JPH04360687A - Dna specific to bovine male and discrimination of bovine male and female - Google Patents

Abstract

PURPOSE:To discriminate bovine male and female simply and in high sensitivity by using bovine male specific DNA having a specific base sequence or a bovine female specific DNA hybridisable with the DNA as a primer and a probe. CONSTITUTION:Peripheral blood is collected from mature Holstein male and female, centrifuged separately, leukocyte fractions are treated with proteinase, RNase and phenol to extract DNAs, which are mixed with primers shown by formula I and formula II and thermostable polymerase, reacted at 60 deg.C for 2 minutes, at 80 deg.C for 2.5 minutes, further at 93 deg.C for 1.5 minute, amplified by repeating PCR operation 30 times, subjected to electrophoresis on agarose gel, a bond existing in male DNA but not in female DNA is selected and the base sequence is determined to give a bovine male specific DNA containing front part of IO-1 sequence shown by formula III or part thereof. The DNA is used as a probe to accurately discriminate bovine male and female.

Description

Detailed Description of the Invention [0001] [Industrial Application Field] The present invention is directed to the use of bovine male-specific DNA.
The present invention also relates to a method for determining male and female cattle using the DNA as a probe. [0002] In recent years, determining the sex of a fetus or embryo has become an important issue. In particular, it is becoming important in reproductive biology fields such as fetal transplantation. For example, approximately 50,000 fetal transplants were reported in 1985 in the dairy and meat industry alone. If the sex of a fetus could be easily determined before it is implanted into the mother's body, it would be possible to increase the number of female offspring, which would be extremely advantageous in the dairy cattle industry. Furthermore, if the sex of a fetus could be easily determined, it would be possible to select offspring with desirable traits, such as the ability to produce more milk or the ability to have more children. [0003] It has been widely known that sex (male or female) is determined by sex chromosomes. In mammals, genes located on the Y chromosome are responsible for the formation of male characteristics, so the sex of an individual mammal depends on whether or not its genome contains a certain DNA sequence. Become. Therefore, sex can be determined by analyzing the Y chromosome-specific genes in the DNA. Alternatively, genetically related sequences,
Specifically, it can also be determined by analyzing sequences related to the Y chromosome. [0004] Conventionally, there have been various reports on methods for determining sex by DNA. For example, many researchers have identified DNA sequences that preferentially hybridize with human male DNA (Kunkel et al.
Science191, 1189-1190 (19
76), Bishop etal, Nature
303, 831 (1983)). However, it is unclear whether these DNA sequences hybridize with DNA sequences from other species other than humans, such as bovines. On the other hand, in Japanese Patent Application Laid-Open No. 63-500214, male DNA
A bovine DNA probe has been described that preferentially hybridizes with. This DNA is said to be useful as a hybridization probe for determining the sex of fetuses and embryos. Also, male-specific DN useful for determining the sex of embryos or fetuses of ruminants belonging to the subfamily Bovidae, especially of the genus Bos.
A in Japanese Patent Application Laid-Open No. 62-273000 describes cows, sheep,
DNA that can hybridize only to the Y chromosome-specific DNA sequence of goats and other ruminants is listed in Special Table 1.
It is disclosed in Japanese Patent No. -500720. [0005] However, the probe described in Japanese Patent Application Laid-Open No. 63-500214, which relates to sex determination of cattle, is too long; It has been pointed out that the described DNA is too short to be used for PCR, etc. Further, Japanese Patent Application Publication No. 1-500720 does not show any practical examples. [0006] In view of the importance of determining the sex of cattle as described above, there is a desire in the industry to develop a method that can easily and highly sensitively determine the sex of cattle, but there is still no sufficient method. The reality is that it has not been discovered. SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a bovine male-specific DNA that can be used to easily and highly sensitively determine the sex of a cow, and a method for determining the sex of a cow using this DNA as a probe. [Means for Solving the Problems] The present inventors have made extensive studies to solve the above problems. As a result, they discovered male-specific DNA of bovines, and further discovered that by using this DNA, it is possible to easily and highly sensitively distinguish between male and female bovines, thereby completing the present invention. That is, the gist of the present invention relates to a method for determining the sex of a bovine, which is characterized by using (1) bovine male-specific DNA and (2) the aforementioned DNA as a probe. [0009] Specific examples of the bovine male-specific DNA of the present invention include the following. ■ A bovine male-specific DNA comprising all or part of the IO-1 sequence (SEQ ID NO: 1), or a bovine male-specific D that can hybridize with the DNA.
N.A. IO-1 (92bp) 5'-ACAGTCACCA GGCACAGGG
C TAAGAACGGC ACACAGTCAC A
CACAAACAC ACACACAAAC
CGGTTGCACA GTCACCAGGG CAC
AGGGCTG AG-3' 0010] ■ IO
-2 sequence (SEQ ID NO: 2) or a bovine male-specific DNA comprising all or a part thereof;
Bovine male-specific DNA that can hybridize with A. IO-2 (142bp) 5'-AGGGGCACAG GGCTGAGAA
A TCAAATTACA CACGAGCAAA A
AAAAACCTCTTGCACAGTT
GCCAGGGGCA CAGGGCTGAG AAC
GGCACAC ACTCACATAC ACAGAC
AATC CGGTTGCACA GTCGC
CAGGG CACAGGGCTG AG-3'00
11] ■ Bovine male-specific DN comprising all or part of the IO-3 sequence (SEQ ID NO: 3)
A, or bovine male-specific DNA that can hybridize with the DNA. IO-3 (170bp) 5'-TACAGTCGCC AGGGCACAG
G
CCAAACAAACCTGTTGCAC
AGTTGCCAGG GCACAGCGCT GAG
TATGGCA CACACTCACA CACACA
CACA AACCGGTTGA ACCGT
TACCA GAGCACAGGG CTGAGAAC
AG CACACACATA CACACACACC-
3'0012] ■ IO-4 array (sequence number: 4)
