JPH08154685A - Bovine and murine sry-related dna and method for utilizing the same - Google Patents

Abstract

PURPOSE: To obtain the subject new Sry-related DNA which is murine and bovine sexual determining gene having a specific amino acid sequence and useful for sexual discrimination, identification of species, determination of the chimerism of a chimera cell, utilization, etc., as a chromosomic marker. CONSTITUTION: This a murine and a bovine sexual determining gene Sry-related DNA is a new DNA comprising a base sequence containing a base sequence represented by the formula or a part thereof (except a DNA having the base sequence containing base Nos. 6645 to 8646 in the sequence) or its complementary chain and useful for sexual discrimination, identification of species or determination of the chimerism of a chimera cell or utilization, etc., as a chromosomic marker. The Sry-related DNA is obtained by separating a genomic DNA from the liver of a mature male mouse according to a conventional method, amplifying the separated genomic DNA according to the polymerase chain reaction(PCR) with a synthetic primer comprising a part of the murine Sry-related gene and cloning the resultant amplified DNA.

Description

Detailed Description of the Invention

[0001]

The present invention relates to mouse and bovine Sr.
y-related DNA, RNA corresponding thereto, method of using the nucleic acid as a probe or primer in sex discrimination and species identification of mouse and bovine, and determination of chimerism of chimeric cells, and method of using the nucleic acid as a chromosome marker Regarding

[0002]

2. Description of the Related Art It is known that all mammals have sex chromosomes XY for males and XX for females, and that male determinants are present on the Y chromosome. Rarely, while holding the XX chromosome, a man,
It is known that there are abnormal cases in which a woman has a XY chromosome.
In particular, in the XY woman, it is considered that a part of the male determinant of the Y chromosome is defective, and the gene SRY was identified by pursuing the defective part (AH Sinclair et al., Nature, 34
6, 240-244, 1990). We isolated a male determinant gene (Sry) corresponding to SRY, introduced it into a mouse embryo, and created a transgenic mouse.In some cases, a mouse with XX, which should be a female originally, became a male, SRY / Sry was thought to be the main body of the sex-determining gene (P. Koopman et al.
al., Nature, 351, 117-121, 1991). But in humans
When SRY was introduced, XX mice did not become male. This may be due to the low homology of SRY / Sry.
SRY / that homology is relatively conserved in evolution
It can only be found in the HMG (high mobility group) box (237 base pairs) in Sry. In fact, human and mouse HM
In comparison of 471 bases sandwiching G, 103 bases on the 5'side, HMG box
Homology of 237 bases and 131 bases on the 3'side are 37.9% and 79.7%, respectively.
% And 42.0%. Therefore, D containing human SRY
Mouse Sry could not be obtained by the method of screening mouse genomic library using NA as a probe, and another method was used (J. Gubbay et al., Natur.
e, 346, 245-250, 1990). In addition, primates (L.-S. Whi
tefield et al., Nature 364, 713-715) also showed that mice and rats (PK Tucker and BL Lundrigan, Natu
re, 364, 715-717, 1993) also revealed that except for the HMG box, there was a great deal of variation, and there were great differences in the nucleotide sequences and lengths depending on the species. In fact, human SRY consists of 204 amino acid residues, whereas in mouse (Mus musculus molossinus), 3
Consists of 95 amino acid residues. Mus musculus with the same mouse
It has 230 amino acid residues in domesticus (C. Peter et a
L., Nature Genet., 6, 245-250 (1994)). It is speculated that these large differences are effective in preventing crosses and maintaining species identity by positively changing SRY / Sry (PK Tucker and BL Lundrigan, Na
ture, 364, 715-717, 1993).

As described above, there are many mutations in SRY / Sry, and
Further homology with SRY / Sry called SOX (SRY box genes)
There is a gene group with (60% or more). Homology of SOX with SRY / Sry is restricted to the HMG box (L.-S. Whitefield et a
L., Nature, 364, 713-715, 1993). These SOX gene clusters hinder the cloning of SRY / Sry. HMG boxes present in multiple genes have low SRY / Sry specificity within the genome, whereas SRY / Sry HMG boxes have only a few tens of homology between species and low species specificity. it is conceivable that. To date, the Sry nucleotide sequence of domestic animals (cattle, sheep, goats, deer, pigs, rabbits) has been investigated using human-derived DNA polymerase chain reaction (PCR) primers, but the target has evolved. It was 146 base pairs in the HMG box, which is relatively highly conserved and has low species specificity (Kaiyama et al., Nippon Kaiho 63, 1059-1065, 1992). Japanese Patent Laid-Open No. 5-
In 76366, 146 bases of bovine HMG box (SEQ ID NO: 2) obtained by using the same human primer pair as Kageyama et al.
The nucleotide numbers 1260 to 1405 of the sequence are shown. Since these are HMG boxes, the human primers were able to obtain a part of the sequences of other animals. Conversely, identification of species is difficult as long as the primers in the HMG box are used. For example, interspecies chimeras (eg, sheep and goats, CB Fehilly et al., Nature, 307, 634-
637, 1984) cannot be used to separate the constituent cells. In addition, even if different kinds of cells or different kinds of DNA are mixed in the reaction, they cannot be distinguished.

As a sex determinant, SRY / Sry can make a genetically female animal a male by producing a transgenic animal (P. Koopman et al., N.
ature, 351, 117-121, 1991). On the other hand, with the advancement of technology and knowledge, it will be possible to make a genetically male animal a female by introducing a ribozyme or an antisense sequence and producing a gene knockdown animal. But in humans
As shown by the fact that even if SRY was introduced, XX mice did not become male, SRY / Sry has high species specificity in terms of mechanism of action, and for developmental engineering applications such as sex control, each species has its own specificity. SRY /
The nucleotide sequence of Sry, especially the highly species-specific sequence other than the HMG box, will be important. However, conventional studies are limited to SRY / Sry sequences near the highly homologous HMG box, determining the nucleotide sequence of a wide region before and after the HMG box, and determining the Y chromosome specificity and regions other than the HMG box. There is no report on an application example utilizing the species specificity of.

[0005]

Accordingly, the present invention is directed to the inclusion of a large region in front of and behind the mouse and bovine HMG boxes.
Its purpose is to provide NA and parts thereof. Also,
The present invention aims to provide RNA corresponding to the DNA. Further, the present invention aims to provide a polypeptide encoded by the DNA.

Furthermore, the present invention is directed to sex discrimination, species identification,
It is an object of the present invention to provide a method for detecting the above-mentioned nucleic acid, which method can be used for determining chimerism of chimeric cells. Another object of the present invention is to provide a Y chromosome marker containing the nucleic acid, and a method for confirming the presence or absence of the nucleic acid region in the Y chromosome and a method for separating the Y chromosome using the Y chromosome marker.

[0007]

[Means for Solving the Problems] As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that the HMG of the Sry gene is
A part of the box was amplified by PCR, labeled with an isotope, and used as a probe to screen a mouse and bovine genomic library.
We have succeeded in obtaining long DNA containing a gene and performing sex discrimination and staining of Y chromosome by utilizing these DNAs or a part thereof, and completed the present invention. The subject matter of the present invention is as follows.

(1) DNA containing the sequence of SEQ ID NO: 1 or a part thereof (excluding the DNA whose base sequence is the base sequence from base number 6645 to 8646 in the above sequence)
Or its complement. (2) An RNA corresponding to the DNA described in (1) above. (3) DNA containing the sequence of SEQ ID NO: 2 or a part thereof
(However, the base sequence from the base number 1261 in the above sequence
Excludes DNA having a nucleotide sequence up to 1405. ) Or its complementary strand. (4) RNA corresponding to the DNA described in (3) above. (5) Selected from the group consisting of DNA containing the sequence of SEQ ID NO: 1 or a part thereof and its complementary strand, DNA containing the sequence of SEQ ID NO: 2 or a part of its complementary strand, and RNA corresponding to the DNA. Y marker containing at least one nucleic acid that is

(6) A polypeptide comprising the amino acid sequence of SEQ ID NO: 3. (7) A method for detecting the presence of a nucleic acid in a biological sample of a mouse using the nucleic acid according to (1) or (2) above. (8) A method for detecting the presence of a nucleic acid in a bovine biological sample, using the nucleic acid according to (3) or (4) above. (9) A method for confirming the presence or absence of the nucleic acid region in the Y chromosome, which comprises staining the Y chromosome with the Y chromosome marker described in (5) above. (10) A method for separating a Y chromosome, which comprises staining the Y chromosome with the Y chromosome marker described in (5) above.

The present invention will be described in detail below. 1. Isolation and Identification of Sry-Related DNA Although the isolation and identification of mouse Sry-related DNA is described below, bovine Sry-related DNA can be isolated and identified in a similar manner. However, the bull used in the preparation of the genomic DNA library may be any strain, and examples thereof include Holstein, Japanese Black cattle, and F1 thereof. The term "Sry-related DNA" refers to a series of DNAs containing a portion of the Sry body transcribed into messenger RNA and 5'side and 3'side sequences directly linked thereto.

(1) Preparation of genomic DNA library First, the liver of a mature male mouse was collected and subjected to a known method (for example, J. Sambrook et al. (Eds.), Molecular Cloning,
2nd ed., Cold Spring Harbor Laboratory Press, pp.
Genomic DNA is obtained according to the method described in 9.16-9.23, 1989). The male mouse used at that time may be of any strain, and examples thereof include C57BL / 6N, AKR / J, 1
29, SWR / J and the like.

The obtained genomic DNA was partially cleaved with the restriction enzyme MboI, and an MboI fragment of about 15 kb containing mouse Sry was inserted into the BamHI site of a vector (eg, EMBL3 phage vector). A genomic library can be prepared by conversion.
Examples of the vector that can be used include EMBL3 phage vector, EMBL4, λDASH (registered trademark) II, λFIX (registered trademark) II, Charomid DNA, Charon4A and the like. To cut genomic DNA with the restriction enzyme MboI, in a known buffer, for example, M buffer (50 mM NaCl, 10 mM Tris-HCl
(pH 7.5), 10 mM MgCl ₂ , 1 mM DTT) or MboI for 200 μg of genomic DNA in the buffer provided with MboI.
Use 6.25 units and treat at 37 ° C for about 1 hour. The obtained DNA fragment was subjected to sucrose density gradient ultracentrifugation to obtain about 1
Collect 5-20 kb of DNA. About obtained in this way
The 15-20 kb MboI fragment can contain mouse Sry. Then, for example, using a commercially available ligation kit, Ba
The phage vector containing the mouse Sry gene fragment is obtained by ligating the MboI fragment of about 15 kb to 20 kb containing mouse Sry obtained as described above to the mHI-treated EMBL3 phage vector. A genomic library can be obtained by introducing this phage vector into a microorganism such as Escherichia coli and culturing the obtained transformant. Specifically, known methods such as J. Sambrook et al. (Ed
s.), Molecular Cloning, 2nd ed., Cold Spring Harbor
Laboratory Press, pp. 2.95-2.107, 1989) by the in vitro packaging method described in Escherichia coli, such as Escherichia coli PLK-17 (Stratagene manufactured by Toyobo Co., Ltd.).
A genome library can be obtained by introducing the above-mentioned phage vector into the above-mentioned vector) and culturing the obtained transformant overnight on NZY agar at 37 ° C.

