JPH08256778A - Bovine y chromosome specific dna and its use as marker common to male and female - Google Patents

Abstract

PURPOSE: To obtain a bovine Y chromosome specific DNA and/or a marker containing a DNA common to male and female useful for distinguishing a sex chromosome from an autosomal chromosome, an X CHR from a Y CHR and a short arm from a long arm in the Y CHR. CONSTITUTION: A female bovine DNA is isolated, a fragment having no regularity in DNA sequences at both ends is prepared from the DNA, is mixed with a DNA fragment obtained by treating a male bovine DNA with II type restriction enzyme and preparing regular ends and reassociated. Only a duplex DNA having regular ends is incorporated into a cloning vector having a restriction end contemporary with the ends, transduced into a host to give a DNA fragment having a male specific sequence, its base sequence is determined to give a sequence of the formula. The DNA fragment is used as a probe, a second and a third DNAs having male specific sequences are isolated from a male bovine DNA genome library, and a fourth DNA providing different association images between male and female and a fifth to a seventh DNAs having sequences common to male and female are obtained by Southern blot method.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a bovine Y chromosome-specific D
Markers including NA and hermaphroditic DNA, methods for identifying or identifying biological samples using them, and bovine markers
DNA using Y-chromosome-specific DNA and a part of male and female common DNA
More specifically, a marker comprising a DNA having at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 or a part thereof,
Using them, it is possible to identify that biological samples such as cells, tissues, and subcellular organs are of bovine origin, distinguish between nucleus and cytoplasm, sex chromosome and autosomal chromosome, and if sex chromosome, X chromosome and Y chromosome. Different from the chromosome, if the Y chromosome, the short arm and the long arm,
If it is a short arm, a method of knowing whether it is a centromere side or an end, and using as a primer a DNA having a part of at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7, 5 ′ adjacent to the selected sequence and / or
Alternatively, the present invention relates to a method for cloning DNA existing on the 3'side.

[0002]

2. Description of the Related Art Among mammals, human and mouse gene research is remarkable, including a genome project. On the other hand, genomic research of livestock including cattle has been slow, and isolation, identification of specific genes and DNA sequences,
Characterization and chromosomal localization (mapping) are awaited. Furthermore, it is also an important issue to isolate and characterize individual genes and DNAs of bovine origin practically and concretely and to develop a method of using them.

We have already isolated and identified several bovine DNAs and found that they are male-specific or common to both sexes (Japanese Patent Application No. 3-352032). It is based on the results of Southern hybridization. Since the Southern hybridization method uses the entire genome, its presence in the genome can be known, but its position on the chromosome is unknown. If the part of the chromosome involved is unknown, interpretation and judgment of the results are limited and the utility value is low. Therefore,
Furthermore, we aimed to expand the utility value by knowing the characteristics of each DNA.

[0004]

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method of using a specific DNA specific to bovine Y chromosome or common to both sexes.

[0005]

Means for Solving the Problems The inventors of the present invention have conducted extensive studies to achieve the above-mentioned object, and as a result, a method for amplifying a defective site
(deletion enrichment) and PERT method (phenol emersion
bovine male-specific DNA fragment isolated by combining the reassociation technique), and the DNA fragment derived from mitochondria common to both sexes and mitochondria that could be cloned during the isolation, their species-specificity, location, By examining their patterns of presence (including copy number), they can be used as markers to identify that biological samples such as cells, tissues, and subcellular organs are of bovine origin, distinguish between nucleus and cytoplasm, and sex chromosomes. And autosomes, X and Y chromosomes for sex chromosomes, short and long arms for Y chromosomes, centromere or end for short arms. The inventors have found that the above can be achieved and have completed the present invention. That is, the present invention provides a marker containing a DNA having at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 or a part thereof. Also,
The present invention relates to a DNA having at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 or a part thereof.
A method for identifying or specifying a biological sample using a marker containing Further, the present invention provides SEQ ID NOs: 1
Using a DNA having a part of at least one sequence selected from the group consisting of 7 sequences as a primer, cloning the DNA existing on the 5'side and / or 3'side adjacent to the selected sequence It also provides a way to do it.

Hereinafter, the present invention will be described in detail. DNAs having the sequences of SEQ ID NOs: 1 to 7 can be obtained as follows. First, cow DNA is separated by a conventional method, and a fragment having no regularity in the DNA sequences at both ends is prepared from the DNA. The length of this fragment is preferably about 1000 bases. In addition, this fragment can be prepared from the above bovine DNA by a conventional method such as sonication.

Separately, bull DNA obtained by a conventional method
Is treated with a type II restriction enzyme to prepare regular ends. Next, mix the cow and bull DNA fragments described above,
Perform a reassociation reaction. DN before or after such mixing
It is necessary as a precondition for the reassociation reaction to denature the A fragment into a single strand by a conventional method. The mixing ratio of the two kinds of DNA is such that the female DNA is in a large excess, specifically 20 to 1000 times, preferably about 50 times. Examples of the method for denaturing the single-stranded DNA of the double-stranded DNA include a method by heat treatment and a method by alkali treatment. The reassociation reaction can be performed under known hybrid conditions.
It is preferable to add a drug such as phenol that promotes the reassociation of A chain.

When re-associated, most of the autosomal and X-chromosome-derived male DNA is common to female DNA, so there is a large excess of female DNA.
It shows complementarity with DNA and reassociates. These are female DNAs with unspecified ends and male DNAs with type II restriction enzyme ends.
Therefore, the end of the sequence unique to the correct type II restriction enzyme cannot be constructed due to the association of On the other hand, the characteristic region of the Y chromosome of male DNA is female D
Since there is no complementary partner in the NA fragment, the original strands reassociate to return to the original double-stranded DNA having the sequence specific to the type II restriction enzyme used at the ends. By incorporating only double-stranded DNA having such regular ends into a cloning vector having a restriction end complementary to the ends by a generally known method, only male-specific DNA fragments are selectively incorporated into the cloning vector. .

The cloning vector that has undergone the above-mentioned integration reaction is introduced into a host, and it is confirmed by using an appropriate marker whether or not the bovine gene has been introduced.
A DNA clone containing DNA having the sequence of is isolated. The marker used here is determined according to the cloning vector to be used, and examples thereof include resistance to antibiotics such as ampicillin and tetracycline, and color development by X-gal. D containing DNA having the sequence of SEQ ID NO: 1
NA clones are isolated by extracting plasmid DNA from each colony, labeling it with radiation by a known method, and cloning by Southern blotting,
When the cloning vector is a phage, a method of cloning can be adopted by using the plaque hybridization method. The base sequence of this cloned DNA can be determined by a generally known method, for example, the Sanger method, and it can be confirmed that it has the sequence of SEQ ID NO: 1.

Further, using the DNA fragment having the sequence of SEQ ID NO: 1 which is the male-specific sequence obtained as described above as a probe, a male-specific sequence is further analyzed from a bovine male DNA genomic library. DNA having certain sequences of SEQ ID NOs: 2 and 3 can be isolated. The sequence of this DNA can also be determined by a conventional method as described above, and it can be confirmed that it has the sequence of SEQ ID NO: 2 or 3.

A large number of clones can be obtained by screening a bovine genomic library using the DNA fragment having the sequence of SEQ ID NO: 1 as a probe. These clones often contain 15-20 kb long bovine Y chromosome DNA. Such a long sequence may include a DNA having a sequence that associates with the X chromosome other than the Y chromosome or an autosomal chromosome. When such a DNA is isolated and purified by a generally known method and then used as a probe for Southern blotting, a difference in the number and density of bands in the electrophoretic image between males and females occurs. As described above, the DNA having the sequence of SEQ ID NO: 4 can be selected as one of the DNAs having the sequences that give different association images between male and female by Southern blotting.

Furthermore, when the male-specific primary clones are searched as described above, clones having a sequence that commonly binds to male and female DNA can be obtained by Southern method. A clone expected to be composed of repetitive sequences can be selected, and DNA can be isolated and extracted by a conventional method to obtain DNA having the sequences of SEQ ID NOs: 5 to 7. These are sequences common to both sexes.

