it further allows enhanced propagation of the desired sex without reduction in the total number of potential progeny.

It would be particularly advantageous to predetermine the sex of progeny by means of insemination of a receptive mare with sperm populations comprising a preponderance of sperm having one or the other sex chromosome constitution, i.e. either the X chromosome (which sperm yield female progeny) or the Y chromosome (which sperm yield male progeny). Such enriched populations of sperm could also be used to great advantage in in vitro fertilisation. In a further very advantageous application, an individual sperm cell of a known sex chromosome constitution can be injected into the cytoplasm of a mature oocyte in vitro (ICSI: intracytoplasmic sperm injection), effecting fertilisation to yield a zygote of known sex. The ability to determine the sex chromosome constitution of populations of sperm cells and of individual sperm cells is an essential prerequisite in such applications.

.wQ 99/02672 PCT/AU98/00533 2 The primary sex of equine species, as in the overwhelming majority of mammalian species, is determined by the presence or absence of the entire Y chromosome or a functional portion thereof The essential portion is a gene known as SRY that is responsible for initiating testis differentiation (9- 11). Secretions of the resultant testis have a dominant influence on the development of secondary sex characters (12).

The sex or presumptive sex of an individual horse can thus be determined by analysis for DNA sequences that are associated uniquely with the equine Y chromosome.

Previous reports of DNA sequences associated with the equine Y chromosome (11,13,14) concern presumptive sequences that are amplified by polymerase chain reaction (PCR; 15,16) from primer oligonucleotides whose sequences are derived from genes known to be Y-linked in other mammalian species, viz. ZFY(13,14) and SRY(11,13). There are no published DNA sequence data for DNA sequences associated with the equine Y chromosome.

Both ZFY and SRY occur in single copy in all mammalian species examined (with the known exception of Mus species, in which two similar Zfv genes have been described; 17) and so, presumably, in the horse. In the context of determining the genetic sex of viable embryos where only a small number of cells is available from a microscopic biopsy, assay sensitivity is a significant consideration. The advantages for embryo sexing of testing for a DNA sequence that is repeated on the Y chromosome have been detailed previously (18,19).

A report of a repeated DNA sequence that is found on the Y chromosome of horses (20) concerns a short DNA sequence element known as Bkm 21-23) that has been reported in many vertebrate species. It is also abundant elsewhere in the genome, to the extent that representatives on the Y chromosome comprise a small minority of the total.

Such a sequence, of itself, has no utility in the diagnosis of genetic sex in microscopic biopsies.

Summary of the Invention The present inventors have now identified specific DNA sequences that are repeated in the Y chromosome of the horse. The nucleic acid isolates correspond to all or part of a DNA sequence found on the Y chromosome of Equus caballus. The present invention therefore provides a number of polvnucleotide isolates capable of specifically hybridizing to samples of WO 99/02672 PCT/AU98/00533 3 nucleic acid derived from horses which contain Y chromosomal DNA sequences.

A procedure similar in essence to that used in the first part of the present invention has been applied previously to animals where it was used to observe, but not isolate or otherwise define, DNA fragments associated with the heterogametic sex of chicken cattle (25) and sheep (26).

Accordingly, in a first aspect the present invention provides an isolated polynucleotide, the polynucleotide having a sequence as set out in any one of SEQ ID NOS: 1 to 4 or 8 to 11, or a sequence which hybridizes thereto.

The polynucleotide sequences of the present invention hybridize specifically to the equine Y chromosome. By "hybridize specifically to the equine Y chromosome" we mean the polynucleotides hybridize to a repeat sequence which is present on the equine Y chromosome in a substantially greater copy number than is present elsewhere in the equine genome.

Preferably, the sequence is present in less than six copies and more preferably in only one copy in the haploid female genome.

In a preferred embodiment the polynucleotide sequence has a sequence as set out in SEQ ID NO: 3 or a sequence which hybridizes thereto.

The polynucleotide sequences of the present invention preferably hybridize to sequences set out in SEQ ID NOS: 1 to 4 or 8 to 11 under high stringency. When used herein, "high stringency" refers to conditions that employ low ionic strength and high temperature for washing after hybridization, for example, 0.1 x SSC and 0.1% SDS at 50 0 C; (ii) employ during hybridization conditions such that the hybridization temperature is 25°C lower than the duplex melting temperature of the hybridizing polynucleotides, for example 1.5 x SSPE, 10% polyethylene glycol 6000 7% SDS 0.25 mg/ml fragmented herring sperm DNA at 65 0

C;

or (iii) for example, 0.5M sodium phosphate, pH 7.2, 5mM EDTA, 7% (w/v) SDS (28) and 0.5% BLOTTO (29.30) at 70 0 C: or (iv) employ during hybridization a denaturing agent such as formamide for example, formamide with 5 x SSC, 50mM sodium phosphate (pH 6.5) and 5 x Denhardt's solution (32) at 42°C; or employ, for example, 50% (v/v) formamide, 5 x SSC, 50mM sodium phosphate (pH 0.1% sodium pyrophosphate, 5 x Denhardt's solution sonicated salmon sperm DNA (50/g/ml) and 10% dextran sulphate (33) at 42°C. See generally references 34- 36.

4 In a further preferred embodiment, the polynucleotide which hybridises under stringent conditions is less than 500 nucleotides, more preferably less than 200 nucleotides, and more preferably less than 100 nucleotides in length.

In a further preferred embodiment, the polynucleotide sequences of the present invention share at least 40% homology, more preferably at least homology, more preferably at least 80% homology, more preferably at least 90% homology and more preferably at least 95% homology with a sequence shown in any one of SEQ ID NOS: 1 to 4 or 8 to 11, wherein the homology is calculated by the BLAST program blastn as described in Altschul, Madden, Schaffer, Zhang, Zhang, Miller, W.

And Lipman, D.J. (1997) "Gapped BLAST and PSI-BLAST: a new generation "of protein database search programs", Nucleic Acids Research 25117):3389- 3402.

15 In a further preferred embodiment, the polynucloetide sequence of the present invention hybridises under stringent conditions to a sequence characterised by nucleotides 990-2497 of SEQ ID NO: 8, 421-1920 of SEQ ID NO. 9, 421 1930 of SEQ ID NO. 10, or 1502-2996 of SEQ ID NO. 11.

i. The polynucleotide of the present invention may comprise DNA or 20 RNA sequences.

The present invention also provides a vector including a polynucleotide sequence according to the first aspect of the present invention and a host cell transformed with such a vector.

In a second aspect, the present invention provides an oligonucleotide 25 probe or primer of at least 8 nucleotides that hybridises specifically to the equine Y chromosome, the oligonucleotide having a sequence that hybridizes to a polynucleotide of the first aspect of the present invention.

In a preferred embodiment the oligonucleotide is at least 10, more preferably at least 15 and more preferably at least 18 nucleotides in length.

In one preferred embodiment the oligonucleotide is derived from the sequence shown in SEQ ID NO:3. In one preferred embodiment the oligonucleotide comprises the sequence: 5'-AGCGGAGAAAGGAATCTCTGG-3' (SEQ ID NO: 12) or 5'-TACCTAGCGCTTCGTCCTCTAT-3' (SEQ ID NO: 13) WO 99/02672 PCT/AU98/00533 derived from nts 6-26 and the reverse complement of nts 184-205, respectively, of the equine male genomic DNA sequence shown in SEQ ID NO: 7.

It will be appreciated that the probes or primers of the present invention may be produced by in vitro or in vivo synthesis. Methods of in vitro probe synthesis include organic chemical synthesis processes or enzymatically mediated synthesis, e.g. by means of SP6 RNA polymerase and a plasmid containing a polynucleotide sequence according to the first aspect of the present invention under transcriptional control of an SP6 specific promoter.

In a further preferred embodiment the oligonucleotide probe is conjugated with a label such as a radioisotope, an enzyme, biotin, a fluorescer or a chemiluminescer.

In a third aspect, the present invention provides a method of determining the sex of a horse, an equine fetus, an equine embryo or an equine cell(s) which method includes analysing a biological sample derived from the horse or the fetus or embryo or the population of cells, for the presence of a polynucleotide according to the first aspect of the present invention.

The equine cell(s) may be, for example, the sperm cells of a horse. In a preferred embodiment they may be populations of sperm cells or individual sperm cells that have been resolved by flow cytometry after staining with the fluorescent DNA-binding dye Hoechst 33342 (37,38).

The equine cell(s) may further be, for example, nucleated fetal cells.

Such cells may be collected by amniocentesis or chorionic villus sampling.

In a preferred embodiment they may be sampled in the peripheral blood of a pregnant mare (see generally reference 39 the disclosure of which is incorporated herein by reference).

In order to minimise the possibility of false negatives, the method is preferably conducted with one or more suitable positive controls.

For example, the biological sample may be simultaneously analysed for the presence of a sequence which is present in approximately equal copy numbers in male and female horses. The biological sample may be analysed, for example. for the presence of a dispersed autosomal repeated sequence.

It will be understood by a person skilled in this field that an analysis to determine whether a sample contains the polynucleotide sequence of the WO 99/02672 PCT/AU98/00533 6 present invention may be performed in a number of ways. For example, the analysis may involve Southern blot hybridization, dot blot hybridization or in situ hybridization tests using probes according to the present invention.

Alternatively, the analysis may involve the technique of polymerase chain reaction (PCR; 16) or ligation amplification reaction (LAR; 40,41) using oligonucleotide primers and probes of the present invention.

The term "polymerase chain reaction" or "PCR" when used herein generally refers to a procedure where minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in references 42 and 43. Generally, sequence information from the ends of the region of interest or beyond needs to be available, such that oligonucleotide primers can be designed; these primers will be identical in sequence or similar in sequence to opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may coincide with the ends of the amplified material. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally references 16 and 44.

As used herein, PCR is considered to be one, but not the only, example of a nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, comprising the use of an established nucleic acid (DNA or RNA) as a primer, and utilises a nucleic acid polymerase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid (see, for example, references 45 and 46).

The terms "ligation chain reaction" or "LCR" or "ligation amplification reaction" or "LAR" when used herein generally refer to a procedure where minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are amplified as described in references 40 and 41. Generally, sequence information from the region of interest needs to be available, such that oligonucleotide pairs can be designed that are complementary to adjacent sites on an appropriate nucleic acid template. The oligonucleotide pair is ligated together by the action of a ligase enzyme. The amount of ligated product may be increased by either linear or exponential amplification using sequential rounds of such template-dependent ligation. In the case of linear amplification, a single pair of oligonucleotides is ligated, the reaction is WO 99/02672 PCT/AU98/00533 7 heated to dissociate the ligation product from its template, and similar additional rounds of ligation are performed. Exponential amplification utilises two. pairs of oligonucleotides, one pair being complementary to one strand of a target sequence and the other pair being complementary to the second strand of the target sequence. In this case the products of ligation serve as mutually complementary templates for subsequent rounds of ligation, interspersed with heating to separate the ligated products from the template strands. A single base-pair mismatch between the annealed oligonucleotides and the template prevents ligation, thus allowing the distinction of single base-pair differences between DNA templates.

LAR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, bacteriophage or plasmid sequences, etc. See generally references 40 and 41.

As used herein, LAR is considered to be one, but not the only, example of a nucleic acid ligase reaction method for amplifying a nucleic acid test sample, comprising the use of an established nucleic acid (DNA or RNA) as a primer/probe, and utilises a nucleic acid ligase to amplify or generate a specific piece of nucleic acid or to amplify or generate a specific piece of nucleic acid which is complementary to a particular nucleic acid (see, for example, references 47 and 48).

In a fourth aspect, the present invention provides a kit for sex determination of a horse, an equine fetus, an equine embryo, an equine cell or a population of equine cells, which kit includes a polynucleotide according to the first aspect of the present invention or an oligonucleotide probe or primer according to the second aspect of the present invention.

The terms "EY.AC6", "EY.AD11", "EY.AI5" and "EY.AM7" as used herein refer to, where provided, the specific DNA sequences set forth in SEQ ID NOS: 1-4 respectively. These terms also include variants where nucleotides have been substituted, added to or deleted from the relevant sequences shown in SEQ ID NOS: 1-4 so long as the variants hybridize specifically to the equine Y chromosome.

Such variants may be naturally occurring variants which may arise within an individual or a population by virtue of point mutation(s), deletion(s) or insertion(s) of DNA sequences, by recombination, gene conversion, flawed replication or rearrangement. Alternatively, such variants may be produced artificially, for example by site-directed mutagenesis, by WO 99/02672 PCT/AU98/00533 8 "gene shuffling", by deletion using exonuclease(s) and/or endonuclease(s), or by the addition of DNA sequences by ligating portions of DNA together, or by the addition of DNA sequences by template-dependent and/or templateindependent DNA polymerase(s).

The EY.AC6 DNA sequence is shown in SEQ ID NO: 1. The sequence, comprising 432 base pairs of nucleotides, was determined from a fragment of DNA that was cloned into plasmid pGEM-T (trademark Promega). The cloned fragment had been recovered from a polyacrylamide gel following electrophoresis and staining of the products of RAPD PCR of male equine genomic DNA with Operon (trademark) primer OPAC.06. The fragment was selected because it was visible as a product of RAPD PCR of male but not female genomic DNA. Homologues of the cloned fragment EY.AC6 have been shown, by its hybridization to Southern blots of genomic DNA from male and female Equus caballus, to be present in both sexes but are repeated at much higher amounts in males, with the haploid female genome containing just one or a small number of copies. The defined sequence EY.AC6 appears to be contiguous with sequence EY.AM7 in the equine Y chromosome since the two sequenced isolates share a region of overlap of 128 bp with 91% similarity.

The EY.AD11 DNA sequence is shown in SEQ ID NO: 2. The sequence, comprising 600 base pairs of nucleotides, was determined from a fragment of DNA that was cloned into plasmid pGEM-T (trademark Promega). The cloned fragment had been recovered from a polyacrylamide gel following electrophoresis and staining of the products of RAPD PCR of male equine genomic DNA with Operon (trademark) primer OPAD.11. The fragment was selected because it was visible as a product of RAPD PCR of male but not female genomic DNA. Homologues of the cloned fragment EY.AD11 have been shown, by its hybridization to Southern blots of genomic DNA from male and female Equus caballus, to be present in both sexes but are repeated at much higher amounts in males, with the haploid female genome containing just one or a small number of copies.

The EY.AI5 DNA sequence is shown in SEQ ID NO: 3. The sequence, comprising 230 base pairs of nucleotides, was determined from a fragment of DNA that was cloned into plasmid pGEM-3Z (trademark Promega).

The cloned fragment had been recovered from a polyacrylamide gel following electrophoresis and staining of the products of RAPD PCR of male equine WO 99/02672 PCT/AU98/00533 9 genomic DNA with Operon (trademark) primer OPAI.05. The fragment was selected because it was visible as a product of RAPD PCR of male but not female genomic DNA. Homologues of the cloned fragment EY.AI5 have been shown, by its hybridization to Southern blots of genomic DNA from male and female Equus caballus, to be present in both sexes but are repeated at much higher amounts in males, with the haploid female genome containing just one or a small number of copies.

The EY.AM7 DNA sequence is shown in SEQ ID NO: 4. The sequence, comprising 285 base pairs of nucleotides, was determined from a fragment of DNA that was cloned into plasmid pGEM-T (trademark Promega). The cloned fragment had been recovered from a polyacrylamide gel following electrophoresis and staining of the products of RAPD PCR of male equine genomic DNA with Operon (trademark) primer OPAM.07. The fragment was selected because it was visible as a product of RAPD PCR of male but not female genomic DNA. Homologues of the cloned fragment EY.AM7 have been shown, by its hybridization to Southern blots of genomic DNA from male and female Equus caballus, to be present in both sexes but are repeated at much higher amounts in males, with the haploid female genome containing just one or a small number of copies. The defined sequence EY.AM7 appears to be contiguous with sequence EY.AC6 in the equine Y chromosome since the sequences isolated share a region of overlap of 128 bp with 91% similarity.

The DNA sequences described herein (SEQ ID NOS: 1-4) were determined by chain-termination DNA sequencing techniques (49) using fluorescence-labelled dideoxynucleotides (50-53).

It will be appreciated by those skilled in the art that the polynucleotide sequences of the present invention are advantageous in that they are present in multiple copies on the Y chromosome, thereby providing greater sensitivity in assays for the presence of a Y chromosome than is possible when the assay involves detection of a unique (single copy) DNA sequence.

