JP3201780B2 - Male-specific DNA of cattle and method of discriminating male and female cattle - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a bovine male-specific DNA.
And a method for discriminating between male and female cows using the DNA as a probe.

[0002]

2. Description of the Related Art In recent years, sex determination of a fetus or embryo has become an important issue. In particular, it is becoming important in the field of reproductive biology such as fetal transplantation. For example, in the dairy and meat industry alone, approximately 50,000 fetal transplants were reported in 1985. If the sex can be easily determined before the fetus is transplanted into the mother, the number of female offspring can be increased, which is extremely advantageous in the dairy cattle industry. Further, if the sex of the fetus can be easily determined, it is possible to select offspring having desirable characteristics such as being able to produce more milk and having more children.

It is widely known that gender (male or female) is determined by sex chromosomes. In mammals, since the gene present on the Y chromosome causes the formation of male characteristics, the sex of an individual mammal depends on whether its genome contains a specific DNA sequence. Become. Therefore, by analyzing the Y chromosome-specific gene in the DNA, it is possible to discriminate between male and female. Alternatively, a genetically related sequence,
Specifically, it can be determined by analyzing a sequence associated with the Y chromosome.

Conventionally, various reports have been made on the method of gender discrimination using DNA. For example, DNA sequences that hybridize preferentially to human male DNA have been identified by many researchers (Kunkel et al, Science 191, 11).
89-1190 (1976), Bishop etal, Nature 303, 831 (198
3)). However, it is unclear whether these DNA sequences hybridize to DNA sequences of other species than humans, for example, bovine. On the other hand, JP-A-63-50021
No. 4 describes a bovine DNA probe that hybridizes preferentially with male DNA. This DNA
Is said to be useful as a hybridization probe for determining fetal and embryo sex.
Also, male-specific DNs belonging to the bovine subfamily, and particularly useful for determining the sex of embryos or fetuses of ruminants of the genus Boss
A: Japanese Patent Application Laid-Open No. 62-273000 discloses cattle, sheep,
DNA that can hybridize only to the Y chromosome-specific DNA sequence of goats and other ruminants is disclosed in
-500720.

[0005]

However, the probe described in Japanese Patent Application Laid-Open No. 63-500214 for discriminating the sex of cattle is too long, and the DNA disclosed in Japanese Patent Application Laid-Open No. 62-273000 is too long. Then, it is pointed out that it is too short to be used for PCR or the like. Japanese Patent Application Laid-Open No. 1-500720 does not show an example of practicality.

[0006] As described above, in the art, in view of the importance of sex determination of cattle as described above, there is a demand for the development of a method capable of discriminating male and female in a simple and highly sensitive manner. The fact is that it has not been found. Accordingly, an object of the present invention is to provide a bovine male-specific DNA capable of easily and highly sensitively discriminating bovine sex, and a method for discriminating bovine sex using the same as a probe.

[0007]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems. As a result, a male-specific DNA of bovine was found, and it was found that discrimination of bovine male and female could be performed easily and with high sensitivity by using the DNA, and the present invention was completed.

That is, the gist of the present invention relates to (1) a bovine male-specific DNA and (2) a method for discriminating between male and female bovines, which comprises using the DNA as a probe.

Specific examples of the bovine male-specific DNA of the present invention include the following. A bovine male-specific DNA consisting of the IO-1 sequence (SEQ ID NO: 1), or the DN
A has a base substitution, deletion or insertion in A
Bovine male-specific DNA capable of hybridizing with NA . IO-1 (92 bp) 5'-ACAGTCACCA GGCACAGGGC TAAGAACGGC ACACAGTCAC ACACAAACAC ACACACAAAC CGGTTGCACA GTCACCAGGG CACAGGGCTG AG-3 '