(2) Selection of clones The genomic library obtained as described above was prepared by a known method, for example, J. Sambrook et al. (Eds.), Molecular.
Cloning, 2nd ed., Cold Spring Harbor Laboratory P
According to the method described in ress, pp. 2.108-2.113, 1989, a suitable probe such as a nucleotide sequence in the HMG box (J.Gub
Bay et al., Nature, 346, 245-250, 1990), a clone containing a BamHI fragment of about 15 kb containing mouse Sry was screened by a plaque hybridization method using a probe having a part thereof. can get. If necessary, the above-mentioned screening is repeated 2-3 times, and further, from the above-mentioned phage clones, a known method such as J. Sambrook et al. (Eds.), P.
p. 2.60-2.81, 1989, ibid.
After decomposing with HI, by agarose gel electrophoresis,
A clone showing a 15 kb fragment should be selected.

(3) Determination of Nucleotide Sequence of DNA Containing Sry Gene One of the clones obtained as described above is selected and the DNA is analyzed by a known method, for example, J. Sambrook et al. (Ed.
s.), Molecular Cloning, 2nd ed., Cold Spring Harbor
Isolate and purify by the method described in Laboratory Press, pp. 2.60-2.81, 1989. Approximately 15 kb BamHI fragment containing Sry gene obtained from the above DNA was further digested with a suitable restriction enzyme,
For example, it is cleaved with EcoRI and the nucleotide sequence of the region containing the Sry gene is determined using the deletion cloning method and the Sanger method (see SEQ ID NO: 1). A polypeptide encoded by the Sry gene can be obtained by expressing the DNA containing the Sry gene as described above in a gene expression system. Specifically, the polypeptide encoded by the Sry gene may be expressed using a commercially available expression vector that uses mammalian or insect cultured cells, yeast, or bacteria. From the viewpoint of easiness of operation, E. coli may be used for expression. In fact, SRY / Sry is expressed in the following vector. pGEX-GT (N. Nasrin et al., Nature, 35
4, 317-320, 1991), pGEX-2T (CM Haqq et al., Pro
c. Natl. Acad. Sci. USA, 90, 1097-1101, 1993), pG
EX-3X (K. Giese et al., Proc. Natl. Acad. Sci. US
A, 91, 3368-3372, 1994), pDS56 (DR Cohen et al.,
Proc. Natl. Acad. Sci. USA, 91, 4372-4376, 199
4), pET-156 (P. Coward et al., Nature Genet., 6, 2
45-250, 1994).

Such a polypeptide can be used for research on the sex-determining mechanism of animals, comparative research on the activity with Sry of other species, and sex reversal. In addition, the above Sry-related DNA
The RNA corresponding to and the complementary strand thereof can be produced using a commercially available vector having an RNA polymerase promoter. Specifically, the T7 promoter and T3
The DNA may be cloned into pKMN or pBlueScript having a promoter, and RNA may be synthesized with T7 RNA polymerase or T3 RNA polymerase. Here, RNA corresponding to the cloned DNA is synthesized, but in order to obtain RNA actually synthesized in vivo, SRY / Sry-related DNA may be introduced into cultured cells of the animal and expressed. (P. Cowar
d et al., Nature Genet., 6, 245-250, 1994). Such RNAs can be used for studies of transcriptional regulation of SRY / Sry genes, studies for translation into polypeptides, and in situ hybridization, Northern hybridization, RNA mapping using radiation- or chemically-labeled RNA.
It can be used as a probe (determining 5'and 3'of messenger RNA).

2. Utilization of mouse and bovine Sry-related DNA and RNA corresponding thereto The Sry-related DNA of the present invention is derived from the Y chromosome, but it cannot be asserted that it is specific to the Y chromosome. SOX gene group with high homology to HMG box of Sry gene (M. Stevanovic et al.
al., Human Mol. Genet., 2, 2013-2018, 1993), for example, when amplifying by PCR, a common signal for male and female may be given depending on conditions. Also, because it is evolutionarily conserved, sheep DNA may be amplified with bovine primers. In general, the longer the DNA, the more specific sequence (here Y chromosome) and non-specific (X
Common with chromosomes and autosomes) Sequences intermingle. Therefore, the obtained Sry-related DNA is, if necessary, HMG box,
It is necessary to divide it into 5'side area, 3'side area, etc. and use it after knowing the characteristics of each.

(1) Sry-related DNA or RN corresponding to them
Method for detecting the presence of A The Sry-related DNA of the present invention or RNA corresponding thereto can be used to detect the presence of the DNA or RNA in a biological sample. Biological samples include mouse and bovine tissues,
Examples thereof include embryos and cells. As a detection method,
DNA containing the sequence of SEQ ID NO: 1 or a part thereof or its complementary strand, DN containing the sequence of SEQ ID NO: 2 or a part thereof
A or its complementary strand, or RN corresponding to them
A, preferably base number 7148-in the sequence of SEQ ID NO: 1
DNA containing 8332 or its complementary strand, DNA containing base numbers 1067 to 1753 in the sequence of SEQ ID NO: 2 or its complementary strand, or RNA corresponding thereto, more preferably base number 7154 in the sequence of SEQ ID NO: 1. D including ~ 7393
NA or its complementary strand, base number in the sequence of SEQ ID NO: 2
A known Southern hybridization method in which DNA containing 1219 to 1459 or its complementary strand, or RNA corresponding thereto is used as a probe or a primer.
In situ hybridization method, PCR method, etc. can be mentioned. Specifically, Southern hybridization can extract DNA from the tissue or cell to determine the presence or absence of homologous DNA.However, when only a small amount of DNA can be obtained, the presence or absence can be confirmed by amplifying a specific portion by PCR. I can know. By preparing sections and specimens without destroying the tissues and cells and performing in situ hybridization, the DN
It is possible to know the presence or absence of A and RNA, and the intracellular localization. Recently developed in situ PCR (BW Heniford et a
L., Nucleic Acid Res., 21, 3159-3166, 1993) can be used to know the existence and localization of DNA and RNA. If in situ hybridization is performed using a chromosome sample, chromosome mapping is possible, and if the position is identified, it can be used as a marker.

As described above, the present invention in a biological sample is
By detecting the presence of Sry-related DNA or RNA corresponding thereto, sex discrimination, species identification, chimerism determination of chimeric cells, etc. can be performed. For example, in the sequence of SEQ ID NO: 1, HMG box base numbers 7154 to 7363
DNA containing the nucleotide sequence of is used for PCR to determine sex in mice, to determine cell sex and chimerism by in situ hybridization, and nucleotide numbers 7555 to 7690.
Sequences with CAG repeats between 11, 12 or 13 can be used to aid in mouse species identification (P. Coward et al.
al., Nature Genet., 6, 245-250, 1994).

Further, for example, in the sequence of SEQ ID NO: 2, HM
D containing the nucleotide sequence of nucleotide numbers 1219 to 1459 that is a G box
NA was used for PCR to identify sex in cattle and in situ
By the hybridization method, the cell sex and chimerism
Available for decisions. SEQ ID NO: 1 has base numbers 2277 to 23
46 (TG)_n, 2419-2450 (AC)_n, 3771 to 3822 (T
G) _n, 5971 to 6032 (AC)_n, 9357-9396 (TG)_nAs
Have short repeats that are useful for sex determination and chromosome mapping
It is desirable to avoid these parts when applying to
Yes. In this regard, SEQ ID NO: 2 does not have this type of repetition,
No matter which part is used, a male-specific probe for cows
Available as a boo.

(2) Use as a Y chromosome marker The Y chromosome can be stained with the Y chromosome marker containing the Sry-related DNA of the present invention or RNA corresponding thereto.
The above-mentioned Y chromosome marker is prepared by chemically labeling Sry-related DNA or RNA corresponding thereto with a biotinylated nucleotide or by labeling with an isotope such as ³² P, ³⁵ S or ¹²⁵ I. You can Alternatively, the Sry-related DNA incorporated into the plasmid may be labeled as described above. The Y chromosome to be stained may be derived from any animal as long as it uses the above-mentioned HMG box sequence, and its origin includes all mammals including marsupials in addition to mice, bovines. be able to.
When the 5'side and 3'side regions other than the HMG box are used as Y chromosome markers, the species specificity is high, so Sry-related DNA
Alternatively, it is preferable that the animal species of the origin of RNA corresponding to them and the animal species of the origin of Y chromosome stained with the marker are the same. By staining the Y chromosome with a Y chromosome marker containing the Sry-related DNA or RNA corresponding thereto as described above, it is possible to detect a deletion or translocation of the DNA region and thereby predict the presence or absence of a malformation. It is also possible to separate the Y chromosome.
The above detection method is selected according to the type of label,
Examples include autoradiography and FISH (fluo
rescence in situ hybridization) method and the like. The Y chromosome can be separated using a flow cytometer. The Y chromosome separated in this way can be used for preparing a DNA library.

3. Deposit of Microorganism Constituting Mouse Sry-Related DNA (SEQ ID NO: 1) of the Present Invention
Escherichia coli HD5α into which the plasmid pUC118 incorporating the two EcoRI fragments A, B, C and D (see FIG. 1) was introduced was designated by accession numbers FERM P-14631, FERM P-14630 and FERM P, respectively.
-14629 and FERM P-14632, November 11, 1994
Deposited at Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science, dated. In addition, the bovine Sry-related DNA of the present invention (SEQ ID NO: 2)
E. coli PLK-17 with EMBL3 phage
Under the accession number FERM P-14633, November 11, 1994
Deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science as of the date below.

[0022]

EXAMPLES The present invention will be specifically described below with reference to examples. However, the scope of the present invention is not limited by these examples.

Example 1 Isolation of Mouse Genomic DNA The livers of 14 adult male mice (Mus musculus, C57BL / 6N) were taken and subjected to a known method (J. Sambrook et al. (Eds.), Molecu.
lar Cloning, 2nd ed., Cold Spring Harbor Laboratory
Genomic DNA was obtained according to Press, pp. 9.16-9.23, 1989). The outline of the method is described below. Shred the liver with scissors
(About 3 to 4 mm square), suspended in a buffer solution, applied to a homogenizer, centrifuged, and then the pellet was treated with Proteinase K.
The mixture was kept at 65 ° C for 30 minutes, shaken at 37 ° C overnight, and extracted with the same amount of phenol solution three times. The aqueous layer was extracted twice with phenol: chloroform and then twice with chloroform. RNase A (10 μg / ml) was added to the aqueous layer and the mixture was treated at 37 ° C. for 3 hours and then extracted once with the same amount of a phenol solution, phenol: chloroform, and chloroform. 2 volumes of ethanol was added to the aqueous layer, and the precipitated DNA was taken with a glass rod, washed with 70% ethanol, dried, and washed with an appropriate amount of TE (10 mM Tris-HCl,
pH 8, 1 mM EDTA). Absorbance was measured at OD ₂₆₀ nm to determine the DNA concentration. Thus, 11.04 μg / μl
1.213 ml of DNA was obtained (total amount 13.4 mg).