This allows detection to be applied with greater facility to very small samples, as in a few cells removed from a viable embryo or cells of fetal origin in peripheral blood of a pregnant mare (39) or sperm cells separated by fluorescence activated cell sorting (38).

The polynucleotide sequences and oligonucleotide primers and probes of the present invention have application, for example, in sexing of embryo WO 99/02672 PCT/AU98/00533 biopsy; fetal sex detection, i.e. by amniocentesis, chorionic villus sampling, fetal cells circulating in peripheral blood of a pregnant mare; analysis of the sex chromosome constitution of an individual sperm cell or of populations of sperm cells; resolution of ambiguities in sexual phenotype; sex analysis of tissues derived from horses (meat, hide, hair, bone, etc. from living or dead horses); and similar applications in related equine species, including extinct or endangered species.

The polynucleotide sequences and oligonucleotide primers and probes of the present invention also have a variety of uses in addition to their use in sexual identification. For example, the sequences may be used to screen recombinant DNA libraries prepared from a variety of mammalian species.

The DNA sequences may be used to deduce similar sequences or genetically linked sequences having similar functionality. The sequences may also be used in chromosome walking or jumping techniques to isolate coding and non-coding sequences proximal to the nucleotide sequence of the present invention.

According to a further aspect of the present invention, there is provided a method for the isolation of Y-chromosomal DNA sequences comprising: pooling equivalent amounts of genomic DNA from a number of male animals of a single species and pooling equivalent amounts of genomic DNA from a similar number of female animals of the same species, with the female animals preferably being related closely to the male animals, e.g. siblings; subjecting equivalent samples of the male and female pooled DNA mixtures to PCR with an arbitrary oligonucleotide primer and resolving the resultant fragments by gel electrophoresis; examining the stained resolved products for fragments that are amplified from male DNA but not from female DNA; recovering said fragment(s) from an electrophoresis gel and isolating individual fragments by molecular cloning; and PCR analysis of samples of male and female genomic DNA using oligonucleotide primers derived from the DNA sequence of said isolated fragment(s).

In a preferred embodiment the method includes the additional step after step (iii) of confirming the male association of fragment(s) by PCR and electrophoretic analysis of equivalent genomic DNA samples from a number WO 99/02672 PCT/AU98/00533 11 of individual male and female animals. Preferably the method also includes an additional step after step (iv) of confirming the isolation of individual male-associated fragment(s) by hybridization of the labelled said fragment(s) with samples of male and female genomic DNA.

The terms "comprise", "comprises" and "comprising" as used throughout the specification are intended to refer to the inclusion of a stated component or feature or group of components or features with or without the inclusion of a further component or feature or group of components or features.

The present invention will now be described, by way of example only, with reference to the following non-limiting drawings and examples.

Brief Description of the Figures Figure 1 shows hybridization analysis of horse breeds, donkey and camel with male-associated sequence EY.AI5. Samples of genomic DNA (2.5 /g) from male and female horses of various breeds (Family Equidae: Equus caballus) as well as Przewalski's horse przewalskii), donkey (E.

asinus) and the camel (Family Camelidae: Camelus dromedarius) as indicated, were digested with Sau3AI. The fragments were resolved by agarose gel electrophoresis, transferred onto positively-charged nylon membrane and hybridized with digoxigenin-labelled probe EY.AI5, as described in the text. The lane labelled M contained DNA standards whose sizes are indicated in base pairs.

Figure 2 shows the sequence of fragments amplified directly from genomic DNA of male and female horses using primers EQYL1 and EQYR1.

Differences between the sequences determined from male and female genomic DNA are indicated by differences between the cloned sequence (SEQ ID NO: 3) and the sequence from male genomic DNA are indicated by The underlined region from nt 6 to nt 26 is the sequence of sexing primer EQYL2; the underlined region from nt 184 to nt 205 is the reverse complement of the sequence of sexing primer EQYR5 (refer to text).

Figure 3 shows hybridization analysis of recombinant phage DNA with cloned male-associated sequences. Samples of DNA (10 gg) of Lambda Fix®II WO 99/02672 PCT/AU98/00533 12 vectors containing equine genomic inserts were digested with restriction enzymes EcoRI and HinduI as shown. Digests were treated at 68 0 C for 15 min then resolved by agarose gel electrophoresis before transfer to positivelycharged nylon membrane as described in the text. The membrane was hybridized with digoxigenin-labelled probes prepared by PCR amplification of cloned inserts from the flanking plasmid primers SP6 and T7. One probe was stripped from the membrane by methods described in the text before hybridization with the second probe. The inserts used as probes were: (a) EY.AD11. A photograph of the gel taken under uv transillumination before DNA transfer is shown. The lanes labelled M contained DNA standards whose sizes are indicated in base pairs.

Figure 4 shows in the sites for restriction enzyme EcoRI in the equine genomic DNA insert 32.3 after excision of the insert, together with its flanking T3 andT7 promoter sequences, from the Lambda Fix®II vector with the restriction enzyme NotI. The position of 4.7 kb subcloned EcoRI fragment 32.3E5 is indicated. The complete sequence of 32.3E5 was determined and, in the positions of previously described sequences EY.AC6, EY.AD11, and EY.AM7 within the subclone are illustrated, as is the relative position of the truncated LINE repeat EY.LINE as defined in the text. There is a close relationship between the DNA sequences of subclone 32.3E5 and subclone 33.1H7 (see Figure 5) which allows 33.1H7 to be superimposed on 32.3E5 as shown.

Figure 5 shows in the sites of restriction enzyme HindIII in the genomic DNA_insert 33.1 after excision of the insert, together with its flanking T3 andT7 promoter sequences, from the Lambda Fix@II vector with the restriction enzyme NotI. The locations of two 3.4 kb subcloned repeated HindIII fragments 33.1H7 and 33.1H2 are indicated although it was not possible to determine which repeat occupied either of the two possible positions. The complete sequences of both fragments were determined and found to have 88-90 identity. In the positions of previously described sequences EY.AC6, EY.AD11, EY.AI5 and EY.AM7 within the subclone 33.1H7 are illustrated, as is the relative position of the truncated LINE repeat EY.LINE as defined in the text. There is a close relationship between the WO 99/02672 PCT/AU98/00533 13 DNA sequences of subclone 32.3E5 and subclone 33.1H7 which allows 33.1H7 (and 33.1H2) to be superimposed on 32.3E5 as shown in Figure 4b.

Figure 6 shows in the sites of restriction enzymes EcoRI and HindIII in the equine genomic DNA insert 36.1 after excision of the insert, together with its flanking T3 andT7 promoter sequences, from the Lambda Fix®II vector with the restriction enzyme NotI. The position of 6.0 kb subcloned EcoRI fragment 36.1E2 is indicated, as is the position of 4.4 kb subcloned HindIII fragment 36.1H7. The complete sequence of 36.1H7 was determined and, in the positions of previously described sequences EY.AC6, EY.AD11, EY.AI5 and EY.AM7 within the subclone are illustrated, as is the relative position of the truncated LINE repeat EY.LINE as defined in the text. There is a close relationship between DNA sequences from base 1378 to base 4355 of subclone 36.1H7 to sequences in subclone 32.3E5 (see Figure 4b) and subclones 33.1H7 and 33.1H2 (see Figure 5b). Sequence in subclone 36.1H7 located 5' to this homologous region encoded inverted and direct repeats of EY.AC6, EY.AD11 and intervening sequence.

Brief description of Sequence Listings SEQ ID NO: 1 shows the sequence of one strand of equine repeat element EY.AC6 comprising 432 complementary base pairs. The sequence is written in single-letter code from the 5'-terminus to the 3'-terminus according to standard practice.

SEQ ID NO: 2 shows the sequence of one strand of equine repeat element EY.AD11 comprising 600 complementary base pairs.

SEQ ID NO: 3 shows the sequence of one strand of equine repeat element comprising 230 complementary base pairs.

SEQ ID NO: 4 shows the sequence of one strand of equine repeat element EY.AM7 comprising 285 complementary base pairs.

SEQ ID NO: 5 shows the sequence of the cloned EY.AI5 sequence WO 99/02672 PCT/AU98/00533 14 SEQ ID NO: 6 shows the sequence of fragments amplified directly from genomic DNA of female horses using primers EQYL1 and EQYR1.

SEQ ID NO: 7 shows the sequence of fragments amplified directly from genomic DNA of male horses using primers EQYL1 and EQYR1.

SEQ ID NO: 8 shows the sequence of one strand of subclone 32.3E5 comprising 4693 complementary base pairs of equine genomic DNA.

Subclone 32.3E5 is an EcoRI fragment of recombinant phage 32.3. The position of the fragment within the phage insert is shown in Figure 4a.

SEQ ID NO: 9 shows the sequence of one strand of subclone 33.1H7 comprising 3430 complementary base pairs of equine genomic DNA.

Subclone 33.1H7 is one of two repeated HindIII fragments of recombinant phage 33.1 and is 88-90% homologous with a second HindIII fragment, 33.1H2 (detailed in SEQ ID NO:10). The position of the repeated fragment within the phage insert is shown in Figure SEQ ID NO: 10 shows the incomplete sequence of one strand of subclone 33.1H2 comprising 3230 complementary base pairs of equine genomic DNA, from 1 to 2122 and from 2342 to 3450 of the subclone. Subclone 33.1H2 is the second of two repeated HindIII fragments of phage 33.1 and is 88-90 homologous with the HindIII fragment, 33.1H7 (detailed in SEQ ID NO: 9).

The position of the repeated fragment within the phage insert is shown in Figure SEQ ID NO: 11 shows the sequence of one strand of subclone 36.1H7 comprising 4355 complementary base pairs of equine genomic DNA.

Subclone 36.1H7 is a HindIII fragment of phage 36.1 The position of the fragment within the phage insert is shown in Figure. 6a.

SEQ ID NO: 12 shows an oligonucleotide probe (EQYL2) derived from SEQ ID NO:3.

SEQ ID NO: 13 shows an oligonucleotide probe (EQYR5) derived from SEQ ID NO:3.

WO 99/02672 PCT/AU98/00533 SEQ ID NO: 14 shows an oligonucleotide primer (EQYR4) derived from SEQ ID NO:3.

SEQ ID NO: 15 shows an oligonucleotide primer (EQYLl) derived from SEQ ID NO:3.

SEQ ID NO: 16 shows an oligonucleotide primer (EQYR1) derived from SEQ ID NO:3.

SEQ ID NO: 17 shows an oligonucleotide primer (EQSIN8) derived from SEQ ID NO:3.

SEQ ID NO: 18 shows an oligonucleotide primer (EQSIN9) derived from SEQ ID NO:3.

SEQ ID NO: 19 shows an oligonucleotide primer (mEQYL2) derived from SEQ ID NO:3.

SEQ ID NO: 20 shows an oligonucleotide primer (mEQYR5) derived from SEQ ID NO:3.

SEQ ID NO: 21 shows an oligonucleotide primer (mEQSIN8) derived from SEQ ID NO:3.

SEQ ID NO: 22 shows an oligonucleotide primer (mEQSIN9) derived from SEQ ID NO:3.

Definitions and Abbreviations ATP BLOTTO skim milk powder bp base pairs ccc covalently closed circular cfu colony-forming units BSA bovine serum albumin WO 99/02672 PCT/AU98/00533 16 Denhardt's solution 0.02% BSA, 0.02% Ficoll 400, 0.02%

PVP

DIG digoxigenin DNA deoxyribonucleic acid dNTP deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP) DTT dithiothreitol EDTAethylenediaminetetraacetic acid g force of gravity h hour(s) LAR ligation amplification reaction LB Luria-Bertani mg milligram(s) 10- 3 gram min minute(s) ml millilitre(s) 10-3 litre kjg microgram(s) 10- 6 gram pl microlitre(s) 10- 6 litre ng nanogram(s) 10- 9 gram nm nanometres 10-9 metre (ref. wavelength of light) nt(s) nucleotide(s) oligonucleotide single-stranded DNA <30 nts PAGE polyacrylamide gel electrophoresis PBS phosphate-buffered saline 100mM NaC1, 2.7mM KC1, 1.75nmM

KH

2

PO

4 4.3mM Na 2

HPO

4 pH 7.4 PCR polymerase chain reaction pg picogram(s) 10- 1 2 gram polynucleotide single- or double-stranded DNA or RNA primer oligonucleotide used to prime PCR probe (labelled) nucleic acid that hybridizes to specific target sequence(s) PVP polyvinylpyrrolidone RAPD random amplification of polymorphic DNA RNA ribonucleic acid rpm revolutions per minute SDS sodium dodecylsulphate SINE short interspersed repetitive element SSC standard saline-citrate 0.15M NaC1, 15mM trisodium citrate WO 99/02672 PCT/AU98/00533 17 SSPE standard saline-phosphate-EDTA 0.18M NaCI, 10mM NaH 2

PO

4 1mM EDTA, pH 7.7 TAE tris-acetate-EDTA 40mM tris-acetate, 20mM acetic acid, EDTA, pH 8.4 Taq Thermus aquaticus TBE tris-borate-EDTA 89mM tris-HC1, 0.89M sodium borate, 2mM EDTA, pH 8.4 TE tris-EDTA 10mM tris-HC1, ImM EDTA, pH TEMED N,N,N',N'-tetramethylethylenediamine temp temperature tris tris(hydroxymethyl)-aminomethane uv ultraviolet V volts vol volume equivalent v/v volume/volume equivalent Detailed Description of the Invention EXAMPLE 1: Preparation Of Genomic DNA From Equine Blood Samples: Equine blood samples were collected into 10 ml EDTA Vacutainers®, placed immediately on ice and delivered to the laboratory within two days. It was found that samples could be stored in a Vacutainer® at 4°C for up to six months without significant loss of yield or quality of DNA extracted therefrom.

Twenty five ml of cold lysis buffer (0.32M sucrose, 10mM tris-HC1, pH 7.5, 5mM MgCl 2 1% Triton X-100) was added to 10 ml of whole blood.

The suspension was centrifuged at 4000 x g for 20 min at 4 0 C and the pelleted cells were resuspended in PBS and recentrifuged. The cells were then suspended in 9 ml of TE. The suspension was adjusted to 25mM EDTA, SDS and 0.1 mg/ml of proteinase K (Boehringer Mannheim) and the lysed mixture was incubated overnight at 37°C with gentle agitation.

The digested sample was extracted with 5 ml of phenol/chloroform (equal volumes of phenol equilibrated with tris-HCl/EDTA (Sigma) and 24:1 (v/v) chloroform/isoamyl alcohol) for 60 min and the mixture was centrifuged at 4000 x g for 25 min at 25 0 C. The aqueous phase was removed from each tube and transferred to a clean tube.

WO 99/02672 PCT/AU98/00533 18 DNA was precipitated by the addition of 2.5 vol ethanol or 1 vol propanol, the supernatant decanted and the DNA pellet rinsed with 0.5 ml of ethanol and air-dried. The DNA was finally dissolved in 2 ml of 0.1 x TE and stored at -20 0

C.

DNA concentrations were determined using a Pharmacia Gene Quant RNA/DNA calculator. The yield was typically 50-250 fng of high molecular weight DNA (estimated by ethidium staining after agarose gel electrophoresis).

Conceptual Basis For Identification Of Male-Associated DNA: The Y chromosome is the sole genetic difference between male and female horses, being present in all nucleated cells of normal males and absent from the cells of normal females. This genetic difference must be reflected in the presence of Y-chromosomal DNA sequences in the male genome that are absent from the female genome. It would be expected that Ychromosomal, male-specific DNA sequences could be identified by a technique that surveys multiple genomic DNA sequences at random (54), by comparing survey data from normal male and female genomes which are in all other respects identical.

It was not possible to obtain isogenic male and female horses, i.e. individuals whose genomes are identical except for the Y chromosome of the male (cf. inbred strains of mice). In the absence of genetic homology, a combination of statistical and genetic techniques was used to generate pseudo-isogenic samples of male and female equine DNA.

DNA was extracted from white blood cells of nine brother-sister sibling pairs and equal amounts of DNA from each of the nine males were pooled to provide a sample of male DNA. Equal amounts of DNA from each of their sisters were pooled to provide a parallel sample of pseudo-isogenic female

DNA.