A bovine male-specific DNA consisting of the IO-2 sequence (SEQ ID NO: 2), or a base in the DNA
A bovine male-specific DNA which has a substitution, deletion or insertion of, and is capable of hybridizing with said DNA. IO-2 (142 bp) 5'-AGGGGCACAG GGCTGAGAAA TCAAATTACA CACGAGCAAA AAAAAACCTC TTGCACAGTT GCCAGGGGCA CAGGGCTGAG AACGGCACAC ACTCACATAC ACAGACAATC CGGTTGCACA GTCGCCAGGG CACAGGGCTG AG-3 '

A bovine male-specific DNA consisting of the IO-3 sequence (SEQ ID NO: 3), or a base in said DNA
A bovine male-specific DNA which has a substitution, deletion or insertion of, and is capable of hybridizing with said DNA. IO-3 (170 bp) 5'-TACAGTCGCC AGGGCACAGG GCTGAGAATT CACACACACA CCCAAACAAA CCTGTTGCAC AGTTGCCAGG GCACAGCGCT GAGTATGGCA CACACTCACA CACACACACA AACCGGTTGA ACCGTTACCA GAGCACAGGG CTGAGAACAG CACACACACATACACACACACATA CACACACAC-3

[0012] A bovine male-specific DNA consisting of the IO-4 sequence (SEQ ID NO: 4), or a base in said DNA
A bovine male-specific DNA which has a substitution, deletion or insertion of, and is capable of hybridizing with said DNA. IO-4 (224 bp) 5'-ACACACACAC ACACACACAC ACACACACTC ACAAATTCCA CAGTCGCCCG GGCACAGGGC TGAGAAGAAC ACATACATAG ACACATACAC ACACAAACTG CTTGCACAGT CGTCAGGGCT CAGGGCTGTG AATGGCACAC ACAAACGGAG ACACGAGCAG ACGCACACACACACACACACACACACGCAC

The bovine male-specific DNA of the present invention may be composed of all of the IO-1, IO-2, IO-3 and IO-4 sequences as described above. As long as it can be used as a probe in the method of discriminating male and female, it may contain a part of the probe. Furthermore, as long as it can be used as a probe in a method for discriminating between bovine sexes, the DN
Those that can hybridize with A are also included in the bovine male-specific DNA of the present invention. Here, as a compound that can hybridize with the DNA, for example, a DNA having a mutation such as a base substitution, deletion, or insertion in a part of the DNA of the IO-1 sequence can hybridize with the DNA. And the like.

To obtain the bovine male-specific DNA of the present invention, for example, the following method can be mentioned. (1) Extraction of genomic DNA A fresh, frozen or fixed sample of formalin obtained from bovine leukocytes, testis, ovaries, liver, etc. is digested with a proteinase or other enzyme, followed by phenol extraction and ethanol precipitation. DNA is extracted by a common method such as the above.

(2) Amplification of genomic DNA by PCR ES8 probe (Ellis et al., Theriogenology 29: 242, 1)
988) 1-20 bp (MI304) and 541-5
An oligonucleotide having a sequence homologous to 60 bp (MI305) is synthesized using a DNA synthesizer (for example, manufactured by ABI), and the obtained oligonucleotide is used as a primer to carry out PCR by a conventional method to obtain bovine genomic DNA. Perform amplification. The nucleotide sequences of these primers are as follows. Primer MI304 (SEQ ID NO: 5): 5′-CACCAGGGCA CAGGGCTGAG-3 ′ Primer MI305 (SEQ ID NO: 6): 5′-ACAGTCGCCA GGGCACAGGG-3 ′ The amount of genomic DNA used for PCR is usually about 1 μg as a standard. The amount is usually about 100 pmol. The conditions for the PCR are as follows: the denaturation step is usually at 93 ° C. for 1.5 minutes, and the annealing step is usually at 60 ° C.
And the extension step are usually performed at 80 ° C. for 2.5 minutes, and PCR is usually performed for 30 cycles in which these steps constitute one cycle.

