FR2648151A1 - CHROMOSOME Y BOVINE-SPECIFIC BOVINE MINISATELLITE - Google Patents

Abstract

The invention relates to a nucleotide sequence specific to the Y chromosome of different species in the bovine subfamily. The sequence consists of a motif of 40 base pairs present in the bovine Y chromosome involving some 100,000 copies. The invention also relates to methods for sexing different bovine subfamily species, which implement said sequence, as well as to nucleic probes and kits for carrying out these sexing methods. The sequence may also be used in the study of the male genealogy of species belonging to the bovine subfamily making use of the polymorphism brought to light by said sequence.

Description

MINI-SATELLITE BOVINE SPECIFIC TO CHROMOSOME Y
VEAL
The possibility of determining the sex of animals in the embryonic state is a subject of study and research of primary importance. This concern is of particular interest in the case of livestock. Indeed, the economic value of livestock often depends on whether the animals are male or female.

 In mammals, a general method of sexing embryos consists in separating, before in-vitro fertilization, the spermatozoa carrying the sex chromosome Y from those carrying the sex chromosome X.

 However, despite extensive research, there is currently no effective sexing method based on the separation of spermatozoa bearing X and Y chromosomes.

 The development of embryo transfer techniques, and embryo preservation techniques, such as cryopreservation, have led scientists to devise sexing methods that can be used with embryos that are at an early stage of development. early, especially before implantation in a female recipient.

 These different processes show varying efficiencies.

 They are based either on a cytogenetic analysis of embryonic cells or on the search for the presence of the H-Y antigen characteristic of the male genome.

 Other methods consist of colorimetric assays indicative of G6PD glucose 6-phosphate dehydrogenase activity (Anderson, G. B. 1986. See bibliography).

 The possibility of sex determination of human embryos using DNA probes specific for the human Y chromosome has also been shown (West, J.D., et al., 1987).

Specific Y sequences in domestic animals would be potentially very powerful tools to determine the sex of preimplantation embryos before they are transferred to a recipient mother, as well as in the search for separation of sperm carrying the chromosome
Y or X. Such a male-specific sequence in pigs has been recently published (McGraw et al., 1988).

 At the molecular level very little is known about the bovine chromosome Y. Livestock have two metacentric sex chromosomes that are easily distinguishable from acrocentric autosomes. The Y chromosome represents only 2.2% of the total chromosome length and is much smaller than the X chromosome.

Leonard et al. (1987) reported a sequence
Y specific bovine of which 2000 to 2500 copies would be present, later mapped on the short arm of the Y chromosome by Popescu et al. (1988).

 Bondioli et al. (1989) isolated three chromosome fragments specific to male bovine and present at 2600, 600 and 20 copies per genome respectively. The nucleotide sequence of these fragments has not been determined. These fragments served as probes in the sexing of bovine embryos and gave reliable results with a sample containing the DNA of two embryonic cells, (approximately 15 ng). The duration of these tests is 8 days, which requires the freezing of the embryos after the removal of the cells.

 European Patent Application 235,046, published on 02/09/1987, describes a DNA probe specific for the male genome of ruminants, for the determination of the sex of embryos, making it possible to obtain a significant result with 50 ng of DNA.

 PCT Patent Application WO 86/07095, published on 04/12/1986, also discloses DNA probes specific for the male genome of bovine animals, which hybridize in a much greater manner with a male DNA sample than with a female DNA sample, when the hybridizations are carried out under the same conditions.

 It has been known, on the other hand, that the human or animal genome comprises hypervariable sequences, consisting of monotonically repeating DNA fragments of which one fraction is polymorphic: the number of repeated DNA fragments differing from one another. individual to another, for a given place in the genome.

 A large part of the genome is composed of these tandem repeat sequences called "satellite DNA". According to the methodology initially used for their study: isopycnic centrifugation, electrophoresis in agarose gels, the satellite sequences were distributed in three classes of different sizes: macro-satellites, midi satellites and mini-satellites. Although macrosatellites appear to be localized in the heterochromatic regions (Singer, 1982), the mini-satellites are distributed throughout the genome with, however, greater concentrations in the proterminal regions (Royle et al., 1988).

The only satellite-satellite so far described has been mapped on the short arm of human chromosome No. 1 in lp (Nakamura et al., 1987b). The function of these satellites, as far as it exists, remains to be determined. The organization of tandem repeat sequences, an important feature of any satellite, depends on a concerted evolution of these repetitions. It is recognized that this concerted evolution would result from a series of unequal "crossing-over" (or any other mechanism conforming to the so-called "card deck" model (Doolittle, 1985)), which are favored by tandem repetitions same. The unequal "crossing over" mechanism, whether between sister chromatids or between homologous chromosomes, is compatible with size polymorphisms as well as with the observed internal site polymorphism for satellites. It is generally estimated that the satellite sequences represent -238 of the bovine genome. Eight cattle macro-satellites were initially described, of which five probably evolved from a common ancestor (Singer, 1982). No satellite studied in the early publications significantly hybridized to the Y or X chromosomes (Kurnit et al., 1973). The generation of genetic fingerprints with a number of minisatellite probes suggests the existence, in this species too, of several families of hypervariable minisatellites (Georges et al., 1988).

Three regions of the human Y chromosome are characterized by satellite sequences: (1) The heterochromatic region of the long arm, essentially formed by DYZ-1 and DYZ-2, the Y-specific representatives of human satellites 3 and 1 respectively (Goodfellow et al. , 1985). (2) A specific DYZ-3Y alpha satellite has been recognized in the centromeric region (Goodfellow et al., 1985). (3)
At least two tandem repeat sequences are mapped in the pseudoautosomal region (Simmler et al., 1987).