Alternatively, Escherichia coli E. coli into which a plasmid having DNAs having the sequences of SEQ ID NOs: 1 to 7 has been introduced.
li DH5α has accession numbers FERM BP-4095 and FERM B, respectively.
P-4089, FERM BP-4090, FERM BP-4091, FERM BP-4092,
December 1991 as FERM BP-4093 and FERM BP-4094
Since it has been deposited at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology on the 12th, these bacteria are cultured in an appropriate medium such as LBA medium and the known alkaline treatment method (J. Sambrook et al.
(Eds.), Molecular Cloning, 2nd ed., Cold Spring H
arbor Laboratory Press, pp. 1.25-1.39, 1989) to isolate and purify DNA. For example, the outline is as follows. Make a culture solution of overnight culture with 5 ml of LBA medium and collect the cells by centrifugation. To this, add 200 μl of Solution I (50 ml glucose, 10 ml EDTA, 20 mM Tris-HCl, pH 8.0),
Suspend the bacteria and transfer to a 1.5 ml Eppendorf tube. 300 μl
Add solution II (0.2 M NaOH, 1% SDS), mix, and mix on ice for 5
Incubate for 200 min and add 200 μl of solution III (3 M sodium acetate, p.
H 4.8) is added and mixed, and the mixture is allowed to stand on ice for 5 minutes. 15,00
Centrifuge at 0 rpm for 5 minutes and transfer the supernatant to another Eppendorf tube.
According to a conventional method, the supernatant is extracted with phenol and washed with chloroform, and by adding the same amount of 2-propanol,
Precipitate the DNA. Collect DNA by centrifugation, wash with 80% ethanol, and dry under vacuum. Add 100 μl of RNase solution (10
μg / ml) and left at 37 ° C for 15 minutes, then 60 μl of 2.5
Add M NaCl / 20% polyethylene glycol and leave on ice for 20 minutes. Centrifuge at 15,000 rpm for 5 minutes, collect DNA, wash with 80% ethanol, vacuum dry, and remove 100 μl of T
Dissolve in E (10 mM Tris-HCl, 1 mM EDTA, pH 8). Take a part of it and measure the DNA concentration.

Sequence Nos. 1 to 7 obtained as described above
In order to know the species specificity of the DNA having the sequence of, the known Southern blotting method can be used. First, DNA is isolated and purified from the desired animal of either sex, cut with a specific restriction enzyme, and subjected to agarose gel electrophoresis to obtain DNA.
Is transferred to a nylon membrane or the like after being made into a single strand, and a DNA probe and a molecule having at least one sequence selected from the group consisting of isotopes or chemically labeled sequences of SEQ ID NOs: 1 to 7 or a part thereof. Allow hybrids to be created. Strong and weak signals are obtained depending on the homology. If only knowing the presence or absence, a dot blot method in which DNA is hybridized without cutting with a restriction enzyme can also be used. An in situ hybridization method for cells may be used.

In addition to the above method utilizing the hybridization of DNA, the PCR method is often used to know the species specificity. This uses a specific part of the DNA having the sequences of SEQ ID NOs: 1 to 7 as 5'side and 3'side primers (usually 15 to 40 bases, preferably 17 to 25 bases), and It is made to react as a template. If the region sandwiched between both primers is amplified, they have commonality and it is determined that there is no species specificity.

In order to know in which chromosome the DNAs having the sequences of SEQ ID NOs: 1 to 7 are present, a method using a hybrid panel can be used. For example, when human-mouse fused cells are made and the culture is continued, human cells are gradually lost, and only one specific cell or a few cells remain. Ideally, one set of panels containing each of the 23 human chromosomes is preferable. The combination may cover all human chromosomes. In any case, the above-mentioned blotting method or PCR method is performed using each fused cell DNA constituting the panel. By doing so, the DNA
You can see which human chromosome is on. This method has drawbacks. One is that it is not easy to create a panel, and it is possible to map on the chromosome, but it is unknown where on the chromosome.

Recently, an in situ hybridization method (FISH, fluorescence) using a fluorescent dye label is used.
In situ hybridization) has made it possible to map directly on the chromosome. Further, when cells are used, various information can be obtained from the nucleus or cytoplasm, and the frequency and distribution state. When the DNAs having the sequences of SEQ ID NOS: 1 to 7 were mapped using the above method, the sequences of SEQ ID NOS: 1 to 4 were almost Y of the short arm of the Y chromosome.
_{In p11} , the sequence of SEQ ID NO: 5 was found to be located in mitochondria, and the sequences of SEQ ID NO: 6 and 7 were found to be autosomal.

Utilizing such characteristics of each DNA, seeds,
It can be used for the identification or identification of biological samples such as male and female, chromosomes and specific parts thereof, and intracellular organs (mitochondria). As an example thereof, DNA having the sequences of SEQ ID NOs: 1 to 7 or a part thereof can be used as a marker. Since the sequences of SEQ ID NOs: 1 to 4 are located almost at _Yp11 on the short arm of the Y chromosome, the sequence of SEQ ID NO: 5 is located in mitochondria, and the sequences of SEQ ID NOs: 6 and 7 are located in autosomal chromosomes, so DNA having the sequence of ~ 4 or a part thereof is a Y chromosome marker, DNA having the sequence of SEQ ID NO: 5 or a part thereof is an extranuclear gene marker, and sequences of SEQ ID NOs: 6 and 7 or a part thereof are The possessed DNA can be used as an autosomal marker.

Generally, when used as a marker, the use of a long sequence increases the sensitivity because the number of labeled portions increases. Therefore, long sequences such as the sequences of SEQ ID NOs: 1 to 4 may be used as they are as probes.
Regardless of the position of the array, it is effective to select half or one tenth thereof as appropriate. On the other hand, the sequences of SEQ ID NOs: 5 to 7 are relatively short sequences, and it is preferable to use them as they are as probes. However, when the copy number of the sequence is large, for example, from the base number 1 to the sequence number 7
In the case of 60, a quarter of 241 base pairs of the original sequence is also effective as a probe. When using a specific portion of the sequences of SEQ ID NOs: 1 to 7 as a primer,
A partial sequence having 15 to 40 bases, preferably 17 to 25 bases may be used. In this case, it is common knowledge about PCR that the 5'side and 3'side primers themselves do not form intramolecular association, and the 5'side and 3'side pair of primers do not form intermolecular association. As such, it is necessary to pay attention to the selection of the base sequence. Furthermore, the length of the PCR-amplified portion sandwiched between a pair of primers is tens to hundreds of base pairs so that it can be easily synthesized by a DNA synthase and the PCR product can be easily detected. It is desirable to set a pair of primers as follows. At present, DNA with a length of 20 to 30 kilobase pairs is also amplified by PCR, but when it is used as a marker, the length that can be reliably amplified and easily detected (tens to hundreds). Base pair) is convenient.

DNAs having the sequences of SEQ ID NOs: 1 to 7 or a part thereof are chemically treated with biotin, bromodeoxyuridine or the like, or a fluorescent substance such as fluorescein isothiocyanate or ³² P, ³⁵ S or ¹²⁵ I. The probe of the present invention can be prepared by labeling with an isotope. Alternatively, the DNA having the sequences of SEQ ID NOS: 1 to 7 or a part thereof incorporated in the plasmid may be labeled as described above. Using the marker of the present invention,
A biological sample can be identified or specified. Examples of biological samples include organs such as lungs, kidneys and livers of mammals such as cows, sheep, pigs and goats, as well as tissues, embryos, cells and DNA extracted therefrom. First, identification or identification as a biological sample is carried out by extracting DNA from the biological sample, if necessary, and then hybridizing it with a DNA having the sequence of SEQ ID NO: 5 or a part thereof. The identification or identification of bovine origin as described in SEQ ID NOs: 1, 2, 3, 4, 6 and 7
Can be carried out by amplification using a DNA having a part of at least one sequence selected from the group consisting of Specifically, a known Southern hybridization method, in sit, in which a DNA having at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 or a part thereof is used as a probe or a primer
u A hybridization method, a PCR method, a method using a hybrid panel, etc. can be used.