(3) Cloning The amplified DNA fragment obtained in (2) is subjected to agarose gel electrophoresis. From the gel subjected to electrophoresis, a band having a chain length estimated from the used primer is cut out to obtain a DNA fragment having a target chain length. Next, the obtained DNA fragment is inserted into a commercially available plasmid, and the plasmid is introduced into a suitable host such as E. coli.
The length of the inserted DNA of the plasmid contained in the transformant is examined, and the clone having the desired inserted DNA is determined as positive.

(4) Digestion with Restriction Enzyme The plasmid DNA of the positive clone obtained in (3) was
By digesting with coRI and PstI, the IO-1 sequence, the IO-2 sequence, the IO-3 sequence and the IO-
Four sequences can be obtained.

The method of discriminating between male and female cattle of the present invention is characterized by using at least one of the male-specific DNAs of the present invention as a probe. Specifically, the male-specific DNA of the bovine of the present invention is used as a probe, and the sex is determined based on whether or not hybridization occurs with the DNA in the sample. That is, if hybridization is recognized, the sample is determined to be male, and if not, female is determined.

The method of hybridization is not particularly limited as long as such a purpose can be achieved. For example, it can be performed by dot blot hybridization. In this case, the male-specific DNA of the present invention can be used by radiolabeling by nick translation using plasmid DNA carrying the DNA as a probe. Specifically, first, alkali denaturation and heat denaturation are performed using a dot blot format, and then the DNA of the sample is adsorbed to a nitrocellulose filter. Pre-hybridization is performed by immersing the filter in a hybridization solution (5 × SSPE, 5 × Denhardt's solution, 0.5% v / v SDS) at 75 ° C. for 1 hour. Then, after the filter was subjected to hybridization with the probe overnight, the filter was
It is performed by washing with an XSSC solution for 1 hour and exposing the X-ray film to -80 ° C overnight.

In the method for discriminating between male and female cattle of the present invention, at least one of the male-specific DNAs of the present invention may be used as a probe. May go.

[0021]

EXAMPLES The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example (1) DNA sample 5 ml of peripheral blood of a mature Holstein cow (one male and one female) was centrifuged. The leukocyte fraction was converted to proteinase K, R
The DNA was extracted by treating with Nase and phenol.
After quantifying the obtained DNA, it was stored at -20 ° C.

(2) Amplification of bovine genomic DNA by PCR The amount of DNA used for amplification is 10 ^{-6 to 1} μg for bovine cell DNA, and 1 μg for bovine cell DNA.
g. ES8 probe (Ellis et al., Theriogenolo
gy 29: 242, 1988) and primers having sequences homologous to 1-20 bp (MI304) and 541-560 bp (MI305) were synthesized using a DNA synthesizer (ABI). The obtained primers were 100 pmo each.
1 was used for PCR. The nucleotide sequences of these primers are as follows. Primer MI304 (SEQ ID NO: 5): 5'-CACCAGGGCA CAGGGCTGAG-3 'Primer MI305 (SEQ ID NO: 6): 5'-ACAGTCGCCA GGGCACAGGG-3' Innis and Gelland (Academic Press
Inc. 3-12, 1990), the primers described above were added to a PCR buffer prepared according to the method described above in a final volume of 100 μl. After adding 2.5 units of heat-resistant DNA polymerase (Taq polymerase, manufactured by Cetus) to each sample, PCR was performed using a programmable heat controller (manufactured by MJ Research) for 2 minutes at 60 ° C. C. for 2.5 minutes at 93.degree.
Operation for 5 minutes) was repeated 30 times. After the completion of the PCR, the reaction product was subjected to 4% agarose gel (Sechem M).
E, manufactured by FMC), stained with ethidium bromide, and developed under ultraviolet irradiation.