or a bovine male-specific DNA that can hybridize with said DNA. IO-4 (224bp) 5'-ACACACACAC ACACACACA
C
AGTCGCCCCGGCACAGGGC
TGAGAAGAAC ACATACATAG ACA
CATACAC ACACAAAAACTG CTTGCA
CAGT CGTCAGGGCT CAGGG
CTGTG AAATGGCACAC ACAAAACAC
TG ACACACACAAC ACACACGAAC
AAACCATTGC ACAGTCACCA
GGGCACAGGG CTGAGGCCAG GG
CACAGGGC TGAG-3' [0013] As mentioned above, the bovine male-specific DNA of the present invention consists of all of the IO-1 sequence, IO-2 sequence, IO-3 sequence, and IO-4 sequence. Alternatively, it may contain a part of any substance that can be used as a probe in a method for determining the sex of cattle. Furthermore, the bovine male-specific DNA of the present invention includes any DNA that can hybridize with the DNA, as long as it can be similarly used as a probe in a method for determining the sex of bovines. Here, examples of things that can hybridize with the DNA include base substitutions in a part of the DNA of the IO-1 sequence,
Examples include those that can hybridize with the DNA even if they have mutations such as deletions and insertions. [0014] The bovine male-specific DNA of the present invention can be obtained, for example, by the following method. (1) Extraction of genomic DNA Fresh, frozen, or formalin-fixed specimens obtained from bovine leukocytes, testes, ovaries, liver, etc. are digested with enzymes such as proteinase to digest the proteins.
DNA is extracted by conventional methods such as phenol extraction and ethanol precipitation. (2) Amplification of genomic DNA by PCR
S8 probe (Ellis et al., Theriogeno
1-20b of 29:242, 1988)
p (MI304) and 541-560bp (MI30
5) An oligonucleotide having a sequence homologous to DN
Bovine genomic DNA is amplified by synthesis using an A synthesizer (for example, manufactured by ABI) and PCR using the obtained oligonucleotide as a primer by a conventional method. The base sequences of these primers are as follows. Primer MI304 (SEQ ID NO: 5): 5'-C
ACCAGGGCA CAGGGCTGAG-3' Primer MI305 (SEQ ID NO: 6): 5'-AC
AGTCGCCA GGGCACAGGG-3' PC
The standard amount of genomic DNA used for R is usually about 1 μg, and the amount of primer is usually about 100 pmol. The conditions for 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 for 2 minutes.
The conditions are usually 80° C. for 2.5 minutes and an extension step, and PCR is usually performed for 30 cycles with these steps as one cycle. (3) The amplified DNA fragment obtained in cloning (2) is subjected to agarose gel electrophoresis. A band having a chain length estimated from the primers used is cut out from the gel subjected to electrophoresis to obtain a DNA fragment having the desired chain length. The obtained DNA fragment is then inserted into a commercially available plasmid, and the plasmid is further introduced into a suitable host such as E. coli. The length of the inserted DNA in the plasmid of the transformant is examined, and a clone having the desired inserted DNA is determined to be positive. (4) The plasmid DNA of the positive clone obtained in restriction enzyme digestion (3) was digested with EcoRI and Ps.
IO-1 sequence of the invention by digestion with tI,
IO-2, IO-3 and IO-4 sequences can be obtained. The method for determining the sex of 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 bovine male-specific DNA of the present invention is used as a probe, and sex is determined based on whether hybridization occurs with DNA in the specimen. That is, if hybridization is observed, the specimen is determined to be male; if not, it is determined to be female. [0019] This hybridization method is not particularly limited as long as it can achieve the above purpose. 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 it by nick translation using a plasmid DNA carrying the DNA as a probe. Specifically, first, using a dot blot format, the sample DNA is adsorbed onto a nitrocellulose filter after performing alkali denaturation and heat denaturation treatments. Prehybridization is performed by immersing the filter in hybridization solution (5x SSPE, 5x Denhardt's solution, 0.5% v/v SDS) at 75°C for 1 hour. The filter was then subjected to overnight hybridization with the probe, after which the filter was incubated 6×
This is done by washing with SSC solution for 1 hour and exposing to X-ray film at -80°C overnight. [0020] The method for determining the sex of cattle of the present invention may use at least one of the male-specific DNAs of the present invention as a probe, but it is also possible to perform dot blot hybridization using a plurality of male-specific DNAs simultaneously as probes. You may go. EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples in any way. Example (1) DNA sample 5 ml of peripheral blood from mature Holstein cows (one male and one female) was centrifuged. Proteinase K, R for leukocyte fraction
DNA was extracted by treatment with Nase and phenol. After the obtained DNA was quantified, 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 male bovine cell DNA, and 1 μg for female bovine cell DNA.
It was μg. ES8 probe (Ellis et al., Th
eriogenology 29:242, 1988)
1-20bp (MI304) and 541-560b of
A primer having a sequence homologous to p(MI305) was synthesized using a DNA synthesizer (manufactured by ABI). 100 pmol of each of the obtained primers was used for PCR. The base sequences of these primers are as follows. Primer MI304 (SEQ ID NO: 5): 5'-C
ACCAGGGCA CAGGGCTGAG-3' Primer MI305 (SEQ ID NO: 6): 5'-AC
AGTCGCCA GGGCACAGGG-3' In
nis and Gelfand (Academi
c Press Inc. 3-12, 1990) to a final volume of 1.
The above-mentioned primers were added in a volume of 00 μl. 2.5
After adding the unit's heat-stable DNA polymerase (Taq polymerase, manufactured by Cetus) to each sample, a PC was used using a programmable heat controller (manufactured by MJ Research).
R operation (operation of reacting at 60°C for 2 minutes, reacting at 80°C for 2.5 minutes, and further reacting at 93°C for 1.5 minutes)
was repeated 30 times. After completion of PCR, the reaction product was
% agarose gel (Sechem ME, 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 of 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, respectively.