The principle by which a biological sample can be identified or specified is that the sequences of SEQ ID NOs: 1, 2, 3, 4, 6 and 7 are derived from bovine, of which the sequences of SEQ ID NOs: 1, 2, 3 and 4 are bovine Y. It is derived from chromosomes and has high specificity for males, the sequences of SEQ ID NOs: 6 and 7 are derived from bovine autosomal chromosomes and highly specific to cattle, and the sequence of SEQ ID NO: 5 is derived from bovine mitochondria. It has a high degree of commonality and is nonspecific to cattle. With the exception of special cases such as red blood cells, cells that compose the living body have mitochondria as energy-producing organs, and mammalian mitochondria have almost the same size and similar nucleotide sequences, The numbers and the order of arrangement are also the same. Therefore, it can be said that the DNA or biological sample that hybridizes with the sequence of SEQ ID NO: 5 is of mammalian origin. On the other hand, the sequences of SEQ ID NOs: 1, 2, 3, 4, 6 and 7 hybridize only to a bovine-derived sample, and thus can be identified or specified as bovine.

Further, a DN having a part of at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7.
A can be used as a primer to clone the DNA present on the 5'side and / or 3'side adjacent to the selected sequence. Inverse-out PCR (T.T.
riglia et al., Nucleic Acid Res., 16, 81-86, 1988)
By using, it is possible to easily clone an unknown DNA adjacent to the upstream of the 5 ′ side and the downstream of the 3 ′ side of the sequences of SEQ ID NOs: 1 to 7. For example, there is a site for the restriction enzyme AatI at the position of base number 27 in the sequence of SEQ ID NO: 1. Plasmid pGEM containing the sequence of SEQ ID NO: 1 (accession number FERM BP
-4095) sequence is --- HindIII, SphI, PstI, SalI, Xba.
I, (base numbers 1 to 1404 of the sequence of SEQ ID NO: 1), SmaI, K
pnI, SacI, EcoRI ---. For example, the restriction enzyme Aat
When cut with I and PstI, PstI to the base number 27 of the sequence of SEQ ID NO: 1 is cut out, and --- HindIII, SphI, PstI cleavage point, AatI cleavage point-base numbers 28 to 14 of the sequence of SEQ ID NO: 1.
04, SmaI, KpnI, SacI, EcoRI ---. On the other hand, when bovine genomic DNA was cut with PstI and AatI and inserted into the above plasmid, various long and short fragments derived from the bovine genome were obtained.
The PstI-AatI fragment becomes the inserted plasmid population. Here, a universal primer having a sequence on the HindIII side (which may be specially synthesized) and a primer having a sequence selected from the vicinity of base number 28 to 1404 of the sequence of SEQ ID NO: 1, preferably the vicinity of base number 28, are used. When synthesized and subjected to PCR, up to PstI upstream of the 5'side of base number 28 will be amplified. This is an example, and it is premised that the PstI site exists at a close position where PCR amplification is possible. Similarly, for example, with AatI and KpnI from base number 28 to 1404, Kp
Cut out up to nI, insert the AatI-KpnI fragment of genomic DNA into this, and use PCR with a primer having a sequence selected from between nucleotide numbers 1 to 28 and a primer having a sequence on the EcoRI side, An unknown region further downstream from the base number 1404 can be cloned.

[0023]

EXAMPLES The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples. Example 1 Isolation of Bovine Genomic DNA About 100 g of male and female bovine (Holstein) liver was obtained from Koshigaya Slaughterhouse (Saitama Prefecture), and 11 g and 24 g of male and female, respectively, were used and a known method (J. Sambrook et. al. (Eds.), Molec
ular Cloning, 2nd ed., Cold Spring Harbor Laborato
ry Press, pp.9.16-9.23, (1989))
I got The outline of the method is shown below. Shred the liver with scissors
(3-4 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, then the same amount of phenol solution, phenol: chloroform,
Extracted once with chloroform. 2 volumes of ethanol was added to the aqueous layer, the precipitated DNA was taken with a glass rod, washed with 70% ethanol, dried, and an appropriate amount of TE (10 mM Tris-HCl, pH 8,
It was dissolved in 1 mM EDTA). Absorbance was measured at OD ₂₆₀ nm and D
The NA concentration was determined. The yield was 7.7 mg for males and 10.4 mg for females. (Example 2) Isolation of sheep, pig, and goat DNA One liver of each male and female sheep was obtained from Koiwai Farm (Iwate Prefecture), and 11.5 g and 10.0 g of each male and female were used, respectively. DNA was isolated and purified. Yield is male 24.3 m
g and 9.3 mg for females.

Each male and female pig liver was obtained from Yuki-Shimodate City Slaughterhouse (Ibaraki Prefecture), and male and female were 10.0 g and 10.0 respectively.
DNA was isolated and purified in the same manner as in Example 1 using g. The yield was 29.3 mg for males and 38.9 mg for females. About 10 ml each of male and female goat blood was obtained from Koiwai Farm, and hypotonic solution (150 mM
After hemolyzing with (NH ₄ Cl / 10 mM KCl / 10 mM EDTA), 2000 r
Centrifuge at pm to collect leukocytes and use PBS (phosphate buffe
It was washed once with red saline) and used for phenol extraction. The following operation is as described in Example 1. Yield is male
0.57 mg and 0.28 mg for females. (Example 3) Isolation and purification of DNA having the sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 SEQ ID NO: 5, SEQ ID NO: 6, and Escherichia coli E. coli DH5α having a plasmid incorporating the DNAs shown in SEQ ID NO: 7 are FER, respectively.
M BP-4045, FERM BP-4089, FERM BP-4090, FERM BP-409
1, FERM BP-4092, FERM BP-4093, and FERM BP-4094 have been deposited at the Institute of Biotechnology, Institute of Industrial Science as of December 12, 1991. These bacteria were cultivated in LBA medium (1 L, polypeptone 10 g, yeast extract 5 g, NaCl 10 g, ampicillin 50 mg), and the well-known alkaline treatment method (J. Sambr
ook et al. (Eds.), Molecular Cloning, 2nd ed., Col
dSpring Harbor Laboratory Press, pp. 1.25-1.39, 19
89) to purify the DNA.

Since the DNAs shown in SEQ ID NOS: 1 to 7 are contained in plasmids, they may be individually cultured and the DNA may be obtained by the same method. It may be used as is with plasmid DNA, or to isolate only each sequence part,
Since it is located in the BamHI site of pUC118 plasmid, it can be excised with the restriction enzymes on both sides of it. Example 4 Bovine Male Specificity of DNA Derived from Bull We have four types of DNA derived from bull (SEQ ID NO:
1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4). First the array number
The DNA shown in 1 was isolated and purified, and a bovine male genomic library was screened using this as a probe to obtain a phage clone. The partial sequence of one clone among them is shown in SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. DNA was obtained (Japanese Patent Application No. 3-352032). Therefore, it can be said that the sequences of SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4 exist at almost the same position on the chromosome (FIG. 1) (T. Kudo et al., J. Repr.
od. Develop., 39, 55-63, 1993). The original sequence of SEQ ID NO: 1 is believed to be located near the sequences of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4. However, since it is a repetitive sequence, it may exist separately from these.