FIG. 1 shows the DNA fragments fractionated by electrophoresis in this manner. That is, FIG. 1 shows the results obtained by subjecting 1/10 of the reaction product to electrophoresis on a 4% agarose gel, staining with ethidium bromide, and developing under ultraviolet irradiation. Lanes 1 to 7 are ¹ , 10 ^-1 , 10 ^-2 , 10
FIG. 3 shows the results of electrophoresis of products amplified from ^-3 , 10 ^-4 , 10 ^-5 and 10 ^-6 μg of bovine male DNA. Lane 8 shows the results of electrophoresis of the product amplified from 1 μg of bovine female DNA. Four unknown bands with arrows in the figure were seen in the male DNA sample.

When this band was analyzed, about 100 b
It was located at p, 130 bp, 180 bp and 230 bp. In addition, bands derived from the remaining primer appeared at other positions. These DNA fragments are 1
Even 0 ^-6 PCR of μg of DNA samples was confirmed. on the other hand,
When 1 μg of the female DNA sample was analyzed by PCR, no such DNA fragment was found.

(3) Cloning and Sequencing of the PCR Product The PCR product was cloned into plasmid DNA pUC119 using the Sm
After binding to the aI site, it was cloned into E. coli MV1184 (see FIG. 2, a is the PstI site, b is the S
maI site, c is the inserted DNA, and d is the Eco site.
RI site). After completion of the cloning, the plasmid DNA was subjected to restriction enzyme analysis with EcoRI and PstI, and the sequence was determined by the dideoxy method of Sanger et al.

As a result, analysis by restriction enzyme
The R product could be divided into four types (FIG. 3). FIG.
Describes the plasmid containing the four PCR products as EcoRI.
5 shows the results of simultaneous digestion with Escherichia coli and PstI, electrophoresis of some of the various components on a 4% agarose gel, and staining with ethidium bromide. * In the figure is a marker.
The nucleotide sequences of these DNA fragments determined by the dideoxy method are shown in FIG. The sizes of these four DNA fragments were 92 bp (I0-1) and 142 bp (I
0-2), 170 bp (I0-3), and 224 bp
(I0-4). These sizes were about the same as the estimates based on electrophoretic mobility.

The base sequences of these four DNA fragments are ES
5 and ES8 did not match. When the base sequence identity between ES8 and the four DNA fragments was examined,
65% of the total sequence for the I0-1 fragment and 5% for the I0-2 fragment
1%, 57% for the I0-3 fragment, 84% for the I0-4 fragment
% Homology was observed. In addition, as apparent from the nucleotide sequence of FIG. 4, all of these four types of DNA fragments had a repeated CA sequence.

(4) Dot Blot Analysis of Male Specificity of DNA Fragments The plasmid DNA holding the four DNA fragments whose sequences were determined as described above was used as a probe and radiolabeled by nick translation. Was analyzed by hybridization. That is, first, a bovine genomic DNA was adsorbed to a nitrocellulose filter after alkali denaturation and heat denaturation treatments were performed using a dot blot format. Pre-hybridization was performed by immersing the filter in a hybridization solution (5 × SSPE, 5 × Denhardt's solution, 0.5% v / v SDS) at 75 ° C. for 1 hour.
The filters were then hybridized overnight with the probes. Subsequently, the filter was washed with a 6 × SSC solution for 1 hour, and then exposed to an X-ray film (manufactured by Konica) at −80 ° C. overnight.

FIG. 5 is a diagram showing the results of the dot blot hybridization analysis thus obtained. That is, 1 μg of bovine male and bovine female DNA was applied to four types of nitrocellulose filters, and four types of ³² P-labeled DNA fragments were hybridized.
In the figure, 1 is the result of hybridization of the I0-1 fragment, 2 is the result of hybridization of the I0-2 fragment, 3 is the result of hybridization of the I0-3 fragment, and 4 is the result of hybridization of the I0-4 fragment. As is apparent from FIG. 5, when these four types of DNA fragments were hybridized with each of the male and female bovine genomic DNAs, all of the four types of fragments showed a positive reaction only with the male genomic DNA. It was found to have male specificity.