RAPD PCR Of Pooled DNA Samples: The pooled mixtures of male and female DNA were surveyed for maleassociated sequence differences by PCR amplification, using decanucleotide primers known as RAPD primers that are available commercially from Operon Technologies.

WO 99/02672 PCT/AU98/00533 19 The method used for RAPD PCR was adapted from a method described previously Each PCR reaction contained 25 ng of equine genomic DNA, RAPD primer (Operon Technologies), 3 units of Taq DNA polymerase Stoffel fragment (Perkin-Elmer), 200LM of each of the four dNTPs (Promega), 10mM tris-HCl, pH 8.0, 10mM KCl and 5mM MgCI 2 in a total volume of l.

Reactions were cycled in a Corbett Research PC-960 Air-Cooled Thermocycler with an initial step of 94°C for 5 min followed by 35 cycles consisting of 94°C for 30 sec then 1 min at each of 57 0 C, 56 0 C, 55 0 C, 54°C and 53°C; on completion of cycling the samples were heated at 72 0 C for 5 min.

Electrophoretic Analysis of RAPD PCR Products: Polyacrylamide gel electrophoresis was used to resolve the products of RAPD PCR, greatly increasing the resolution of fragments relative to that attainable by agarose gel electrophoresis. Silver staining enhanced the sensitivity of detection compared with uv fluorescence of ethidium bromide.

DNA amplification products were resolved by polyacrylamide gel electrophoresis (PAGE) in a Bio-Rad Mini-Protean II. The polyacrylamide gels were 10% acrylamide and 2% bis-acrylamide in TBE buffer containing 10% urea and 5% glycerol. Ammonium persulphate (0.15% w/v) and TEMED (0.15% v/v) were used to initiate and catalyse polymerisation.

The 0.5 mm gels were cast on Gel Bond PAG backing film (FMC; 56).

Samples (2 pl) of PCR reaction product were mixed with 1 pl of loading buffer (40% urea, 3% Ficoll 400, 10mM tris-HC1, pH 3mM EDTA, 0.02% xylene cyanol, 0.02% bromophenol blue), loaded into pre-formed slots and electrophoresed in TBE buffer at 300V for min. Resolved DNA fragments were visualised by silver staining (57).

In total, 216 different Operon RAPD primers were used to screen the pooled pseudo-isogenous samples of male and female DNA, of which yielded clear, reproducible results for both pooled samples.

Identification Of Male-Associated DNA Fragments: Nineteen of the 216 tested primers were found to amplify a fragment from the male DNA pool that was either less intense than a fragment of similar size in the female DNA pool or apparently absent from the PCR WO 99/02672 PCT/AU98/00533 products of the female DNA pool. To determine whether candidate fragments were indeed amplified from the DNA of all males and only males, primers yielding candidate male-associated fragments from the pooled DNA samples were used for RAPD PCR of DNA isolated from a number of individual males and females. A fragment amplified differentially from pooled male DNA could arise from an autosomal polymorphism in one or two individuals, a possibility confirmed by the occasional observation of differential RAPD PCR fragments from the pooled female DNA sample.

The 19 candidate primers were used to amplify individual DNA samples from four male and four female horses. Unambiguous maleassociated fragments were evident in the products from five of these primers: OPAC.06 (5'-CCAGAACGGA-3'), OPAD.11 (5'-CAATCGGGTC-3'), OPAI.05 GTCGTAGCGG-3'), OPAM.01 (5'-TCACGTACGG-3') and OPAM.07 AACCGCGGCA-3'). The sizes of the differential fragments were estimated at approximately 460bp, 530bp, 240bp, 320bp and 300bp, respectively.

Isolation Of Male-Associated DNA Fragments: For each of the five candidate male-specific fragments, a slice containing the fragment was cut from the silver-stained polyacrylamide gel and allowed to stand in 20-50 jil of 0.1 x TE at room temp for 60 min.

Eluted DNA was re-amplified under the conditions described above for RAPD PCR using the relevant RAPD primer and 1 Al of excised fragment solution as template. Reactions were cycled in a Corbett Research PC-960 Air-Cooled Thermocycler with an initial step at 94 0 C for 2 min followed by 35 cycles of 94°C for 30 sec then 55 0 C or 60 0 C for 1 min; on completion of cycling the samples were heated at 72 0 C for 2 min.

It was necessary to confirm that the re-amplified DNA samples corresponded in electrophoretic mobility with the candidate RAPD fragments and that each contained a male-associated DNA fragment. Accordingly, each re-amplified sample was electrophoresed on a polyacrylamide gel adjacent to the products of RAPD PCR of male and female genomic DNA with the relevant primer, and with the products of RAPD PCR from female DNA mixed with the re-amplified sample.

In each case, the re-amplified fragment migrated similarly to the fragment associated differentially with male DNA. In further confirmation, each of the re-amplified samples was labelled with digoxigenin and the WO 99/02672 PCT/AU98/00533 21 resultant probes were hybridized to Southern blots of male and female horse genomic DNA that had been digested with the restriction enzyme Sau3AI.

Each re-amplified fragment showed an unequivocal male-associated pattern of hybridization (data not shown; refer to "Colony Screening By Dot Blot Hybridization" for details of probe preparation, hybridization and detection).

In each case the probe also hybridized with female genomic DNA, implying that the fragments may not be associated uniquely with the Y chromosome and/or the sample included contaminating non-Y-chromosomal DNA.

Re-amplified PCR products were electrophoresed in 1% LMP agarose (Sigma) in 0.5 x TBE buffer. The material recovered from PCR with the OPAI.05 primer was visualised by illumination at 302 nm of an ethidium bromide-stained gel. The material recovered from PCR of equine genomic DNA with the OPAC.06, OPAD.11, OPAM.01 and OPAM.07 primers were visualised by staining with crystal violet (58).

A minimal portion of gel containing the desired fragment was excised and melted at 70 0 C in a 1.5 ml microcentrifuge tube. The molten gel slice was diluted with three volumes of TE and extracted with an equal volume of phenol (saturated with TE) at 70 0 C for 2 min. The tube was transferred to ice for 2 min then centrifuged at 14,000 rpm in an Eppendorf 5414C microcentrifuge for 4 min at room temp and the aqueous phase removed into a clean tube. The phenol phase was back-extracted with 50 Al of TE and this was combined with the original extracted aqueous phase.

DNA was precipitated by the addition of 0.1 vol of 3M sodium acetate, pH 5, and 2.5 vol ethanol. The tube was stored overnight at -20 0 C then centrifuged at 13000 rpm in an Eppendorf 5414C microcentrifuge for 30 min at 4 0 C. The supernatant was decanted carefully, the DNA pellet was rinsed with cold 75% ethanol and centrifuged briefly. The pellet was dried in a vacuum desiccator for 10 min and the DNA was finally dissolved in 20 /A of TE and stored at 4°C.

Ligation Of PCR-Amplified Male-Associated Fragments Into Plasmid Vector: Fragments resulting from PCR with RAPD primers OPAC.06. OPAD.11, OPAM.01 and OPAM.07 were ligated into plasmid pGEM-T (a linearised derivative of pGEM-3) using the pGEM-T vector cloning system (Promega) according to the supplier's instructions.

WO 99/02672 PCT/AU98/00533 22 Fragments resulting from PCR with RAPD primer OPAI.05 were cloned by blunt-end ligation into the plasmid vector pGem-3Z (Promega). The vector was linearised by digestion with restriction endonuclease SmaI (New England BioLabs) in NEBuffer 4 (New England BioLabs) then treated with calf alkaline phosphatase (New England BioLabs). The digested plasmid DNA was purified by electrophoresis in 1% LMP agarose (Sigma) in 0.5 x TBE buffer. The gel was stained with crystal violet a minimal portion of gel containing the linear plasmid was excised and DNA was recovered as described above.

The gel-purified OPAI.05 male-associated RAPD material was treated with T4 DNA polymerase (New England BioLabs) according to the supplier's instructions then heated at 65 0 C for 15 min. The cooled sample was then treated with T4 polynucleotide kinase (New England BioLabs) according to the supplier's instructions, then again heated at 65 0 C for 15 min.

The gel-purified linear vector (approx. 10 ng) and PCR fragments (approx. ng) were ligated with 3 Weiss units of T4 DNA ligase (Promega) in 50 Al of Promega DNA ligase buffer (30mM Tris-HC1, pH 7.8, 10mM MgCl 2 DTT, 0.5mM ATP) at 4°C for 14-16 h.

Transformation With Recombinant Plasmids: Single colonies of Escherichia coli strain DH5a (fragments from RAPD PCR with primers OPAC.06, OPAD.11, OPAM.01 and OPAM.07) or strain XL1-Blue (fragments from RAPD PCR with primer OPAI.05) were inoculated into 200 ml of LB broth and grown in a shaking incubator at 37 0 C to an optical absorbance of approx. 0.3 at 550 nm (3-4 The cells were collected by centrifugation at 3000 rpm for 5 min at 4°C in an Eppendorf 5414C microcentrifuge, resuspended in 30 ml of cold 0.1M MgC12 and placed on ice for 20 min. The cells were collected by centrifugation as before and the pellet suspended in 1 ml of cold 0.1M CaCl2. Glycerol was added to 15% and the competent cells were stored at -70 0

C.

For transformation, 50 A1 of competent cells was thawed and mixed with 5 Il of ligation reaction, placed on ice for 20 min, heat-shocked at 42°C for 45' sec then returned to ice for 5 min. The transformed cells were allowed to recover by incubation at 37°C for 1 h in 500 .l of SOC medium (2% bacto-tryptone, 0.5% bacto-yeast extract, 10mM NaC1, KC1, 10mM MgCIl, 0lmM MgSO 4 20mM glucose) and were then WO 99/02672 PCT/AU98/00533 23 plated onto LB agar containing ampicillin (100/g/ml), X-gal and IPTG (10/M) for overnight culture at 37°C. Transformation efficiency was 2 x 107 cfu/g plasmid (with ccc pGEM-T).

Colony Screening By PCR: White colonies were selected and incubated overnight in 500 /L of LB broth. Inserts in recombinant plasmids of the cloned cells were analysed by PCR amplification from primer sites flanking the cloning site. One 1 l of the cell suspension was mixed with 2.7MI each of the SP6 (5'-ATTTAGGTGACACTATAGAATAC-3') and T7 (5'-ATTATGCTGAGTGATATCCCGCT-3') primers (both from Bresatec Custom Oligos), 200gMI of each of the four dNTPs, 1.5mM MgCl 2 100mM tris-HCl, pH 8.3, 500mM KCl and 1 unit of Taq DNA polymerase (Boehringer Mannheim) in a final volume of 25 Al.

Reactions were cycled in a Corbett Research PC-960 Air-Cooled Thermocycler with an initial step at 94°C for 2 min followed by 35 cycles of 94 0 C for 20 sec, 50°C for 20 sec and 72 0 C for 30 sec; on completion of cycling the samples were heated at 72 0 C for 2 min.

Colony Screening By Dot Blot Hybridization: Colonies that were found by PCR to contain a recombinant insert of appropriate size appropriate to the size of the male-associated fragment generated from genomic DNA by RAPD PCR) were labelled by incorporating 8AM digoxigenin-11-dUTP (DIG-dUTP; Boehringer Mannheim) in colony PCR reactions, as described above.

A replicate dilution series of male and female horse genomic DNA samples (1 jg, 250 ng, 100 ng and 10 ng of each) were denatured in 0.2 ml of 0.4M NaOH, 10mM EDTA and heated at 100°C for 10 min. The samples were applied to positively-charged nylon membrane (Boehringer Mannheim; 59) with a Vacuum Blot Manifold (Gibco-BRL). Each well was washed with 500 gl of 0.4M NaOH and the membrane was neutralised by 3 x 10 min washes in 2 x SSC.

-DNA hybridizations were performed according to the DIG System User's Guide for Filter Hybridization (Boehringer Mannheim).

Membranes were prehybridized at 50°C for at least 2 h in 10 ml of DIG Easy Hyb hybridization buffer (Boehringer Mannheim, cat. no. 1603558) in glass WO 99/02672 PCT/AU98/00533 24 hybridization bottles (Hybaid) placed in a Eurotherm 91E Rotating Hybridization Incubator (Model 310; Robbins Scientific).

DIG-labelled probes were prepared as described above from the inserts of recombinant plasmids, using the SP6 and T7 primers. Each was added to 4 ml of DIG Easy Hyb solution (Boehringer Mannheim) at a concentration of 50-100ng/ml and denatured at 68 0 C for 10 min. The prehybridization solution was replaced by the probe solution and hybridization was conducted in the rotating incubator at 50 0 C for 14-16 h.

The membrane was then removed and washed for 3 x 10 min at low stringency (2 x SSC, 0.1% SDS, 25 0 C) followed by 2 x 10 min at high stringency (0.2 x SSC, 0.1% SDS, 68°C). The washed membrane was rotary-incubated for 1 h in 2 x blocking solution (Boehringer Mannheim, cat. no. 1585762) containing 1 x maleic acid buffer (Boehringer Mannheim, cat. no. 1585762).

Anti-DIG antibody labelled with alkaline phosphatase (Boehringer Mannheim, cat. no. 1093274) was added to the blocking solution at a concentration of 0.075 units/ml and rotary incubation continued for a further min.

The membrane was then washed for 2 x 15 min in 1 x wash buffer (Boehringer Mannheim, cat. no. 1585762) and transferred to 1 x detection buffer (Boehringer Mannheim, cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat.

no. 1685627) was diluted 1:100 in detection buffer and 1 ml was added per 150 cm 2 of membrane. The substrate solution was spread evenly between clear transparency sheets and the signal was detected at room temperature using X-ray film (AGFA Eurix RP1) with intensifying screens (Dupont Quanta

III-T).

Differential intensity of hybridization to male and female DNA samples indicated probes derived from clones containing a male-associated fragment from RAPD PCR with each of primers OPAC.06, OPAD.11, OPAI.05 and OPAM.07 (data not shown). Attempts to recover a cloned male-associated fragment from RAPD PCR with primer OPAM.01 were unsuccessful.

EXAMPLE 2: Sequence Analysis Of Cloned Male-Associated Fragments: DNA sequencing was performed using dideoxy sequencing chemistry utilising the ABI PRISM"' Dye Terminator Cycle-Sequencing-Ready Reaction WO 99/02672 PCT/AU98/00533 Kit (ABI Perkin-Elmer) with AmpliTaq DNA polymerase, according to the manufacturer's instructions (ABI Perkin-Elmer). Products of sequencing reactions were analysed according to the manufacturer's instructions on an ABI A373 sequencer at the University of Queensland DNA Sequence and Analysis Facility.

The sequence of cloned inserts of recombinant plasmids, derived from recovered products of RAPD PCR with each of primers OPAC.06, OPAD.11, and OPAM.07, that hybridized differentially with male DNA on dot blots are shown in SEQ ID NOS: 1, 2, 3 and 4, respectively. These inserts are known henceforth as EY.AC6, EY.AD11, EY.AI5 and EY.AM7, respectively.

Hybridization Analysis Of Cloned Male-Associated Fragments: Samples of genomic DNA (2.5 pg) from nine male and nine female horses were digested with 5 units of Sau3AI (New England BioLabs) in NEBuffer (New England BioLabs 100nmM NaCI, 10mMI bis-tris-propane-HC1, pH 7.0 at 25 0 C, 10mM MgCl 2 1mM dithiothreitol) and 0.1mg/ml BSA in a final volume of 25 pl.

The digested samples, together with a DIG-labelled DNA molecular weight marker mix (Boehringer Mannheim, cat. no. 1218603), were electrophoresed in 1% agarose at 70V for 3 h in 0.5 x TBE. Resolved fragments were capillary-transferred overnight in 0.4M NaOH to a positivelycharged nylon membrane (Boehringer Mannheim; 30). Following transfer, the membrane was neutralised with 3 x 10 min washes in 2 x SSC.

All hybridizations were performed according to the DIG System User's Guide for Filter Hybridization (Boehringer Mannheim).

Membranes were prehybridized at 50°C for at least 2 h in 10 ml of DIG Easy Hyb hybridization buffer (Boehringer Mannheim, cat. no. 1603558) in glass hybridization bottles (Hybaid) placed in a Eurotherm 91E Rotating Hybridization Incubator (Model 310; Robbins Scientific).