 In the present application, a bovine satellite sequence located in the pericentric region of Y choromosome is described. The first steps of the isolation of this sequence could be carried out thanks to a sequence described by SHIN et al. (1985).

This publication describes a DNA sequence isolated in the Drosophila Per gene, which repeats monotonically and encodes about 50 threonines and alternating glycines.

 The good functioning of this Per gene is particularly responsible in a Drosophila 3, certain periodic behaviors.

The DNA sequence encoding this Per gene also has a high degree of polymorphism (Yu,
Q., et al. , 1987).

 In the article by SHIN et al. referenced above, it has been shown that this "minisatellite" region of the Per gene was able to hybridize with DNA samples from different species of vertebrates, such as chicken, cat, mouse and man.

 Thus, it has been possible to clone a fragment of surties called pSP64.2.5EI, partially homologous to the Per gene, whose nucleotide sequence comprises a mini-satellite region comprising the motifs ACNGGN and TCAGGC in which N represents any one 4 nucleotides adenine, cytosine, guanine and thymine (Shin et al 1985).

 As a probe, this particular piece of DNA, pSP 64.2.5EI, recognizes a large number of polymorphic sequences in humans and different animal species, giving rise to genetic fingerprints (Georges et al., 1987b & 1988).

The use of this probe on cattle DNA, which has been restricted by one of the restriction enzymes HaeIII, HinfI, or MboI, presents in females a typical genetic fingerprint image whereas the image in the males are masked by an intense signal (Figure 1). It has thus been shown that this probe, already known for its ability to detect mini-satellite DNA regions in the human or animal genome, was also able to detect the presence of the Y chromosome in a ruminant DNA sample. (see French patent application 88/10706).

This phenomenon has suggested to the inventors that pSP64.2.5EI restores specific Y sequences in cattle outside the different autosomal loci.

 Following this fortuitous observation, the inventors used this "per" -like clone pSP64.2.5EI in the first step of isolating the sequence likely to be specific to the bovine chromosome Y. They then cloned and characterized this DNA sequence, which has a size of 4.2kb and which was called, btDYZ-1, and mapped in the centromere Y region.

 The btDYZ-1 sequence was digested with the restriction enzyme EcoRI, and the 1.2 kb EcoRI fragment of this btDYZ-1 sequence, called Y1.2, was sequenced.

 It was thus found that this fragment is formed of a pattern (called "X") of 40 base pairs repeated 17 times and whose divergence between the units is 27%. The X motifs are separated from each other by TG-rich sequences (called "Z") of variable length. The X and Z designations are defined more precisely below.

Using the btDYZ-1 or Y1.2 segments as probes in hybridization experiments, the inventors then demonstrated that these segments of Y chromosome-specific DNA make it possible to sexate bovine animals from 100 picograms of DNA. bovine (the equivalent of about 15 cells); that these DNA segments recognize a sequence present on the Y chromosome in the number of about 100,000 copies; by in situ hybridization that the cloned DNA segments correspond to a pericentric zone of the bovine Y chromosome; that the cloned segments, used as a probe, demonstrate a restriction fragment size polymorphism (RFLP), for example with Hae
III, specific to the Y chromosome and make it possible to determine parent-based relationships.

only on the transmission of Y material; - that the new molecular probes are usable for the sexing of other bovine species.

In addition, as soon as the precise sequence of Y1.2 was elucidated, the chain poly-chain reaction (PCR) technique could be used to amplify the sequence. The presence of chromosome DNA
Y bovine was thus detected from 0.5 μg of purified bovine genomic DNA. The amplified sequences were directly visualized by fluorescence after addition of ethidium bromide.

The subject of the invention is an isolated nucleotide sequence specific to the Y chromosome of the various species of the subfamily of bovine animals, characterized in that it comprises - the sequence
Aa - [(X) p- (Z) n- (W) q] m-Bb in which
X = CNNTTNTCAGCCCTGTGCCCTGGNRAYTGTGNAANNNGTT
N = any of the 4 thymine nucleotides
(or uracil), guanine, adenine and cytosine;
R = purine;
Y = pyrimidine, or a fragment thereof provided that at least 12 consecutive nucleotides are present, or a sequence having at least 70% homology therewith;
Z = a TG-rich nucleotide sequence containing at least 50%, preferably 80% TG, w = any nucleotide sequence that is not X or Z;
A and B = nucleotide sequences substantially free of sequences present in the bovine Y chromosome;
p> l
nO
q # 0
mrl
a = O or i
b = 0 or 1 and the X, Z and W sequences may be in the order indicated o0 in a different order; provided that the X sequence represents at least 1 *, preferably at least 30X, of the total length of the [(X) p- (Z) n- (W) q] m portion and that when the values of p, not; q and / or m are greater than 1, the multiple copies of the X, Z, W and / or [XZW] sequences respectively may be identical or different; - the sequence A @ - [S] -Bb in which
S = the sequence complementary to the sequence (X> p - (Z) n - (W) q).

or a sequence which hybridizes with the sequence [(X) p- (Z) a- (W) q].

 The invention also relates to a nucleotide probe and its use in a method for the detection of the presence of Y chromosome-specific DNA of different species of the bovine subfamily, said probe comprising the sequence [(X) p- (Z) n- (W) q] n. as defined above, and possibly the appropriate means for the detection of hybrids formed. Any length of probe may be used, the length being chosen or determined by the method of preparation of the probe. If, for example, the probe is manufactured by chemical synthesis, the latter may advantageously be made so as to obtain said probe consisting of up to +100 motifs of the X sequence.

