JPS63503272A - Y-specific DNA hybridization probe and its use - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Y-specific DNA hybridization solopes and their uses TECHNICAL FIELD The present invention belongs to the field of genetics, and specifically relates to Y-specific DNA, especially testis-determining flags. Regarding the analysis of r-zum regarding the presence or absence of ment.

Background technology In mammals, the first sex-determining signal is the X chromosome. per cell Regardless of the number of X chromosomes, mammalian embryos with X chromosomes develop testes and X staining. Ovaries develop in embryos that do not have color bodies. XY, XXY.

XXXY and XXXY embryos develop testes and IO.

Ovaries develop in xx, xxx and xxxx embryos.

The testes or ovaries of the embryo establish a male or female hormonal environment, which Determine other sexual phenotypes, including the sex of organs and external reproductive organs (Wilao 4 J, D.

Others: The Metabolic Ba5is of engineering Disease.

5th edition, N0w Yark). i.e., all phenotypes, sexes, X staining It is initially determined by a gene or group of genes on the body. The inheritance on this X chromosome The offspring or gene complex is called testis determining factor (TDF).

H) Regarding the X chromosome, many structural specificities are revealed by optical microscopy of stained mitotic chromosomes. Detected by microscopic observation. The presence of testicular determinants on the human X chromosome have been inferred from the relationship between these abnormal karyotypes and phenotypes (Bu hler, E. M.: Human Genetics.

55:145-175, 1980: Davia, R.M.:,y.

Med, Genet, 18: 161-195.1981).

In general, evaluation of structural specificity of chromosomes involves treatment of the chromosomes (e.g., with quinacrine). Or staining by Giemde method, controlled thermal denaturation) '# minute horizontal stripes C detected after @Light) A chromosome banding method using patterns (bands) is used (VOge l, F, & Motulsky, A, G, : HumanGeneti cs:, problems and approaches, Spring er-verlag, pp. 23-31.1982).

A normal male is 46. Has an XY karyotype. That is, one X and one Y dye There are a total of 46 chromosomes, including chromoplasts. A normal woman is 46. xx, 2 X notes Chromosomal body 'i: I: Umami There are a total of 46 chromosomes. There are many mutations associated with X chromosome abnormalities. For example, a XX male has a 46.XX karyotype and a testicular mass. is a sterile male with no ovaries. The total number of chromosomes is 46, and the sex chromosomes are is also an X chromosome. There is approximately 1 case of xx1s out of 125,000 men.

In addition, apparently normal 46. If you have an XY karyotype, you are a female. This gonadal development Incompetent females have female external reproductive organs, a uterus, trumpet tubes, and poorly developed ovaries. There is. 45. Many of the characteristics of Turner syndrome, a condition usually associated with have symptoms. 46. with the Turner phenotype. Many of the XY females are modic, 45 , has an X cell lineage.

Testicles 45. Although sterile male X is extremely rare, among these individuals It is currently unknown at what rate masculinization occurs.

Chromosome banding methods have limited accuracy, resulting in abnormal X chromosome structures. Regarding construction, it is often quite inaccurate. For example, XX male is the maleness of the X chromosome It has been speculated that it has a determined small part, but conventional chromosome banding methods can't detect it. DNA hybridization of clone rhi Y sequence By using it as a reaction probe, XX males have Y-specific DNA. These males may not necessarily be the same due to the YDNA in their genes. (Guellaen, G-et al.: Nature , 307: 172-173 + 1984 m Page*D, O, are Biature, 515: 224-226 el 985). this At that time, the nature of the testicular determinant had not been clarified, and TDP was It is still unclear whether it exists in the short arm (Y), the centromere region, or the long arm (Y, ). It is clear.