10 μg each of the bovine, ovine, goat and porcine male and female DNAs obtained in Examples 1 and 2 were completely digested with the restriction enzyme EcoRI (manufactured by Nippon Gene) according to the instructions, and 0.8% agarose ( Manufactured by Takara Shuzo,
Electrophoresis was performed at 70 volts for 3 hours using TAE (Tris-acetate-EDTA) buffer with SeaKem GTG). As a DNA length marker, a HindIII degradation product of λDNA was used. After the electrophoresis, the gel was stained with an EtBr (ethydium bromide) solution for 0.5 hour and photographed (a in FIG. 2). Treat the gel with 0.25 M HCl for 15 minutes, then add it to an alkaline denaturing solution (0.5 M NaOH, 1.5 M NaCl).
Soak for 15 minutes, then neutralize (1.5 M NaCl, 0.5 M Tris-HCl
(pH 7.2)) for 30 minutes. The denatured DNA in the gel is a known method (J. Sambrook et al. (Eds.), Molecular Cloning,
2nd ed., ColdSpring Harbor Laboratory Press, pp.
According to 9.31-9.57, 1989), nylon membrane (Hibond N,
Amersham). Filters should be treated with 0.9M NaCl, 0.09M prior to hybridization as described in the instructions.
Incubated in sodium citrate, 0.2% Ficoll, 0.2% polyvinyl-pyrrolidone, 0.2% bovine serum albumin, 0.5% sodium dodecyl sulfate, 100 μg / ml heat-denatured salmon sperm DNA (hybidization solution) at 65 ° C. for 3 hours. . The bovine DNA shown in SEQ ID NO: 1 was labeled with ³² P-α by the random primer method (Amersham kit).
A probe labeled with -dCTP (Amersham, 3000 μCi / m mol) was added, and hybridization was performed at 65 ° C. for 16 hours. The filters were then washed twice in 2 x SSC (0.3 M NaCl, 0.03 M sodium citrate), 0.1% sodium dodecyl sulfate at room temperature for 10 minutes twice and the same solution at 65 ° C for 15 minutes.
Washed twice. An autoradiogram of the result of this Southern blotting is shown in FIG. 2b. Here, lanes 1 and 10 are size markers (HindIII degradation product of λ phage),
Lanes 2 and 3 are holstein DNA, lanes 4 and 5 are porcine DNA,
Lanes 6 and 7 are sheep DNA, lanes 8 and 9 are goat DNA, odd numbers are male and even numbers are female. As shown in FIG. 2b,
The sequence of SEQ ID NO: 1 has bovine (lane
It hybridized only with 2, 3) and did not hybridize with male and female DNA of pigs (lanes 4, 5) and sheep (lanes 6, 7). Smears are seen in goats (lanes 8 and 9),
Since there was no clear band, it was judged to have no specificity. Since there is a high possibility that different species will not hybridize here, washing with mild conditions of 2 x SSC resulted in banding in cow DNA (Fig. 2b, b).
Lane 2). However, when washed under a more strict condition of 0.1 x SSC, it reacts only with bull's DNA and becomes male-specific (Japanese Patent Application No. 3-352032). It is also shown in Example 5 below that the sequence SEQ ID NO: 1 is male specific. (Example 5) Position of the sequence of SEQ ID NO: 1 on the bovine Y chromosome Since the DNA shown in SEQ ID NO: 1 was shown to specifically hybridize to the DNA of bull, it was used next to in situ hi-hi Hybridization (D. Pinke et al.,
Proc. Natl. Acad. Sci. USA, 83, 2934-2938, 1986). The specific method is FISH (fluorescence in situ hyb
ridization) method, known method (edited by Masaaki Hori, Yusuke Nakamura,
Lab Manual Human Genome Mapping, Maruzen, pp. 118-
133, 1991).

[0027] According to the standard method (Tissue Culture Technology, Tissue Culture Technology, Asakura Shoten, pp. 174-177, 1982), from Dulbecco's modified minimal medium (DMEM, GIBCO, 10 Cultured cells were obtained using 100% calf serum and gentamicin (100 μg / ml). 300 before preparation of the chromosome sample
The cells were cultured for 10-12 hours with the addition of μg / ml thymidine, and the medium was replaced. The cells were further cultured in fresh medium for 4-6 hours, but 0.02 μg / ml colcemid was added 20 minutes before preparation of the specimen. The medium was once removed, treated with 0.05% trypsin solution for 3 minutes, the original medium was returned thereto, and slow pipetting was performed to selectively collect the rounded dividing cells. Fix these cells with fixative (methanol:
Fix with acetic acid = 3: 1), so-called air-drying method (edited by the Japan Tissue Culture Society, tissue culture technology, Asakura Shoten, pp. 42-46, 198).
According to 2), a chromosome sample was prepared. Specimens were stored at -70 ° C and used as needed. Chromosomes in 100% formamide
It was denatured by treating at 70 ° C for 2 minutes and used in the following tests.