DIG-labelled DNA probes were prepared as described above from the four recombinant plasmids containing inserts EY.AC6, EY.AD11, EY.AI5 and EY.AM7. Each was added to 4 ml of DIG Easy Hyb solution (Boehringer Mannheim) at a concentration of 50-100ng/ml and denatured at 68 0 C for min. The prehybridization solution was replaced by the probe solution and hybridization was conducted in the rotating incubator at 50 0 C for 14- 16 h.

WO 99/02672 PCT/AU98/00533 26 The membrane was then removed and washed for 3 x 10 min at low stringency (2 x SSC, 0.1% SDS, 25 0 C) followed by 2 x 10 min at high stringency (0.2 x SSC, 0.1% SDS, 68°C). The washed membrane was rotary-incubated for 1 h in 2 x blocking solution (Boehringer Mannheim, cat. no. 1585762) containing 1 x maleic acid buffer (Boehringer Mannheim, cat. no. 1585762).

Anti-DIG antibody labelled with alkaline phosphatase (Boehringer Mannheim, cat. no. 1093274) was added to the blocking solution at a concentration of 0.075 units/ml and rotary incubation continued for a further 30 min.

The membrane was then washed for 2 x 15 min in 1 x wash buffer (Boehringer Mannheim, cat. no. 1585762) and transferred to 1 x detection buffer (Boehringer Mannheim, cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat. no.

1685627) was diluted 1:100 in detection buffer and 1 ml was added per 150 cm 2 of membrane. The substrate solution was spread evenly between clear transparency sheets and the signal was detected at room temperature using X-ray film (AGFA Eurix RP1) with intensifying screens (Dupont Quanta II-

T).

The male-differential hybridization pattern using EY.AI5 indicated that this sequence is present in multiple copies in the DNA of all male horses surveyed. A homologous sequence is present in the female genome but is much less abundant, where the relative intensity and pattern of hybridization are suggestive of just one or a few copies.

In order to confirm that the cloned fragment EY.AI5 represents a canonical genomic repeated element, a DIG-labelled probe was prepared by direct PCR of male genomic DNA using primers EQYL2: 5'-AGCGGAGAAAGGAATCTCTGG-3' (SEQ ID NO: 12) and EQYR4: 5'-TTCGTCCTCTATGTTGAAATCAG-3' (SEQ ID NO: 14) derived from the sequence of EY.AI5 (nts 6-26 and the reverse complement of nts 173-195, respectively, in SEQ ID NO: 3; both primers provided by Bresatec Custom Oligos).

'The hybridization patterns with both probes are similar, although the direct genomic probe appeared to hybridize relatively more strongly with fragments smaller than 900 bp in both male and female DNA, suggesting that WO 99/02672 PCT/AU98/00533 27 genomic representatives of the repeat include sequences that are not part of the cloned EY.AI5 fragment.

The four cloned sequences EY.AC6, EY.AD11, EY.AI5 and EY.AM7 were subsequently DIG-labelled and hybridized with Southern blots of male and female genomic DNA that had been digested with nine different restriction enzymes. All showed male-specific hybridization patterns but also hybridized with female DNA, albeit to a significantly lesser extent.

These data demonstrate that each of the four sequences is repeated many times in the male genome and hence, by comparison with hybridization to female DNA, on the Y chromosome.

The striking similarity of hybridization patterns with all four probes to fragments cut by restriction enzymes having a six-base recognition sequence (KpnI, EcoRI, HindIII, BamHI) implies that all four cloned fragments are components of a single long-range tandem repeat in the equine Y chromosome. Sequence analysis of the four cloned fragments revealed overlap between EY.AC6 and EY.AM7 (SEQ ID NOS: 1 and consistent with this interpretation.

Of the four cloned sequences, EY.AI5 showed the greatest quantitative difference between male and female DNA. Restriction patterns suggest that it has a basic repeat unit in the genome of approximately 230bp (TaqI and RsaI digests), consistent with the length of the sequenced isolate (SEQ ID NO: 3).

Using the conditions described above, the cloned sequence EY.AI5 was DIG-labelled and hybridized with Southern blots of male and female genomic DNA that had been isolated from a variety of horse breeds and digested with Sau3AI (Figure Hybridization patterns were similar for all breeds examined, including the subspecies known as Przewalski's horse. This confirms the sex-differential occurrence of EY.AI5 sequences throughout the species Equus caballus.

EXAMPLE 3: Conceptual Basis For Discriminatory PCR-Based Sexing Assay: Each of the four male-associated DNA sequences is clearly present in the equine Y chromosome since each shows a male-specific hybridization pattern. but none is unique to the male genome. Considering the four candidates as targets for a diagnostic test for the equine Y chromosome, the EY.AI5 fragment appears to offer most promise in that it shows the greatest differential between abundance on the Y chromosome and WO 99/02672 PCT/AU98/00533 28 elsewhere. Accordingly, further studies focused on this sequence in an attempt to develop PCR conditions that would provide absolute discrimination between male and female equine DNA by utilising potential differences between the sequence on the Y chromosome and its homologue(s) elsewhere in the genome.

The fact that the EY.AI5 sequence is repeated on the Y chromosome implies that it is not represented by a single, definable sequence; repeated DNA elements invariably show sequence heterogeneity 60). Cloning of PCR-amplified sequences yields single, specific representatives that, in addition to intrinsic sequence variations, may additionally contain errors due to incidental in vitro and in vivo mutagenesis.

Furthermore, EY.AI5-primed sequence(s) present in female genomes must be analysed to allow identification of possible sequence differences between it/them and Y-chromosomal representatives.

Analysis Of EY.AI5 Sequences In Male And Female Genomic DNA: For the above reasons, samples of genomic DNA from individual male and female horses were amplified by PCR from a pair of primers specific to the sequence EY.AI5. Primers EQYL1: 5'-GTCGTAGCGGAGAAAGGAATC-3' (SEQ ID NO: 15) and EQYR1: 5'-AGCGGACTGTTCCGTTTCGG-3' (SEQ ID NO: 16) derived from the sequence of EY.AI5 (nts 1-21 and the reverse complement of nts 206-225, respectively, in SEQ ID NO: 3) were used to amplify genomic DNA targets from a male and female horse (both primers provided by Bresatec Custom Oligos). The products were sequenced directly from these primers, without cloning, to allow sequence analysis of the bulk population of repeated elements.

The sequence data (Figure 2) show minor variations between the individual (cloned) representative EY.AI5 and the bulk sequence population in the male.

Two regions in fragments derived directly from the male genome differ from the equivalent regions in female-derived fragments (nts 1-30 and nts 162-220). These regions of sequence divergence were chosen as the annealing targets for PCR primers designed to discriminate between EY.AI5 sequences in male and female genomic DNA.

WO 99/02672 PCT/AU98/00533 29 EXAMPLE 4: Development Of PCR-Based Equine Sexing Assay: A primer pair was derived from the sequence data of Figure 2 for specific detection of equine Y-chromosomal DNA. These primers are EQYL2: 5'-AGCGGAGAAAGGAATCTCTGG-3' (SEQ ID NO: 12) and 5'-TACCTAGCGCTTCGTCCTCTAT-3' (SEQ ID NO: 13), derived from nts 6-26 and the reverse complement of nts 184-205, respectively, of the male genomic DNA sequence shown in Figure 2 (underlined). These two regions exhibit significant sequence differences between male and female genomes.

Amplification of equine genomic DNA samples (15 pg to 2 ng) from these primers (both primers provided by Bresatec Custom Oligos) yielded a product of approximately 200 bp from male DNA samples and no detectable product from female DNA samples (data not shown). Analysis of genomic DNA samples from ten unrelated horses (data not shown) confirmed that PCR amplification from these primers provides an accurate means of detecting the presence of Y-chromosomal DNA sequences.

In a diagnostic assay for genetic sex, no detectable product of PCR amplification from male-specific primers may result not only from a female sample but from PCR failure or loss of sample. The possibility of false negative results must be minimised. For this reason, a duplex PCR assay was developed in which a 121 bp (approximately) fragment of a dispersed autosomal repeated sequence (SINE; 61) was amplified simultaneously with the Y-specific target.

Primers used to amplify the SINE element were EQSIN8: 5'-GCCCAGTGTTTCGTTGGTTCG-3' (SEQ ID NO: 17) and EQSIN9: 5'-CATAGTTGTATATTCTTCGTTGTGG-3' (SEQ ID NO: 18), derived from nts 53-72 and the reverse complement of nts 148-172, respectively, of the ERE-1 SINE sequence family (61).

Duplex PCR amplifications were mutually optimised by inclusion of a common m13 sequence at the 5'-termini of all four primers (62,63). The two primer pairs used for duplex equine sexing by PCR were: WO 99/02672 PCT/AU98/00533 sexing primers: mEQYL2 5'-GCGGTCCCAAAAGGGTCAGTAGCGGAGAAAGGAATCTCTGG-3' (SEQ ID NO: 19) 5'-GCGGTCCCAAAAGGGTCAGTTACCTAGCGCTTCGTCCTCTAT-3' (SEQ ID NO: control primers: mEQSIN8 5'-GCGGTCCCAAAAGGGTCAGTGCCCAGTGTTTCGTTGGTTCG3' (SEQ ID NO: 21) mEQSIN9 5'-GCGGTCCCAAAAGGGTCAGTCATAGTTGTATATTCTTCGTTGTGG-3' (SEQ ID NO: 22) Duplex PCR reactions for internally-controlled assay of equine genetic sex were conducted in plastic capillary tubes (for use with the Corbett Research FTS-1 thermal cycler) containing 50mM KC1, 10mM Tris-HC1, pH 8.3, 0.001%(w/v) gelatin, 2mM MgCl 2 100LM dATP, 100ApM dCTP, 100AM dGTP, 100AM dTTP, 0.2pM mEQSIN8, 0.2/pM mEQSIN9, 0.45gM mEQYL2, 0.451l mEQYR5 (all four primers provided by Bresatec Custom Oligos) and units of AmpliTaq (Perkin-Elmer) in a total volume of 10 il.

Samples were placed in a Corbett Research FTS-1 capillary thermal cycler and subjected to a heating program of 94*C for 30 sec, 69 0 C for 30 sec and 72°C for 30 sec for a total of six cycles, followed by an additional cycles of 94°C for 30 sec and 72 0 C for 30 sec, then finally held at pending analysis by agarose gel electrophoresis.

The products were electrophoresed in 2% agarose gel in TAE buffer (Boehringer Mannheim) at 100V for approximately 30 min, then stained with ethidium bromide and visualised under uv irradiation.

Duplex PCR amplification of equine genomic DNA samples resulted in a single visible fragment of approximately 160 bp from female horse DNA, resulting from amplification of SINE elements. Male DNA gave rise to a similar band and an additional band at approximately 240 bp, resulting from WO 99/02672 PCT/AU98/00533 31 amplification of Y-chromosomal EY.AI5 elements. No product was seen in the absence of DNA.

The duplex PCR sexing assay described above is clearly able to identify and discriminate between male and female DNA samples, from 5 ng to as little as 20 pg (approximately equivalent to the amount of DNA in three cells).

EXAMPLE Application Of Duplex PCR Sexing Assay To Horses Of Various Breeds: Samples of DNA isolated from male and female horses of various breeds were analysed by duplex PCR as described above.

Duplex PCR with the sexing and control primers was able to identify and discriminate between DNA samples from male and female horses of all breeds, with similar results for all breeds including the subspecies known as Przewalski's horse.

EXAMPLE 6: Application Of Duplex PCR Sexing Assay To Whole Blood Cells: The preceding examples illustrate successful application of the described duplex PCR sexing assay to small DNA samples. For ease of utility it is desirable to conduct the assay on small numbers of cells without the necessity to isolate DNA from them. White blood cells were used to establish appropriate assay conditions.

Blood samples were withdrawn into Vacutainer®CPT" tubes with sodium citrate (Becton Dickinson). Tubes were kept upright at room temperature and processed within 2 hours of collection.

Each tube was centrifuged in a swinging bucket rotor (Sigma 3K18 with 11133 rotor) at 1900g for 30 min at 24°C. Approximately 60-70% of the clear plasma layer was removed then the remaining liquid above the gel matrix, substantially free of red blood cells, was transferred into a clean tube.

The sample was diluted to 10 ml with PBS and centrifuged at 300 x g for min at 24°C. Supernatant was removed and the pellet resuspended gently in ml of PBS and centrifuged under the same conditions. The pellet was again suspended in 10 ml of PBS then centrifuged at 100 x g.

The cell pellet was resuspended in 250 1l of PBS and the suspension counted by haemocytometer to determine the concentration of nucleated WO 99/02672 PCT/AU98/00533 32 cells. The suspension was finally diluted in PBS to a concentration of 106 nucleated cells/ml.

Samples of cell suspensions from male and female horses were serially diluted in PBS containing 50mM DTT and appropriate dilutions were subjected to two successive freeze/thaw cycles: tubes containing the samples were initially floated on liquid nitrogen for 1-2 min until frozen, then transferred to a water bath at room temperature until the suspension thawed.

The tubes were placed in a boiling water bath for 15 min then cooled on ice.

Duplex PCR reactions were conducted in plastic capillary tubes (for use with the Corbett Research FTS-1 thermal cycler) containing 50mM KC1, Tris-HC1, pH 8.3, 0.001%(w/v) gelatin, 2mM MgCl 2 100/M dATP, 100/M dCTP, 100/zM dGTP, 100M dTTP, 0.2pM mEQSIN8, 0.2vM mEQSIN9, 0.45AjM mEQYL2, 0.45/xM mEQYR5, 0.5 units of AmpliTaq and 2 1d of treated cell suspension in a total volume of 10 1 Samples were placed in a Corbett Research FTS-1 capillary thermal cycler and subjected to a heating program of 94°C for 30 sec, 69 0 C for 30 sec and 72°C for 30 sec for a total of six cycles, followed by an additional 21 cycles of 94 0 C for 30 sec and 72 0 C for 30 sec, then finally held at pending analysis by agarose gel electrophoresis.

The products were electrophoresed in 2% agarose gel in TAE buffer (Boehringer Mannheim) at 100V for approximately 30 min, then stained with ethidium bromide and visualised under uv irradiation.

As before, no bands were observed in the absence of equine DNA whereas bands are clearly visible with 20 pg of DNA from a female horse (single band at approximately 160 bp) and a male horse (two bands, at approximately 160 bp and 240 bp).

Samples of white blood cells containing approximately 5 cells to 100 cells each yielded one (160 bp) or two (160 and 240 bp) bands, consistent with their origin from female or male horses, respectively.

EXAMPLE 7: Application Of Duplex PCR Sexing Assay To Equine Embryos: Eight embryos were recovered from five mares approximately eight days after fertilisation and each was immediately split into four or more sections depending on the size of the blastocyst (each sample contained an estimated maximum of 50 cells). Splitting was performed by WO 99/02672 PCT/AU98/00533 33 micromanipulation in 50 1l of PBS. Each section of the blastocyst was collected with 2 AL of 4% BSA (Miles Pentex crystalline, cat. no. 81-001- 4; 1,2) and transferred into 7.5 Al of deionised water. The sections were stored frozen at -20 0

C.

The thawed suspension of each embryo section, containing approximately 15-50 cells, was made 20mM in DTT and dispensed randomly into tubes numbered from 8 to 39. The 32 samples were from this stage processed 'blind' by a second individual who had not been involved with embryo collection, splitting or sample preparation.

Each sample was subjected to two successive freeze/thaw cycles. For each cycle the tubes containing the samples were floated on liquid nitrogen for 1-2 min until frozen, then transferred to a water bath at room temperature until the suspension thawed. The tubes were finally placed in a boiling water bath for 15 min then cooled on ice.

Duplex PCR reactions were conducted in plastic capillary tubes (for use with the Corbett Research FTS-1 thermal cycler) containing KC1, 10mM Tris-HCl, pH 8.3, 0.001%(w/v) gelatin, 2mM MgCl 2 100M dATP, 100/.M dCTP, 100gM dGTP, 100AM1 dTTP, 0.2/M mEQSIN8, 0.2gM mEQSIN9, 0.6/pM mEQYL2, 0.6G/M mEQYR5, 0.5 units of AmpliTaq and 9.5 Al of embryo cell suspension in a total volume of 20 l.

Samples were placed in a Corbett Research FTS-1 capillary thermal cycler and subjected to a heating program of 94°C for 30 sec, 69 0 C for 30 sec and 72 0 C for 30 sec for a total of six cycles, followed by an additional 21 cycles of 94°C for 30 sec and 72 0 C for 30 sec, then finally held at pending analysis by agarose gel electrophoresis.