Testicular determinants are not only genes on the X chromosome. However, the sentinel Y chromosome Genetic analysis of the species has been hampered by its haploid shape. different from other chromosomes in the nucleus Therefore, Y does not have a homologue that joins with the homolog, and it is possible, if not completely impossible, that the Y gene This is because it makes it difficult to study the genetic linkage of offspring. On the mouse or human X chromosome At least part of the reason why relatively few genes have been identified in the I can explain it. Attempts to establish a Y-linkage of certain traits are based on sex-restricted expression. True Y-linked inheritance is difficult to identify and often inconclusive. However However, in addition to the male determinant, there is evidence for the presence of many C genes on the X chromosome. Ru. For example, it is clear that the structural gene for antigen 12E7 is located on the human X chromosome. It is being done. The structure or regulatory locus of the H-Y antigen has been determined in both mice and humans. However, it is probably present on the X chromosome. Two genes are involved in the weakly fluorescent proximal region of Yq (barrier). I have gout (Yq11). Genes that affect spermatogenesis and fang length This is a gene that affects tooth size. However, its exact location is unknown. current At this point, we will identify the gene in question on the X chromosome, determine its location, and determine the location of the gene in question on the X chromosome. Detection of abnormalities, presence or absence of specific regions of chromosomes, gonads, their sex, etc. There is no satisfactory method for correlating the phenotypic effects of

Disclosure of invention The invention described herein is directed to the normal Determination of the location on the chromosome, determination of the location of the male sex (testis) determinant on the X chromosome, and It is based on the construction of a physical or deletion genetic map of the normal human X chromosome. . Genes located on the X chromosome play an essential role in human male sexual development, and The presence of chromoplasts is usually associated with testicular development. Conversely, X-chromosome abnormalities (e.g. deletions) (losses, rearrangements) are often accompanied by defects such as sexual dysfunction and mental retardation.

Currently available techniques, such as light microscopy analysis of stained chromosomes, It is not possible to definitively determine the presence or absence of specific segments of chromosomes.

h) The deletion map of the X chromosome is determined by hybridization with the Y-DNA probe. More abnormal karyotype? Individuals with or having a structurally abnormal X chromosome determined by cytogenetics consists of an interval n defined as the part of the X chromosome whose presence or absence has been shown in individuals with . Individuals whose denominated DNA was tested had cytogenetics on the long arm (Yq) of the The short 1lFiI (Yp) of the X chromosome with a visible deletion in It is said that Yp has a cytogenetically detectable deletion and Yq has a normal appearance. The hybridization results from these samples indicate that this chromosome Useful for constructing deletion maps for the long and short arms.

This map facilitates the selection and cloning of the Y-specific DIJA sequence, which is the subject of the present invention. It is the foundation of the training. The present invention provides methods for using the above sequences in the analysis of sex chromosome materials. It is the law. This map is extremely useful for clinical diagnosis and for the evaluation of any Y function required. Considered useful. by currently available methods (e.g. chromosome banding). Even if an anomaly cannot be detected using existing maps, structural abnormalities can be detected using existing maps. The normal X chromosome was systematically characterized by D11A/% hybridization. It is possible to For example, testis-determining factor (TDF) is the short arm of the X chromosome (Y The location is determined in p).

According to the method of the present invention, a region of a normal X chromosome is present in DNA from a subject. Y-specific cloned as a DNA probe to clearly establish whether An array is used. Using the deletion map of the normal X chromosome, chromosome banding However, even in this case, the X chromosome, especially structurally Systematically Characterizing Abnormal Y Chromosome YDNA Hybridization Can be done. Cloned Y-DNA sequence as hybridization probe , analysis of the structural characteristics of the X chromosome, and the existence of KY-specific DNA in the r-tum of individuals. determination of whether or not the Determination of the location of existing or missing sequences on Y-DNA, genetic abnormality and its associated effects. In particular, one or more types of Y ha Hybridization probes can be used to detect and characterize X-chromosome abnormalities in individuals. The short arm (Yp) of the X chromosome in samples taken from patients, the centromere Determination of the presence of region or long arm (Yq) specific sequences, male sex determinant, X staining Detection of body regions as well as regions that determine the occurrence of spermatogenesis, as well as other regions of interest (e.g. For example, chromosomal changes that make individuals with gonadal hypoplasia clearly more susceptible to gonadal neoplasms region, the region responsible for the expression of the H-Y antigen).

DNA prepared from blood or other tissues from the subject is injected with one or more types of Y-characteristics. When analyzed by hybridization with isomeric proso, the results are e.g. It is possible to establish the presence or absence of specific regions of the X chromosome that are of clinical or diagnostic interest. Ru.

Brief description of the drawing Figure 1 shows normal and) X staining constructed based on DNA hybridization studies. This is a deletion map of chromoplasts.