The sequence of SEQ ID NO: 1 incorporated in the plasmid (pGEM, Stratagene) was directly labeled with biotin (biotin-14-dATP) using a commercially available kit (BioNick Labeling System, GibcoBRL). This was treated with 70% formamide (in 2 x SSC) at 75 ° C for 10 minutes to denature it, and then it was allowed to act on the above denatured chromosome. That is,
100 μl of the probe solution was added to the slide on which the chromosome sample was prepared, covered with parafilm, and kept at 37 ° C. for 18-20 hours in the dark. Slides in 4% Block Ace ^TM (Dainippon Pharmaceutical)
It was treated at 37 ° C for 15 minutes. 40 μl of 0.02 mg / ml FIT here
C (fluorescence isothiocyanate) bound avidin
(avidin-FITC, manufactured by Boehringer Mannheim) at 37 ° C, 45-6
Treated for 0 minutes. This solution contained 2 μl avidin-FITC (5 mg / m
l) was added to 500 μl of 1% Block Ace ^™ (in 4 x SSC) to prepare. Wash slides with 4 x SSC and wash with 12.5% DABCO (1,4
-diazabicylo [2,2,2] octane, Sigma) and 0.75 μg
PBS containing / ml propidium iodide
It was sealed with (phosphate buffered saline). The sample was observed with an Olympus epi-illumination fluorescence microscope (BH-2, G excitation, using O-515 filter). The photo was taken with Fuji's Provia slide film. As a result, as shown in FIG.
1 is almost Y _p11 (DiBerardino et al., Cyt
ogenet. Cell Genet., 53, 65-79, 1990), that is, between the centromere and the middle part of the short arm. (Example 6) Position of SEQ ID NO: 2 on bovine Y chromosome In the same manner as in Example 5, in situ hybridization was carried out using the DNA shown in SEQ ID NO: 2 as a probe. As a result, as shown in FIG. 4 (arrow), it was shown that it exists at the Y _p11 position of the Y chromosome, which is exactly the same as the sequence of SEQ ID NO: 1. (Example 7) Position of the sequence of SEQ ID NO: 4 on the bovine Y chromosome The sequence of SEQ ID NO: 3 is a sequence existing adjacent to the sequence of SEQ ID NO: 2 and is specific to male DNA (Japanese Patent Application Flat 3
−352032). Therefore, it is in the same position as the sequences of SEQ ID NO: 1 and 2. On the other hand, the sequence of SEQ ID NO: 4 exists on the Y chromosome as a sequence adjacent to the sequences of SEQ ID NOs: 2 and 3, but a similar sequence was found in the female DN by Southern blotting.
It was suggested that A also exists (Japanese Patent Application No. 3-35203).
2). Therefore, as in Example 5, the DNA shown in SEQ ID NO: 4
Was used as a probe for in situ hybridization. As a result, as shown in FIG. 5 (arrow), it was shown that it exists at the exact same position of the Y chromosome as the sequences of SEQ ID NOs: 1 and 2. This is probably because the FISH method has a lower sensitivity than the Southern method, and thus a signal was specifically detected on the Y chromosome with high specificity. This usually happens:
The sequence of SEQ ID NO: 1 is male-specific by the FISH method (FIG. 3), but it is also shown by the Southern method that it hybridizes to females when the conditions are relaxed (FIG. 2b). (Example 8) Location of the sequence of SEQ ID NO: 5 The sequence of SEQ ID NO: 5 is a part of mitochondrial DNA by homology search (S. Anderson et al., J. Mol. Biol., 15
6, 683-717, 1982) and 311 of its sequence number 12401-12711.
It was found to correspond to base pairs. Sequence number 5 is 3
It was 05 base pairs, and when the two were compared, the sequence of SEQ ID NO: 5 showed deletion of 7 bases, insertion of 1 base, and substitution of 2 bases. The homology was almost 98%. In addition, the sequence of mouse mitochondrial DNA (MJ Bibb et al., Cell., 167-1
80, 1981), corresponding to base number 12036-12345, 310, with deletion, insertion, and substitution of bases, and the homology was almost 78%. Mitochondria were expected to be present in the cytoplasm, and in situ hybridization was carried out using the DNA shown in SEQ ID NO: 5 as a probe, as in Example 5.
As a result, it was shown that they were scattered in the cytoplasm as shown in FIG. 6 (only a part is shown by an arrow). Mitochondrial DNA has low species-specificity and can be commonly used in mammals as a cytoplasmic probe or marker. (Example 9) Location of the sequence of SEQ ID NO: 6 The sequence of SEQ ID NO: 6 is a common sequence for both sexes, but the Southern blot image shows that the copy number is smaller than that of the sequences of SEQ ID NO: 5 and SEQ ID NO: 7. Show. FIG. 7 shows the results of FISH using the sequence of SEQ ID NO: 6 as in Example 5. Signals have been shown to be scattered around the autosomal centromere or its vicinity (triangle mark), the middle part of the chromosome (short arrow), or the vicinity of the end part (long arrow), but the number of the chromosome cannot be identified. could not. (Example 10) Sequence-existing position of SEQ ID NO: 7 The sequence of SEQ ID NO: 7 is a DNA existing in both males and females. Comparing the sequence of SEQ ID NO: 5 with the signal intensity in Southern blotting, this is stronger. Since mitochondrial DNA is thought to exist in the cells in the number of thousands to 10,000, it is expected that the sequence of SEQ ID NO: 7 has a higher copy number. Therefore, in the same manner as in Example 5, in situ hybridization was performed using the DNA shown in SEQ ID NO: 7 as a probe. As a result, as shown in FIG.
That is, it was shown that at least 48 out of 58 centromeres existed. The copy number varied depending on the autosomal chromosome, and varied from those that were not detected to those that showed many signals. However, individual chromosomes were not identified. No signal could be detected on the sex chromosome (arrow). (Example 11) Species specificity of the sequence of SEQ ID NO: 7 Using the DNA shown in SEQ ID NO: 7 as a probe, DNA of cattle, pig, sheep, and goat male and female was used in the same manner as in Example 4 to carry out the Southern method. went. For the DNA blot, the nylon membrane (high bond N, manufactured by Amersham Co.) of FIG. 2b used in Example 4 was washed and used. Wash with 0.4 N NaOH (45 ° C) for 30 minutes as described in the manual, and then wash with 1 MT.
It was neutralized with ris (pH 7.4). The washing of the probe after hybridization was carried out as described in Example 4, except that 0.1 x SSC was used. The result is shown in FIG. Hybridize only to holstein DNA in lanes 2 and 3, then lanes 4 and 5
Pig DNA, lanes 6, 7 sheep DNA, lanes 8, 9 goat
It did not hybridize to DNA (odd males, even females). Therefore, it can be said that the sequence of SEQ ID NO: 7 is specific to bovine DNA. (Example 12) Presence position of two fragments contained in the sequence of SEQ ID NO: 7 In the sequence of SEQ ID NO: 7, despite cleavage with MboI, M
There is a boI site. Therefore, EMBL (European Molecula
A search for DNA registered in the Gene Bank of the r Biology Laboratory (using Hitachi software and DNASIS) revealed that 60 out of 241 base pairs from the sequence of SEQ ID NO: 7 were bovine α satellites (M. Pech et al. ., Cell, 18, 883-893, 1
979), but the homology was found to be high. No homologous sequence was found in the remaining 180 base pairs. Therefore, the DNA containing the sequence of SEQ ID NO: 7 sandwiched between SacI and KpnI was cut with MboI, treated with Klenow enzyme to smooth the cross section of the DNA, and subsequently cut with SacI and KpnI. By doing so, three DNA fragments of a vector plasmid having 60 base pairs with blunt end and SacI cross section, 180 base pairs with blunt end and KpnI cross section, and SacI and KpnI cross section were prepared. S part of this DNA
It was ligated to a vector treated with acI and HincII (BlueScript SK-, Stratagene) and the same vector treated with KpnI and HincII (Takara Shuzo, ligation kit according to the instructions). Infected with E. coli DH5α, cloned, extracted DNA, cut with SacI and KpnI,
By applying agarose gel electrophoresis, it was confirmed that the former contained 60 base pairs and the latter contained 180 base pairs of DNA. When the nucleotide sequence was determined, it was shown that the desired DNA was cloned. When both of them were subjected to in situ hybridization in the same manner as in Example 5, the same image (FIG. 8) as that shown by the sequence of SEQ ID NO: 7 was obtained for 60 base pairs containing α satellite. Therefore, the signal shown in FIG.
It seems to be derived from base pairs.

On the other hand, using DNA containing 180 base pairs,
The result of performing the FISH method is shown in FIG. Signals were found on the centromeric part of the autosomal chromosome (short arrow) and on the autosomal chromosome (long arrow), but the chromosome number and position could not be identified. In general, the strength of the signal obtained depends on the number of repeats (more is stronger), homogeneity of repeats (more homologous is stronger), length of the DNA sequence (longer is stronger), and experiment to obtain the signal. It depends on the conditions (in particular salt concentration, temperature, and time). A strong signal was obtained with a short 60 base pairs and a strong signal was not obtained with a long 180 base pairs, and the main reason for the weak signal seems to be that the number of repeats in the former is extremely large.

[0030]

INDUSTRIAL APPLICABILITY The present invention provides a marker containing a DNA having at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 or a part thereof. Using the marker of the present invention, cells, tissues, identification or identification of biological samples such as intracellular organs, for example, identification of the biological sample is bovine, nuclear and cytoplasmic, sex chromosome and autosomal distinction, If it is a sex chromosome, it is different from the X chromosome and the Y chromosome,
If it is the Y chromosome, it is possible to know the distinction between the short arm and the long arm, and if it is the short arm, whether it is on the centromere side or the end. In addition, when the cloning method of the present invention is used, 5'of the sequences of SEQ ID NOs: 1 to 7
One can clone unknown DNA flanking upstream and 3 ′ downstream.

[0031]

[Sequence list]

SEQ ID NO: 1 Sequence Length: 1404 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 Repeat sequence SEQ located on chromosome _{Y p11 GATCCTCTCA CCTGTCCCAG TCCCACAGGC CTGGGCTGAT GGGGTAGGCT GGCGTGTGTT} 60 CTCACCTGGG GGCTGGATGA GAACAGAGCT ACTTTGAAAG CGCCTCTGTA CAATAAATGG 120 GCAGACTTCA ATTCCTCTGG CACTGGTGAG TCATGGCTCT TAGCGGGACA CTGTTCTTAG 180 CATCTCCGGA TTGAGAGATG TTTCGTCTCT AAATCTCTCT CTACACAGAG AAGTCCAGGT 240 TTGTGTCACA TTTGGACACG TGGGCAACAG TCTTTTATGT GAAGCGCGGA ACTCGTTATT 300 TCCCGAAAGA ATACATGCAT TAAGTTTTCT GCCAAGAAAT GTGGCATTCT CTCTGTTTCA 360 CCTGTGAAAA TCTCACCACA AACTTCCCAA AAGGGAAATG GAATTCTCTA TATGTTATAT 420 GAATCATGGG TGCACGTGTT ATTTGCCGGC ATGAAATTTG GAATTCATTATCTGATCTATGACTCATTCATTCAGGATCATCGACCAGACCAGATGACCAGATGACCAGAGAA TGTTGA GTACAGATTT CCTTACATGT CTTTATCTTT 660 CCTCTGTAAG AAAATCGTGG CAGAGACTTA GTCCATGAGC TAATGAACTG TTTATATTTC 720 ATGTAGGAGA ATCTGGGTCG TGATTTTCCT ACATGAAACA TGCAGTTCTC TGTATTTAAT 780 GAATGAACAT CCGGGCACCG ATTTTCCTGC CTTGAATATG GGTTTCTCTA CGTCGGCATT 840 TGAAACTCCA GGCACGGAGC TTTTCCCCGT GAAATATGAA ATTCTGGTAT TTCACGTTGA 900 ATTGGGCATA AACAAGGTAA CACGAAATAC GGAATTTTTC ATCTTTCTCA TTTGAAAATC 960 TGTGTGCTGA CTTGCGTTAT GAGTTACAGA ACACTGTATA TGTAACAACT GAAAGAATTG 1020 GCCTCGATTT TCCTACACAA AATATGGAAT TTTCTATTTC ATACATAAAG TGATTTTCAC 1080 ACTTGAGGTA TGAAGTCACT GTCAATGAAA ACACGAAGTC AATCCTTGTT CAAGGTGATG 1140 AACAGGTAAG TCAAAAACCT TGAGCATGTG ACCACACCTG AAAGGGAACC CTGAATGTTC 1200 TGAAACTCTG AGACTGGATA AAACCTGGTC TTTTATGCAA AATGGGGGGA CAGAAGTACT 1260 AACTAAGAGC CCTAACCCCA AGACCTGGAT GTTTCCTCTT TAGCCACACT CACTCTTGAG 1320 CAGGGTTGCT TTAAAAAGCC ACATGTAAGA ACAATCAGTC TGGGGACAGC GGACAGCCGT 1380 GGTCAGCCGG CCTGGTGTGA ATTC 1404