This figure shows the results of electrophoresis of products amplified from 10-3, 10-4, 10-5, and 10-6 μg of bovine male DNA. Lane 8 is the electrophoresis result for the product amplified from 1 μg of female bovine DNA. Four unknown bands marked with arrows in the figure were seen in the male DNA specimen. [0024] When this band was analyzed, it was found that approximately 100b
p, 130bp, 180bp and 230bp positions. Bands originating from residual primers also appeared at other positions. These DNA fragments are 1
It was also confirmed by PCR of 0-6 μg of DNA sample. On the other hand, when 1 μg of female DNA sample was analyzed by PCR, no such DNA fragments were found. (3) Cloning and sequencing of PCR products The PCR products were converted into plasmid DNA pUC119 S
After binding to the maI site, it was cloned into E. coli MV1184 (see Figure 2, a is the PstI site, b is the SmaI site, c is the insert DNA, and d is the Ec
oRI site). After cloning was completed, the plasmid DNA was subjected to restriction enzyme analysis using EcoRI and PstI, and then the sequence was determined by the dideoxy method of Sanger et al. As a result, restriction enzyme analysis revealed that PC
The R products could be divided into four types (Figure 3). FIG. 3 shows the results of simultaneously digesting plasmids with four types of PCR products with EcoRI and PstI, subjecting a portion of each type to electrophoresis on a 4% agarose gel, and staining with ethidium bromide. * in the figure is a marker. The base sequences of these DNA fragments determined by the dideoxy method are shown in FIG. The sizes of these four types of DNA fragments are 92 bp (I0-1) and 142 bp (I0-1), respectively.
0-2), 170bp (I0-3), and 224bp
(I0-4). These sizes were approximately the same as estimated values based on electrophoretic mobility. [0027] The base sequences of these four types of DNA fragments are ES
The base sequence did not match that of 5 or ES8. When we examined the degree of identity between the base sequences of ES8 and four types of DNA fragments, we found that
65% of the total sequence for the I0-1 fragment and 5 for the I0-2 fragment.
1%, 57% for the I0-3 fragment, and 84% for the I0-4 fragment.
% homology was observed. Moreover, as is clear from the base sequences in FIG. 4, all of these four types of DNA fragments had repeating CA sequences. (4) Dot blot analysis of male specificity of DNA fragments Plasmid DNA carrying the four types of DNA fragments whose sequences were determined as described above was used as a probe, and after radiolabeling by nick translation, the following Analysis by hybridization was performed. That is, first, using a dot blot format, bovine genomic DNA was adsorbed onto a nitrocellulose filter after performing alkali denaturation and heat denaturation treatments. Prehybridization was performed by immersing the filter in hybridization solution (5x SSPE, 5x Denhardt's solution, 0.5% v/v SDS) at 75°C for 1 hour. The filter was then subjected to hybridization with the probe overnight. Subsequently, the filter was washed with 6xSSC solution for 1 hour, and then exposed to X-ray film (manufactured by Konica) at -80°C overnight. FIG. 5 is a diagram showing the results of the dot blot hybridization analysis obtained in this manner. That is, 1 μg of male and female bovine DNA was applied to four types of nitrocellulose filters, and each of the four types of DNA fragments labeled with 32P was hybridized. In the figure, 1 is the result of hybridization of the I0-1 fragment, 2 is the I0-2 fragment, 3 is the I0-3 fragment, and 4 is the result of hybridization of the I0-4 fragment. As is clear from Figure 5, when these four types of DNA fragments were hybridized with male and female genomic DNA, all four types of fragments showed a positive reaction only to male genomic DNA, and these DNA fragments It was revealed that this species has male specificity. Effects of the Invention In the present invention, in PCR using primers obtained from ES8, the expected amplification of ES8 did not occur, and four types of DNA fragments of the present invention (92 bp, 142 bp,
bp, 170bp, 224bp) were obtained. Although the base sequences of these amplified DNA fragments partially matched the base sequence of ES8, they were not completely identical to either ES5 or ES8. It is known that ES8 constitutes one unit of approximately 600 copies of repetitive sequences present on the bovine Y chromosome (Bondiol
i et al. Theriogenology 31:95-1
04, 1989), and are thought to be included in a group with similar base sequences, although there are some differences. Therefore, it is highly likely that the four types of DNA fragments of the present invention amplified with primers MI304 and MI305 are derived from the above-mentioned similar base sequences. [0031] Furthermore, the base sequences of the four types of DNA fragments all contained repeated CA sequences. This repeat sequence is ES8
accounted for a large portion of the base sequence. In this regard, Kashi et al. (Genomics. 7:31-3
6 (1990), when a probe with a 10-nucleotide sequence (TG) was digested with a restriction enzyme (HaeIII or MboI) and then hybridized to male and female bovine genomic DNA, a male-specific band was detected at a position of several kb to several tens of kb. It is reported that this has happened. From these findings, it is inferred that the repeated CA sequence is a base sequence unique to the bovine Y chromosome. As described above, these four types of DNA fragments are male-specific, and by using them as primers and probes, it is possible to easily and highly sensitively distinguish between male and female cattle. [Sequence Listing] Sequence Number: 1 Sequence Length: 92 Sequence Type: Number of Nucleic Acid Chains: Double-stranded Topology: Linear Sequence Type: Genomic DNA Origin: White blood cells of peripheral blood of adult Holstein cows Sequence ACAGTCACCA GGCACAGGGC TAA
GAACGGC ACACAGTCAC ACACAA
ACAC ACACACAAAAC 60CGGTT
GCACA GTCACCAGGG CACAGGGC
TG AG
92 SEQ ID NO: 2 Sequence length: 142 Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin: Adult Holstein cow peripheral blood leukocyte sequence AGGGGCACAG GGCTGAGAAA T.C.A.
AATTACA CACGAGCAAA AAAAAA
CCTC TTGCACAGTT 60GCCAG
GGGCA CAGGGCTGAG AACGGCAC
AC ACTCACATAC ACAGACAATC
CGGTTGCACA 120GTCGCCAGGG
CACAGGGCTGAG