The products were electrophoresed in 2% agarose gel in TAE buffer (Boehringer Mannheim) at 100V for approximately 30 min, then stained with ethidium bromide and visualised under uv irradiation.

No bands are observed in the absence of equine DNA whereas bands are clearly visible with 20 pg of DNA from female DNA (single band at approximately 160 bp) and male DNA (two bands, at approximately 160 bp and 240 bp). Bands are clearly visible with both 125 and 5 (approximately) white blood cells from a female horse (single band at approximately 160 bp) and a male horse (two bands, at approximately 160 bp and 240 bp).

Products resulting from assay of embryo sections showed relatively weak signals which were variable in intensity; this was found subsequently WO 99/02672 PCT/AU98/00533 34 to result from sub-optimal PCR conditions due to a pH shift caused by the BSA used in the collection of embryo sections.

Two individuals, who had not been involved in earlier stages of the analysis, independently called the sex of each embryo section from the assay results. The calls of both individuals were in complete agreement and are shown in Table 1.

Table 1 shows the analysis of sex of embryo biopsies by duplex PCR.

Embryos were recovered from mares approximately eight days after fertilisation, cut into four or more sections and the sections frozen at -20 0

C.

The thawed sections, each containing approximately 15-50 cells, were dispensed randomly into tubes numbered from 8 to 39 and the 32 samples were analysed 'blind' by duplex PCR, as described in the text. At the conclusion of assay, two further individuals independently called the sex of each assay result. The calls of both individuals were in complete agreement.

F is female diagnosis, M is male diagnosis and NR is no result. The data have been rearranged for clarity.

For every embryo, all four sections from the same embryo were called as the same sex, with the exception of sample 34 which yielded no result (neither male-specific band nor control band was visible, confirming the value of including primers for an internal control). The probability of such a result arising by chance is 10 7 These data provide statistical validation of the duplex PCR sexing assay for embryo sections.

WO 99/02672 WO 9902672PC/AU98/00533 Table 1.

Einbryo no. Estimated no.cells Sapl I Sex called 1 50 12 F 1 50 39 F 1 50 20 F 1 50 33 F 2 45 10 MV 2 45 13 Mv 2 45 30 Ii 2 35 24 Ivi 3 30 31 F 3 35 17 F 3 45 37 F 3 35 19 F 4 50 36 hI 4 50 8 NI 4 50 21 NI 4 50 38 I 50 27 F 30 16 F 50 35 F 50 29 F 6 50 25 NvI 6 50 14 I 6 50 23 IVI 6 50 11 NI 7 50 32 I 7 50 28 I 7 50 9 I 7 50 22 8 15 26 NI 8 25 15 I 8 40 18 I 8 40 34 NR~ WO 99/02672 PCT/AU98/00533 36 EXAMPLE 8 Identification Of Long-Range Repeat In The Equine Y Chromosome: To investigate the inter-relation, repetition, conservation and genomic environment of the four described sequence elements associated with the equine Y chromosome, these elements were used as probes to identify recombinant bacteriophage in an equine genomic library.

Equine Genomic Library: A male horse genomic DNA library in the Lambda Fix@II vector was obtained from Stratagene (cat. no. 946701). The estimated titre of the library after a single round of amplification was 2.0 x 109 plaque forming units (pfu)/ml. Phage and host bacteria were cultured according to methods detailed in the instruction manual provided with the library (Stratagene).

Screening Of Equine Genomic Library With EY.AI5 Probe: For the first round of screening a total of 25,000 to 30,000 plaques grown in host E. coli XL1-Blue MRA (P2) were present on each 150 mm plate of growth medium. Duplicate plaque lifts were made from each plate and DNA was bound to uncharged Nylon Membranes for Colony and Plaque Hybridization (132 mm diameter) purchased from Boehringer Mannheim (cat. no. 1699083) according to protocols outlined in the DIG System User's Guide for Filter Hybridization (Boehringer Mannheim). After uv cross-linking (Bio-Rad GS Gene Linker), membranes were prehybridized at 42 0 C for at least 2 h in 10 ml of DIG Easy Hyb hybridization buffer (Boehringer Mannheim, cat. no. 1603558) in glass hybridization bottles (Hybaid) placed in a Eurotherm 91E Rotating Hybridization Incubator (Model 310: Robbins Scientific). A DIG-labelled EY.AI5 probe and a control probe for a 121 bp (approximately) fragment of a dispersed autosomal repeated sequence (SINE; 61) were prepared as described above from the inserts of recombinant plasmids, using the SP6 and T7 primers. Each was added to 10 ml of DIG Easy Hyb solution (Boehringer Mannheim) at a concentration of 25-50ng/ml and denatured at 68 0 C for 10 min. The prehybridization solution was replaced by the probe solution and hybridization was conducted in the rotating incubator at 42 0 C for 14-16 h.

The membrane was then removed and washed for 3 x 10 min at low stringency (2 x SSC, 0.1% SDS, 250C) followed by 2 x 10 min at high WO 99/02672 PCT/AU98/00533 37 stringency (0.2 x SSC, 0.1% SDS, 68°C). The washed membrane was rotary-incubated for 1 h in 2 x blocking solution (Boehringer Mannheim, cat.

no. 1585762) containing 1 x maleic acid buffer (Boehringer Mannheim, cat.

no. 1585762).

Anti-DIG antibody labelled with alkaline phosphatase (Boehringer Mannheim, cat. no. 1093274) was added to the blocking solution at a concentration of 0.075 units/ml and rotary incubation continued for a further min.

The membrane was then washed for 2 x 15 min in 1 x wash buffer (Boehringer Mannheim, cat. no. 1585762) and transferred to 1 x detection buffer (Boehringer Mannheim, cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat.

no. 1685627) was diluted 1:100 in detection buffer and 1 ml was added per 150 cm 2 of membrane. The substrate solution was spread evenly between clear transparency sheets and the signal was detected at room temperature using X-ray film (AGFA Eurix RP1) with intensifying screens (Dupont Quanta

III-T).

Fifty plaques of the 100 (approximately) plaques that gave positive signals in duplicate were selected from the 300,000 that were screened and each was removed in a small agar plug; the plugs were stored in SMT buffer at 4 0

C.

A second round of screening was conducted at reduced plaque density (approx. 3,500 pfu) for 20 of the positives. Methods for plaque lifts and hybridizations were identical to those used in first round screening. Sixteen independent clones positive for EY.A15 probe (on duplicate filters) were selected for further investigation. These were: 31.1, 31.2, 31.3, 31.4, 31.5 (originating from five positive plugs selected from plate number 31 in the first screening round), 32.3, 33.1, 33.2, 33.4, 34.1, 34.2, 34.3, 34.5, 36.1, 36.2 and 36.3 (similarly selected from plates 32, 33, 34 and 36, respectively).

Isolation Of DNA From Recombinant Phage: DNA was isolated from five positive clones using host strain E. coli XL1-Blue MRA growing at 37 0 C overnight in 50 ml LB medium supplemented with 0.3% glycerol and 10 mM MgSO4 DNA (200 pg) was isolated using anion-exchange resin under appropriate salt and pH conditions WO 99/02672 PCT/AU98/00533 38 (QIAGEN Lambda Maxi Kit; cat. no. 12562) in accordance with the methods supplied by the manufacturers.

Southern Hybridization Of Phage Inserts With EY.AI5 And EY.AD11 Probes: Twenty Ag of recombinant phage DNA was incubated overnight with units of restriction enzyme EcoRI or HindIII. All restriction digests were carried out with enzymes supplied by Boehringer Mannheim or New England Biolabs in buffers as supplied by the manufacturers and in accordance with their instructions. Aliquots of 10 Ag of digested DNA, together with a DIGlabelled DNA molecular weight marker mix (Boehringer Mannheim, cat. no.

1218603), were electrophoresed in 1% agarose at 80V for 5 h in 0.5 x TAE. Resolved fragments were capillaly-transferred overnight in 0.4M NaOH to a positively-charged nylon membrane (Boehringer Mannheim; Following transfer, the membrane was neutralised with 3 x 10 min washes in 2 x SSC then DNA was uv cross-linked to the membrane (Bio-Rad GS Gene Linker).

All hybridizations were performed according to the DIG System User's Guide for Filter Hybridization (Boehringer Mannheim). Membranes were prehybridized at 42 0 C for at least 2 h in 10 ml of DIG Easy Hyb hybridization buffer (Boehringer Mannheim, cat. no. 1603558) in glass hybridization bottles (Hybaid) placed in a Eurotherm 91E Rotating Hybridization Incubator (Model 310; Robbins Scientific).

DIG-labelled DNA probes were prepared as described above from the two recombinant plasmids containing inserts EY.AD11 and (Example Probe was added to 10 ml of DIG Easy Hyb solution (Boehringer Mannheim) at a concentration of 25-50ng/ml and denatured at 68°C for min. The prehybridization solution was replaced by the probe solution and hybridization was conducted in the rotating incubator at 42 0 C for 14- 16 h.

The membrane was then removed and washed for 3 x 10 min at low stringency (2 x SSC, 0.1% SDS, 25°C) followed by 2 x 10 min at high stringency (0.2 x SSC, 0.1% SDS, 68 0 The washed membrane was rotary-incubated for 1 h in 2 x blocking solution (Boehringer Mannheim.

cat. no. 1585762) containing 1 x maleic acid buffer (Boehringer Mannheim, cat. no. 1585762).

WO 99/02672 PCT/AU98/00533 39 Anti-DIG antibody labelled with alkaline phosphatase (Boehringer Mannheim, cat. no. 1093274) was added to the blocking solution at a concentration of 0.075 units/mi and rotary incubation continued for a further min.

The membrane was then washed for 2 x 15 min in 1 x wash buffer (Boehringer Mannheim, cat. no. 1585762) and transferred to 1 x detection buffer (Boehringer Mannheim, cat. no. 1585762) for 5 min.

The chemiluminescent substrate CDP-Star (Boehringer Mannheim, cat.

no. 1685627) was diluted 1:100 in detection buffer and 1 ml was added per 150 cm 2 of membrane. The substrate solution was spread evenly between clear transparency sheets and the signal was detected at room temperature using X-ray film (AGFA Eurix RP1) with intensifying screens (Dupont Quanta

III-T).

Following detection of the positive signals for EY.AI5 probe (Figure 3a), the probe was stripped from the membrane by treatment with 0.1% SDS in 0.2M NaOH at 68 0 C for 20 min. The membrane was then hybridized using the methods detailed above with the probe for the EY.AD11 sequence (Figure 3b). Most restriction fragments positive for EY.AI5 were also positive for EY.AD11. Phage 31.1 failed to exhibit hybridization with either probe.

Phage restriction fragments were selected for sequence analysis based on their hybridization to both probes. The fragments were alike in size to genomic repeated elements identified by using the same two probes for Southern analysis of equine genomic DNA digested with EcoRI and HindIII.

Fragments selected for sequencing were: a 3.3 kb HindIII fragment from phage 33.1 (similar in size to a HindIII fragment in both male and female genomic DNA at an intensity ratio of approximately a 4.4 kb HindIII fragment from phage 36.1 (similar in size to a HindIII fragment in both male and female genomic DNA at an intensity ratio of approximately 20:1); a 4.7 kb EcoRI fragment from phage 32.3 (similar in size to a genomic EcoRI band which hybridizes to both probes at low intensity in the male and at a barely detectable level in the female); and a 6.0 kb EcoRI fragment from phage 36.1 (present in male genomic DNA but not detected in the female).

WO 99/02672 PCT/AU98/00533 Restriction Mapping Of Recombinant Phage Inserts: Inserts of equine genomic DNA flanked by T3 and T7 promoter sites were excised from the Lambda Fix®II vector by digestion with NotI. This cassette (1 g) was subjected to partial digestion with either EcoRI for page 32.3 and 36.1) or HindIII (for phage 33.land 36.1). The partial digestion fragments, together with fragments of 1 DNA digested with HindIII and EcoRI as size markers (Boehringer Mannheim; cat. no. 528552), were electrophoresed through a 1% agarose gel at 80V for 5 h in 0.5 x TAE with ethidium bromide; the gel was then overlaid with a scale ruler and photographed with uv transillumination (302 nm).

The resolved fragments were transferred to an uncharged membrane (Hybond-N, Amersham cat. no.RPN303N) with 20 x SSC following depurination in 0.25M HC1, denaturation in 0.5M NaOH, 1.5M NaCl and neutralization in 1M Tris (pH 1.5M NaCl The membranes were probed successively with biotinylated oligonucleotide probes for the T3 and T7 promoter sequences, respectively (New England Biolabs cat. nos. 1227-BT and 1223-BT); membranes were stripped before the second hybridization by treatment with 0.1% SDS in 0.2M NaOH at 68 0 C for 20 min.

Prehybridization was done in glass hybridization bottles (Hybaid) placed in a Eurotherm 91E Rotating Hybridization Incubator (Model 310; Robbins Scientific) in 10 ml phosphate buffered 7% SDS solution with 1% (w/v) BSA and 0.5 mg/ml carrier DNA Biotinylated oligonucleotide probe was added to a final concentration of 10 ng/ml. Hybridizaton temperatures were 59 0 C for the T7 probe and 49 0 C for the T3 probe. Post-hybridization washes were in 5 x SSC, 0.1% SDS at 25 0 C for 15 min, then in 5 x SSC. 0.1% SDS at 60 0 C for 15 min. Membranes were blocked with 10 ml of 1 x Blocker SDS, 125mM NaC1, 25rmM sodium phosphate (pH at 0 C for 15 min. Alkaline phosphatase-conjugated streptavidin (Boehringer Mannheim; cat no. 1093266) was added to the blocking mixture to a concentration of 1 unit/ml and the membranes were treated for a further min. Post-treatment washes were in 1 x Blocker for 10 min then in two changes of 0.1 x Blocker for 15 min followed by detection with CDP-Star and autoradiography as outlined above.

Sizes of hybridizing fragments were estimated by comparison with positions of control size markers run concurrently in the gel. The resulting ladder of DNA fragments corresponded to the distance from the T3 or T7 WO 99/02672 PCT/AU98/00533 41 promoter site, respectively, and successive restriction sites (analagous to the ladder generated from labelled primers with dideoxy DNA sequencing).

Because the T3 and T7 promoter sites flank the two ends of the insert, complementary maps were obtained, allowing confirmation of the position of restriction sites.

Analysis of complete digestion products on an ethidium bromidestained gel provided additional information regarding distances separating all adjacent cleavage sites. Restriction maps of phage clones 32.3 (EcoRI), 33.1 (HindIII) and 36.1 (HindIII and EcoRI) are shown in Figures 4a, 5a and 6a, respectively.

Subcloning Of Restriction Fragments From Recombinant Phage: Recombinant phage DNA (7.5 gg) was digested with 20 units of EcoRI (for phage 32.2 and 36.1) or HindIII (for phage 33.1 and 36.1) for 6h at 370C.

Restriction fragments were resolved by electrophoresis in 1% agarose in x TAE buffer containing ethidium bromide. Bands were visualized with uv transillumination (302 nm) and the selected fragments were excised. DNA was recovered using silica-gel membrane technology (QIAquick Gel Extraction Kit; QIAGEN cat. no. 28704) in accordance with the manufacturer's instructions.

The cloning vector was phagemid pBluescript®II KS+ (Stratagene) which had been linearized by digestion with either HindIII or EcoRI then treated with shrimp alkaline phosphatase (Boehringer Mannheim; cat. no.

1758250). Vector DNA was purified as above by gel electrophoresis and the QIAquick gel extraction technique. The linear vector (approx. 20 ng) and phage restriction fragments (approx. 20 ng) were ligated with 1 Weiss unit of T4 DNA ligase (Boehringer Mannheim) in 10 Al of T4 DNA ligase buffer, as supplied with the enzyme, at 15 0 C for 16 h. Ligation reactions were used to transform 100 1l of competent E. coli DH5a cells.