SEQ ID NO: 2 Sequence length: 1655 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bos taurus Strain name: Holstein Sequence features: A repetitive sequence located on bovine Y chromosome _Yp11 isolated using SEQ ID NO: 1 as a probe.
And in the vicinity of SEQ ID NO: 4 SEQ GAATTCGTGA GATGCAGATC TAGACGTGCT TATAATTTTA AATGCTTGTA TTAGAAAAGA 60 ATAAAAGCCT AAGCCAGTGG AAAGAGAAAT GGCAAACTCC GTCAAGGCTT TCAGCAAAGG 120 CCACATAGAG GAAACGTGAG GGAGGGGTGT GATTAGCCGG TGCAAACTTC TTGCTGTCAC 180 ATCTTGTGTT CTTGAGGTGA AGTGGTGGCC AGCTAACTGT GTCCTGCAAT CCTCAAGGGA 240 AACAAGTATG ATTCCCTATC CCCTGAAAGA GATTCCCCAG ATTGACTTTC ACCCTCCAAG 300 GTCCTGGTTG CGAGAAGGGG GTCCCCGTAT AGCCTGGTAG TCCCAGCTGG AAGAGGCAGG 360 TCTCAGTTCT CTGTGCCCTC CTCTTACCAA GGCCCCCACA CACTGCCCGG CCACCTGCCA 420 TGAGGGAGCC AGGTACCCAG CACGCAGCTG ACCCTCAGCC TCCTCCGTCT ACCCAAATGG 480 GGAGCTCGGT CCTGTAGACT GTGGCCCATG GACATTGCCA CAACCATTCG GACATGGAGG 540 TGGGCATGGG ACACGGTGGC CGTTGCTTTG AAACCTGGGC CAGGCCGCTG AGTGGCTCCA 600 GCAAGGCCTC CGGGACTCTG CAAGACACAG CCTTTCCTCA CGCTTGTCCA AGCACCACAG 660 TTCATTACCC AGCCCAAGGC CCAAGGCTGG AAGGCCTGTG GGAAGGCTTT GATATCTCTC 720 TCAGTGCATT CAGCTGAGAG GCTTTCTGTT GGTGGTGTTG GGTTTGCGAA CTTCCCTAAT 780 GGTCTTGGCT GAGCTGCAGC TCCGTTTTCC ATCCTGCCTC TTTTACAGTG TCTTCCTGGA 840 TGAGCTGAGT CAACCTGTGA CTAAAAGATG GTAACGTGGA CCATCGGGGG CCATTTCCAC 900 TGGCCCGATG ACCACATCCT ACTCTCTTTC TATAACGCAG GGCTTTCAAC GTGACAATGT 960 CGTGTGATGT AAAAGCCTTC ATCTTCCTCG AACTCAAGTA TTTAACTCAG CATACTCTCC 1020 TCAAGAACAT GTATTGCATA CAGGGCTACC ATTTATTTTT TCACTGCCGG AATTATACTG 1080 GACATGTGAT GTACTTCCAA CCATTAATTT GGGGCCATAG TCACGTTACA GTGTTTCTTT 1140 GGTTTCTGCT GTGCGTGAAT CAAGTTTTAG CTGTACGTGT ATCCCGTCAA TTTTGGATTT 1200 CCTTCCCATT TAGGCCACCA CAGAGCAGTC GAGTCCCTTT CTTCTTTCAG TTTTTCTTTT 1260 TATTAAATTC AACTGGGGGT TAATTATGAT ACAATATTGT AATGGCTTCT GCCATGCATC 1320 AACATTAATA AGCCATAAGT ACACAGGGGT CCCAGCTATC CTGGAGCCGC TCCCACATCC 1380 CTGCCTACGC TATTCTTCGG GATTGCTCCA GAGCACTTGC TCTGCATTCC CTGATTCATG 1440 CAGATGAAAA CCAGAAAGAT GATTTTTCTG CATAAAATAT GCAATTGTCC TTCTTTCACA 1500 TGTGAGAACA TGGGTGCAGA GGTTTCTACC TGAAATAGGA AATCGGTATT TCATGTATGA 1560 AAATCGACTT GCATCATTTT CTTCATCATA TGTGGAATCT TGTGTCTCCC AGACGTATAT 1620 CTGTGCACAC GTTTTCCTAC ATGAGTGGGG AATTC 1655