(Example 2) Partial amplification of mouse Sry gene using PCR The nucleotide sequence of the HMG box of mouse (129 strains) Sry gene is known (J. Gubbay et al., Nature, 346, 245-250, 199).
0). A part of this was synthesized as a primer for PCR. An automatic DNA synthesizer (ABI, Model 391) was used for the synthesis. Its base sequence is as follows.

5'-CCATGTCAAGCGCCCCATGA-3 '(SEQ ID NO: 1
5'-GTAAGGCTTTTCCACCTGCA-3 '(base numbers 7156 to 7175 of the sequence of SEQ ID NO: 7) (complementary strand of base numbers 7268 to 7287 of the sequence of SEQ ID NO: 1)

PCR using the mouse DNA obtained in Example 1
When this was carried out, the expected 132 base pair DNA was amplified. The conditions of the PCR performed here are as follows. Mouse DNA 500 ng, 2 μM each of the above primers, DNA synthesis substrate
dNTPs 2 mM each, thermostable DNA synthase (Tth, Toyobo) 2
U, total 10 μl of reaction solution attached to the same enzyme, total 100
μl was used, and the reaction was performed at 94 ° C. for 1 minute, 55 ° C. for 1 minute, and 72 ° C. for 1 minute for 35 cycles. The PCR reaction solution was electrophoresed (100 V, 30 minutes) on 1% agarose (Seakem, GTG), stained with ethidium bromide solution (EtBr solution, 0.5 μg / ml) for 30 minutes, and then the DNA band was excised. It was Ultra Free C3HV
(Manufactured by Millipore), and DNA was extracted according to the instruction. The DNA solution was treated once with each of the same amount of phenol solution, phenol: chloroform, and chloroform, the DNA solution was concentrated to 50 μl using 2-butanol, and the DNA was precipitated with 2 times the volume of ethanol, which was then added to 80% ethanol. 15 after washing and vacuum drying
It was dissolved in μl of TE. A part of the sample was subjected to electrophoresis together with a DNA marker, and it was confirmed that a predetermined 132 base pair single band was detected. Also, the DNA concentration could be known by comparison with the marker DNA. Thus, a part of the HMG box of the mouse Sry gene could be obtained.

Example 3 Identification of Length of BamHI Fragment Containing Mouse Sry Gene 10 μg of mouse DNA obtained in Example 1 was used as a restriction enzyme BamHI.
(Nippon Gene Co., Ltd.)
TAE (Tris-acet) with 1% agarose (Takara Shuzo, 03)
(ate-EDTA) buffer, and electrophoresis was performed at 100 V for 10 hours. As a DNA length marker, a HindIII degradation product of λDNA was used. After electrophoresis, stain the gel with EtBr solution for 1 hour,
I took a picture. The gel is an alkaline denaturing solution (0.5 M NaOH,
Soak for 30 minutes in 1.5 M NaCl, and then neutralize solution (1.5 M NaCl,
It was soaked in 0.5 M Tris-HCl (pH 7.2) for 30 minutes. Denaturation in gel
DNA was prepared by known methods (J. Sambrook et al. (Eds.), Molecu
larCloning, 2nd ed., Cold Spring Harbor Laboratory
Press, pp. 9.32-9.37, 1989), and transferred to a nylon membrane (Hybond N, manufactured by Pharmacia).

1 of the mouse Sry gene obtained in Example 2
³² P-α-dCTP (Amersham, 3000 μCi / m mol) by random primer method (Amersham kit)
Labeled with. Using this as a probe, Southern blotting was performed by the designated method (Pharmacia). When the result was analyzed by autoradiography,
It was found that a BamHI fragment of mouse genomic DNA containing the HMG box was present at around 15 kb.

(Example 4) Isolation of clone of genomic DNA containing mouse Sry gene From Example 3, mouse Sry was shown to be in a BamHI fragment of about 15 kb. Therefore, in the same manner as in Example 3, 10 μg of mouse genomic DNA was digested with BamHI and subjected to GTG agarose gel electrophoresis, and a portion of about 15 kb was excised from this. DNA was recovered from the gel as described in Example 2. On the other hand, EMBL3 DNA (Strata
1 μg (2 μl) was purchased, mixed with 1 μg (1 μl) of the BamHI fragment containing mouse Sry described above, and ligated using a ligation kit (Takara Shuzo). This is an in vitro packaging kit (Giga-PackI
I Gold, Stratagene) was used as a phage. This was suspended in 10 mM MgSO ₄ , inoculated into Escherichia coli PLK-17, and plated on NZY agar together with 3 ml of soft agar. After overnight culture at 37 ° C., a genomic library consisting of about 1.2 × 10 ⁶ cells was obtained.

This library was prepared by a known method (J. Sambr
ook et al. (Eds.), Molecular Cloning, 2nd ed., Col
d Spring Harbor Laboratory Press, pp. 2.108-2.117,
1989), the HMG box probe used in Example 3 was used for screening by the plaque hybridization method, and 51 coulombs showing a positive reaction were obtained. These clones were rescreened by the plaque hybridization method to obtain 16 positive clones. Furthermore, from each phage clone, a known method (J. Sambrook et al. (Eds.), Pp. 2.60-2.81,
DNA was isolated in the same manner as above), digested with restriction enzyme BamHI, and analyzed by agarose gel electrophoresis to obtain 6 clones showing a fragment of about 15 kb.

Example 5 Nucleotide Sequence Determination of DNA Containing Mouse Sry Gene One of the clones obtained in Example 4 was selected and
Known methods (J. Sambrook et al. (Eds.), Molecular
Cloning, 2nd ed., Cold Spring Harbor Laboratory P
ress, pp. 2.60-2.81, 1989). This was cut with various restriction enzymes to prepare a restriction map. The positions of typical restriction enzymes are shown in FIG. Of these, 4 fragments (A, B, C, D) cut with EcoRI were introduced into the plasmid vector pUC118. Its length is A: 2.3 kb, B: 2.8 kb, C: 3.5 k.
b, and D: 1.5 kb. Using each deletion cloning kit (Takara Shuzo Co., Ltd.), defective clones having various lengths were taken and the nucleotide sequences were determined. The method was based on an automatic sequencer (ABI, model 373A) using the Sanger method. The determined nucleotide sequence is shown in SEQ ID NO: 1.

This sequence was analyzed by DNA search system (DNASIS, Hitachi software). The sequence giving the longest polypeptide is from base number 7148 to 8332 and consists of 395 amino acid residues. It contains 240 bp from base number 7154 to 7393.
There is an HMG box sequence (within the C fragment). Base number 6445-86
The sequence of 46 is the sequence of mouse 129 strain (Peter et al., Natur
e Genet., 6, 245-250, 1994) except for 1 base. The only difference is that the base number 8336 of SEQ ID NO: 1 derived from C57BL / 6N is A, and that of 129 lines is C.

Example 6 Male and Female Properties and Species Specificity of A, B, C, and D Fragments Male and female mouse genomes obtained by the method described in Example 1
10 μg of DNA was taken, completely digested with EcoRI, subjected to agarose gel electrophoresis as described in Example 3, and transferred to a nylon membrane (Pharmacia). about this,
Southern hybridization was performed according to the instruction using probes labeled with ³² P-α-dCTP for the DNAs of the A, B, C, and D fragments. The result is shown in FIG. a, b, c, and d are autoradiograms using A, B, C, and D fragments as probes, respectively. Only the A fragment gave a male-specific signal, while the B, C, and D fragments smeared in both sexes. In b and c of FIG. 2, there is a male-specific band in the smear, but it is difficult to recognize because it is hidden by a strong signal around it. The cause is (AC) _n and
It appears to be in a short repeat such as (TG) _n . As described above, the B, C, and D fragments are derived from the Y chromosome, but do not necessarily give a male-specific signal because the sequences common to both sexes are mixed.

As shown in FIG. 2, only the A fragment was male-specific to the EcoRI fragment of mouse genomic DNA. So for A fragment, mouse, rat, Holstein, F
₁ decompose genomic DNA of cow and chicken with HindIII,
When Southern blotting was performed, a single male-specific band similar to that shown in FIG. 2 was obtained only in mice. It was male-specific in rats, but only smears were seen, not bands. No signal was seen in both males and females in Holstein, F ₁ cows and chickens. Thus, the male-specific A fragment of mouse was highly specific to the mouse species.

Example 7 Isolation of Genomic DNA Containing Bovine Sry Gene and Determination of Nucleotide Sequence According to the method described in Example 1, bovine (Holstein)
DNA was extracted and purified from the liver of. 100 μg of the fragment was partially digested with 6.25 units of the restriction enzyme MboI (Takara Shuzo), and the portion having a length of 15-20 Kbp was subjected to a known method (J. Sambrook).
et al. (Eds.), Molecular Cloning 2nd ed., Cold Spri
ng Harbor Laboratory Press, pp. 9.24-9.28, 1989)
Was collected and incorporated into the BamHI site of the EMBL3 phage vector (Stratagene) to prepare a bovine genomic library. This library consisted of approximately 500,000 clones (T. Kudo et al., J. Reprod. Develop., 3
9, 55-63, 1993).

On the other hand, according to the method described in Example 2,
HMG box of human SRY (J. Gubby et al.,
Nature, 346, 245-250, 1990)
Labeled with ³² P-α-dCTP by the method described in Example 3,
The genomic library was screened by plaque hybridization. The primers used in this PCR are as follows, and the length of the obtained DNA is 214 bp.

5'-AAGCGACCCATGAACGCATTCATCGTGTGG-3 '
(Base numbers 113 to 142 in Fig. 2 of J. Gubby's reference) 5'-GAGGTCGATACTTATAATTCGGGTATTTCTCTCTGTG-3 '(complementary strand of base numbers 290 to 326 above)

As a result of the primary screening, 39 clones were obtained. Further secondary screening was performed to obtain 6 clones. DNA was extracted from this, digested with various restriction enzymes, and Southern blotting was carried out using human HMG box as a probe to examine which fragment contained Sry. As a result, it was revealed that Sry was present in a short fragment of about 3 Kb obtained by digestion with BamHI and HincII. This DNA
The fragment was introduced into the plasmid vector pBlueScript SK (-), and the nucleotide sequence was determined as in Example 5. The base sequence is shown in SEQ ID NO: 2. This sequence is a DNA search system (D
NASIS, Hitachi software). The sequence giving the longest polypeptide is from base number 1067 to 1753 and consists of 229 amino acid residues. Among these are base numbers 1220 to 1459 to 24
There is an HMG box sequence consisting of 0 bp. Of these, 146 bp of base numbers 1260 to 1405 is the same as the sequence shown in JP-A-5-76366. The amino acid sequence of the longest polypeptide containing the HMG box is shown in SEQ ID NO: 3.