Single colonies of E.coli strain DH5a were inoculated into 250 ml of LB broth and grown in a shaking incubator at 37°C to an optical absorbance of approx. 0.5 at 550 nm. The cells were collected by centrifugation at 3,000 rpm for 5 min at 4°C in an Eppendorf 5414C microcentrifuge, resuspended in 30 ml of cold 0.1M MgC1, and placed on ice for 20 min. The cells were collected by centrifugation as before and the pellet suspended in WO 99/02672 PCT/AU98/00533 42 1 ml of cold 0.1M CaCl2. Glycerol was added to 15% and the competent cells were stored at For transformation, a 100 .l aliquot of competent cells was thawed and mixed with 10 Al of ligation reaction, placed on ice for 30 min, heat-shocked at 42°C for 2 min then returned to ice for 2 min. The transformed cells were allowed to recover by incubation at 37 0 C for 1 h in 900 bl of SOC medium bacto-tryptone, 0.5% bacto-yeast extract, 10mM NaCl, KC1, 10mM MgCl 2 10mM MgSO4, 20mM glucose) and were then plated onto LB agar containing ampicillin (100 /g/ml) for overnight culture at 37 0 C. Eight or sixteen colonies were selected from ligations of each vector/insert pair.

A volume of 3 ml of LB broth containing ampicillin (100 zg/ml) was inoculated with each colony and bacterial suspensions were harvested after 16 h at 37°C in a shaking incubator. Miniprep DNA was prepared from each suspension (Wizard Plus Mvinpreps, Promega cat no. A7500). Purified plasmid DNA was compared with uncut vector by gel electrophoresis to identify clones with plasmids containing an insert of appropriate size.

DNA from these clones was digested with either HindIII or EcoRI, as appropriate, and clones with excisable inserts of the correct size were used for sequence analysis. Insert-containing plasmid clones were as follows: 32.3E1 and 32.3E5 (pBluescript®II with 4.7 kb EcoRI fragments from recombinant phage 32.3); 33.1H2, 33.1H3, 33.1H4, 33.1H6, 33.1H7 and 33.1H8 (pBluescript®II with 3.3 kb HindIII fragments from recombinant phage 33.1); 36.1H1, 36.1H2, 36.1H4, 36.1H6, 36.1H7, 36.1H8, 36.1H9 and 36.1H10 (pBluescript®II with 4.4 kb HindIII fragments from recombinant phage 36.1): and 36.1E2 and 36.1E8 (pBluescript®II with 6.0 kb EcoRI fragments from recombinant phage 36.1).

Single colonies of E. coli DH5a with plasmids 32.3E5, 33.1H2, 33.1H7, 36.1H7, 36.1H1 and 36.1E2 were used to prepare bacterial suspensions in 50 ml LB broth with ampicillin (100 Lg/ml). Preparations of 100-200 pg of plasmid DNA were purified from these suspensions according to supplier's instructions using a QIAGEN Plasmid Kit and QIAGEN-tip 100 resin columns (QIAGEN; cat. no. 12144).

WO 99/02672 PCT/AU98/00533 43 Sequence Analysis Of Subcloned Phage Fragments Hybridizing To Probes And EY.AD11.

DNA sequencing was performed using dideoxy sequencing chemistry utilising the ABI PRISIITM BigDye Terminator Cycle Sequencing Ready Reaction Kit (ABI Perkin-Elmer) with AmpliTaq DNA polymerase according to the manufacturer's instructions (ABI Perkin-Elmer). Where apparent secondary DNA structure in insert 36.1H7 impeded the terminator sequencing reaction, the BigDye Primer Cycle Sequencing Ready Reaction Kit (T7) (ABI Perkin-Elmer) was used. Products of sequencing reactions were analysed according to the manufacturer's instructions on an ABI A377 sequencer at the Australian Genome Research Facility located at The University of Queensland.

A total of 112 sequencing reactions with an average read length of 700 bases were undertaken. Approximately 20 kb of novel equine genomic DNA sequences were recorded then analysed, firstly for homology to sequences described herein as EY.AC7, EY.AM7, EY.AD11 and EY.AI5, and secondly for homology to sequences available worldwide in GenBank (National Center for Biotechnology Information, Maryland, USA [NCBI]) and similar DNA and protein databases.

Primers for T3 and T7 promoter sequences flanking inserts in pBluescript@II vector (Bresatec Custom Oligos) were used for the initial sequencing steps. Sequences were extended from their 3' extremity with 19- 24-mer oligonucleotide primers (Bresatec Custom Oligos) designed for this purpose from known sequence data, then matching this extended sequence to the primary data. Replicate, overlapping and complementary strand sequencing assured the accuracy of the final genomic DNA sequences.

Computer software used to construct contiguous DNA sequences was SequencherTM 3.0 (Gene Codes Corporation) for Macintosh@.

Eight 4.4 kb HindIII fragments subcloned from phage 36.1 proved to be identical, differing only in the orientation of the insert in the vector. This conclusion was based on 100% identity observed within blocks of at least 400 bases of sequence. Application of this criterion established that the two kb EcoRI fragments from phage 36.1 were also identical. Two subcloned EcoRI fragments of 4.7 kb from phage 32.3 were not identical.

Mapping and restriction analysis of phage clone 33.1 (see Figure indicated the presence of two similar 3.3 kb HindIII fragments. Partial WO 99/02672 PCT/AU98/00533 44 sequencing of six subcloned 3.3 kb inserts supported this view. Inserts in plasmids 33.1H7, 33.1H3, 33.1H4 and 33.1H6 were identical; inserts in plasmids 33.1H2 and 33.1H8 were identical to each other but exhibited 88similarity with the former group. Accordingly, inserts in plasmids 33.1H7 and 33.1H2 were sequenced independently.

Sequencing of phage DNA selected for hybridization with EY.AI5 and EY.AD11 sequences (Example 2) revealed that these and the sequences EY.AC6 and EY.AM7 (Example 2) were components of a long range repeat unit in the equine Y chromosome. The structure of the repeat as it was found in equine genomic DNA inserts in phage 32.3, 33.1 (twice) and 36.1 is shown in Figures 4b, 5b and 6b.

Plasmid 32.3E1 contains an insert, possibly part of a pseudogene, with limited homology to open reading frames of a number of unrelated genes, as determined by a database nucleotide and protein translation search (GenBank BLAST 2.0; blastn and blastx programs; NCBI).

The repeat element identified in equine genomic DNA comprises sequences which include the aforementioned repeats EY.AI5, EY.AD11, EY.AC6 and EY.AM7. In addition, EY.AI5 sequence featured as part of a 1500 bp (approximately) unit, hereafter referred to as EY.LINE, found to be approximately 50% homologous at the protein level (GenBank BLAST blastx search: NCBI) to regions of open reading frame 2 (ORF2) in a mammalian long interspersed repeated element (LINE: GenBank accession nos. U93574 (human) and AB012223 These elements, when functional, are 6.0 kb in size and contain a 5' untranslated region (UTR) with an internal promoter, two open reading frames (ORF1 and ORF2) and a 3' UTR that terminates in a polyA tail. ORF1 encodes a nucleic acid binding protein and ORF2 encodes a protein with endonuclease and reverse transcriptase activities. The EY-LINE sequence is located at nucleotides 990- 2497 of SEQ ID NO: 8, 421-1920 of SEQ ID NO. 9, 421 1930 of SEQ ID NO.

10, and 1502-2996 of SEQ ID NO. 11.

LINEs are highly repetitive DNA sequence elements capable of retrotransposition that pervade mammalian genomes. Most are functionally inactive due to truncations, rearrangements and nonsense mutations (67).

LINEs are present in all mammals that have been studied (including marsupials) and are thought to be derived from a common ancestor. When cloned elements from a particular species are compared there may be WO 99/02672 PCT/AU98/00533 differences between individual sequences. Southern hybridizations of restricted genomic DNA give species-specific patterns when hybridized with a LINE probe (68).

The aforementioned four DNA sequences associated with the equine Y chromosome, when used as probes, detect this EY.LINE repeated element.

This element is, in turn, a part of a larger repeated element of 3.5 kb (minimum length) that is evident from comparison of Figures 4b, 5b and 6b.

This explains the similarity between Southern hybridizations using the different sequences as probes. That the EY.LINE described here is specific to Equidae is illustrated by Figure 1 where no hybridization of EY.AI5 is detected to restricted genomic DNA of the camel. The male specificity of EY.

LINE in Equus spp. is illustrated clearly by the data of Figure 1.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