[0030]

According to the present invention, in the PCR using primers obtained from ES8, the expected amplification of ES8 does not occur, and four kinds of DNA fragments of the present invention (92 bp, 142 bp) are used.
bp, 170 bp, 224 bp). Although the nucleotide sequences of these amplified DNA fragments were partially identical to those of ES8, they were not completely identical to ES5 or ES8. Since ES8 is known to form one unit of a repeat sequence of about 600 copies present on the bovine Y chromosome (Bondioli et al., Th.
eriogenology 31: 95-104, 1989), and is likely to be included in a group having similar nucleotide sequences with some differences. Therefore, it is highly likely that the four kinds of DNA fragments of the present invention amplified with the primers MI304 and MI305 are derived from the similar nucleotide sequences described above.

In addition, the base sequences of all four DNA fragments contained a repetitive CA sequence. This repeat is ES8
Occupies a considerable part of the nucleotide sequence. In this regard, Kashi et al. (Genomics. 7: 31-36 1990) describe (T
G) A probe having a 10-base sequence was replaced with a restriction enzyme (HaeII).
I or MboI) after digestion with the male and female bovine genome DN
It is reported that when hybridized to A, a male-specific band was detected at a position of several kb to several tens kb. From these findings, it is inferred that the repetitive CA sequence is a nucleotide sequence unique to bovine Y chromosome. Thus these four
Species DNA fragments are male-specific, and by using them as primers and probes, bovine sexes can be distinguished easily and with high sensitivity.

[0032]

[Sequence list] SEQ ID NO: 1 Sequence length: 92 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin: Peripheral blood leukocyte sequence of mature Holstein cattle ACAGTCACCA GGCACAGGGC TAAGAACGGC ACACAGTCAC ACACAAACAC ACACACAAAC 60 CGGTTGCACA GTCACCAGGG CACAGGGCTG AG 92

SEQ ID NO: 2 Sequence length: 142 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: Leukocyte sequence of peripheral blood of mature Holstein cattle AGGGGCACAG GGCTGAGAAA TCAAATTACA CACGAGCAAA AAAAAACCTC TTGCACAGTT 60 GCCAGGGGCA CAGGGCTGAG AACGGCACAC ACTCACATAC ACAGACAATC CGGTTGCACA 120 GTCGCCAGGG CACAGGGCTG AG 142

SEQ ID NO: 3 Sequence length: 170 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: leukocyte sequence of peripheral blood of mature Holstein cow TACAGTCGCC AGGGCACAGG GCTGAGAATT CACACACACA CCCAAACAAA CCTGTTGCAC 60 AGTTGCCAGG GCACAGCGCT GAGTATGGCA CACACTCACA CACACACACA AACCGGTTGA 120 ACCGTTACCA GAGCACAGGG CTGAGAACAG CACACACATA CACACACACC 170

SEQ ID NO: 4 Sequence length: 224 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin: peripheral blood leukocyte sequence of mature Holstein cattle ACACACACAC ACACACACAC ACACACACTC ACAAATTCCA CAGTCGCCCG GGCACAGGGC 60 TGAGAAGAAC ACATACATAG ACACATACAC ACACAAACTG CTTGCACAGT CGTCAGGGCT 120 CAGGGCTGTG AATGGCACAC ACAAACACTG ACACACACAC ACACACGAAC AAACCATTGC 180 ACAGTCACCA GGGCACAGGG CTGAGGGCAG GG

SEQ ID NO: 5 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence CACCAGGGCA CAGGGCTGAG 20

SEQ ID NO: 6 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ACAGTCGCCA GGGCACAGGG 20

[Brief description of the drawings]

FIG. 1 is a diagram showing the results of electrophoresis of 1/10 of the reaction product on a 4% agarose gel, staining with ethidium bromide, and developing under ultraviolet irradiation. Lanes 1-7
Are ¹ , 10 ^-1 , 10 ^-2 , 10 ^-3 , 10 ^-4 , 10
¹ shows the results of electrophoresis of products amplified from ^-5 and 10 ^-6 μg of bovine male DNA. Lane 8 is bovine female D
It is an electrophoresis result about the product amplified from 1 microgram of NA.