(Example 8) Amplification of DNA containing bovine Sry gene using PCR and species and sex specificity In the sequence of SEQ ID NO: 2, nucleotide numbers 885 to 902 and nucleotide numbers 1074 to 1091 are 5'side sequences, Base numbers 1220 to 1239 and base numbers 1440 to 1459 are HMG box sequences, base numbers 1507 to 1
524 and nucleotide numbers 1696 to 1713 were selected as the 3'side sequence, and the sequence or the complementary sequence was used as a PCR primer and synthesized as follows.

5'-side primer 5'-GCTGACTGCCAGGACGTA-3 '(base numbers 885 to 902 of the sequence of SEQ ID NO: 2) 5'-CATCGTCGTTCAATACTC-3' (complementary strand of base numbers 1074 to 1091 of the sequence of SEQ ID NO: 2)

HMG box sequence primer 5'-GACCACGTCAAGCGACCCAT-3 '(base numbers 1220 to 1239 of the sequence of SEQ ID NO: 2) 5'-CCTCTTGGCTCTCCGACGAG-3' (complementary strand of the base numbers 1440 to 1459 of the sequence of SEQ ID NO: 2)

3'-side primer 5'-GCATGTAGAGACATTGCA-3 '(base numbers 1507 to 1524 of the sequence of SEQ ID NO: 2) 5'-TTGTTACAGGGAAAGTCC-3' (complementary strand of the base numbers 1696 to 1713 of the sequence of SEQ ID NO: 2)

As the DNA template for PCR, the genomic DNAs of male and female bovine, sheep, goat and mouse obtained in the same manner as in Example 1 were used. PCR reaction composition is 400 ng genomic DNA,
A 0.05 mM dNTP mixed solution, each primer was 0.02 μM, 1 unit of Tth DNA polymerase (manufactured by TOYOBO, Tth-501), and the reaction amount was 20 μl. PCR uses Thermal Cycler (Cetus), and after heating at 94 ° C for 4 minutes, 94 ° C for 1 minute, 60 ° C for 1 minute, 72 ° C.
40 cycles were performed in 1 minute. Add 3 μl of the gel loading dye to 10 μl of the PCR product, and then use 0.5% TBE (Tris-borate-E) on 2% agarose gel (Seakem, Nuesive 3: 1).
Electrophoresis was performed using DTA) buffer. After electrophoresis, the gel was stained with EtBr solution and photographed under UV irradiation.
The results are shown in Figure 3, where lane 1 is a size marker (123 bp multiples), lanes 2 and 3 are holstein and lane.
4 and 5 are F ₁ cows (a cross between Holstein females and Japanese Black cattle males),
Lanes 6 and 7 are sheep, lanes 8 and 9 are goats, lanes 10 and 11
Is a mouse, DN is male with even lanes and female with odd lanes
A. Lane 12 is distilled water.

Holstein and F ₁ for 5 ′ primer
A specific band (207 bp) was always detected only in bovine males,
It was possible to distinguish between male and female (Fig. 3, a, lanes 2, 3 and lanes 4, 5). In other animals (lanes 6 to 11), a thin band with poor reproducibility was sometimes detected, which was considered to be the result of nonspecific reaction due to subtle changes in PCR conditions. Thus, despite the Sry-related DNA sequence, HM
The 5'side sequence near the G box was found to have high bovine specificity.

With the HMG box primer, a specific band (241 bp) was detected only in males of Holstein and F ₁ cows, and male and female could be discriminated (FIG. 3, b, lanes 2, 3).
And lanes 4, 5). However, in sheep, both males and females showed the same bands as cow and goat males (lanes 6 and 7),
Gender discrimination was impossible. However, there was a difference between males and females in goats as in cows, indicating that it can be used for sex discrimination (lanes 8 and 9). In males and females, a short band common to both males and females of cattle, sheep, and goats was seen, and it was not male-specific, so sex discrimination was not possible (lanes 10 to 1).
1). This short band increases the annealing temperature from 55 ℃ to 6 ℃.
When it is raised to 0 ℃, it becomes thin and disappears.
It is highly possible that DNA with HMG sequences such as OX gene is amplified, and it can be said that the specificity is low.

Holstein and F ₁ for 3 ′ primer
A specific band (207 bp) was detected only in bovine males (Fig. 3, c, lanes 2, 3 and lanes 4, 5). No male-specific band was amplified in sheep and mice. A male-specific band was sometimes detected in goats (lane
7), it was close to the detection limit and was extremely thin compared to the bovine signal. As there are large changes within the mouse species, the 3'sequence is richer in livestock compared to the 5'primer,
This sequence can be said to be bovine specific.

(Example 9) Sex determination of bovine embryo using PCR Using the three pairs of primers used in Example 8, bovine genomic DNA (200 pg) as a control, sex determination of bovine embryo by PCR was performed. It was The conditions are as in Example 8. Bovine prolapsed blastocysts were taken in 10 μl of distilled water, heat-denatured for 2 minutes in boiling water, and then 5 μl each for 2 minutes, one for sex discrimination by the primer described in Example 8 and the other for sex discrimination kit. (IT selector, XY selector) was used for gender discrimination. The results using the 5'side, HMG box, and 3'side primers described in Example 8 are shown in Fig. 4, respectively. Here, lane 1 is a size marker (a multiple of 123 bp), lanes 2 to 9 are results obtained by using the primers described in Example 8, lane 2 is distilled water, lane 3 is bovine male genomic DNA, and lane 4 is Bovine female genomic DNA, lanes 5-9 are the results using half of 5 bovine embryos. Lanes 10 to 17 are PCs using an XY selector
R results show lanes 10-12 are lanes 2-4, lanes 13-1
7 corresponds to lanes 5 to 9, respectively. XY selector has a male-specific band of 275 bp and a male-female common band of 102 bp
give. Five embryos were analyzed and all were consistent with the results using the XY selector, with 5'primer male, female,
Male, male, male (Figure 4, a, lanes 5-9 and lanes 13-1)
7), male, female, female, female, female (FIG. 4, b, lanes 5 to 9 and lanes 13 to 17) with HMG box primer, and male, female, male, male, male (FIG. 4) with 3 ′ side primer. , C, lane 5 ~
9 and lanes 13-17). Therefore, it was shown that all three can be used as sex discrimination primers.

Example 10 Ten bovine morula stage embryos were collected and a kit (Quick Prep Micro mRNA purification kit,
(Manufactured by Pharmacia) was used to prepare mRNA, and the same kit (First-strand cDNA Synthesis Kit, manufactured by Pharmacia) was used to synthesize cDNA according to the instruction manual. To 33 μl of this reaction solution, add 10 μl of PCR solution (final concentration of 10 mM Tri
sa-HCl (pH 8.9), 1.5 mM MgCl ₂ , 80 mM KCl, 0.1% NaC
l, 0.1% Triton X-100, 50 μM dNTP, 0.2 μM DNA primer, 2 U Tth DNA polymerase)
μl was used for PCR. PCR 94 ° C 1 minute, 60 ° C 1 minute, 72 ° C
One minute, 35 cycles. The primer is SEQ ID NO: 2
It was selected from and was synthesized by an automatic DNA synthesizer.

5'-GCTGACTGCCAGGACGTA-3 '(base numbers 885 to 902 of the sequence of SEQ ID NO: 2) 5'-CATGCATCGGGTTGCATA-3' (complementary strand of base numbers 1494 to 1511 of the sequence of SEQ ID NO: 2)

This primer gives a PCR product of 627 bp. As a positive control for RT-PCR, bovine actin gene (JL Degen et al., J. Biol. Chem., 258, 12153-1) was used.
2162,1983) was used. The primer sequences are as follows, giving a 356 bp PCR product.

5'-TCTGAACCCCAAGGCCAACCGT-3 '5'-CCATCTCCTGCTGGAAGTCCAA-3'

The results of RT-PCR are shown in FIG. Here, in Lanes 1 to 7 and 8 to 13, the above sequence in the sequence of SEQ ID NO: 2 and the above sequence in the actin gene were used as PCR primers, respectively. Lane 1 is 123 b
The size marker of p-series DNA, lane 2 is the DNA shown in SEQ ID NO: 2 (about 3 kb of BamHI / H prepared in Example 7).
incII fragment) containing the plasmid vector pBlueScript SK
(-) DNA (200 pg), lane 3 contains Holstein male DNA (20 pg)
0 pg), lane 4 is Holstein female DNA (200 pg), lane
5 is morula (about 16 cells) in the presence of RT, lane 6 is morula in the absence of RT, and lane 7 is distilled water. As expected, lane 5 gave a band of the same length as lanes 2 and 3. As expected for the actin gene used as a positive control in lanes 8 to 13 corresponding to lanes 2 to 7, lane 9,
10 and 11 gave a 356 bp band positive.

(Example 11) Utilization of Sry-related sequence as mouse Y chromosome marker As shown in Example 6, among the A, B, C, and D fragments,
Only the A fragment showed clearly male-specific Southern hybridization. Therefore, in situ hybridization (D. Pinkel et al., Proc. Natl. Acad. Sci. USA, 8) was performed using an A fragment that specifically hybridizes to male DNA.
3, 2934-2938, 1986). The specific method is FISH (flu
Orescence in situ hybridization) was performed by a known method (edited by Masaaki Hori and Yusuke Nakamura, Lab Manual Human Genome Mapping, Maruzen, pp. 118-133, 1991). The A fragment incorporated into the plasmid pUC118 was labeled with biotin as it was using a commercially available kit (BioNick Labeling System, Gibco BRL). Standard method (edited by Japan Tissue Culture Society,
According to the technique of tissue culture, Asakura Shoten, 1982), a chromosome sample derived from bone marrow of MS / Ae mouse was prepared and hybridized in situ with a biotin-labeled A fragment, and FITC (fluo
rescence isothiocyanate) -bound avidin (avidin-
FITC, manufactured by Boehringer Mannheim). As a result, as shown in FIG. 6 (a color photograph was copied to a black and white photograph), the A fragment was bound to the short arm Yp of the Y chromosome (arrow). This indicates that the DNA region corresponding to the A fragment is located on the short arm of the Y chromosome. Therefore, the A fragment can be used as a marker for this site on the Y chromosome.

(Example 12) Use of Sry-related sequence as bovine Y chromosome marker As shown in Examples 7 and 8, the bovine Sry-related DNA shown in SEQ ID NO: 2 has high specificity to bovine male DNA. . Therefore, as described in Example 11, in situ hybridization was performed using the DNA shown in SEQ ID NO: 2 as a probe. However, chromosomal specimens are prepared from cultured cells derived from a fetal bovine (edited by the Japan Tissue Culture Society, tissue culture technology,
Asakura Shoten, 1982). As a result, the bovine Sry-related DNA shown in SEQ ID NO: 2 has a long arm Yq12 of the Y chromosome as shown in FIG.
Was bound to the terminal part of the (arrow). This indicates that the DNA region corresponding to the bovine Sry-related DNA shown in SEQ ID NO: 2 is located at the end of the long arm of the Y chromosome. Therefore, the bovine Sry-related DNA shown in SEQ ID NO: 2 can be used as a marker for this site on the Y chromosome.