1XIA"% An/nIVIC-71) 46 PCT/AU98/005 SEQUENCE LISTINGS <110) WU Li Dance Company Pty Ltd <120> Determination of genetic sex of equine species by analysis of Ychromosomal DNA sequences <130> 91176 '33 <150> <151> P078 02 1997-07-09 <160> 22 <170> Patentln Ver. SEQ ID NO: 1 <211> 432 <212> DNA <213> Equus caballus <400> 1 ccagaacgga gagaaagcaa tgaaactctg atcacggtga tggtctatta aactagaaga gcgagaagag attccgttct gcaggccttt aactatccta tgtacttgga ccatgttccc aaggaagaca gatcaaacag agggacaagc gg ccaagttcga cctggaaagg ggttgccagg caagttggcc tacccagaaa actctgatta actgagagga ggagaaagtt gggaggagtc acaacttagg gcatggttac tagctttgac caggcataag gaacattctc agaaacctac agacaggtct tatctgaggt agtctgagaa aaacagaagt aaaggactcc atctgttcaa aagaaatcca gagatggctc tgtttatgac ttgccctggc cgtgcacaga ttgccaagaa gtctatgggg SEQ ID NO: 2 <211> 600 WO 99/02672 WO 9902672PCT/AU98/00533 <212> DNA <213> Equus caballus <400> 2 caatcgggtc acttccaagg tccaaataca aacgtaaagc ggcccacatc cctgtggggg cagaacgggt gacagagggc caaccatgat aaggaaatca cagaataacg attgaaaatc tgaagaggaa gctagcgtcc ttcccaagat agaaggcaag gcagatcaca atacataagt aatattccaa tgggacctgc acatacagct acctcaaaag cactaccaag atgatgttcc caggatacag tgcccaggtt ttagcactga agcaattgtg tctatgaaag caaggtggac gtgggggctg tcttactgat agaacccgat tactaatcac gttggtccaa ctgatgggtg tgggaaatag aaggtcggtt catctgatac ttggcagagt aaagagatta gctacagaag ccgtgctaag cttcacgaat aagtaaaagt gcccagccct agatctcatg atcaatacgc act tccacaa gctaagagat gagaacgtga ggtagaccat cccaggaacc aatccaaaca cagggccggc cagtctttca ggacggtgaa cccagaaaat aggaagaagg gacccgattg SEQ ID NO: 3 <211> 230 <212> DNA <213> Equus caballus (400> 3 gtcgtagcgg tttgccggag ataggccaag aacatagagg agaaaggaat ctctggattc catgcaatcc cagtcaaagt ggcagccata agatagaaga gagaatccta aagtgtctag ccagcaacaa gagcccctga 120 gaatcctcag gaaaacgaac aaagcaggac ggatcacact ttctgatttc 180 acgaagcgct aggtaccgaa acggaacagt ccgctacgac 230 SEQ ID NO: 4 <211> 285 <212> DNA <213> Equus caballus <400> 4 WO 99/02672 WO 9902672PCT/AU98/00533 aaccgcggca tgcttaaggc aacaagtttc ctcctctcag agaatcagag tcgactagtc tcttcattgt Cctaatgaga tccttctgga ctttggatta agaaggacac tgtaggtctg ataaggccca agtccggcct cgtgtttgca aagattgaat cagcatggcc tccccataga cttgaacaga tgacaatgtt tgcttgtccc tctcttctcg cttattgcca aggattcctt tcctatgcca tctgtttgat ctcttgcagt ttctgtgccg cggtt SEQ. ID NO: <211> 230 <212> DNA (213> Equus caballus <400> gtcgtagcgg tttgccggag ataggccaag aacatagagg agaaaggaat ctctggattc catgcaatcc cagtcaaagt ggcagccata agatagaaga gagaatccta aagtgtctaa ccagcaacaa gagcccctga 120 gaatcctcag gaaaacgaac aaagcaggac ggatcacact ttctgatttc 180 acgaagcgct aggtaccgaa acggaacagt ccgctacgac 230 SEQ ID NO: 6 (211> 217 <212> DNA <213> Equus caballus <400> 6 kcggagggag agagagatag caaggaatcc aaggaccack gaatgtatgt attgcatgca atcccagtca aagtggcagc catatttgcc aagagagaat cctaaagttt ctagccagca acaagagccc ctgaataggc 120 tcaggaaaac gaacaaagca ggacgtatca cactttctga tttcaaccta 180 cggtaggtac cgagacggat cgtccgc 217 SEQ ID NO: 7 <211> 220 <212> DNA <213> Equus caballus <400> 7 gtagcggaga aaggaatctc tggattccat gcaatcccag tcaaagtggc agccatattt gccagagaga tagaagagag aatcctaaag tttctagcca gcaacaagag cccctgaata 120 WO 99/02672 WO 9902672PCT/AU98/00533 49 ggccaaggaa tcctcaggaa aacgaacaaa gcaggacgta tcacactttc tgatttcaac 180 atagaggacg aagcgctagg taccgaaacg gaacagtcgc 220 SEQ ID NO: 8 <211> 4693 <212> DNA <213> Equus caballus <400> 8 gaattcatca aatttgccaa caggaccacc tcgaatttgg cgggtactca gtgcttcgtt gtgcaaattc aaaaacagca catacggtga ttcaagaaac agcacatcaa agggagatag agggtccagg aaattgcaag gtcagttttt agacaggctc aaacggcagc ggaggtcaaa agcgggctct gaagcccttt ccaatcctct gacgtcacag gagttgggct caagcagcat acatgaaagg ggccggccgg aggggtgggc cgcgtggaat ggccaggagt ccaagggggt ttaacagcaa atacaagagc aacacaacca attacaagtt acgaaaaaca cagcttgcca aggtgaaagc tatgtcaatc aaaaggaaaa agaaggatat tccatagcgg agaaaggaat tttgccagag agatagaaga ataggccaag gaatcctcag aacatagagg acgaagcgct cacagaccca tgcaacagaa catttgcgac cagggagcca atttgtctcg gaaaagcatt gtgtccatga actggattgc agtttctgat tgacctaggc accaagattc acaaaattac ctgagaagac gcgaacagat tgctagaacg cctgggctca gtccatgacg tggccccgac attcaatacc tggtgcagcg cctctgggtg gtagaggcag attgccagca gcaaagaata gtaccatgaa gaaaaccagc tttcagacct ttctggattc gasaatccta gaaaaygaac aggtaccgaa tcgagagagc agaggacaca ctcaggatgt taggatagca acgaaagcac tccctgggtg catgacgagt atcacacctg attgaaagtc ctgggaagtg attgccagga aaacaacctg cattgcgctc gtgaggtecg agcctcctca atgggaaggg gatgtgagca gaaaaaggga gaagatctct aaacctttgg acgtatctaa catgcaatcc aagtttctar aaagcaggac acggmacagt agagactaac gtggaccaag caaagcaacc aagagcacaa tcacacctcg tgaaggagaa atactgtgga aataatccac agagaaagcc tgagctatga cagcagacac gcatcctcta aagtgcaggt cagcttctga ccactgcttc acgttagata tagcgatgca agctaaaaac atgaagaacc ggagagaaat gaattaacac cagtcaaagt ccagyracaa gtatcacact cgcgggctcc tcaaatatac gattgtctct cagatgacca 120 cgagtgacag 180 gcctagagaa 240 tggactgata 300 aaa gaaaaaa 360 gatgaaagat 420 acaagccaaa 480 gacagctcga 540 acacttatga 600 cacagtgcca 660 tgccaatgga 720 gtcagtggcc 780 agtcattctg 840 gggccagact 900 cttctgccaa 960 aaacaaagaa 1020 ttagcaaaac 1080 tcaagaagac 1140 actggagacg 1200 ggcagccata 1260 gagcccctga 1320 ttctgakttc 1380 aaaacaggca 1440 atggacagcc 1500 ccaataaact 1560 gtgcigggaa gcctgcatag ccacaggaac acaacgagag tagaccatga tgttccacct 1620 WO 99/02672 WO 9902672PCT/AU98/00533 ggcaaaggca ccacctgaaa ttttaggaga agacctaggc gaagcctgtc tgacagggca aatgcagacg cttctgccca cctgggagtg gatgtttgca atcaactcac acgtctccac gatacagaga tttctcccaa cacccgcatt aagtcttagt tggtcagaat ggctataagg gagaagggaa gcctcctgca gcggtgtgga actgcctcag cggggccgtg tttagacaaa agttcaccac agccttattc ggacgaatag agaaggacat aggatgcaat ccagccattt aaaaggcccc agggacatag aaagcactaa ccaacaggtg ggcggtggcg agggtgtaag ggcttaagct ggtgaagagg tctgaaagac ataggatatt aatcccaaac gtgggtccag aaattgaaca tccaaggatg agacccattc caaatccatg cccttggacc aaacccaaaa atcatccaaa caggcccaca gtcagggccg gccctgtggt cctcactctt tcacagcccg atgggacggt gcggacagag cgccacagaa aaacaaccat caaaggaaga aggaaggaaa gatgacacga tcattgcgca caggactttc tctggggaag gagccagggt aatcttacag caagttcgag gagaaagtta ctggaaaggg ggaggagtca gttgtcagga caacttagcc aagattaaag agagtgctct agggcagcaa gtcaaggaaa aagcacacat aagaaaaaaa taagatctaa ccaatgcaaa aggcccacag ctgctggtgg aaatttaaga gaactcggaa ccgctctcca gggctatagc gtgaccacca tcaaataccg gcgcaggaca agatgatgag atgtcaacta gtaacgcagg aatgacgaca gaaaatcacc aagaagaatg cattaccgtc tcttcccaag ggagtgggca ggtgcagatc gtcataaaga gggaatatta acatgggacc tcagaggatt gtccagtcac ggaagaaacc gaaaccgaca gactggtctg ttctgaggtt ccctgaatgg ttgccatctg aggagacaag ccatcgactc cggacgaggg acaagcaaat agagggccaa tccaaaccgc gaaaacaaca cagtgtaaac acccatgtcc acacaaccgc agacttggaa cacacatggc gaatggctgg tagcatcaca tttgattggg gcacaagtgt cacggaagtc gttaaggcaa tacagctctg tcaaaaggct cttaccaaga catgatgtta ttcaggccac agtgccctcg acattagcac agtagcaatt caatctatga tgccaaggtg gcctgggcaa ttggccctct agtgtctaga agaaaatcag agatggcact ctttatgaca cacacttgaa tctgagccgc aaacaaacgg actgcaaaga gtgaaaagcc gaaaacctgg caggttcatg aatgacatag agtgtcagag gggtgcagcc cataggatcc taaagacatg gccgcctggt ataccactca gagggtttca cttacaagga ggtgcccaga gtggtgaggt caaatcgatg taaattcacc ggagctaaaa tactgatgct gaccttgatc ctacttaata aggttggtcc ttccgcttgg tgttaggaaa gtgagggtcc aggcatctgg gacttggcag cttcaacttg ccccaagaca gtgaacggag aaaaaggtaa gaaactctgt tcagggcgaa accgtgaaac 1680 ctatttggaa 1740 gaccacctca 1800 cagcacaaca 1860 cacagatgca 1920 gcaaaagatg 1980 aaaacttgtt 2040 cagctcactg 2100 aggaggtgga 2160 actaggccaa 2220 agctattcct 2280 tgcaccgctg 2340 gccgagcaag 2400 gcgggaacaa 2460 gggtaagtga 2520 gaagataaaa 2580 ggcaaagcgg 2640 catgtgattc 2700 aggatgcaaa 2760 tacaatcaaa 2820 gagattagag 2880 acagaagggt 2940 cgtggttaag 3000 accttcacaa 3060 aaaagtacaa 3120 gtggcccggg 3180 tagagatttc 3240 attatcagca 3300 tacacttccc 3360 agtgctagga 3420 ggagggagtc 3480 taagatataa 3540 caggcctttc 3600 actatcctac 3660 gtgcttggag 3720 catgtccccc 3780 WO 99/02672 WO 9902672PCT/AU98/00533 aagatggccg tacccagaaa actctgattc actgagagga gaaacttgat gccttaagca tgcctccttt gcgtcacggg aaatgaatgg gtgtgctgaa ggcttgtgtg gattactgat acaattctgt cacacactat catggttaca cagctttgac caggcataag gaacatcgtc tggaaacacg taatccaaag cctaggcccc ggcccactgg agccctgtca aatcaccctt ctcactttgg tacgtgcttt ttccagaaat agacataaag gtctgagtat aaagagaaag aaagaaatcc atctattcaa aggtcggact tcacgaagga tagcattctt atacatgcta tgcttggggt ttccagaggg ctccaatgaa gacgccatat atagacatgc taagcccgta tggcctaggc cgggtacaga ttggcaagaa gtctagggga tgggccttat tctcttgagg tgaagatcag atgcgtccca aggtctcttt cttggttcct attctctccc ggaattaagc cagggctgag actagacgga tgggctacta aactggaaga gcgagaagag ggccatgctg cagacctaca acattgaata tctcactttc agaaatgtct gttgggaacg ttgtgaaggc cctacctctt tggctgaaaa gctgtaggtc atctagggca aaggatgaca gatcaaacag agggacaagc atgcaagcct gtgtccttct ggagagtcga cataactctg tggaagcctt gcctctccaa tgccctctca cccgtatatg ttagaacatt aaacaaatgg accittcaact 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 4440 4500 4560 4620 gtcaggggca agttcgaacc tttccaaatc cacaaaaaag acagaaaaat *atcattcctg 4680 gagagtggaa ttc 4693 SEQ ID NO: 9 <211> 3430 <212> DNA <213> Equus caballus <400> 9 aagcttgccg gacaaacaac ccacactgcg cggtgaggtc gagcctcctc gatgggaagg agacgtgagc agaaaaacgg agatgtctat tgtttgggga aatggaagaa gcaatcccag tttctagcca ggacagcaga ctggcctcct ctcaactgca cgcagctttg accactgctt gacgttagat acatcgatgc aagctaaaca gaagaacctt gagaaattta ttaacacact ttaaagtgga gcaagaagag cacacaatta ctacactgtg ggtttccaat tgtcggtggc cagtcattct agggccagaa acttctgcca aataaacaaa agcaaaacag agaagacaca ggaaacgtcc agccttcttt cccctgaata tgaccaatcc ccagagttgg agaacatgaa ccgtggtggg ggggcaggcg ttccacagca aaacagaacc aataaacaca gtgaaagcta aggaaaaaga atagcagaga gccagarata ggccaaggaa tctgacgtca gctcaagcag agaggccgcc ccaggtggaa tccgagggcg aatacaggag aataacaagt gcttcccagt tgttaatcga aggatattcc aaggaatctc tagaagagag tcctcaggaa cagtggcccc catattccag 120 ggtggtgcag 180 tcctggggtt 240 tgtagaggct 300 cattgccggc 360 tgcaaagaac 420 acgaagaaga 480 aaatagcaaa 540 gcgacctagg 600 tggattccag 660 aatcctaaag 720 aacgaacaaa 780 WO 99/02672 WO 9902672PCT/AU98/00533 gcaggacgta gtacagtcca cccaactcaa acaaacgaga acgagagtag tgaatggcgc cccatctgtc gagacaagaa atcgactcaa gacgaggggt aagccaatga agggccaaca caaaccgcaa cagcaacaat gtgtaaatgg tccatgtcct ctcaacccgc acttggaagc caatggcata tgaggtttag aaggagaaga tgcccaaagg tggtgagtgc aaatcgatgg aaattcacct ggctgaaaga ctgatgctac cttatccatg atcaccttca ccaaaagtac gtggcccggg tagagatttc ttatcaatac gtcactttcc agtgctagga tcacactttc gtcccaaaaa acatacatgg gtctctccaa accatgatgt acttgaaacc tgagccgccc gcaaacggga ggcaaagaca gaaaagccca aaacctgcgc ggtgcatgaa tgacatagca tgtcagaggg gtgcagccac ttaggatcca taaagacatg cqcctggtgc ccactcattg gtgaagtgaa aaaarlcaagc caaaaaaggg ctgtgattcg ggatgtaaat acaatgaaaa gattagagaa agaagggtag cttagcccag cgaataatcc aagtcaggga cctcactctt atggacrctc gccccagaaa acaaaggaag gatgacacgg tgatttcaac caggcacaca acagcccatt aaaacggtgc tccacctggc cgggaacttn acttggaaga ccacctcaaa gcacaacaac aagatacaat aaaagatgga aacttgttca gctaactgag gaggtggaga taaggcaagc gcgattcctc tgcatcgctg ccaacaagga cggaccagat caggcccagg actaacccaa ccgttttgga gcttacgctg ctgaaagaca atccaaaaag ggcgaacttt agcattccaa gaacccacgg aaacaggccc cggcgctgcg tcacagcacg ggacaaaggk agccaaccat naggaagaaa taattgggca gtggggaagg atagagcacg gacaaatgta tgcgaccagg tgggaagcct acacgcacca ttaggagaag agcctgtctg tgcagacgct tgggagtgga caaatcacac cacagagact ccctcattaa ttcagaatgg gaagggaagc aatgtngiac gggggcgtgt agttcaccac agaatggaga gccatccagc aaatagtgaa caggtggccc aggagaaagg gagaagagga tcggatattc gggtccggaa caaagatgga atacatgaag aaaacgttac acatcttccc gggagcgggc ggtgcagatc yataaatagg gagaatattc acatgggacc tcagaggact tccagccact aagcgctagg acactataga gagccaagag gcacagccac tctgaaattg acctaggcag actcggcaag tctgcccagt tgtttgcaaa gtctccacaa tctcccaaga gtctgaggcc ctataaggag ctcctgcact tggccacaga ttagccaaag agcttactcc agaacatggc cattgtggcc ttccatagca ttggacaatt gatgatnagg tgtcgcctac taatgcaggg tgacggcata aaatcacctc gggaatacta cgtccatgat aagttcaggc aaatgcccat acattagcac tagcaattgt caatcta tga tgccaaggtg gcctgggcaa tggccctttc tactgaaacg 840 gageccagag 900 gacacagtgg 960 atggtacaca 1020 attcaagccc 1080 agcgctctkk 1140 ggcagcaaag 1200 caaggaaacc 1260 gcacacatcg 1320 gaaataaaac 1380 agatctaaag 1440 aatgcaaatc 1500 accgacagga 1560 gctggtggca 1620 aatttaagaa 1680 aaatcggaaa 1740 gctctccaag 1800 tatagccaca 1860 accagatggt 1920 cgtcacttac 1980 tgattggggg 2040 cacaagtgcg 2100 acggaagtct 2160 ttatggtaat 2220 cagctctggg 2280 aaaaggctta 2340 ccatgagaac 2400 gttactacta 2460 cacgggtcgt 2520 gttccgcttg 2580 tgttagqaaa 2640 gaagggtcag 2700 aggcatctgg 2760 gacttggaag 2820 ttcaacttgg 2880 cccaagacta 2940 aagggagtcc aggactttct WO 99/02672 WO 992672PT/AU98/00533 aagagataag aggcctttcc caatcctacc tgcttggagg atgtccccca agggagacat atcaaacaga gggacaagca attcaagctt agccagggtt aagttcgagg tggaaagggg ttgtcaggac agatggccgc acccaaaaac ctctgattcc ctgagaggag atcttacagg agaattttag gaggagtcag aacttagcct atggttatag agctttgaca aggcataaga aacattgtca gaagaaacca aaacttacaa actggtctga tctgaggttg tctgaggatt aacagaaagc aa gga at cct tctgttcaag gtgtctagag gaaataaaga gatggctctg tttatgacat ggc -ctaggct gggcacagaa tggcaagaag tctatgggga agaatggagc aaaaggaaaa gaactctgtg cacggtgaac gggctattaa actggaagag cgagaagaga ggccatgctg 3000 3060 3120 3180 3240 3300 3360 3420 3430 SEQ ID NO: <211> 3450 <212> DNA <213> Equus caballus <400> aagcttgccg gacaaacaac tcccattgcg cggtgaggtc tgaggctcct agatgggaag cagatgtgag tagaaaaacg aagaagatct gcaaacgttt ctaggaatgg tccattcaat taaagtttct acaaagcaga acacggcaca cagagcccaa agcggacaaa gcacaacgag agccctgaat ctttgccatc ggacagcaga ctggcctcat ttcaactgca cgcagattct caccactgct ggacgttaca aacagcgatg gatgctaaaa ctatgaagac ggggaaagaa aagaattaac cccagtcaaa agccagcaat acgtatcaca gtccgggccc ctcaaacata ggagagtctc agtagaccat ggcacacttg tgtctgagcc cacacaatta ctacacagtg ggttgccaat gggtcggtgg tcagtcattc tagggccaga cacttctgcc acaaacaaac acttagcaaa attcaagaag acactggaaa gtggcagcca aagagcccct ctttctgatt aaacacagac catggacggc tccaataaac gatgttccac aaaccgtgaa acctatttgg tgaccaatcc cgagagttgg ggaacatgaa acaggggtgg cggggcaggc cgtccacatc aaaacacaac aaacaaaaaa acaggtgaaa acacaaggaa cgaccatagc tcttcgtcag gaataggcca tcaacataga acacagaccc ccatttgcga gctgctggga ctggcacacg act tgtagga aagaagcctg tctgatgtca gctcaagcag acggccggcc gccgggtgga gtccgcgtgg aaatacagga caattacaag cacagcttgc gctatgtcaa aaagaaggat ggagaaggga agagatagaa aggaatcctc ggacgaagcg atgcaacaga ccaaggagcc agtctgaaca cagaacctga gaagacctag tctgactggg cagtggcccc catattcaag ggtggtgcag atcctggggg ttgtagaggc gcattgccag ttgaaaagat cagtacgaag tagaaaacca ataccgggac atatctggat gagagaatcc aggaaagcaa ctaggtaccg atcgagagcc aagaggagac gccacatggt aatggattaa gcagagtgct caagggcagc 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 WO 99/02672 WO 9902672PCT/AU98/00533 aaaggagaca aaccatcgac atcggactaa aaacaagcca aaagagggcc aatccaaacc aggaaaacaa actgtaaacc tccatgtccc cacaaccgcc agacttggaa gccacacaat ccagaatggc ccgtaggatc acatttgatt gatgaggcaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nyacntcaaa atactaccaa catgatgttg ttcaggccac gtgaccacgt cattagcact gtagcaattg aatctatgaa gccaaggtgg cctgggcaac tggccctctc gtgtctagag gaaatccaga gatggctctg tttgtgacat ggcctagcct gggcacagaa agaaacaaac tcaatgcaaa gggtgaaaag atgaaaacct aacaggtgca gcaatgacat ttgtcagaga ggtgcagcca ataggatgcc taaagacatg gccaccctgg ggcaaaccac tgggaggctt tcactttcaa gggggtgccc atgggtgtga nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn aggcttactg gagaacctga ctactaatca aggttggtcc tccgcttggg gtaaggaaat tgagggtccg ggcatctggt acttggcaga ttcaacttgg cccaagacat agaatggagc aaaaggaaaa aaactctgtg cacggtgaac gggctattca actggatgag gggaccacct gacagcacaa cccaaagata gggcaaaaga tgaaaacttg agcagctcac ggaggtggag ctatgccaag gctattcctc tgcaccgctg tccccagcaa tcagcgggaa ttaggggaag ggagaagata agaggcaaag cnnnnnnnnn nrinnnnnnnn nnnnnnnnrnn nnnnnnnnnn atgcgacaga tccatgctaa ccttcaggaa aaagtcaagt tggcccgggc agagatttca ttatcaatac acacttcccc gtgctaggag gatggagtcc aagagataag aggccattcc ctatcctacc tgcttggagg atgtccccca agggggacat atcaaacaga caaatgcaga caactgggag caatcaactc tggacacaga_ ttcaccctca tgtggtcaga agaagggaag cggtgtggaa gggggcgtgt agttccccac gggacgaatg caaaggatgc tgaaacaggc aaagaagaac cggggccatg nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn agggtagcca acccaggaac taatccaaac ccagtttcgg ctcactcttt tgggacggtg accccagaaa aaaggaagaa atgacaggat aggactttct agczcagggta aagttcgagg tggaaagggg ttgtcaggac agatggaggc acccaaaaac ctctgattcc cgcttctgcc tggatgtttg acacgtctcc gatttctccc ttadgtctga atggctataa ccgcctgcac ctgcctcagg ttagccaaag agccttactc gagaaggaac aatccagcca cccagggaca taaccaacag ggggaggagt nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn tticcaaatac caacggaaaa aggcccacaa ccctgtggtg cacagcacgg cggacagagg agcatccatt ggaaggaaaa cattgggcat ctggggaagg atcttacagg agaaagttag gatgagtcag aacttagccg atggttacag agctttgaca aggcataaga cagtcaagga caaagcacac acaagaaaaa aagaagatct ggccaatgca ggagtccgac tgctggtggc aatttaagaa aactcggaaa ccgctctcca atgggctata tttgtgagca tagtcaaata gtggcgctgg gacagagata nnnrinnnnnn nnnnnnnnnn nnnnnnnnnn nnrinnnnnnn atgtagagga tgttaccgtc cttccccaag gatcgggcaa gtgcagatca tcataaagaa agaatattcc catgggacct ctgaggatta tccagccact gaagaaacca aaaccgacaa actggtctga tctgaggttg tctgagcatt aacagaaagc agggaatcct 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 WO 99/02672 WO 9902672PCT/AU98/00533 tggcaagaag cgagaagaga gggtcaagca ctgacaggag aacatttccc tctgttcaag 3420 actataggga ggccatgctg attcaagctt 3450 SEQ ID NO: 11 <211> 4344 <212> DNA <213> Equus caballus <400> 11 aagcttgaat tgcttgtccc tctgcctgat atgtctccct tgggggacat cctccaagca ggtgattact atcaggttct atcagtaaga acttgggacc tccagccatt cagggacata ccaacaggtg gagggtgaaa tggggaagag cataggatgt agtggtccag tcaaaggatg aaatacatga ggaaaatgtt cctcatcttc gtggtggagc gcacgggtca aggtcataaa cagatgtgat tagaaaaccg aagaagatct cagcatggcc tctcttctcg ctcctccagt ttaatagccc gttcaccgtg cacagagttt agtaacatca ctttgtagta cttttcaggt tgccaaggtg tgtgaccatc gtcaaacacc gcgctggaca gagatgatga gatgtcacct tctaatgcag aatgactcca gaaaatcacc agaggaatac accgtccatg ccaagatcag gggcaagtgc gatcccatta aggacgttag cacaacgatg gaagctaaaa ctatgaagaa tccccataga cttcttgcca ttctgtgccc agcctaggcc atgtcataaa cagagccatc tggagggtag ttcctcttca gattttctgg gtcttggcag agaatgggtg ataggattca tttgattggg ggcacaagtg acacggaagt ggttatggca taaagctctg tcaaaagtct tacaaagaga atgttactac gccacaggtt ccacgttccg gccttgt tag ataaggcaga cacttctgcc acaaaaaaac cttgaacaga aggattcctt gctttctgtt aatcctcaga caacctcaga tcagaccagt cctttcagtt tgtatctgga tacacttccc agtgctagga ggagggtttt ctttcaagga ggtgcccaga tgtggtgagg ctaaatcgat ataaattcac ggggctgaaa tagtgatgcg acctgatccg taatcacctt ggtccaaaag cttgggtggg gaaatagaga tttccacagc aaaaczacaac aaaaacaaaa tgacagtgtt tcttatgcct tgtcaaagct ctgtaaccat aggctaagtt ctgactcctc tgttcctgga atggtgtacc caaagcaaga gatgagaaca aggggaagtg gaagatataa ggcaaagccg gcctatgatt gaggatgtaa ctacaatcaa gagattagag acagaagggt tgctaagccc catgaataat tacaagtcag cagggcttca tttcttggac aaatacagga cacttacaag cacagcttgc ggtatgtcaa ctcctctcag ggaatcagag gtttctgggt gcggccatct gtcctgacaa ctcctttcca cttagcactg cttctgtcgc aggaaggaga aaggatgcaa aaacaggccc gaagcactaa ggcagtgggg cggcttacgc atctgagaga aaatccaaaa acggtgaact agaccattcc aggaaccaac ccaaacaggc ttccggctct gtctttcaca ggtcggacag gcattcccag ttgcaaagat cactacgaag tctaaaacca 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 ccttagcaaa acaggtgaaa WO 99/02672 WO 9902672PCT/AU98/00533 acaaacgttt ggggaaagaa ctaggaatgg aagaattagc tccatgcaat cccagtcaaa taaagtttct agccagcaag acaaagcagg acggatcaca aaacggcaca gtccgggCc cacagcccaa ctcaaacata agtggacaaa ggagagtctc acaagaatag accatgatgt tggccacttg aaccgtgaac ctgacccgcc tatttggaag aacaaacggg accacctgaa atgcaaagac agcacaacac cgaaaagccc aaagatacaa aaaatctggg caaaagatgg ggtgcatgaa aacttgttca catagcagct cacttgtggt tcagagagga ggtggagagt cagccagtag gccaagcggt gatccagcta ttcctcgggg gacatgtgca ccgctgagtt tcctggtgcc gagcaaggga taccagtcag tgggaacaaa agagttttat gggaagtgaa ttacaaggag aagataaaaa gtgcccagag gcaaagcggg gtggtgagga catgtgattc taaatcgatg agcatgtaaa taaattcacc tacaatcaaa gggctgaaag agattagaga actgatgcta cagaagggtg ctgatcagtg ctaagcccag atcaccttcg cgaataatcc tccaaaagta caagtcaggg gggtggccag ggcctcactc attcaagaag acacttgaaa gtggcagcca aaaagcccct ctttctgatt aaaaacaggc catggacacc tccaataaac tccacctggc ttgtaggaga caggctgtct atgcagacgc ctgggagtgg tcaactcaca acacagagat ccctcattag cagaatggct aggaagcctc gtgaaactac gcgtgtttag caccacagcc cgaatgtaga ggttgcaatc acaqgaccca aagcactaac gcggtggggg ggcttatgct tgtgaaagac aatccaaaaa aggtgaactt accattccaa gaaccaacgg aaacaggccc aagccctgtg tttcacagca acacaaggaa cgtccataga tatttgccag gaataggcca tcAacataga acacagaccc ccatttgcga gggctgggaa agacgcccac agacctagca gactgggcaa ttctgcccag atgttagcaa cgtctccaca ttctcccaag tctaaggcca ataaggaggc ctgcactgct ctcagcaatt ccaaaaaact ttactcccgc aggaacatgg cagtcatttg gggatatagt caacaggtgg agggttaaag ggtgaagagg atagggtgtt ggggtccaga ccaaggatgg atacttgaag aaaacgttac acatct tccc ggggagcggg cgggtgcaga taagaaggat ggagaaagga agagatagaa aggaatcctc ggacgaagcg atgcgacaga ccagggagcc gcctgacagc tgaatggatt gagtgctctt gggcagcaaa tcaaggaaac agcacacatc agaaaaaaaa aagatctaag atgcaatcca agacaggaaa ggtggcagtg tcagaatccg cggaaacaca tctccaagac gctatagcca cgaccaccag caaataccgt cgctggacat acttgatgag atgtcaccta ctaatgcaag atgacggcat aaaatcacct aggaatacta catccatgat aagttcaggc caagggccaa tcacattagc attccgggac 1680 atctctggat 1740 gagagaatcc 1800 aagaaaacga 1860 ctaggtactg 1920 atcgagagcc 1980 aagaggagac 2040 cacatagaac 2100 caagccctga 2160 tgccatctgt 2220 ggagacaaga 2280 catcgactca 2340 ggatgagggg 2400 caagccaatg 2460 agggccaaca 2520 accgcaatga 2580 acaacaagtg 2640 taaacgggtg 2700 tgtaccatag 2760 aactcctaaa 2820 ttggaagccg 2880 cacaatggca 2940 aatggcttgg 3000 agcataacac 3060 ttgattgggg 3120 gcacaagtgt 3180 cacggaagac 3240 gttatggcac 3300 acagctctgc 3360 caaaaggctt 3420 ccaagagaac 3480 gttactacta 3540 cacatgttgg 3600 cgttccgctt 3660 actgttagga 3720 aatagagatt tcatgggacg gtgcggacag aggacataaa gaagtagcaa ttgtgagggt 3780 WO 99/02672 PTA9/03 PCT/AU98/00533 cctttatcaa ggaacacttc agagtgctag tgggagggag cataagagca accctttcca tatcctacct gctgggaggt tgtcccccaa ctgagacata tacgccccaa cccaaaggaa gagatgactg tccaggactt agagccaggg agttcgagga ggaaaggggg tgtcaggaca gatggccgca cccagaaaaa aaaagcaacc gaaggaagga gatcattgca tctctgggga tatcttacag taaagttcga aggagtcaga acttagactt tggttactgt gct t atgacagtat aatcatggga catcagagga aggtccagcc ggaagaacca aaccgacaag ctggtctgag ctgaggttgt gtgaggattg tccaatctat cctgccaaga ttgcctgggc acttatccct gtgtctagag aaatccagaa atggctctga ttatgacata gcctaggctg gaaggcatct tggacttggc aacatcaact ctctccaaga agaatggaca aaaggaaaac aactctgtgt acggcgaaca ggctattaaa 3840 3900 3960 4020 4080 4140 4200 4260 4320 4344 SEQ ID NO: 12 <211> 21 <212> DNA <213> EqLUS caballus <400> 12 aogcgagaaa ggaatctclg g SEQ ID NO: 13 <211> 22 212 DNA <213> Equius caballus <400> 13 tacctagcgc ttcgtcctct at SEQ ID NO: 14 <211> 23 <212> DNA <213> EquuIs caballus <400> 14 Itcoiccl ct atollgaaat cag SEQ ID NO: WO 99/02672 WO 9902672PCT/AU98/00533 58 <211> 21 <212> DNA <213> Equus caballus <400> gtcgtagcgg agaaaggaat c 21 SEQ ID NO: 16 <211> <212> DNA <213> Equus caballus <400> 16 aocgygactgtl tccgtttcgg SEQ ID NO: 17 <211> 21 212 DNA <213> Equus caballus <400> 17 gcccagtgtt tcgttggltc g21 SEQ ID NO: 18 <211> <212> DNA <213> Equus caballus <400> 18 catagttgta tattcttcgt tgtog SEQ ID NO: 19 <211 >41 <212> DNA <213> EquuILS caballus WO 99/02672 PCT/AU98/00533 59 <400> 19 gcggtcccaa aagggtcagt agcggagaaa ggaatctctg g 41 SEQ ID NO: <211> 42 <212> DNA <213> Equus caballus <400> gcggtcccaa aagggtcagt tacctagcgc ttcgtcctct at 42 SEQ ID NO: 21 <211> 41 <212> DNA <213> Equus caballus <400> 21 gcgtccaa aagggytcaat gcccagtgti tcgtgagttc a 41 SEQ ID NO: 22 <211> 212 DNA <213> Eqtius caballus <400> 22 gcgg9tcccaa aagggtcagt catagttgta tattcttcgt tgtggl WO 99/02672 PCT/AU98/00533