142 SEQ ID NO: 3 Sequence length: 170 Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin: Adult Holstein cow peripheral blood leukocyte sequence TACAGTCGCC AGGGCACAGG GCT
GAGAATT CACACACACA CCCAAA
CAAA CCTGTTGCAC 60AGTTG
CCAGG GCACAGCGCT GAGTATGG
CA CACACTCACA CACACACACA
AACCGGTTGA 120ACCGTTACCA
GAGCACAGGG CTGAGAACAG CA
CACACATA CACACACACC
170 SEQ ID NO: 4 Sequence length: 224 Sequence type: Number of nucleic acid strands: Double stranded Topology: Linear Sequence type: Genomic DNA Origin: Adult Holstein cow peripheral blood leukocyte sequence ACACACACAC ACACACACAC A.C.A.
CACACTC ACAAATTCCA CAGTCG
CCCG GGCACAGGGC 60TGAGA
AGAAC ACATACATAG ACACATAC
AC ACACAAAAACTG CTTGCACAGT
CGTCAGGGCT 120CAGGGCTGTG
AAATGGCACAC
ACACACAC ACACACGAAC AAACC
ATTGC 180ACAGTCACCA GGGC
ACAGGG CTGAGGCCAG GGCACAG
GGC TGAG
224 SEQ ID NO: 5 Sequence length: 20 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids 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 acids Synthetic DNA Sequence ACAGTCGCCA GGGCACAGGG