[0055]

According to the present invention, mouse and bovine HMG
DNA and a part of it were provided, including the extensive region around the box. Focusing on the fact that the Sry gene is a sex-determining gene, the DNA shown in SEQ ID NO: 1 can be used for research on sex control in mice as experimental animals.
In addition, the DNA shown in SEQ ID NO: 2 can be used for preliminary tests in mice or studies on bovine sex control based on other findings. Information regarding these sequences serves as basic data for producing transgenic animals and gene knockout animals. Further, the present invention provides RNA corresponding to the above DNA. These RN
A can be used for cell sex discrimination by in situ hybridization and examination of SRY / Sry gene expression.

Further, the present invention provides a polypeptide encoded by the above DNA. Such a polypeptide can be used for sex reversal, etc., as well as research on the sex determining mechanism of animals, comparative research on the activity with Sry of other species. Furthermore, the present invention provides a method for detecting the nucleic acid, which can be used for sex discrimination, species identification, determination of chimerism of chimeric cells, and the like. Further, the present invention provides a Y chromosome marker containing the nucleic acid, and a method for confirming the presence or absence of the nucleic acid region in the Y chromosome using the Y chromosome marker. This makes it possible to detect the deletion or translocation of the nucleic acid region and predict the presence or absence of malformation.
Furthermore, the present invention provides a method for separating a Y chromosome using the Y chromosome marker containing the nucleic acid.

[0057]

[Sequence list]

