Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-03-22
2002-09-24
Fredman, Jeffrey (Department: 1655)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200, C536S024310, C536S024300, C536S023100
Reexamination Certificate
active
06455252
ABSTRACT:
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 (i.e. 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 recovers 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 (1,2) 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: intra-cytoplasmic 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.
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 (3-8). 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 (5′-G.A.C/T.A-3′; 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 polynucleotide isolates capable of specifically hybridizing to samples of 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 (24), 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 (i) employ low ionic strength and high temperature for washing after hybridization, for example, 0.1×SSC and 0.1% (w/v) SDS at 50° 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×SSPE, 10% (w/v) polyethylene glycol 6000 (27), 7% (w/v) SDS (28), 0.25 mg/ml fragmented herring sperm DNA at 65° C.; or (iii) for example, 0.5M sodium phosphate, pH 7.2. 5 mM EDTA. 7% (w/v) SDS (28) and 0.5% (w/v) BLOTTO (29.30) at 70° C.: or (iv) employ during hybridization a denaturing agent such as formamide (31), for example, 50% (v/v) formamide with 5×SSC, 50 mM sodium phosphate (pH 6.5) and 5×Denhardt's solution (32) at 42° C.; or (v) employ, for example, 50% (v/v) formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% (w/v) sodium pyrophosphate, 5×Denhardt's solution (32). Sonicated salmon sperm DNA (50 &mgr;g/ml) and 10% dextran sulphate (33) at 42° C. See generally references 34-36.
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 60% 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, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. And Lipman, D. J. (1997) “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Research 25(17):3389-3402.
In a further preferred embodiment, the polynucloetide sequence of the present invention hybridises under stringent conditions to a sequence characterised by nucleotides 9
Harrison Bruce Thomas
King Brian William
Murphy Kathleen Margaret
Reed Kenneth Clifford
Wade Nicholas Michael
Einsmann Juliet C.
Fredman Jeffrey
Nixon & Vanderhye
Wu Li Dance Company Pty Ltd.
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