20

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the results of subjecting 1/10 of the reaction product to electrophoresis on a 4% agarose gel, staining with ethidium bromide, and developing under ultraviolet irradiation. lanes 1-7
are respectively 1, 10-1, 10-2, 10-3, 10-
4 shows the results of electrophoresis of products amplified from 4, 10-5, and 10-6 μg of male bovine DNA. Lane 8 is the electrophoresis result for the product amplified from 1 μg of female bovine DNA.

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

[Figure 3
] Figure 3 shows plasmids with four types of PCR products.
This figure shows the results of simultaneous digestion with oRI and Pst I, electrophoresis of a portion of each species on a 4% agarose gel, and ethidium bromide staining. M is a marker.

[Figure 4] Figure 4 shows four types of DNA fragments, each 92 bp (
I0-1), 142bp (I0-2), 170bp (I
0-3) and 224 bp (I0-4).

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

Claims (1)

[Scope of Claims] [Claim 1] Bovine male-specific DNA comprising all or part of the IO-1 sequence (SEQ ID NO: 1), or a bovine male-specific DNA that can hybridize with the DNA. Male-specific DNA. IO-1 (92bp) 5'-ACAGTCACCA GGCACAGGG
C TAAGAACGGC ACACAGTCAC A
CACAAACAC ACACACAAAC
CGGTTGCACA GTCACCAGGG CAC
AGGGCTG AG-3' [Claim 2] I
Bovine male-specific DNA comprising all or part of the O-2 sequence (SEQ ID NO: 2), or the D
Bovine male-specific DNA that can hybridize with NA. IO-2 (142bp) 5'-AGGGGCACAG GGCTGAGAA
A TCAAATTACA CACGAGCAAA A
AAAAACCTCTTGCACAGTT
GCCAGGGGCA CAGGGCTGAG AAC
GGCACAC ACTCACATAC ACAGAC
AATC CGGTTGCACA GTCGC
CAGGG CACAGGGCTG AG-3' [
3. A bovine male-specific D comprising all or part of the IO-3 sequence (SEQ ID NO: 3).
NA, or bovine male-specific DNA that can hybridize with the DNA. IO-3 (170bp) 5'-TACAGTCGCC AGGGCACAG
G
CCAAACAAACCTGTTGCAC
AGTTGCCAGG GCACAGCGCT GAG
TATGGCA CACACTCACA CACACA
CACA AACCGGTTGA ACCGT
TACCA GAGCACAGGG CTGAGAAC
AG CACACACATA CACACACACC-
3' [Claim 4] IO-4 sequence (SEQ ID NO: 4
) or a bovine male-specific DNA that can hybridize with the DNA. IO-4 (224bp) 5'-ACACACACAC ACACACACA
C
AGTCGCCCCGGCACAGGGC
TGAGAAGAAC ACATACATAG ACA
CATACAC ACACAAAAACTG CTTGCA
CAGT CGTCAGGGCT CAGGG
CTGTG AAATGGCACAC ACAAAACAC
TG ACACACACAAC ACACACGAAC
AAACCATTGC ACAGTCACCA
GGGCACAGGG CTGAGGCCAG GG
CACAGGGC TGAG-3'.Claim 5. A method for determining the sex of cattle, which comprises using at least one of the DNAs according to any one of claims 1 to 4 as a probe.