SEQ ID NO: 1 Sequence length: 10266 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Biological name: Mus musculus Strain name: C57BL / 6J Sequence features: Y chromosome-derived DNA sequences containing the mouse Sry gene: GAATTCATCT GCAATTTCCC TCCCAGCTCT GTTAAGCTTG GGTTCTGGGC CTACTTATAA 60 TCTTACAACT TGGAGCTGAG TGGCCAGGTT GAGAAGGCTT AAAATGAACT GGGGTTGGGG 120 GGGGGGGGGT AGGTGGATAA AAGATAGGGA GGGGTTAGTG GTTAGTGGGT GAGGGAATGA 180 TTGCCTCAGG TGGGTGGATC AGGAAGTGGG CCCAGGCCCT TTGTCATTTC CTTTCAGAAA 240 CTTATCATAG GTTTTGAAGA CCTTAAAATT GAAAGTTTAA AAATGTGGCC ATTATCTGAG 300 AATGTGGCTA CATTCGAGTG GATAACTGTT TTTGGTGGAC TAAGACAGAG TTTTAGAGTC 360 CATCTTTAAG TCTGGCTTAA ATTTGACATA TCAGGCAGGA CTGGTAGGGT GTTTTATCCC 420 ACTGCCTCCT AAGTTTTTGG CAAAGATGAA AATGAGCCCG ATGTGGCACC CCAATACTCA 480 TAAGACGATG GTGGGACTTT GTCATGTCAG TCAAGAACTG GGAGGTGCCT AGAACCTCCA 540 GGACATTGTG ATCCAGAGAA GGAATGAGAA GCTCCCACAG CTTCTGGGAT CTGGTCAGGG 600 CACC TCTCAG TCAGACCAGG ATGGACAGTT ACCTTGTCCT GCACAATTTG GGTGTGTGAT 660 GATAGCAATC CAATGGAAAA AGCTCTCTTA TCCTGAGGTT CAGAGACTCT TGAAGAAGAG 720 GTCCGAGGAA GAGTCTTAGG AAGATGGATG GGGAGGACTC AAAGGACAGC TGAGCAAACT 780 GTGATGAAGC TGGGCTGACA GAGAGTACTA ATGAAGTCCC AGGAGCTGAA ATTGCCTTCA 840 TAAGAAAGCA GAGGCTACAA GAGGCCAGAG ATCTAGGTCA CTTAAGTAAC CATTAGCCTA 900 TGTAATGTGC TCAGAGAGTG TGTGTCCTTC CATTTGAATG ATTGCAGAGG AACATAAGAA 960 AAATGAGGCA GAGAGGCAGA CAGACTCAAA TTCATCAGGC TGACAGTGGT TTATTGGAAC 1020 AGCAGTGAGA GGTGTCAATA TTTTGGCAGC ATGAAGCTTA AGTGTACTTA ATTGTGATGA 1080 GACAGGCTGA CTCCATGACA GGCTTCAAAT AGGCAGTTCA GGGGATTATG CCTAAAACCC 1140 CAGGCCTTAA GCAACTAATC ACAAAATGTT CCTCCACTTG GCCTAAAGTA AGGCCAGTGG 1200 GTCAGCAACA GTTTCCAGCC CTCACACCCA GGTAATGTTC TGGAATGTCC CTGAGAGATA 1260 GAGTAGGATA AGGTCACCTA GCCAGGAATC CCCTGAGTGC ACATTAAATC AGGCCTAACT 1320 CACTCTGGTG TCTCTCTATC TTAATGGGAG ATCCCAGTAT GCAGGACTTT GGGAGAATGA 1380 AACAACCTTT GCGTTTACAT ACTGTTTGAG TCTGGGGTAT CATTCTTCAG TGAATCATGG 1440 ACCCTTACTT TTGGG TGCTC ATTCTGTGAT TCACTCTAGT ACCCCCCCCC AACCCCCGAG 1500 AGTGGAGGCT TTGTCTGATT GGTAAGTCCA GGTGCCTGGT TTTGTTTCAT CTTTTCCATA 1560 TCTATGTTTC TATTTCTCCG TTAATTCTGT TTCCGTTTTA ATCAGAAGGC CAAGAGGATC 1620 AAGGCCGACT CATAGTTACC TGGCCAGGGG TAATGGAAGA CTCTCCCAGA TTCCCTACCA 1680 GAAGCAGGAG AACTTGCTAG TTCTCATCTG AATCCTGAAC TGGCTGTGGG TCTATGGGCC 1740 TCCCATGAGC AGGGAGGAAG ACCATGTAAG ATAGCCTTTT GGTTTTGGTT TTGGTTTTCT 1800 GGGAATGTCC TTTTGTGTTT GTGTGTATTT TGTTTGTCCT TTTTGTCCTC TTGTACAATT 1860 TTTTCCTTTT TTAATTCCTA GGATGGAAAG GCATAAAGCA CTGCACTGGA CCTGATCCTG 1920 AACCATTTTA AGGACTTTAA CAGAGTCATA GAAGACGTGG GTCTGCTTGG TTGTCCCTAC 1980 AAACTAACCA TCTTTTGCAG GAATGAGTGG CCCACCTATA ACCTGAGGAG GGGACTTTCC 2040 ATCTCCCCAT CATTTTTTGG GTGAATACTT GGGGCCACCT GGACCAAGTG CCATACATCA 2100 CCATGTGGCA ATACCTGGTG GAGAGGCCCT CTGTTTGGAT GAGAACTTTT TCCTTACAGG 2160 CCTCACCACC GCACACCCTT CTCCCTCTAA AGGAGGGTCA GCCTTGGCCC CTGCCCAACC 2220 AGCACAGGCA GAGGGAAGAA CAGCCCCCTG ACTCCTCTAA CTTCACTTGG AATTCATGTG 2280 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTATGTGTG TGTGTGTGTG 2340 TGTGTGTTTG TGTGTGTCTT TTTGTGTGTG ATGTGTGATT GGCGATTGTC TTTGCTTTGT 2400 ACACACACTT TACTTTGTAC ACACACACAC ACACACACAC ACACACACAC AGAGTGTTTA 2460 TTGATATGTA AATCACTGGA TTCAGTTTAC AGAGATTCAG ATGTCGTTTG GAATATGATA 2520 GCTGCTAGCA GTACATGTTG CTTGTTCAGC TTTGCTTCCT GATTTAGATG TGTGCCCACT 2580 GTTGCTTTAG GTTGCTTCAG GCCTGCCTAA CCACACCACC TCCTACATCC AAAGTCTCAG 2640 CAGGCACCCC AGGCCTGGCA TCTCTCACGA ATGGTACAGT TCTCAATTCC AGAATCCCTC 2700 ACCACTAACC CTACATGGAT TTGGAGGAGT TAGCAAGAAA GGCCTCCTAC ATGGGGCAAA 2760 GGACTGGCCA GTGATTTGTC CTAGACAGAG GCAGGCTCTA GGTCAACATA CTTCTCACAC 2820 CTGCATTCCA CACCTCCTGA CCTTGGACAG CTAACACTGA TCTTTTCTCA GAATGTCAGG 2880 GAAAGCAGAA GTCTGGAATC TCCCACGGAT GGGAAGTGCA AAAGTCCCCC TGCAAAATTG 2940 AAGGTCACAT AGGCTAATAG AACTGTCCAG GTCCTGGGCT ACATAGACCT AGGGACCCTG 3000 GGTGTTTCCT CTATGCCCAT CAGCCTCCCT CTTCCCAAAA TGGTTACAAA TGTCAATCAT 3060 GCTCTTGATT GCCTTCTCAG CACAGGACAA GTGCCAGCCT CATTTGGGTG ACTGGTGGCC 3120 CCTCCTCCTT GCCCTGTTTC CCCACA GCAG CTCTCAGACA AGAGGTTCAT GGGTTTTGTC 3180 CTGAGGACAC CTGGCTGTTC TCTATCTTGC CAACTCTCAA CCTGGCATGA CCCCCTCCTG 3240 CTCTTAATAT CAAAGCTGTG CATAGGGCAA GCACCCAGGC TTTCCTAGAA ATGCATCCTG 3300 TGTCACAATG GAATGAAGCT GTCAGGGCCT TAAATGTCTT GACTTGGTTG AGCAGAGGTT 3360 ATATGCAGAT CTTTCTCGCG GGAATACACA CTCCTTCTGT TTTGTTGACC TCCATAAATA 3420 CTTTATGTTC CAATTTTAAA TGCTCACTGG CCATCACCTG AACAGATTTC CTCTTTCCCT 3480 TTCCACATAA TGAGTCTCAT GATTTTCTTT CCCAACCTCA CTGTATTTGG AAATCAAAGT 3540 TTGCCAACAC TAATGTTTTA GAAATCCTTT ATTAATGGCA TTTGAATTAT TCCTAAAGAT 3600 GAACAACTGA GAATGACATA ATCACAAGCT ATGATTCTGG CTCAGTAAGT AAGAGCACTT 3660 GGACTTCATT TAGAGCTTTC TATTATCTTT GTAACAAGCA GGCATTGAAG CCAGGTGCTA 3720 TGTAAGCTTT CCAGAAGAAC AGTATCCCAG GGAATACTTT TTGCTAATGG TGTGTGTGTG 3780 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGATGGCAAT TGTCTTTGCT 3840 TTGCTTTCAA TTGCAAGTAT CAGAGGGCAA ACAACCTCAT AGTCCCTGGT CAGTAGGGAG 3900 TTAGCATCCA GGGGACAACA ACTTCATATT TCAGAGATAA AAGAAAGGTC TTTAGTTAAA 3960 CCAAAATCAA CTAATACCAG TTTAATTTTA T AGTTTGTGT CATTAAATGT TTTGAAACCC 4020 TGGACATATT TTTGTTCAAA AGGGAAAAAT AATGCTCCTT GTCACAAATC CAGAGTTTCA 4080 ATATACTCTT TGTGGGAATA CAAAACAAAA CAAAAACAAA CAAACAAACA AACAAAAAAA 4140 CAAGGATCTT CGTATTTACA TTGTCCTTTA TCCTGTCTAG TTTAGATTTT CAGTGAAATC 4200 TCATGCAATT TGCTTGCAAC TCTCCTCCTT AATATGTTTG TTTGCTCTAA AACATTAAAA 4260 GTTGGGTGGT AGGACAAAGT ATTTCAGGAC TCCTTGAGCT GTCCTGTTCT GTAAGCCCCG 4320 TCTTCACCAT TGTTCTAGAA GTTTCTTCCC AGATATTGGT GCTGTGGTCA GTGCCTTGGT 4380 TTGGATTTCT TGGTGTGGCC CATTGAAAGT GCATAGCAGG AAGTATCAGG AGAGGTATGT 4440 GTGTAGGGGG ATGGCATCTT ACAAAAGGAC AGCATCTCAT GAGCTCTCCA AGGATAGCCA 4500 CCAATCCCAG CACTCACCTC TAAGCAGATA GTCGAGTGAA CATTCTGGAG AATGCTAAAG 4560 GTATATAAAT CTTAGGCAGA ACACTGCTTG CAAAATTCAA GCCAGAATTA ACTCGAAAGC 4620 TCTCTTGCAC AAGGATACTC TGTGCACAAA GATGAAGGAG AGAGAGGAGG AGGAAGAAGA 4680 AAGAGGAGGA GGAGGAGGAG GAGGAAGAAG AGGAGAAAGA AAGAGGAGGA AGAAAGAGGA 4740 GGAGGAGGAG GAGGAGACAG AGGGAGATAT GTAGCAAGTA GGTACAGGTT GAAAAGAGGA 4800 AGCCTGTGGA ATGCCAAAGG ATCAATGAGG TCCTTGT CCT GTATCTATGG CTCTCTGCAT 4860 ACAATTCACA CAGAGGAGAA TAAAATCCAC AAATGACATG GAGAAACAAT AGCAGAAACT 4920 CTCCAGAGAA AGAGACATAA GCACCCATTG GGATGGATAT ATTTGTATTC ATTTGTGTTA 4980 GAAATCTATC CAAAGGCTTA TAAAGTCTTA TTGATTGTTG GCATTGAAAT GGTGCTGAAA 5040 CATACCCTTA AAAGGAAAAC TAACCACTTA GAAGTCCAGT AGTGCCAGCT AACTGTTCCA 5100 TTGAATTCTA GAGAACAGTC TTTTAACTGA TTTACAGAAA ACAATACATG TAAGTTACTG 5160 TTCACAATAC ATAATTTTGA AAATCAACTA AAACTCAAAG GTCTTAAGAC CATGTAGAAC 5220 AGTAGTTTAA TTGTTTCCAA GAGTTCAATA CATCAGATAA TACACCATGT TGTTGTATAG 5280 TATTTAGTGT ATAAGATGTC ACAGTTGAGG CTCCTTGGCT GCTATTTTCT TCCAACCTAT 5340 GTTTTAGAGT TGACAGGGCT ACATAATCAA TTCAAAAGTT CATGGGTAAC AGAGTTATTA 5400 TGAGGGAAGC ATCGAGGGAG ATTAATGTGA TGAAATTGGA ACAGGCTAAC TGAAGGTCTG 5460 GGTATGTAAC ATGGTGTTAA CAAGCCTATG TGTAGTTCCT TGGTCTTTCT TAAAGAAACT 5520 GTTGCAGGGG TTGCTGTGTA CACTCTTTAT GTCTCTTTAA ACTTGTTTGT AAGAACAAGA 5580 GTGGGTAACT TTGAACCATT CCTTAGCTTA ACAATCTGGC AGTTGAGTTA ATGTGCAGAT 5640 GGCCATTCAT TCATCCCACA TATACTTGCC CTCCCCTTCA CC TTTTGTTT TATTCCAATC 5700 ATTTTGTATA TGTGGTGTCT TAAGTGTTGG TACATCCACT TAGCTTGACT ATTTGTCCAT 5760 TTTGTTTCTG ATTCTTAGCT AGCCCTTGAA AACACAGCTT TGCTGTATGT CAATAAAACA 5820 GTCTATTCTT TAGAGATAGA TAATAAAAGA GAAGCAAAAT GTGACTCCTT ATGCATAAAG 5880 GCTTATTTTA CCAAAAGATA TAGAGAGCTC ATAGCATAGT TCATAGAACA ACTGGGCTTT 5940 GCACATTGTG GAGGAGAACT AAAATGTCAT ACACACACAC ACACACACAC ACACACACAC 6000 ACACACACAC ACACACACAC ACACACACAC ACTCACAAGA CTTTACATAC TCATGATGTA 6060 GTTTTGTGTG CTAGAGAGAA ACCCTGATAA ACAGAAAGAT GCATTTGATC AACAAAGGAG 6120 TGTTTTGCAT ACTAAAGAGA CACAGCCTGT GTTTTCACAG TATTAAGTTT GGTATGTTTT 6180 TCAAAGATGA GTATTGAAGA CTTACAGATG GTTTGCATGG TGATGCTGTA AGGAAATGAC 6240 AGCCAGCCCT GACCCGATGG CACTGTGTTT TTGCCTGCTC AGAAATGAAC TACTGCATCC 6300 CAGTCATTAG AATGCTCAAC CTGGATGATT TTTACCATGA AAGAATTTTT GCTATTATGT 6360 AAGAAATTTG TATACATTAC TTTTATGATG CATTGATATG ACCAAGAAAG GAAGCAAAAG 6420 TTCTTTTATG GTGTAGATAT TTATACTGGG CTATAGAGCC AGAACAGACT ATTTCTTAGT 6480 TTTTTCCATG CTGCCTGCCG TAGTAGACTA TGATACTAAT GATAGGTT CC ATGCTTATCA 6540 TTTTTAACCC TTTCCACTAC TTTTGCAAGC TCGTTAATTT CTCACGTTAG TCCATCCTAA 6600 ACATTAGGTA CGTTACAGGG CAAAAGAACT CATCAGTATG TTCTTTGTAT CTTAATACTT 6660 GTTGTGACAG AATGATCAAA GAAAAAGCGA TATATTTTTA AGGAGTGTGA AAGTGCAGTC 6720 TCTAACATTC ACAAACTTGA GGGGTTTTGC TTGACAGTAT CTAGGTTCAT GAGATGGCTT 6780 CAAAGTAGAT TAGTTGGGCT GGACTAGGGA GGTCCTGAAG GAGGAGGGAT AAATATTTTC 6840 TTACACACGT TAAATATTAA AATCTTTGTT ACAAGCACAT TTTGGTCAGT GGCTTTTAGC 6900 TCTTACACTT TAAGTTTTGA CTTCTGTATC TGTCTGTCTC TGTCTGTCTC TGTCTGTCTG 6960 TCTGTCTTTG TCTGTCTGTC TGTTTCTCTT TGCATCACAT CTCTGTCTTT TTGGTTGCAA 7020 TCATAATTCT TCCATTATGT TCAGCATTTC AGATCTTGAT TTTTAGTGTT CAGCCCTACA 7080 GCCACATGAT ATCTTAAACT CTGAAGAAGA GACAAGTTTT GGGACTGGTG ACAATTGTCT 7140 AGAGAGC ATG GAG GGC CAT GTC AAG CGC CCC ATG AAT GCA TTT ATG GTG 7189 Met Glu Gly His Val Lys Arg Pro Met Asn Ala Phe Met Val TGG TCC CGT GGT GAG AGG CAC AAG TTG GCC CAG CAG AAT CCC AGC ATG 7237 Trp Ser Arg Gly Glu Arg His Lys Leu Ala Gln Gln Asn Pro Ser Met CAA AAT ACA GAG ATC AGC AAG CAG CTG GGA TGC AGG TGG AAA AGC CTT 7285 Gln Asn Thr Glu Ile Ser Lys Gln Leu Gly Cys Arg Trp Lys Ser Leu ACA GAA GCC GAA AAA AGG CCC TTT TTC CAG GAG GCA CAG AGA TTG AAG 7333 Thr Glu Ala Glu Lys Arg Pro Phe Phe Gln Glu Ala Gln Arg Leu Lys ATC CTA CAC AGA GAG AAA TAC CCA AAC TAT AAA TAT CAG CCT CAT CGG 7381 Ile Leu His Arg Glu Lys Tyr Pro Asn Tyr Lys Tyr Gln Pro His Arg AGG GCT AAA GTG TCA CAG AGG AGT GGC ATT TTA CAG CCT GCA GTT GCC 7429 Arg Ala Lys Val Ser Gln Arg Ser Gly Ile Leu Gln Pro Ala Val Ala TCA ACA AAA CTG TAC AAC CTT CTG CAG TGG GAC AGG AAC CCA CAT GCC 7477 Ser Thr Lys Leu Tyr Asn Leu Leu Gln Trp Asp Arg Asn Pro His Ala ATC ACA TAC AGG CAA GAC TGG AGT AGA GCT GCA CAC CTG TAC TCC AAA 7525 Ile Thr Tyr Arg Gln Asp Trp Ser Arg Ala Ala His Leu Tyr Ser Lys AAC CAG CAA AGC TTT TAT TGG CAG CCT GTT GAT ATC CCC ACT GGG CAC 7573 Asn Gln Gln Ser Phe Tyr Trp Gln Pro Val Asp Ile Pro Thr Gly His CTG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG TTC CAT AAC CAC 7621 Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Phe His Asn His CAC CAG CAG CAA CAG CAG TTC TAT GAC CAC CAC CAG CAG CAG CAG CAG 7669 His Gln Gln Gln Gln Gln Phe Tyr Asp His His Gln Gln Gln Gln Gln CAG CAG CAG CAG CAG CAG CAG CAG TTC CAT GAC CAC CAC CAG GAG AAG CAG 7717 Gln Gln Gln Gln Gln Gln Gln Phe His Asp His His Gln Glu Lys Gln GAG TTT CAT GAC CAC CAC CAG CAG CAA CAG CAG TTC CAT GAC CAC CAC 7765 Glu Phe His Asp His His Gln Gln Gln Gln Gln Phe His Asp His His CAC CAC CAC CAG GAG CAG CAG TTC CAT GAC CAC CAC CAG CAG CAA CAG 7813 His His Gln Glu Gln Gln Phe His Asp His His Gln Gln Gln Gln CAG TTC CAT GAC CAC CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG 7861 Gln Phe His Asp His Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln TTC CAT GAC CAC CAC CAG CAG AAG CAG CAG TTC CAT GAC CAC CAC CAC 7909 Phe His Asp His His Gln Gln Lys Gln Gln Phe His Asp His His His CAC CAA CAG CAG CAG CAG TTC CAT GAC CAC CAG CAG CAG CAG CAG CAG 7957 His Gln Gln Gln Gln Gln Phe His Asp His Gln Gln Gln Gln Gln Gln TTC CAT GAC CAC C AG CAG CAG CAG CAT CAG TTC CAT GAC CAC CCC CAG 8005 Phe His Asp His Gln Gln Gln Gln His Gln Phe His Asp His Pro Gln CAG AAG CAG CAG TTC CAT GAC CAC CCC CAG CAG CAA CAG CAG TTC CAT 8053 Gln Lys Gln Gln Phe His Asp His Pro Gln Gln Gln Gln Gln Phe His GAC CAC CAC CAC CAG CAG CAG CAG AAG CAG CAG TTC CAT GAC CAC CAC 8101 Asp His His His Gln Gln Gln Gln Lys Gln Gln Phe His Asp His His CAG CAG AAG CAG CAG TTC CAT GAC CAC CAC CAG CAG AAG CAG CAG TTC 8149 Gln Gln Lys Gln Gln Phe His Asp His His Gln Gln Lys Gln Gln Phe CAT GAC CAC CAC CAG CAG CAA CAG CAG TTC CAT GAC CAC CAC CAG CAG 8197 His Asp His His Gln Gln Gln Gln Gln Phe His Asp His His Gln Gln CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG CAG TTC CAC GAC CAG CAG 8245 Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Phe His Asp Gln Gln CTT ACC TAC TTA CTA ACA GCT GAC ATC ACT GGT GAG CAT ACA CCA TAC 8293 Leu Thr Tyr Leu Leu Thr Ala Asp Ile Thr Gly Glu His Thr Pro Tyr CAG GAG CAC CTC AGC ACA GCC CTG TGG TTG GCA GTC TCA TGAAACTGGC 8342 Gln Glu His Leu Se r Thr Ala Leu Trp Leu Ala Val Ser CCTTTTCTCC TACTATGCCA ACACTCCCCC TTGCTATGAT TTTTAAGTCT GAGATTCCTA 8402 GTTGGTTTAG ATACTGACTT TTCTCCTGTC ATGAAAAAGG TCCTGCTCCT TCCTTTTGGG 8462 TTCTAAAATG TGTGTAAAGA TGTGTAGTGT GTAAGGAAGG AGAAAGCTTA GAAATGCTTC 8522 TACATAGTTT TTGTTTAACA TAGTACTAGT TGCATTGTTG TGGCATTTAA TCCACAGAAG 8582 AAACCAGAGA TTTACACTCA GATCCAATTT AGATGAAATG CATTTATTCT TAGGCTAGCA 8640 CAAAGACAGC AAACAGCTTT GTGGTCAATA CAGCATGCCA TGCTAAAGTG GGGGCTAGGG 8700 GTGAGTAAGC TGGAGTTTTA AAGGAAACAT CAGAATGACA CATTGTAATG ACAAATATCC 8762 AGCTGGGCAA GAAAATTCTT TTCATTCCTG TTATGTAGTA ATAAAAGACC ATTTCATCCC 8822 CTTTTCCACA GATAATCAGG TTTTACAGGA ACAAAGACAG CTGTGAATTC CTCACAGCCT 8882 ATAAGCCTAT AGTAATCTTA CTCTTTGGCC AATAGGATCT CCCAGGCATC CCAAGGCAAA 8942 GGTCAAGGAC TCTAACTAGA TGCCTGGCTG CATGTAAAAT TTCTTTAGCC ACCACCGGAG 9002 ACATATATAT ATATATATAT GTGTATATAT ATATATAATA TGTAGGTATG TATATAATCA 9062 TTCAGGTTGA GCATTCTAAT GACTGGGATG CAGTAGTTCA TTTCTGAGCA GGCAAAAACA 9122 CAGTGCCATC GGGTCAGGGC TGGCTGTCAT TTCCTTACAG CATCACCATG CAAACCATCT 9182 GTAAGTCTTC AATACTCATC TTTGAAAAAC ATACCAAACT TAATACTGTG AAAACACAGG 9242 CTGTGTCTCT TTAGTATGCA AAACACTCCT TTGTTGATCA AATGCATCTT TCTGTTTATC 9302 AGGGTTTCTC TCTAGCACAC AAAACTACAT CATGAGTATG TAAAGTCTTG TGAGTGTGTG 9362 TGTGTGTGTG TGTGTGTGTG TGTGTGTGTG TGTGTATGAC ATTTTAGTTC TCCTCCACAA 9422 TGTGCAAAGC CCAGTTGTCC TATGAACTAT GCTATGAGCT CTCTATATCT TTGGGTAAAA 9482 TAAGCCTTTA TGCATAAGGC GTCACATTTT CCTTCTCTTT AATAACCTAT CTCTAAAGAA 9542 TAGACTGTTT TATTGACATA CAGCAAAGCT GGGTTTTCCA AGGGCTAGCT AAGAATCAGA 9602 AACAAAATGG ACAAATAGTC AAGCTAAGTG GATGTACCAA CACTTAAGAC ACCACATATA 9662 CAAAATGATT GGAATAAAAC AAAAGGTGAA GGGGAGGGCA AGTATATGTG GGATGACTGA 9722 CTGGCCATCT GCCCATTACC TCACCTGCCA GATTGTTAAG CTAAGGCATG GTTCACAGTT 9782 ACCCACTCTT GTCCTTCCAA CCAAGTTTAA AGAGACATAA AGAGTGTACA CAGCAACCCC 9842 TGCAACAGTT TCTTTAAGAA AGACCAAGGA ACTACACATA GGCTTGTTAA CACCATGTTA 9902 CATACCCAGA CCTTCAGTTA GCCTGTCCCA ATTTCATCAC ATTAATCTCC CTCGATGCTT 9962 CCCTCATAAT AACTCTGTTA CCCATGAACT TTTGAATTGA TTATG TAGCC CTGTCAACTC 10022 TAAAACATAG GTTGGAAGAA AATAGCAGCC AAGGAGCCTC AACTGTAACA TCTTATACAC 10082 TAAATACTAT ACAACAACAT GGTGTATTAT CTGATGTATT GAACTCTTGG AAACAATTAA 10142 ACTACTGTCC TACATGGTCT TAAGACCTTT GAGTTTTAGT TGATTTTCAA AATTATGTAT 10202 TGTGAACAGT AACTTACATG TATTGTTTTC TGTAAATCAG TTAAAAGACT GTCCTCTAGA 10262 ATTC ...... .......... .. ........ .......... .......... .......... 10266