Another object of the invention is a method for detecting the presence of Y-chromosome-specific DNA from different species of the bovine subfamily in a bovine DNA sample, characterized by the following successive steps - specific amplification of at least a portion of the Bovin Y chromosome-specific DNA by the polymerase chain reaction (PCR), said amplification being effected via at least one nucleotide primer, said primer (s) ) nucleotide (s) being selected to be either identical to or complementary to a segment of the sequence [(X) p- (Z) n- (W> qii defined above); segment containing at least 12 consecutive nucleotides of the X sequence, capable of hybridizing with this segment, detection of the presence of specifically amplified DNA
The sequence W can be any nucleotide sequence that is not X or Z, for example, linkers, adapters, etc., and does not harm the specificity of the probe, provided that the X sequence represents at least minus 1%, and preferably at least 30S of the total length of the portion t <X) p- (Z> a- (W) qiu.

Sequences A and B may be any identical or different nucleotide sequences, which are essentially free of sequences present in the Bovin Y chromosome, for example plasmid sequences. The high specificity of the X sequence for the bovine Y chromosome provides a differential signal between male and female AD, even when the X sequence represents only a very small proportion of the entire sequence. The highly repetitive nature of the sequence allows the use of a range of sequences defined in the above general formula, the smallest sequence that can be used being a sequence of 12 consecutive nucleotides of the X sequence.

 By "hybridizable" is meant sequences having at least 708 homology and hybridizing under non-stringent conditions.

 The characterization of Y1.2 and, as a result of btDYZ-1, revealed that its organization presents a repeating tandem pattern reminiscent of sequences of satellite families, characterized nevertheless by an unusual inter-pattern divergence. This divergence affects the two parts of the repetition pattern: a 40 base pair unit suitably called "X" and having a mean unit divergence of 27%, and a TG rich part, referred to as "Z" convenience, of very variable length, which could result from skidding during replication. These characterization experiments were carried out on the Y1.2 sequence. The highly repetitive nature and the very high copy number of the repeated Y-chromosome motif have been interpreted to mean that the total sequence byDYZ-1 has the same structure as Y1.2. A more extensive study of the total sequence of btDYZ-1 may reveal a higher degree of organization with greater conservation between repeats comparable to that observed in human alpha satellites (Willard and Waye, 1987). If not, the inter-repetition divergence could then reflect a poor homogenization of the tandem repeats possibly resulting from the hemizygocity of the Y chromosome.

 The number of copies of the repeated motif was estimated to be 1 x 105 copies per Y chromosome. Assuming that the average length of the repeating motif is 60 base pairs, the bt-DYZ-1 satellite extends over approximately 6Mb, or one-tenth of the bovine Y chromosome.

 No resemblance was found between btDYZ-1 and the following 6 bovine satellite sequences 1706, 1709, 1711a, 1711b, 1715 and 1720b. However, one would have expected a homology with one ve these bovine satellite sequences by analogy with the human specific Y-satellite DYZ-3 obviously related to the human alpha satellite family.

 The genetic polymorphism evidenced by btDYZ-1 in the subfamily of cattle, which is assumed to result from rearrangement of internal restriction sites by unequal crossing-over between sister chromatids or any other mechanism related, is in fact a valuable tool in the study of male genealogy.

 The possibility of sexing embryos of different species of the subfamily of bovines before implantation is of obvious interest. The high number of copies of the repeated motif as well as the centromeric location make btDYZ-1 a particularly suitable probe for this purpose. Indeed, one would expect to find other highly repeated sequences from the hetero chromatic region of the long arm of the Bovin Y chromosome by analogy with other species. Nevertheless, significant losses of long-arm heterochromatin have been reported in phenotypically normal men, making sexing by these probes risky (Gtodfellow et al., 1985).

 The inventors have shown that with the probe of the invention, a differential signal between males and females can be obtained with S 100 μg of DNA, which corresponds to +15 cells. By depositing on a smaller surface, this limit can be reduced further, for example, if some embryonic cells are deposited on nitrocellulose or nylon membranes, a technique which has been called "embryo blot", and proceeding as for colony hybridization. (See examples, "Dot Blot" hybridizations). In this way a differential signal can be obtained with at least 50 μg of DNA. The probe of the invention can be used to carry out hybridizations in situ. This in situ hybridization technique not only makes it possible to determine the vex of a member of the bovine subfamily, but can also be used in the context of the invention for karyotype and chromosomal localization studies.

 In a preferred embodiment of the invention, the chain polymerization reaction (PCR) is used to amplify the male DNA present in a sample, while taking advantage of the natural amplification of the target sequence. In this way, a differential signal can be obtained from a single cell which could be isolated by suction after piercing the zona pellucida.

 The basic technique of P.C.R. is well described in the literature [eg, R.K. Saiki et al. 1988].

 For P.C.R., it is preferable to use two primers. At least one of the primers must correspond to the sequence of 40 bases, X, or to a fragment comprising at least 12 consecutive bases of the sequence X. By "corresponding" is meant in this context "to be identical" or "to be complementary "or" to be able to hybridize with ".

It can also be envisaged to use a primer which corresponds to the junction region between the two X and Z sequences, provided that at least 12 consecutive nucleotides of the X sequence are present. The use of a primer that contains only nucleotides of the Z sequence is to be avoided due to the fact that sequences rich in
TG are also present in the genome of female bovine. Such a primer could therefore lead to the amplification of female DNA, and the obtaining of a differential signal between male and female would therefore be compromised.

 Preferably, two primers are used for the PCR which each correspond to at least 12 consecutive nucleotides of the X sequence, and more preferably at least 12 nucleotides which form the 5 'and 3' ends respectively of the X sequence. this way, no female DNA is amplified.

 The probe (s) can (be) synthesized chemically from the known sequence.

 In a particularly preferred embodiment of the invention, the DNA amplified by PCR is visualized by fluorescence after addition of ethidium bromide (EtBr). This technique, put together by the inventors and called "Drop Stain", has proved particularly suitable for amplifying the sequence of the invention. This method involves the addition of EtBr directly to the reaction mixture of PCR, without having to separate the differently sized fragments on an agarose gel. However, under certain conditions, the use of such a gel facilitates the interpretation of the results obtained.