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Claims (24)

1. An isolated polynucleotide, the polynucleotide having a sequence as set out in any one of SEQ ID NOS: 1 to 4 or 8 to 11, or a sequence which hybridises thereto, wherein the polynucleotide hybridises specifically to the equine Y chromosome.

2. An isolated polynucleotide, the polynucloetide having a sequence characterised by nucleotides 990-2497 of SEQ ID NO: 8, 421-1920 of SEQ ID NO. 9, 421 1930 of SEQ ID NO. 10, or 1502-2996 of SEQ ID NO. 11 or a sequence which hybridises thereto, wherein the polynucleotide hybridises specifically to the equine Y chromosome.

3. An isolated polynucleotide as claimed in claim 1 in which the sequence shares at least 60% homology with a sequence shown in any one of SEQ ID Nos 1 to 4 or 8 to 11, wherein the homology is calculated by the blastn program as herein described.

4. An isolated polynucleotide as claimed in claim 3 in which the sequence shares at least 80% homology with a sequence shown in any one of SEQ ID Nos 1 to 4 or 8 to 11, wherein the homology is calculated by the blastn program as herein described. An isolated polynucleotide as claimed in claim 3 or claim 4, wherein the sequence shown in any one of SEQ ID Nos 1 to 4 or 8 to 11 is characterised by nucleotides 990-2497 of SEQ ID NO: 8, 421-1920 of SEQ ID NO. 9, 421 1930 of SEQ ID NO. 10, or 1502-2996 of SEQ ID NO. 11

6. An isolated polynucleotide as claimed in claim 1 which has a sequence as set out in SEQ ID NO: 3 or a sequence which hybridises thereto.

7. A vector including a polynucleotide sequence as claimed in any one of claims 1 to 6.

8. A host cell including a vector as claimed in claim 7. 'W 67

9. An oligonucleotide probe or primer of at least 8 nucleotides that hybridises specifically to the equine Y chromosome, the oligonucleotide having a sequence that hybridises to a polynucleotide as claimed in claim 1 or claim 2. An oligonucleotide probe or primer as claimed in claim 9 which is at least 10 nucleotides in length.

11. An oligonucleotide probe or primer as claimed in claim 10 which is at least 18 nucleotides in length.

12. An oligonucleotide probe or primer as claimed in claim 11 which includes a sequence selected from: AGCGGAGAAAGGAATCTCTGG, (SEQ ID NO: 12) or TACCTAGCGCTTCGTCCTCTAT (SEQ ID NO: 13). 9

13. An oligonucleotide probe as claimed in any one of claims 9 to 12 in which the probe is conjugated to a detectable label.

14. An oligonucleotide probe as claimed in claim 13 in which the label is selected from a radioisotope, an enzyme, biotin, a fluorescer or a chemiluminescer. 25 15. A method of determining the sex of a horse, an equine fetus, an equine embryo or an equine cell(s) which method includes analysing a biological sample derived from the horse, fetus, embryo or cell(s) for the presence of a polynucleotide sequence as set out in any one of SEQ ID NOS: 1 to 4 or 8 to 11, wherein the presence of the polynucleotide in multiple copy number is indicative that the biological sample is derived from a male.

16. A method according to claim 15 wherein the multiple copy number is greater than 5 copies in the haploid genome.

17. A method according to claim 15 or claim 16 in which the biological sample includes one or more sperm cells. WO 99/02672 PCT/AU98/00533 68

18. A method according to claim 15 or claim 16 in which the biological sample includes nucleated fetal cells.

19. A method according to claim 15 or claim 16 in which the analysis involves Southern blot hybridisation, dot blot hybridisation or in situ hybridisation. A method according to claim 19 in which the analysis involves the use of an oligonucleotide probe as claimed in any one of claims 9 to 14.

21. A method according to claim 15 or claim 16 in which the analysis involves the polymerase chain reaction or ligation amplification reaction.

22. A method according to claim 21 in which the analysis involves the use of an oligonucleotide primer according to any one of claims 9 to 12.

23. A kit for sex determination of a horse, an equine fetus, an equine embryo, an equine cell or a population of equine cells, which includes a polynucleotide as claimed in any one of claims 1 to 6 or an oligonucleotide probe or primer as claimed in any one of claims 9 to 14.

24. A method for the isolation of polynucleotides which are specific for the Y chromosome which includes: pooling substantially equivalent amounts of genomic DNA from two or more male animals of a single species and pooling substantially equivalent amounts of genomic DNA from a similar number of female animals of the same species; (ii) subjecting substantially equivalent samples of the male and female pooled DNA mixtures to PCR or LCR with an arbitrary oligonucleotide primer and resolving the resultant amplified polynucleotides by gel electrophoresis; and (iii) isolating polynucleotide(s) from the gel that are amplified from male DNA but are not amplified from female DNA.

25. A method according to claim 24 in which the male and female animals in step are siblings. WO 99/02672 PCT/AU98/00533 69

26. A method according to claim 24 or claim 25 which includes the additional step of confirming that the isolated polynucleotide(s) from step (iii) is specific for the Y chromosome by using the isolated polynucleotide, or fragment thereof, as a primer in PCR reactions performed on DNA samples isolated from individual male and female animals, wherein the presence of an amplified product following the PCR reaction on DNA isolated from the male, but not the female, is confirmation that the polynucleotide is specific for the Y chromosome.

27. A method according to claim 24 or claim 25 which includes the additional step of confirming that the isolated polynucleotide(s) from step (iii) is specific for the Y chromosome by using the isolated polynucleotide, or fragment thereof, to probe samples of male and female genomic DNA, wherein a hybridisation signal indicative of multiple copy number in the male DNA, but not the female DNA, is confirmation that the polynucleotide is specific for the Y chromosome.

28. A method according to claim 27 wherein the multiple copy number is greater than 5 copies in the haploid genome.