SEQ ID NO: 3 Sequence length: 3068 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bos taurus Strain name: Holstein Sequence features: A repetitive sequence located on bovine Y chromosome _Yp11 isolated using SEQ ID NO: 1 as a probe.
And in the vicinity of SEQ ID NO: 4 SEQ GAATTCCTCC ACCTGCCAGG ACAAAGTGTG CGGGGTGGGG TGGGTGTGAA ACCTCTTTAC 60 AGAAGAGCTG TCCTTTCCTG TGTTAGGGAT AGACCATGCC CTCTCCCCTC CCCAGTCACC 120 TCCCCTCCAC AATCATCAGT GTCCCCTGCC TGACCTCCAA GTTGGTCATG AAGTGAAGCA 180 TGTCTGCATC TTGCTTGCTC ATCAAAACTG ACATTTGGGG GTGGTTCATA AACTGCTTTA 240 GGTCAAGGAG CCTCTAGATG CTAGAGGTAA AAGTGCTGGA AGAACAAAGC TAGCCTAAAG 300 AGTAGAATGG CCCTCCCTGT TTGACTTCAA CTTGCTACTG GTTAACTCGT CACATTTCGG 360 TTCACGCGGG GCTATATGAA TGCCGCACAA GGTCCAAGCT GTGCCCATGA ATGCCCTACC 420 ACGAGGAGGT TCTCTCCTAA CCGGATAAGC TTCATCTCAG CCCCTTGAAA GTTAGCTTAC 480 TTCGGGAAAA CAAGCACTCC TAGCTAGAAC CCGTAGACCA CTTACACTTC CCCACCCCTC 540 CCCCATTCCA GACAAGCCTT CTCTCCGAAG AGACCCTGGT GCTAATGACA ATTCTGATAG 600 GGACCTCTCA AAGTATAGCC CAACTATCAA TTTGGGCTAG CCAGAACAGC AAGGACATCA 660 CACCTACGAT CAGGAACAGA TATGCAGGAC GGTAACCAAT ACCCACCATC CAGAAAAGTA 720 AATACGGTAC CAGAACTGCC AGATCTGTAA ACTGTTCCTG CCATAGCCCG GCTTAAACAA 780 ACAAACAAAC ACAAAACTCA CAAGCACCAC ACCAAGGGAT ACAACTTCGA CCCAGAAGCC 840 ACGGATGCCC TGGATGATGG CGCTTCTGTG CTCTAGATGC ACCTTCCGCC GCTGACTGGT 900 CTTATGTTTC AGGCGAGCAT GCGCCTTTCG GCTTTCTTAT TCACGGGCTC CAGCTCCAGC 960 TGCAGGGCCG CCAGCGCTCC AGTGCAGGCC GGTCACTCAG GGCTCCGGGC CTTGGATGGT 1020 CGTGGACATG CTCCTCCGAG GACACCTGCT CCTGCTCCTC GTATGCCACC ACCTCCACCT 1080 CCTCCATGAC GTCCAACACC AGCAGCTGGA ACTCCTGCCC GAGTCCCGCC ACCTCTCCCT 1140 CCTCCACAGC CGCACCTTCC TCCACTGCCT CCACCCAGAA GAGGGCGGCC TCCTCGCCTG 1200 GCGCACCACC TGCGGCCTGC ATCGCCTCTG CCGCCCCCAC CGGACTGGAA GGCCAGGGCC 1260 CCTCCCTCAT GAAGACCCGG GCTCACAACT AAGATCCAGG ATCCCGGAGT CCTGCCGCCT 1320 TCCTCTGGCC CCGTCTCACT CTCCATGGTA TTCTCGCCGA AGCCCGGAAC TGCAAACAAG 1380 CTGGACGCTA GAGCACGGCA GACGGCCCTC ACGGCCCGCC GGCGCAGTCT ACGTGGTCCT 1440 ACTCCCGACG GTTACTGGGC AAGAGCCAGG GAACGGCGAA AGGGAGAGAC GCATGCGCAG 1500 AACCCTCTGC CACCAGGCCG CCTACTATGG CGACGGGAAG GGGCTGAACT CTCCACCCTG 1560 ACCTCCTGAC CTCATCCCAG AACCAATCAA ATGGAGTCTA AAGCGGTTCG CTCCTGGACA 1620 CGCCCCTTGG AATCCTGGGC CCTCTTGCCA CCTGTGACAG GCGCCAAT GT TGGCCCAGCG 1680 CAAAGTGGGC GTGGCATGCA AGCCTTTTGC CTGCCTAGCA GTGCAGCCGC GCCCGAGCAG 1740 CGACTGGGAG GCCAGGGCCA CCTGAAGCTG CAACAGTCCT CAAGCTTGAA GTTGCCCCTG 1800 GGGCAGCGCG CCCGTGTGCT CAGGGACACA CTCAGGAAGA CAGGGTTCCT GGGTACCTCA 1860 GGGACAGAGT TGCTCTCCTC CGATCCAAAC CGCAAGCCAC GGGGTGGGGT GGGGGGAGTA 1920 GGAGGGAGGC GGGCAGGGTA GGGGGGTTTG GGGGAGGAGT GCGCGGTGGA GGGGGATGGG 1980 GAAGCGGGGC GGGGCGGTGG GGGCGGGCTT ACGCGACCTC ACCCATGTGC ACCTGCTGCA 2040 GAGTCATGGC TAAACTTGTG CCTTAGGTTG AGCAGTTGTC AGGAATGCAG CCCTCCTCTG 2100 AGGAAGATGC AGAGCACTTT CCTCGGCTAG CTGTCCAAGC CCCACGATGC AGCCGCAAGC 2160 CCAACCAGCC TGGAGCCCCT GGCTACGATC TGTCAGCCCT CGGCTTTCCC CCGGCCCGTG 2220 GTTAGGGCAG GCGCCACGTA GCTAAGGATG TGCGCCTGCA GATGCACTTG CCAGAGGCAG 2280 AGCTATTCCT GCAAGTGTTT CAAGTGGACC AAAGTGCTCC TCCTGACCAT TAGTTACTCC 2340 TACGTCCGGG CCGCCTAGCT CAGGTGGGCA CACTCAGGTT AAGTGCCTGG ACAGTTTCCA 2400 CTGCTCACGC TCTTTCTCCT AGGATGTCTG CTGTTTCACA CTGAGCACCT ACCTCCCTTA 2460 GTCCTGACAC ACTCACAGGG CCACCACAGC CCACCCCACC CCCCACACCC CCC CACAAAC 2520 ACACACAAAC ACGTGACACA AGTACACACA TGCGTGCAAC ACACAGAGCC ACAAATGCCA 2580 ACAGGTGTTT CAGAGACTGA AGAACCAGTG GTTCGCATGA AATGATAGAT GTCTTTATTT 2640 CTAACGGGAA AATGTTCATT CTGATCTTCA GATGTGAAAT ATAGAGAAGT CCATCTTCCA 2700 TGAGGGAGAA TCATGCCTAG ATTTGGATAC AGGAAGTATA GAGGAACTCC TTATTTCATG 2760 GTGGACAGTC CTTGATTTAT GCATGAGAAA TACAAGGAGT TAAGTATTTC ACGAAGAAAC 2820 AGTGGTGCCC AGAATTTCAT TTATGAAATA GAGATAATTC CAATTCCATA TTGGTTCACC 2880 AGATCTTAAT TTCACACTTC ATGTCAGTAA ACTTTCATTG AGATCTTCAT ATACGAAATG 2940 TATACAATTT CAGGATTCAA GAAGGGAATT GTAGGCTCAA CTTTTCATAT GTAAAATATA 3000 TAGAATTCCA AATTTCATGC CGGCAAATAA CACGTGCACC CATGATTCAT ATTACATATA 3060 GAGAATTC 3068

SEQ ID NO: 4 Sequence length: 2104 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bos taurus Strain name: Holstein Sequence features: A repetitive sequence located on bovine Y chromosome _Yp11 isolated using SEQ ID NO: 1 as a probe.
And in the vicinity of SEQ ID NO: 3 SEQ GAATTCACTG CAAAGGAAAA ATAAAAAGCA TCAGCTGCAT GCGTGCGTGT ATTGTTTGGG 60 CAGGTCCTCA GGACAAACAC TTCAGCTGCA TGTACGAGTG TCAGTTCACC GCGTGGGAAT 120 TACCAGTCTG TGGAGATTCA TAGAGGGGGC TAAGTCGTAT GTTGGGGTTG TGTCAGGGGG 180 TGACGTTCTT CCTGTGGGGT GGGGGTTGGG GAGGACGGAG AAGGAAGATG AGGGGCCTAT 240 TGACTGTGGG TCTGGCAATT TTACAAGGGA AATGTGGCTG TTCGTGGAAA GGGTCTGGTG 300 AACAAGGGCC AGAGATTGAA ACAGTGGAAT GCAATTCTGT TGGAAGGTGG TGGCCCAGGT 360 ATCTTCTCTG TTGCCTATCA TCAGACACTT TAATAAGGGT ACAGAACAAA GGAAAAGCCT 420 TATCATTGAA CTCGCTATGA ACCTCGAAGG CCACATGTGT ATGAGTGTGT ATATGAGTGC 480 ACGGTCAGCT TTGCTGAGAG TCTGCTTCCC AGAGCTAGGG TTCCAGGTAA CATCTTAGGG 540 CATTGTGTAT AAGGTATATG GAGGGGGGCT GTGGCCTAGG GAGAAAGTGA GGGGTGGGAA 600 GGCAGTGAGG ATACTAGTTT CTGGCAAAGG TGACCAGGGG AAAAGAGAAA TTGCAAGGAA 660 TCAGATAAAG GAAAACAGAT CCCGTCGTAG ATGTTAGAGC TCATGTGAAC TCTGTTATTT 720 TTTCCAAGAA CTCAGCTTCG GTATAGCGTG TCCAGGGGCA GAAGTGAGGG GAAAGAGGCT 780 GTTAGGGTGA GCCTATGCAG CCATGTGGTC TGTGTCCCAT GCTGCTGCTG CTGCTGCTGC 840 TGCTGCAGCT CAGGGGTCAA TATCCCTTTT TCCAGGAAGG CTTAAGGTCT TTGCCCAAGG 900 CCCTCTGTCA GGCCAAGCTG TAGAAAAAGC ATTTTTTCTC ACCAGCCCTC TGGTGACAGT 960 TTCTCTTCCT TGTGGAATGG CTCAGCACAG AATGTTTTCA ACCACAAAGG AGCTGTCCTT 1020 CATCTAGGGC TTTGCACTAC TGTGTGGGCC AAGGGCAGTG GGAACAGCTG GAGCACATGG 1080 AGCTCACTGA GGGACTGGTC GGCACTTAGG GTGGTCCTTC TGAAGTAGAT GACTGCTTTC 1140 TGGAATCGCG TCCACCTCTG GATTGAGCAA AAGACAGACT GGCACAAATG GAGTAGGCAG 1200 AGTTCAACGT CTGTCCCCCT ACTGTTGTCC TCTCATCCCA GATAGAAGGG CTCCACCGTG 1260 GGCATGCCTG AGGGTGATGT CCCACATGAA GTTCTGTCTC GCATGAAGTT CTCACTCTAC 1320 AACTTTTCTC CCTGAAGCAG CATCTCTTTT GAGGGCAAAG GGTAAAGTTC ACAGGTTTTA 1380 TGGATGTCCT CTTCACGGTG CCCTGCATGA GATCTCCTGC CCAGGGCCAG CTTTTGCATT 1440 TGACCATGTA CACCCCAGCT TTGCCCCTTC TCCAGAGACG CATCTACAGC GCTAGAAGGC 1500 AAGGCTTGGC CGTCTCTGGG TGCTTTCTGA ACAAGCAGCC CAGGAGACAC TGGAAACATG 1560 TCTTCTTACT TTCTGAATCT GTCAACGTCC AAGAATCCGT GGGGCTCCGG CAGGCTGGTA 1620 CCTGTCTTTC CCAGTCCTAG TTCAAAACAA AACAAAGCAG AAACTATG GT GTGATAACTC 1680 GACAGCGGGT GTGAGATGGC ATACTGGGGC ATGGTTTTCG GGACCTTAGA CTGGGCTCAT 1740 TCTAGGGACT GCATGGCATT TACCCACATT CAGAAGTTTC CGTAAGGCAT TGCAAAGAAG 1800 TTAAAGACAA GAAAAGTTTA GGACAGGGTC CCCCAGTGCC TAATATTTCC TGCCAGTGCT 1860 TCAGGACCAG GGTTGGCATT AGCTCTAACC TGTCCTCGAG TGGAGTGCAG AGATAGTCTT 1920 CGTTGACTGG ATGGTCATCC TCTGGAGACT TGCCGTGACT TGTGTCCCAG GCTCTCTAGG 1980 GCTTTCACTT GGGCCCTAAA GGCTAGGTGC TCTTAGAGTT GACTGCACAC TTGGATGTGT 2040 CGTGTAAGGT TTTCTTCCGC CGAATATCGA TGCGTGTGAG ACTCGCCGGT TTAGTGATGA 2100 ATTC 2104