Figure 1a is a deletion map showing inversion polymorphism.

Figure 2 shows 1. Normal female a (FIJ28), xx male a (%J12), 2 cases of xYq ( →TaqI digestion D from male (Example 25 and Example 26) and normal male (9129) (1) Hybridize two types of labeled probes to the rayx fur. This is an autoradiogram showing the results of fusion.

Figure 3 shows normal male C example 29), normal female (example 28) and XXjla (example 10). ) Carano Taq Neeliase (1 (D This is an autoradiogram showing the results of noibridization of the tube.

Figure 4 is a genetic deletion map of the human X chromosome.

Figure 5 shows 45. X/46. xy male; control female; and 45. x/46. xy tsB target color I) Has DI digested DNA with Y% heterogeneity 2.1 kb Ha Probe Y 4' 31-HlnfA hybrid that detects the θ■ fragment 1 is an autoradiogram showing the results of diization.

BEST MODE FOR CARRYING OUT THE INVENTION Most of the X chromosome, the only haploid human chromosome, does not participate in meiotic recombination. No. As a result, based on the recombination distance between markers (the genetic map of the X chromosome Construction is impossible. However, in nature, extensive X-chromosome deletions occur. . H) Attempt to estimate the location of testis-determining factor (TDF) on the X chromosome is determined based on the relationship between these abnormal karyotypes and the sexual phenotype of individuals with the abnormal karyotypes. It's been coming. However, the accuracy of these chromosome banding studies is extremely limited. The structure of the abnormal X chromosome is highly inaccurate.

The location of the TDF remains controversial.

Until the construction of the X chromosome deletion map published herein, TDF was It is also clear whether it exists in the arm (Yp), centromere region or long arm (Y, ). There wasn't.

The present invention uses DNA ( When analyzing the presence of homologous DNA sequences (i.e., sex chromosomes), hybridization This invention relates to X-chromosome sequences that can be used as fusion probes. With its use, In particular, the Y-DNA in an individual (i.e., the DNA that occurs on the normal X chromosome) to clearly establish the presence or absence of the DNA sequence in question on the X chromosome; It is possible to determine the current location. For example, the Y-specific D IJ A of the present invention Probes can be used to detect the male sex-determining region, centromeric region, and diagnostic or Sex chromosomal regions of interest for clinical relevance (e.g. Areas thought to be susceptible to substances, areas that already affect sperm shape, H-Y antigen This makes it possible to amortize the existence of regions (areas that are responsible for the expression of Kono Yonna Y Specific probes are used to analyze a set of chromosomes that appear cytogenetically normal. Can be used. Homology for chromosomes that appear to be cytogenetically normal X chromosomes For example, consider the existence of TDF. Furthermore, the Y-specific DNA probe It can be used to evaluate chromosome structure during the fetal period. In other words, detection of X chromosome abnormality and identification is possible.

Below, 1) r-tum DN A from normal individuals and individuals with sex chromosome abnormalities Construction of a deletion map of the Y chromosome based on hybridization studies of and characteristics, and 2) known to be present on normal human I-Y chromosomes. Hybridization probe for detecting DNA sequences homologous to the sequence The use of Y-specific DNA as a buffer will be described.

The physical or deletion map of the Y chromosome from a normal human is It was constructed by analyzing and comparing DNAs from individuals with chromosomal abnormalities. map is shown in Figure 1.

For each chromosome, hive into sadane transfer of restriction-digested r-tum DNA. The presence or absence of 120 Y-DNAs was tested by redization (Sout Hern.

E, M: J, Mo1. Blol,, 9 B: 503-517, 19 75).

The majority of individuals tested were XX male, XO male or XY female, or cytogenetic. Scientifically determined, he had a structurally abnormal Y chromosome. Normal male (46,XY ) and a normal female e(46,XX) were also analyzed.

Most Y-DNA sequences used as hybridization probes were Na tional Laboratory Gene’Li1) rar7 proj Obtained from ECT (LOs Angels) and made from Taflow sorted Y chromosome. Derived from the library.