 EtBr fluorescence DNA detection only works when there is a minimum amount of DNA present in the sample. Figure 12 (c), 3 shows that at least 50 ng of DNA should be present to give a visible signal.

In the method of the invention, only one tenth of the final reaction volume of the PCR (which originally contains 100 μg or 10 μg of purified genomic DNA,
Target DNA) is measured, which means that initially, 10 Da or 1 Da of target DNA is applied to the agarose gel or is present in a microtiter well. A visible signal from such a sample can therefore be due only to the amplification of the target DNA.

 Too high concentrations of primer (2.0 μg / 100 μl each) or target DNA (2100 μg / 100 μl) can give ambiguous results when using the "Drop Stain" method (see Fig. 12 ( b): A3 - A8 and B5 - B8).

Under these conditions, the results can be analyzed and interpreted more precisely by electrophoresis on an agarose gel. Too high amounts of primer give rise to a visible signal corresponding to a discrete band of low molecular weight on the gel (see Fig. 12,, H6, H7,
H8, H14, H15 and H16), and can lead to non-specific hybridizations (and thus to nonspecific amplifications) that are characterized by a slight drag in the female specimens (Fig. 12 (a), lanes 4, 6, 8, 14 and 16).

 It should be noted that when 0.1 μg of each primer is used there is no residual primer in samples that contain male genomic bovine genome (Fig. 12 ', H5 and H13) and DNA trainee specifically amplified is visible on the agarose gel (Fig 12, lanes 5 and 13). On the other hand, in the presence of female genomic bovine DNA, no amplified DNA is visible but a discrete low molecular weight band, which corresponds to the primers, is evidenced.

 Thus, by using an agarose gel, it is possible to differentiate between male and female DNA, even with high amounts of primer (0.1 μg / 100 μl each). On the other hand, the method "Drop Stain" where there is no separation of fragments according to their size on a gel, can not be used for sexing with such a quantity of primer because a visible signal is produced at both by amplified DNA and primers (Fig. 12b, A5, A6, B5 and B6).

 Outside, the advantage of using low concentration of primer and mentioned in the previous paragraphs, that is to say - the DNA of the primers does not produce a background fluorescent signal which would contribute to the signal EtBr (Fig. 12 *, H1 - H4 and H10 - H12), small amounts of primers also seem to offer another advantage - the average molecular weight of the DNA fragments synthesized during PCR increases when the Initial primer concentration decreases to a minimum concentration. A comparison between the molecular weight range synthesized with 1.0 μg of primer on one side and with 0.1 μg of primer on the other side (see Fig. 12 (a) lanes 13 and 15) 10 μg of target DNA male being present in each case, shows very clearly this phenomenon: the range of sizes obtained goes from <3kb in the first instance, to between 2 and 10 kb in the second situation. Ethidium bromide forms complexes with DNA by intercalating between base pairs of ADS, and excitation of these complexes with 6'V waves gives a fluorescence signal. The more DNA bases there are, the more EtBr is inserted and the stronger is the signal. As explained above, a low concentration of primer leads to the synthesis of longer fragments. As a result, a stronger signal is obtained with a low initial concentration of primer.

 The concentration of target DNA also plays an important role in amplification: when the target DNA is present at less than 100 μg, non-specific pairings between the primers and the target DNA do not seem to occur. take place, and no female DNA is amplified (Fig.

12 ', lanes 12, 14 and 16).

These observations can therefore be summarized as follows - low concentration of primer ('0.05 μg)
no background noise, and
very strong signal.

low concentration of target DNA (of the order of <100 μg)
r lack of non-specific pairing between primer and target DNA, therefore, lack of amplification of female DNA.

 The inventors have applied these procedures, in the specific amplification of male DNA to a sample which contains only the DNA corresponding to a single cell (~ 6 μg DNA) and could subsequently determine the sex of the donor bovine donor. from this amount of DNA.

 Normally, PCR results in a reaction mixture which, on a gel, gives one or more discrete bands corresponding to well-defined molecular weights. This phenomenon is due to the amplification of one or more specific loci which are flanked by the pairing sites of the primers used, these sites being present in single copy.

On the other hand, one cycle of the PCR serves in turn as primers for the next cycle. This gives a population of DNA molecules of different lengths (which is characterized on an agarose gel by a drag instead of the distinct bands normally observed) with an average molecular weight which increases as the number of cycles increases; small molecules disappear because they served as primers; This phenomenon, which may be termed "self-priming", may be accompanied later by possible non-specific matches of the TG-rich regions synthesized during the first cycles to the many TG-containing regions that are found in most genomes.

 It should be noted that the specific amplification of male DNA depends entirely on the initial specific pairing of the primers to the 40 bp male sequence. Since this initial pairing can not take place with female DNA, the 40 bp unit is not present, no strand of DNA is primed and the Taq polymerase is unable to synthesize DNA.

 To summarize: the choice of the primer <from the 40 bp unit of the sequence Y1.2), and the conditions applied (ratio of concentration of primer / target DNA, concentrations of the solutions, temperature, duration and number of cycles) are essential for the specific amplification of male DNA from small amounts of target, enomic DNA.

In this way, a clear differential signal between male and female is obtained with the method "Drop
Stain ".

 This detection technique with ethidium bromide called by the inventors "Drop stain", therefore avoids having to resort to expensive and inconvenient methods of separation by gel electrophoresis, and allows to determine very quickly the sex of a bovine from an extremely small amount of DNA.

 In addition, since the amplified sequences consist of alternating Pu-Py regions, which adopt a so-called Z-DNA conformation under appropriate salt conditions, another way of rapidly detecting the target DNA would be an ELISA-type test. use of anti-Z-DNA antibodies. This procedure would significantly reduce the time required for a sex diagnosis in relation to the time required for a probe hybridization procedure.