SEQ ID NO: 5 Sequence length: 305 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bos taurus Strain name: Holstein Sequence feature: is part of the mitochondrial DNA of the bovine sequence is that differ from the known sequences GATCTGTTGG AGGTTCAGGT ATTGAGATTT GTTAGAAACC ATGCTATTGC TAAAATGAAA 60 CCAATGTCGC CGATGCGGTT ATATAAGATT GCTTGTAGGC TGCTGTGTTT GCATCTGTCG 120 TCCGTATCAT CATCCGATGA GTAGATGATA TGATTCCGAC GCCTTCTCAG CCAATGAATA 180 GCTGGAAGAG GTTGTTTGCG GTTACAAGGA TGAGCATAGT ATGAGGAATA GGAGTAGGTA 240 TTTGAAGAAT TTGTTAATAT TGGGGTCTGA GTGTATATAT CATATTGAGA ATTCTATAAT 300 AGATC 305

SEQ ID NO: 6 Sequence length: 373 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bos taurus Strain name: Holstein Sequence feature: is a repetitive sequence on autosomes of cattle, copy number is small, the sequence position has not been determined GATCAAGCTA AAGAAATCTT CCTTATTTGA AAGGCCGAAA GACAAGCAAG CAAGCAAACA 60 AAACAAAAAC CCACATCCCT AAATCAAACT ATTGCCCTAG CGCAAAACAG AAGCAGATTT 120 CACAGCCCGA GGTCAGCCTA GCAGGCTTGA GAAAGAATGT TTAATTATAA ACTAAAACAA 180 CAGGATTCCA AAGGATAAGC GGTCCTGCAG GGAAATCTAT TCGTTTTGAT TTTTTCTCTC 240 TCAGTGTCCT GTCAAATAGA AGCCACTTGT AGAATCCGCT AAACATTGTT CTGCATTAGA 300 AATATTGCAT ATTGAGCGCA CACACACACA CCACACACAT ATACAGAGGA AAGACTTACT 360 CTTGGGAATG ATC 373

SEQ ID NO: 7 Sequence length: 241 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Bos taurus Strain name: Holstein Sequence features: Sequences 1-60 are part of bovine satellite DNA and are located in most centromeric centrosomes.
61-241 has a low copy number and is presumed to be located in the centromeric part of one autosomal chromosome and located in another autosomal chromosome TCTAGTTCTG CCACTTCAGC TTGCTCCATC TTCTCTGTTG ACCCACATGC ATATTAAGAT 240 C 241

[Brief description of drawings]

FIG. 1 is a diagram showing the positional relationship of the sequences of SEQ ID NO: 2 (a), SEQ ID NO: 3 (b), and SEQ ID NO: 4 (d).

FIG. 2 shows bovine, porcine, ovine and caprine male and female DNA digested with restriction enzyme EcoRI, electrophoresed and then stained (a), and Southern blotted with the DNA shown in SEQ ID NO: 1. (b) is an X-ray photograph (autoradiograph) showing the result of (b) Southern blotting with the DNA shown in SEQ ID NO: 7 after washing.

FIG. 3 is a photograph of the organism morphology showing the results of in situ hybridization of the sequence SEQ ID NO: 1.

FIG. 4 is a photograph of the morphology of the organism showing the results of in situ hybridization of the sequence SEQ ID NO: 2.

FIG. 5 is a photograph of the organism morphology showing the results of in situ hybridization of the sequence of SEQ ID NO: 4.

FIG. 6 is a photograph of the organism morphology showing the results of in situ hybridization of the sequence of SEQ ID NO: 5.

FIG. 7 is a photograph of the organism morphology showing the results of in situ hybridization of the sequence of SEQ ID NO: 6.

FIG. 8 is a photograph of the morphology of the organism showing the results of in situ hybridization of the sequence SEQ ID NO: 7.

FIG. 9 is a photograph of the morphology of an organism showing the result of in situ hybridization of the sequence of nucleotide numbers 61-241 of SEQ ID NO: 7.

Front Page Continuation (72) Inventor Shizuharu Sato 1-2 Kubogaoka, Moriya-cho, Kitasoma-gun, Ibaraki Itoham Co., Ltd. Central Research Laboratory (72) Inventor Shinzo Sudo Kubogaoka, Moriya-cho, Kitasoma-gun, Ibaraki 1-2, Central Research Laboratory, Itoham Co., Ltd. (72) Inventor, Toyoro Nakamura 1-2-2 Kubogaoka, Moriya-cho, Kitasoma-gun, Ibaraki Central Research Laboratory, Itoham Co., Ltd.

Claims (9)

[Claims] 1. At least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 or a part thereof.
A marker containing DNA. 2. Having at least one sequence selected from the group consisting of the sequences of SEQ ID Nos. 1 to 4 or a part thereof.
The marker according to claim 1, which comprises DNA and is used as a Y chromosome marker. 3. A DNA containing the sequence of SEQ ID NO: 5 or a part thereof, which is used as an extranuclear gene marker,
The marker according to claim 1. 4. A DNA containing at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 6 and 7 or a part thereof, which is used as an autosomal marker,
The marker according to claim 1. 5. At least one sequence selected from the group consisting of the sequences of SEQ ID Nos. 1 to 7 or a part thereof.
A method for identifying or specifying a biological sample using a marker containing DNA. 6. The method according to claim 5, wherein a marker containing a DNA having a part of at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 is used as a primer. 7. At least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 4 or a part thereof.
A marker containing DNA is used as a probe.
The described method. 8. The method according to claim 7, further comprising using a marker containing a DNA having the sequence of SEQ ID NO: 5 or a part thereof as a probe. 9. A DNA having a part of at least one sequence selected from the group consisting of the sequences of SEQ ID NOs: 1 to 7 is used as a primer, and 5 ′ adjacent to the selected sequence.
Method for cloning DNA present on the 3'side and / or 3'side.