That is, Y Clone fragments of chromosomal DNA (obtained by complete digestion of DNA) I used it after searching. Analysis of randomly selected Y-DNA-containing clones revealed that the staining Deletion spacing was restricted on the chromoplast. The DNA sequence of interest was removed from the λ phage and Recloned into cilasmid (eg 1.σC8 or σC13).

Other DNA sequences used as probes were described by Page et al. (Nature, 31 1:119-123.1984) and B15hop et al. (, r, Mo1. Biol.

175: 403-417.1984: Nature, 3Q3:831 ~852.1983). The disclosure of this report is incorporated herein by reference. Introduced as a matter of reference.

The DNA to be tested is peripheral leukocytes, cultured skin fibroblasts (EBV) From the murine lymphoblasts, the well-known million @ (Kunkel, L, M, et al.: Pro c, NatlAcad+Sci.

USA, 7±:1245~i 249.1977;ve rgnaud, G , et al.: Br, Med,, r,, 289: 73-76゜1984) It was prepared accordingly. It is then treated with restriction endonuclease Taq or EII: 5outhθrn's method as described above. Hybridization is performed with the selected radiolabeled DNA probe, and Y-specific A specific restriction fragment was detected.

A study of 77 DNAs with abnormal karyotypes or structurally abnormal Y chromosomes It was revealed that 47 of these cases had some, but not all, of the Y chromosome. .

In other words, in each of these 47 cases, there is a constant presence in normal (46,XY) males. Some, but not all, Y-specific restriction fragments were detected. These 47 cases In only 19 gA, the deletion was detected cytogenetically. cytogenetically detected The resulting Y deletion was also revealed by hybridization. Ta. Changes in the pattern of the Y-D IJ A gene locus observed in these individuals The simplest explanation is based on the deletion map of the Y chromosome shown in FIG. child The map is explained based on 2 cases out of 47 cases (all cases, each case?: 1 Y cut point) It is something to do. In other words, does the deletion map in Figure 1 represent 2 out of 47 cases? In all cases except hybridization data that corresponds to a single contiguous portion of the Y chromosome. ing.

The map has 10 deletion intervals (named IA to 7). Each of the intervals shown in is an absent portion of the Y chromosome, or a difference between two individuals or between two classes of individuals. is defined as For example, QIA is the Y chromosome present in class 1A XX males. This is the part. Class IAXX males are the only Y-specific fragments tested. 1 fragment (a fragment derived from and detecting an interval of 1 person) ) and don't have it. Interval 1B is class IBXX; This is the part excluding the part present in Las IAXX males. Ding, that is, class I B XX males contain the same Y-specific fragment that is present in class I AXX males. , and additional fragments (derived from and detecting interval 1B). ment). Similarly, interval 2 indicates Y staining present in class 2xx males. This is the interval obtained by excluding the part present in class IBXX males from the color body part. class 2 xx males carry an additional 13 Y-specific fragments than class IBXX males. I have it. Future studies using YW heterologous DNA probes will improve these intervals. One or more of them are actually two or more than six with small intervals or It is conceivable that it is divided into subunits (for example, interval 1 is divided into 1A and 1 (It is divided into two intervals named B). divided into two subunits Interval 3 is the portion of the Y chromosome present in class 3xx1s Note. This maleness is All fragments present in Las 2XX males plus 22 other fragments. ■Do. Interval 4A is present in class 4XX males but not in class 3Xx males. This is the part of the Y chromosome that does not exist.

Individuals with Yq with cytogenetically detectable deletion and Y with normal appearance (X (in Yq) and Yp with a cytologically detectable deletion and an apparently normal Obtained in hybridization studies of individuals with Y9 (XYp(-) female) It is possible to create a deletion map for Yp and Y from the data obtained. The centromere is 2 Assigned to interval 4B based on two observations. First, 4B is deleted but independently It is the only interval present on all separated Y chromosomes. Second, certain Y-specific It is known that the reverse sequence is located at the centromere 1cIQ by 5 intu hybridization. (Wolf, 7. et al.: J. Mo1. Biol.

182:477-485.1985). This repetitive sequence is a Y-specific DNA protein. Using the lobe, it was determined that it is currently located in interval 4B.

Previously available methods (e.g. chromosome banding) have not been able to determine the genetic basis. There are some transsexual syndromes for which it has been impossible to determine or explain. for example, 46, xx karyotype, male individual; 46. xx karyotype 1 bisexual individual; 45. x karyotype, male individuals (designated XO); and individuals with XY karyotype, female.