 Having a specific Y sequence could also facilitate the search for methods of separating sperm carrying the Y chromosome of sperm carrying the X chromosome, separation which remains an important goal of breeding. The probe of the invention can be used in separate ports to verify the reliability of these separation methods.

FIG. 1 shows Southern blot of Southern blot obtained with bovine genomic DNA digested with
HaeIII and the pSP64.2.5EI probe related to "Per".

 Figure 2 shows the "Dot Blots" of various amounts of male and female bovine DNA with the btDYZ-1 (a) probe, and with genomic bovine DNA (b). A positive signal is obtained even with 100 μg of male DNA, while the female DNA samples were still negative. The fingerprint obtained with total genomic DNA as a probe shows that comparable amounts were used for each sex.

 Figure 3 shows the sequence Y1.2 so as to bring out its organization in patterns repeated in tandem. Each repeated pattern consists of two distinct parts: a 40 base pair long unit whose consensus sequence is illustrated, as well as the deviations of this consensus sequence for each unit; and a TG-rich sequence of highly variable pattern pattern length. It should be noted that the consensus sequence of 40 bases is CNNTTNTCAGCCCTGTGCCCTGGNRAYTGTGGNAANNNGTT in which only those nucleotides that are conserved due to 280% have been retained. Nucleotides that are stored at <80% have been replaced by "N" except for positions 25 and 27, which have been replaced by R (= purine) and Y (= pyrimidine) respectively.

 Figure 4 shows the estimate of the number of.

copies of the repeated motif by male genome. Defined amounts of male DNA and Y1.2 were hybridized to the Y1.2 probe, allowing the copy number to be estimated at about 105. Equimolar vector amounts (ds M13mp18) served as a control for possible contamination of the Y1.2 probe. the probe with the ADW's
M13.

Figure 5 is a histogram which shows the distribution of in-situ hybridized in situ btDYZ-1 silver grains on metaphase bull chromosomes. The histogram was done according to the method of
Fries et al.

 Figure 6 shows a bovine metaphase plate spread before and after hybridization with the btDYZ-1 probe. Plaque was stained with quinacrine mustard (left) and Giemsa (right) after hybridization. The arrow indicates grains specific to the Y chromosome. The triangles indicate accumulations of grains in interphase nuclei and probably represent signals from Y sequences.

Figure 7 shows the polymorphism of the btDYZ-1 satellite in the Belgian Blue-white bovine race. The image obtained clearly shows that the bull # 5 has a variant of btDYZ-1 different to the other 4. The
DNA were digested with HaeIII and the probe used was btDYZ-1. The size markers are in Kb.

Figure 8 shows the conservation of the btDYZ-1 sequence in the Bovinae family. A clear differential signal is obtained between males and females with the btDYZ-1 probe on 100ng spots of genomic DNA in all tested species of the Bovinae subfamily. No differential signal was observed in other mammals, for example other
Bovidae like sheep.

 Figure 9 shows the sequence Y1.2, the underlined parts being the sequences used as primer in P.C.R.

Figure 10 (a) and (b) show images
Polaroïd Drop Stains obtained after the reaction
PCR One-tenth of the total reaction volume of the
PCR was measured.

 FIG. 11 shows the fragments obtained by the EcoRI digestion of btDYZ-1, and the fact that the Y1.2 sequence constitutes the 1.2kb EcoRI fragment produced by this digestion.

 Figure 12 (a), (b) and (c) show the results obtained after the specific amplification of the male DNA by means of PCR.

 Figure 13 is a schematic of the successive steps and cycles of the PCR.

EXAMPLES
In the examples that follow, the following materials and methods are used
DNA extraction, restriction and hybridization of
Southern blot
The procedures for extraction of ADS, restriction by enzymatic digestion and hybridization of
Southern blots are described elsewhere (Georges et al., 1987). Hybridization of "Dot Blots" was performed according to Anderson and Young (1985): a nitrocellulose sheet was immersed in water, transferred onto Whatman 3MM paper and transferred for 30 minutes into a 20 x ssc solution. The filter is dried at room temperature.

 The bovine genomic DNA sample is applied to the filter in the form of 1 μl samples in TE buffer (10 mM Tris-HCl, pH 8, 1 mM EDTA, pH 8).

 The samples are allowed to dry at room temperature.

 The DNA is denatured by placing the filter for 5 minutes on a Whatman 3 MM paper sheet saturated with 1.5M NaCl, 0.5M NaOH.

Then, the filter is transferred for 30 seconds onto a 3 m Whatman saturated paper with
0.5M Tris-HCl, pH 7.4, then on a Whatman 3 paper
MM saturated with 1.5M NaCl, 0.5M Tris-HCl, pH 7.4 for 5 minutes.

 The filter is air dried and cooked at 800C for 2 hours.

 Prehybridization, hybridization and washing conditions are stringent, defined below.

 In order to allow normalization of the signal obtained with the probe, duplicates of the filters are hybridized under the same conditions with 500 ng of bovine genomic DNA, radioactively labeled with the 32 phosphorus isotope by the method of Feinberg et al.

 (1983).

Cloning of btDYZ-1:
500 μg purified male bovine DNA was digested to completion with MboI and size-separated by centrifugation in a 10-40% sucrose gradient.

After dialysis and precipitation, the> 2kb fractions were ligated into a BamHI cleaved and dephosphorylated pUC13 vector. Cloning is done by transformation into DH5α competent bacteria (Hanahan, 1985). Colony hybridization is performed according to standard procedures.

Sequencing of ADA ':
DNA sequencing is done using T7
DNA Polymerase (Pharmacia) involved in the chain termination reaction, after subcloning into the Mi3mpl8 vector and the production of unidirectional progressive deletions by exonuclease III.