SEQ ID NO: 2 Sequence length: 2840 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Biological name: Bos taurus Strain name: Holstein Sequence characteristics: Bovine Y chromosome-derived DNA sequence containing the Sry gene: GTCAACTTTC AAGTTTGCCT TATGGATTTA TTTGGAAGAA GGCTTCACTT ATTTCATTTT 60 GTAAAGCCTT ACTTTTCAAT TTTCTCCACA GTTTGAACTA CAATATAAAA CAATTGATGG 120 TTACAATTTA GAAATTTAGA TTTATAAAGC TTTTTTTTTT GTTTTGTTTT ATTTAGTTTT 180 GATCATCTGT TAGGTAAAAG TGCAGAAGAG AGTGATGGAT ATTACTTCTG CACAGCCTAC 240 GCATCTAAGA CACCACACAC CAACCCCCAC CCCCACCCTC CCACTTTTCC TACTCCCCAA 300 CCGTCAGAAC AAAAAAATAA AAATCCAGTC CCTTTGAAGT GTTATATTAA CACCAGGGAA 360 CTGCTTGGGT ACCAAGGTTA TGTGTTTTTT CTTTTAATGG AACAGGTTTT TAATCTAATT 420 TTAGTTGTAT CTGAGATTGC CTGTTATGCATGATCATGATGACTGACTGACATCGACGAGAC CATGACTAGAGA 480 CGTTAGCAG AAATATATAA CAGGATTGGG CTGTTTTTCA TCTTTTTTTG TTTAAGATAA 660 CATTCTCTTA CATTCATAAC CGTAGACAAT TTGCTAGTAT GTCTGCTGCA CCTTCATCCT 720 TTGAAATAAG TATATGAAAA TAACTTCATA ATGACACTTT TTGTATTTTT AAGCAGGTGT 780 TAGCACATTC ACAAATTCTG ATTAGATGTA AACAAAGAAG AAAGCAGAGC CTTAATATCC 840 TGTTAAGTAG CTTTGCTTGA GAAAGAGTAG GTTGATGGGT TTGGGCTGAC TGCCAGGACG 900 TATTGAGGGG AGGTATTGGG GGCGGAGAAA TAAATATTTC ACTGTATATA TTGCACTAAG 960 TCAGTCTGTG GTAAGAACAA CTTATGAATA GCACCATAAT TTTTAGAACG CTTACACCGC 1020 ATATTACTTC CTCCCCTTTT AAACAGTGCA GTCGTATGCT TCTGCT ATG TTC AGA 1075 Met Phe Arg GTA TTG AAC GAC GAT GTT TAC AGT CCA GCT GTG GTA CAG CAA CAA ACT 1123 Val Leu Asn Asp Asp Val Tyr Ser Pro Ala Val Val Gln Gln Gln Thr ACT CTC GAC TTT AGG AAA TCT TCC TTG TGC ACA GAC AGT CAT AGC 1171 Thr Leu Ala Phe Arg Lys Asp Ser Ser Leu Cys Thr Asp Ser His Ser GCA AAT GAT CAG TGT GAA AGG GGA GAA CAT GTT AGG GAG AGC AGC CAG 1219 Ala Asn Asp Gln Cys Glu Arg Gly Glu His Val Arg Glu Ser Ser Gln GAC CAC GTC AAG CGA CCC ATG AAC GCC TTC ATT GTG TGG TCT CGT GAA 1267 Asp His Val Lys Arg Pro Met Asn Ala Phe Ile Val Trp Ser Arg Glu CGA AGA CGA AAG GTG GCT CTA GAG AAT CCC AAA ATG AAA AAC TCA GAC 1315 Arg Arg Arg Lys Val Ala Leu Glu Asn Pro Lys Met Lys Asn Ser Asp ATC AGC AAG CAG CTG GGA TAT GAG TGG AAA AGG CTT ACA GAT GCT GAA 1363 Ile Ser Lys Gln Leu Gly Tyr Glu Trp Lys Arg Leu Thr Asp Ala Glu AAG CGC CCA TTC TTT GAG GAG GCA CAG AGA CTA CTA GCC ATA CAC CGA 1411 Lys Arg Pro Phe Phe Glu Glu Ala Gln Arg Leu Leu Ala Ile His Arg GAC AAA TAC CCG GGC TAT AAA TAT CGA CCT CGT CGG AGA GCC AAG AGG 1459 Asp Lys Tyr Pro Gly Tyr Lys Tyr Arg Pro Arg Arg Arg Ala Lys Arg CCA CAG AAA TCG CTT CCT GCA GAC TCT TCA ATA CTA TGC AAC CCG ATG 1507 Pro Gln Lys Ser Leu Pro Ala Asp Ser Ser Ile Leu Cys Asn Pro Met CAT GTA GAG ACA TTG CAC CCC TTC ACA TAC AGG GAT GGT TGT GCC AAG 1555 His Val Glu Thr Leu His Pro Phe Thr Tyr Arg Asp Gly Cys Ala Lys ACC ACA TAC TCA CAA ATG GAA AGC CAA TTA AGC CGG TCA CAG TCC GTG 1603 Thr Thr Tyr Ser Gln Met Glu Ser Gln Leu Ser Arg Ser Gln Ser Val ATC ATA ACC AAT TCA CTC CTG CAA AAG GAG CAT CAC AGC AGC TGG ACA 1651 Ile Ile Thr Asn Ser Leu Leu Gln Lys Glu His His Ser Ser Trp Thr AGC CTG GGC CAC AAT AAG GTA ACA TTG GCT ACA CGG ATT TCG GCG GAC 1699 Ser Leu Gly His Asn Lys Val Thr Leu Ala Thr Arg Ile Ser Ala Asp TTT CCC TGT AAC AAA AGC TTA GAG CCT GGA CTT TCT TGT GCT TAT TTT 1747 Phe Pro Cys Asn Lys Ser Leu Glu Pro Gly Leu Ser Cys Ala Tyr Phe CAA TAT TGACTTCCTT ACTCTCGCTA ACAAAGGCGC TCTTTATCTC AATTTTACTA 1803 Gln Tyr CAGTTTCACC TGCGACTTAA TTTTATAAGA AGCCCAATAA GTAAGTACAT TTAACATGTA 1863 AAGAATTCAG ACTTTCCAAT ATAACTGGTC CTCTGTTAAT CAGTTCTTTC TATAAGAGTA 1923 CTTTTTTGTA AAAAATTATC TTAACAGCAC CAAAACTGCT TGAGTTCAAA GATCATCTAT 1983 TTTNCCTAGT AATGGCACAA TTTTTATATT TCTAATTTTA ATTGTTCCAG AGATTGGTCA 2043 TTAGTTAGAT GGTAGCATAT ATTAATAATC TGGTAATGGC CACTATAGAC AATATAACTT 2103 TTTATTCTAA ATACTGTAAC TCCAAACTAT AGTACTTTCA GAAATACTCA CAAATTCATG 2163 GTACAGAGTA AAAAATCTCG TACTTGGATG GAAAGCTCCC TACATTCTAA AGCACTTTCT 2223 GGTA CAAGGT CTGTCCTTTG GTCTTTCTAG CTACTTTCCA CCTCTTTGTA AATTGCAGGT 2283 AATTAGTGAA TATATATGCA TATCCTATCT CCTTTCACAG TTTATAGTCT TTTGTTCCCA 2343 CTGTGAAAAA GCCAGTTTAG CTGCCATTTG CAACTCAGTT CCTTTAGTCA TACAAAACTG 2403 CAAGCTTTGA CCTGTGTTTT GAGGCGATTA TAACATCCAT CCAGTATTTA ATTAGCACCT 2463 GTGAGACCCA AGGTTTCATC TCTTTTCTGA AAATTTCCTT TTAATCACTG GCAATAAATA 2523 CACTTGTTTC CATTTTCACT TAAGTTTTGC ATTCTTGGAG GGAGAAAACA AAAAATAAAG 2583 TGCCTTCATA TCAAGAATAT AAATTATTCA GATTATGTGG CATGGGGGAT GGGATAAACA 2643 AGATCCTGTC TGATTAATCT GATGAGGTGT CAGTGAAAAT GTAAATCAAA GGTGTTTAAA 2703 AATTTGCAAA TAGGCTAAAG TAGAAAAATT GGCAGCTTGC AAAGGAAGCA TCCCTTTTTT 2763 GGAATGAAAA TAAGTCCATG GTGA ... .... .......... 2840