FIG. 2 is a diagram showing a state in which a PCR product was ligated to Escherichia coli MV1184 at the SmaI site of plasmid DNA pUC119 and cloned. In the figure, a is P
stI site, b is Sma I site, c is inserted DNA
And d indicates EcoRI site

FIG. 3 shows a plasmid containing four PCR products.
Digest with EcoRI and Pst I at the same time.
It shows the results of electrophoresis on an agarose gel and ethidium bromide staining. M is a marker.

FIG. 4 shows four DNA fragments, each 92 bp.
(I0-1), 142 bp (I0-2), 170 bp
It is the figure which showed the base sequence of (I0-3) and 224 bp (I0-4).

FIG. 5 shows the results of a dot blot hybridization analysis. In the figure, 1 is the I0-1 fragment, 2 is the I0-1 fragment.
0-2 fragment, 3 is the result of hybridizing the I0-3 fragment, and 4 is the result of hybridizing the I0-4 fragment.

────────────────────────────────────────────────── ─── Continuation of front page (56) Reference US Pat. No. 4,960,690 (US, A) Animal Biotechnology, Vol. 2, No. 1, p. 61-73 (1991) (58) Field surveyed (Int. Cl. ⁷ , DB name) C12N 15/09 C12Q 1/68 GenBank / EMBL / DDBJ (GENETYX)

Claims (5)

(57) [Claims] [Claim 1] IO-1 sequence (SEQ ID NO: 1) consisting essentially of a male-specific DNA in bovine or bases in the DNA,
Bovine male-specific DNA having substitutions, deletions or insertions, and capable of hybridizing with the DNA. IO-1 (92 bp) 5'-ACAGTCACCA GGCACAGGGC TAAGAACGGC ACACAGTCAC ACACAAACAC ACACACAAAC CGGTTGCACA GTCACCAGGG CACAGGGCTG AG-3 ' 2. A bovine male-specific DNA consisting of the IO-2 sequence (SEQ ID NO: 2) ,
Bovine male-specific DNA having substitutions, deletions or insertions, and capable of hybridizing with the DNA. IO-2 (142 bp) 5'-AGGGGCACAG GGCTGAGAAA TCAAATTACA CACGAGCAAA AAAAAACCTC TTGCACAGTT GCCAGGGGCA CAGGGCTGAG AACGGCACAC ACTCACATAC ACAGACAATC CGGTTGCACA GTCGCCAGGG CACAGGGCTG AG-3 ' 3. A bovine male-specific DNA consisting of the IO-3 sequence (SEQ ID NO: 3), or a base in the DNA .
Bovine male-specific DNA having substitutions, deletions or insertions, and capable of hybridizing with the DNA. IO-3 (170 bp) 5'-TACAGTCGCC AGGGCACAGG GCTGAGAATT CACACACACA CCCAAACAAA CCTGTTGCAC AGTTGCCAGG GCACAGCGCT GAGTATGGCA CACACTCACA CACACACACA AACCGGTTGA ACCGTTACCA GAGCACAGGG CTGAGAACAG CACACACACATACACACACACATA CACACACAC-3 4. IO-4 sequence (SEQ ID NO: 4) consisting essentially of a male-specific DNA in bovine or bases in the DNA,
Bovine male-specific DNA having substitutions, deletions or insertions, and capable of hybridizing with the DNA. IO-4 (224 bp) 5'-ACACACACAC ACACACACAC ACACACACTC ACAAATTCCA CAGTCGCCCG GGCACAGGGC TGAGAAGAAC ACATACATAG ACACATACAC ACACAAACTG CTTGCACAGT CGTCAGGGCT CAGGGCTGTG AATGGCACAC ACAAACGGAG ACACGAGCAG ACGCACACACACACACACACACACACGCAC 5. A method for discriminating between male and female bovines, comprising using at least one of the DNAs according to claim 1 as a probe.