Chromosome preparation and identification
Chromosomal plate smears were prepared from fibroblast cell lines by the mitotic detachment technique. The fibroblastic cell line was established from male bovine fetal tissue. Chromosomes were labeled and identified according to Fries et al. (1988).

Preparation of the probes:
For in situ hybridization experiments the 4.2-kb insert was labeled with three tritiated nucleotides (Shin et al., 1985 & Fries et al., 1986).

The specific activity of the probes was 10 dmp / μg.

In situ hybridization
Hybridization of probes with chromosome preparations according to Fries et al. (1988) was based on the method of Harper and Saunders (1981). The spreading plates of the chromosomal plates are immersed in an Ilford K2 emulsion to perform the autoradiographs and are developed after 24 hours of exposure to 4 C.

HYBRIDIZATION CONDITIONS
In the examples, the following hybridization conditions were applied
A - Non-strinqent conditions
prehybridization:

<tb> 35% <SEP> formamide
<tb> 6 <SEP> x <SEP> SSC
<tb> 5mM <SEP> EDTA
<tb> 0.025% <SEP> PLE <SEP> (powder <SEP> of <SEP> milk <SEP> skim)
<Tb>
incubation for 2 hours at room temperature
room.

Hybridization
same prehybridization with the DNA probe.

 incubation 12 hours at 420C.

Washing
1 X 1 hour 650C 2 X SSC
0.1% SDS
B - Stringent conditions Prehybridization

<tb> 50% <SEP> formamide
<tb> 6 <SEP> X <SEP> SSC <SEP> (20X)
<tb> 5mM <SEP> EDTA <SEP> (0.5M)
<tb> 0.25% <SEP> PLE
<tb> 100ug / ml <SEP> DNA <SEP> sperm <SEP> herring
<tb> (10 mg / ml) <SEP> denatured. <SEP> 5 <SEP> min. <SEP> to <SEP> 1000C
<Tb>
Hybridization: same prehybridization, no herring sperm DNA with the DNA probe.

incubation for 12 hours at 42 ° C., with moderate agitation.

 Wash 2 X 20 min. Temp. amb.

4 X 20 min. 650C 1 X 20 min. 650C 2 X 20 min. 650C

<tb><SEP> 2 <SEP> X <SEP> SSC
<tb><SEP> 0.1% <SEP> SDS
<tb> 2 <SEP> X <SEP> SSC
<tb> 0.1% <SEP> SDS
<tb> 1 <SEP> x <SEP> ssc <SEP>
<tb> t0 <SEP> 1% <SEP> SDS
<tb> 0.1 <SEP> X <SEP> SSC
<tb> 0.1% <SEP> SDS
<tb> 1) Cloning of btDYZ-1
In order to isolate the specific Y sequences, 500 μg of male DNA was completely digested with MboI and the> 2-kb fragments isolated in a silica gradient and then cloned into pUC13. As predicted from the images in females, beyond this size limit, very few autosoinal fragments were cross-reactive. In addition, by virtue of the expected size distribution for cleavage by
MboI this class should be heavily enriched in specific Y sequences. If the length distribution of restriction fragments is exponential, according to Bishop et al. (1983)> 2-kb fragments would represent + 10- of the total MboI fragments (ie +104 fragments) of which +20 would be Y specific, which was suggested by Southern blots. As a result +1 positive clone was expected on 500 screened clones. This simplified estimation does not take into account the decrease in the cloning efficiency with the size of the fragments to be cloned.

 After two rounds of screening with the pSP64.2.5EI probe under non-stringent conditions, ten clones were retained on +2500 colonies initially.

These clones were in turn used as probes on "Southern blots" of cleaved male and female DNAs.
MboI. At high stringency, two of these clones gave a specific sex signal. One of these, called btDYZ-1, gave a very intense signal in males and on these bases it was retained for further analysis. Figure 2 shows "dot blots" of male and female DNA hybridized with the bt probe
DYZ-1 under stringent conditions. After 16 hours of exposure a positive signal is visible even on 100 μg of male DNA, the female DNA spots remaining completely negative. At least 3 of the remaining 8 clones gave hypervariable specific locus signals (VNTR) and will be described later.

2) Organization of tandem renovations
The restriction analysis of clone btDYZ-1 shows that the insert is 4.2-kb long and does not have restriction sites for XbaI, AccI, SalI, PstI,
SphI, SstI, KpnI and SmaI, but has two internal EcoRI sites producing three fragments of 0.9, 2.1 and 1.2-kb respectively (Fig. 11). The Shard
EcoRI-SstI of 1.2-kb was subcloned into M13mp18 giving a clone that was named Y1.2. This clone was then completely sequenced by the dideoxynucleotide ring termination method, after production of unidirectional progressive deletions by exonuclease III. FIG. 3 shows that this fragment is formed of a tandem repeating unit 17 composed of two distinct parts: a unit of 40 base pairs whose average divergence between the units is 27% separated by a second part rich in TG of very variable length (12 to 63 base pairs).

 This repeat sequence is present at about 105 copies per male genome, estimated by comparing the Y1.2 DNA stain intensity with that obtained with male bovine DNA, using a probe made from the EcoRI fragment of 1.2-kb (Fig.

4), and under stringent conditions.

 The cross hybridization observed initially between clone pSP64.2.5EI related to the btDYZ-1 clone, under non-stringent conditions, could therefore be due to the "in-phase" abundance of TGTG quadruplets present in the sequence of pSP64.2.5. EI (Shin et al., 1987).