SEQ ID NO: 3 Sequence length: 229 Sequence type: Polypeptide Number of chains: 1 Strand Topology: Linear Sequence type: Peptide Origin Biological name: Bos taurus Strain name: Holstein Sequence characteristics: Polypeptide sequence designated by No. 1067 to No. 1753 of SEQ ID NO: 2 Met Phe Arg Val Leu Asn Asp Asp Val Tyr Ser Pro Ala Val Val Gln 1 5 10 15 Gln Gln Thr Thr Leu Ala Phe Arg Lys Asp Ser Ser Leu Cys Thr Asp 20 25 30 Ser His Ser Ala Asn Asp Gln Cys Glu Arg Gly Glu His Val Arg Glu 35 40 45 Ser Ser Gln Asp His Val Lys Arg Pro Met Asn Ala Phe Ile Val Trp 50 55 60 Ser Arg Glu Arg Arg Arg Lys Val Ala Leu Glu Asn Pro Lys Met Lys 65 70 75 80 Asn Ser Asp Ile Ser Lys Gln Leu Gly Tyr Glu Trp Lys Arg Leu Thr 85 90 95 Asp Ala Glu Lys Arg Pro Phe Phe Glu Glu Ala Gln Arg Leu Leu Ala 100 105 110 Ile His Arg Asp Lys Tyr Pro Gly Tyr Lys Tyr Arg Pro Arg Arg Arg 115 120 125 Ala Lys Arg Pro Gln Lys Ser Leu Pro Ala Asp Ser Ser Ile Leu Cys 130 135 140 Asn Pr o Met His Val Glu Thr Leu His Pro Phe Thr Tyr Arg Asp Gly 145 150 155 160 Cys Ala Lys Thr Thr Tyr Ser Gln Met Glu Ser Gln Leu Ser Arg Ser 165 170 175 Gln Ser Val Ile Ile Thr Asn Ser Leu Leu Gln Lys Glu His His Ser 180 185 190 Ser Trp Thr Ser Leu Gly His Asn Lys Val Thr Leu Ala Thr Arg Ile 195 200 205 Ser Ala Asp Phe Pro Cys Asn Lys Ser Leu Glu Pro Gly Leu Ser Cys 210 215 220 Ala Tyr Phe Gln Tyr 225

[Brief description of drawings]

FIG. 1 is a restriction enzyme EcoRI map of a phage clone containing the DNA shown in SEQ ID NO: 1.

FIG. 2 shows A (a), B (b), C (c) shown in FIG.
, And an X-ray photograph (autoradiograph) showing a Southern blot image of D (d) fragment against mouse genomic DNA.

[FIG. 3] FIG. 3 shows the use of three types of primers (5'-side primer, HMG box primer, 3'-side primer) having sequences present in the bovine Sry gene for the genomic DNA of several animals. It is a photograph of electrophoresis showing the result of PCR.

FIG. 4 is a PCR of bovine embryo using three kinds of primers (5 ′ side primer, HMG box primer, 3 ′ side primer) having a sequence present in bovine Sry gene.
3 is an electrophoretic photograph showing the results of performing.

FIG. 5 is a photograph of electrophoresis showing the results of PCR (RT-PCR) of bovine morula with a primer having a sequence present in the bovine Sry gene.

FIG. 6 shows a sequence containing the mouse Sry gene (FIG. 1).
2 is a photograph of the morphology of an organism showing that the A fragment of the) is on the short arm of mouse Y chromosome.

FIG. 7 is a photograph of the morphology of an organism showing that the sequence containing the bovine Sry gene (sequence of SEQ ID NO: 2) is on the long arm of the bovine Y chromosome.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Internal reference number FI technical display location C12Q 1/68 A 9453-4B

Claims (22)

[Claims] 1. A DNA containing the sequence of SEQ ID NO: 1 or a part thereof (provided that the base sequence is the base number 66 in the sequence).
Excludes DNA having a nucleotide sequence from 45 to 8646. ) Or its complementary strand. 2. An RN corresponding to the DNA of claim 1.
A. 3. A DNA containing the sequence of SEQ ID NO: 2 or a part thereof (provided that the base sequence is the base number 12 in the sequence).
The DNA having the nucleotide sequence from 61 to 1405 is excluded. ) Or its complementary strand. 4. An RN corresponding to the DNA of claim 3.
A. 5. A DNA containing the sequence of SEQ ID NO: 1 or a part thereof and its complementary strand, a DNA containing the sequence of SEQ ID NO: 2 or a part of its complementary strand, and the DN.
A Y chromosome marker containing at least one nucleic acid selected from the group consisting of RNA corresponding to A. 6. A polypeptide comprising the amino acid sequence of SEQ ID NO: 3. 7. A method of using the nucleic acid according to claim 1 or 2 to detect the presence of the nucleic acid in a biological sample of a mouse. 8. The method according to claim 7, which comprises a step of performing hybridization using the nucleic acid according to claim 1 or 2. 9. The method according to claim 7, comprising a step of amplifying a DNA derived from a mouse biological sample by using the DNA according to claim 1 as a primer. 10. A method comprising the steps of collecting a biological sample of a mouse, producing a cDNA from the collected material using a reverse transcriptase, and amplifying the cDNA using the DNA of SEQ ID NO: 1 as a primer. 7. The method according to 7. 11. The method according to claim 7, which is used for sex determination of a mouse. 12. The method according to claim 7, which is used for identifying a species of a mouse. 13. The method according to claim 7, which is used for determining chimerism of chimeric mouse cells. 14. A method for detecting the presence of a nucleic acid in a bovine biological sample using the nucleic acid according to claim 3 or 4. 15. The method according to claim 14, which comprises the step of performing hybridization using the nucleic acid according to claim 3 or 4.
The described method. 16. The method according to claim 14, which comprises a step of amplifying a DNA derived from a bovine biological sample by using the DNA according to claim 3 as a primer. 17. A bovine biological sample is collected, cDNA is prepared from the collected sample using reverse transcriptase, and D of SEQ ID NO: 3 is prepared.
15. The method of claim 14, comprising the step of amplifying the cDNA using NA as a primer. 18. The method according to claim 14, which is used for sex determination of cattle. 19. The method according to claim 14, which is used for bovine species identification. 20. The method according to claim 14, which is used for determining chimerism of chimeric bovine cells. 21. A method for confirming the presence or absence of the nucleic acid region in the Y chromosome, which comprises staining the Y chromosome with the Y chromosome marker according to claim 5. 22. A method for separating a Y chromosome, which comprises staining the Y chromosome with the Y chromosome marker according to claim 5.