3) Pericentrial Location (Hybridization in situ)
The distribution of silver grains on metaphase chromosomes hybridized with btDYZ-1 and after autoradiography is shown in the histogram of Figure 5. A metaphase plate before and after hybridization is shown in Figure 6. In total 610 grains were located on or touching chromosomes, in 34 prephotographed metaphasic plates. Of these 268 grains were associated with the Y chromosome which represents 2.1 * of the length of the haploid bovine genome (existing in only one copy per cell). Ypl3q12 bands were associated with 148 grains, indicating a concentration of grains in the centromeric region of the Y chromosome. A statistical evaluation of the distribution of silver grains per unit chromosome and assuming a distribution of
Poisson, indicates that the signal of btDYZ-1 was significant for the Ypl3-q12 region.

4) Polymorhism and Evolutive Conservation
The DNA of 5 bulls of the Belgian White Blue breed.

restricted by HaeIII were hybridized under stringent conditions with the btDYZ-1 probe. The image obtained (Figure 7) clearly shows that 4 of them share a variant of the Y chromosome while the latter has a different variant.

The genetic polymorphism of the btDYZ-1 satellite is a tool that allows us to study the genealogy of bovine males. Such a study could also be extended to the different species of the subfamily Bovines. Indeed, a clear image m le-spFcifique is obtained in the bulls of the Bleu Blanc race
Belgian (Bos Taurus), Zebu (Bos Indicus), Yack (Bos
Mutus), Gaur (Bos Gaurus), American Bison (Bison
Bison), and the African buffalo (Svncerus Caffer) (Figure 8). All of these species belong to the subfamily Bovines, having diverged about 25 x 10 'years or less, up the evolutionary tree leading to Bos Taurus. On the other hand, no differential signal between males and females could be observed in other mammals including sheep, even when less severe hybridization and washing conditions were used.

5) amplification with P.C.R.

The primers used were two oligonucleotides 18 bases long each. These sequences were chosen from the sequence
Y1.2 (see Fig. 9, primers being underlined) and chemically synthesized with a "DNA synthesizer" [Model 381A, Applied Biosystems Inc., Foster City, CA.
USA.].

These sequences are illustrated below primer (S368) 5 'AGGGCACAGGGCTGAGAA 3' primer (S370) 5 '

GT. 3 '
PCR was performed in tubes
Eppendorf 500 l in a final volume of 1001, which consists of 50mM KCl, 10 mM Tris (pH8), 1.5 mM
MgCl 2, 0.01% (w / v) gelatin, 200 μM each of dATP, dCTP, dTTP and dGTP, 90 nM (0.05 g) each of the two oligonucleotide primers and 2.5 units of Taq
Polymerase [Perkin Elmer Cetus, Norwalk, USA] and the whole was covered with mineral oil.

Various amounts (100-0.1 g) of purified bovine genome DNA (male and female) were subjected to 40 cycles in a DNA Thermal Cycler [Perkin Elmer Cetus
Instruments, Norwalk, CT, USA]. To begin, the samples are heated to 930C for 1 minute, then cycles which consist of a denaturation step for 1 minute at 930C, a 1 minute recaption step at 550C, and a polymerization step at 720C for 2 minutes. minutes, are triggered. After 40 complete cycles, the samples are stored at 720C for 6 minutes, and the PCR is terminated by cooling to 150C (see Fig. 13).

 For a first visualization after agarose gel electrophoresis, transfer buffer (4 μl) was added to 10 μl (1/10) of the PCR reaction mixture (100 μl) and was subjected to horizontal electrophoresis on a 0.7% agarose gel in a TAE buffer (40 mM Tris-acetate, 2 mM EDTA, pH +7.8). After migration for + 2 hours at + 100 volts, the gel was stained for 20 - 30 minutes in a solution of 1-2 μg EtBr / ml TAE Buffer and then visualized with a U.V. transluminator.

 . The technique "Drop Stain" allows the immediate visualization of amplified DNA after the PCR reaction described above without having to go through the step of separation of fragments on agarose gel. The detection of specifically amplified bovine chromosome Y DNA is carried out in 96-well microtiter plates to which is added 10 μl of PCR reaction mixture (which represents one-tenth of the total reaction volume), followed by 15 μl of bromide solution. ethidium (3.3ug / ml 10mM Tris-HCl, 1mM EDTA, pH 8.0). The amplified DNA is visualized immediately on a U.V.

 (302 nm) (Fig. 12 (b)).

 A standard curve was obtained by placing specific amounts (0, 25, 50, 100, 250 and 500 ng) of purified genomic bovine DNA in the 96-well microtiter plates and adjusting the volume to 10 μl by the addition of 10mM Tris-HCl, 1mM EDTA, pH 8.0 (Fig. 12 (C)). The visualization was done by "Drop Stain" as indicated above.

 A Polaroid photo of "Drop Stain" (FIG. 10 ") shows that the use of the primers of the invention in a PCR reaction allows the specific amplification of bovine chromosome DNA from 0.5 g of DNA. purified bovine genome and gives a clear differential signal even with this very small amount of DNA.This signal is reproducible, as shown in Figure 10b, when the DNA from male and female cattle is subjected to this test.

 This experiment can be repeated using, as DNA sample, a single bovine embryo cell obtained, for example, by suction, after piercing the zona pellucida (Bondioli technique et al.

1989).

 This method of "Drop Stain" thus avoids the need to use expensive and time-consuming techniques of separation by gel electrophoresis, because no female DNA is amplified.

The amplification of 0.5 μg of bovine male DNA makes it possible to obtain a very clear signal even from one tenth of the total reaction mixture of the PCR (FIG. 10a).

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Claims (23)

 1. Nucleotide sequence containing a Y-chromosome-specific part of the different species of the subfamily Bovine, characterized in that it comprises - either the sequence  Aa - [(X) p- (Z) a- (W) g] m-Bb in which X = CNNTTNTCAGCCCTGTGCCCTGGNRAYTGTGNAANNNGTT  N = any of the 4 thymine nucleotides  (or uracil), guanine, adenine and cytosine;  R = purine;  Y = pyrimidine, or a fragment thereof provided that at least 12 consecutive nucleotides are present, or a sequence having at least 70% homology therewith; Z = a TG-rich nucleotide sequence containing at least 50%, preferably 80% TG, W = any nucleotide sequence that is not X or Z; A and B = nucleotide sequences-essentially free of sequences present in the bovine chromosome Y;  prl  n> O  q 2 O  m> 1  a = O or 1  b = 0 or 1 and the X, Z and W sequences may be in the indicated order or in a different order; provided that the X sequence represents at least 1%, preferably at least 30%, of the total length of the [(X) p- <Z) n- (W) qj portion. S = the complementary sequence of the sequence f <X) p - 'Z) 0 - (W) q j. and that when the values of p, n; q and / or m are greater than 1, the multiple copies of the X, Z, W and / or [X-Z-W] sequences respectively may be identical or different; or the sequence Aa- [S] -Bb in which or a sequence which hybridizes with the sequence [(X) p- (Z) @ - (W) q] m.  2. Sequence according to claim 1, characterized in that a, b, n and q all take the value O, and p and m each take the value 1.  3. Sequence according to claim 1, characterized in that it comprises the sequence Y1.2 as illustrated in Figure 3.  4. Sequence according to claim 1, characterized in that the Z sequence has a length of the order of 12 to 63 nucleotides.  5. Nucleotide probe for the detection of the presence of Y-chromosome-specific DNA of different species of the bovine sub-family in a bovine DNA sample under conditions that allow hybridization between the probe and the DNA bovine, characterized in that said probe comprises the sequence [(X) p- (Z) - (W) q) m of claim 1 and optionally suitable means for detecting the hybrids formed.  6. A method for detecting the presence of Y-chromosome-specific DNA of the different species of the bovine subfamily, characterized in that a bovine DNA sample and a living probe are brought into contact with claim 5, under conditions that allow hybridization of the probe and the DNA sample.  7. Method according to claim 6, characterized in that it comprises the following steps - fixing the biological sample on a suitable support, the DNA of said sample being made accessible to further hybridization, - the realization of a pre-hybridizing said sample obtained in the preceding step so as to reduce the nonspecific adsorption of the probe on the support, - bringing said sample obtained in the preceding step into contact with said nucleic probe, bringing it into contact with each other; being carried out in a medium and conditions suitable for allowing hybridization of the probe with the sample, - washing of the support containing the hybrids formed, - detection of a specific signal of the Y chromosome in the case of DNA originating from 'a male.  8. Process according to claim 6, characterized in that the DNA sample consists of a DNA fragment, an entire embryo, embryonic cell (s) or sperm. .  9. Method according to any one of claims 6 to 8, characterized in that the hybridization conditions are stringent.  The method according to any of claims 6 to 9, characterized in that the DNA sample contains at least 50 μg of DNA, and more particularly about 100 μg.  11. A method for detecting the presence of Y-chromosome-specific DNA of different species of the bovine subfamily in a bovine DNA sample, characterized by the following successive steps - the specific amplification of at least one part of the bovine chromosome Y specific DNA by the polymerase chain reaction (PCR), this amplification being carried out via at least one nucleotide primer, the nucleotide primer (s); s) being chosen to be either identical or complementary to a segment of the sequence [(X) p ~ {Z). ~ (W) q]. defined in claim 1, this segment containing at least 12 consecutive nucleotides of the X sequence, being capable of hybridizing with this segment, the detection of the presence of specifically amplified DNA.  12. Method according to claim 11, characterized in that the specific amplification is effeotue via two nucleotide primers.  13. Method according to one of claims 11 or 12, characterized in that the DNA sample consists of a DNA fragment, one or embryonic cell (s) or spermatozoa.  14. Method according to claim 12, characterized in that the primers are the sequences  5 'AGGGCACAGGGCTGAGAA 3'; 5 'GGCGACTGTGCAAC

GT 3 '  15. Process according to claims 11 and 12, characterized in that the biological sample contains at least 0.5 μg of DNA.  16. The method of claim 14, characterized in that the concentration of the primers is about 90 nM (0.05 ug / 100ul)  17. The method according to claims 11 and 13, the amplified DNA is visualized by the addition of ethidium bromide  18. Isolated nucleotide sequence, capable of acting as a primer in the chain polymerization reaction (PCR) for the amplification of Y chromosome-specific DNA of the different species of the subfamily Bovines, characterized in that it is identical or complementary to a segment of the sequence [(X) P- (Z) D- (W) a] defined in claim 1, this segment containing at least 12 consecutive nucleotides of the sequence X, or in that that it is able to hybridize with this segment.  19. Use of the sequence according to any one of claims 1 to 4 for determining the sex of embryos of different species of the subfamily Bovine  20. Use of the sequence according to any one of claims 1 to 4 for the verification of the reliability of the separation methods of sperm bearing sex chromosome Y and those bearing sex chromosome X.  21. Use of the sequence according to one of claims 1 to 4 for studying the genealogy of species belonging to the subfamily of cattle by exploiting the polymorphism demonstrated by said sequence on the Y chromosome.  22. Kit for the sexing of different species of the subfamily of cattle characterized in that it comprises - at least one probe as defined in claim 5, - the buffer solutions and compounds necessary for the preparation of the media of hybridization and prehybridization, possibly a control male DNA.  23. Kit for the sexing of different species of the subfamily of bovine characterized in that it comprises - at least one primer sequence as defined in claim 19, - the four deoxynucleotides dATP, dCTP, dTTP or dUTP and dGTP buffer solutions and compounds necessary for the affection of the polymerase chain reaction, possibly a control male DNA, optionally appropriate means for detecting this amplified DNA.