Using the Y-DNA specific probe and homologous DNA sequence detection method of the present invention, The genetic basis for these cosmetic abnormalities can be determined.

A, XX male For example, an XX male is a sterile individual who is otherwise phenotypically male and karyotypically male. is 46. It is xx. In other words, it does not have a Y chromosome and is clearly (cytogenetically) Definitely contains normal female chromosomes. However, by definition, XX male The gonads are composed exclusively of testicular elements. The testes of such individuals were traditionally This is thought to be due to the presence of male-determining chromosomal DNA, which cannot be detected by this method. Y special The heterologous DNA probe contains a homologous sequence (j, etc.) in the genomic DNA of 46,XXi. ie, the presence of a Y-specific sequence). 12 [] different Hybridization of Y-DNA probe to Sudden Transfer By doing so, 46. Detection of certain Y-specific sequences in r-tums of XX males I was able to do it.

Based on the Y-specific sequences present, xxts can be divided into six classes. came. In other words, Y[→XX male and YDNA sequences were not detected. There are 5 classes of Y (+) x x male that were detected.

Y (→xx, t#: Y-specific DNA sequences present in the notes are in intervals 18 to 4A in Figure 1. is an array in . Y! Hybridization using a heterologous DN A probe Based on chromosomal studies, they have been assigned to Yp, the short arm of the Y chromosome. In this study , interval 1 person tested Y (+) x x male and VcXYq (→ all male It was revealed that this is common to both. As a result, xX1sNote is the Y-guided TDF If we assume that we have testes due to the presence of , TDF will exist in interval 1.

This assignment of TDF to Yp is consistent with most karyotype-phenotype relationships.

Y-specific DNA sequences can also be obtained using the probes and methods described herein. One case tested was 47,xxxXX male. Part of Y chromosome present The portions are similar, if not identical, to those present in the Class 3xx male notes.

As mentioned above, one class of XX males (named Y (→), whose Y DNA sequence is Until now, Y-specific DNA probes have also been used by chromosome banding. It has not been detected by I.I hybridization either.

Y(→XX male) is even smaller than the part seen in class IAY(+) male. However, there is a possibility that it has a Y chromosome part. In addition, Y(→XX male is autochromatic It is thought to be the result of a somatic or X chromosome mutation.

And Yuji 1” 1 standing 2 XX notes are 46. xx karyotype, and the gonads include both testicular and ovarian elements. Y Even with the use of specific DNA probes, the denominator of both XX and Y-D in the six cases tested was No NA was detected. Y　(-)　x　x Regarding maleness, both XX notes are IAY (+) due to the presence of a smaller Y chromosome segment in males or due to normal It is unknown whether this is due to chromosomal or X chromosome mutations, but the latter is more likely. .

C, XO male XO males are sterile, but are otherwise phenotypically male a, 45. x karyotype . It lacks part of the Y chromosome, as determined by chromosome banding. XO male The properties were investigated using the pair of Y-D IJA probes described above. These pros In DNA hybridization studies using It is clear that less than 3% of fibroblasts are mosaic with a low-grade XY sex chromosome composition. It was made clear. This mozuiku (later confirmed by determination of cell karyotype on the same page) , was detected and quantified using a probe that detects Y-specific repeat sequences.

This analysis is detailed in Example 4. I think that maleness is due to the XY cell lineage.

Six other XO males determined by DNA hybridization were Y-stained. It has a body spacing of 1A to 4B (class I XXq (→same as male). As shown in the figure. In other words, even banding studies performed by skilled cytogeneticists do not show Y material. were not detected at all, but DNA hybridization revealed that these three columns When the xo male smiles, all or at least most of the cells contain the entire short arm and It was revealed that it has centromere and centromere. In two of these cases, l　n　8　 i/S hybridization (i.e. metaphase stage chromosomes and radiolabels) (hybridization of DNA probes) and retrospective cytogenetic studies , the Y material is autosomal, and in both cases there is strong evidence that the 15pK translocation occurred. Obtained. In one case, YDNA was not revealed by pure cytogenetics. Nevertheless, the YDNA zolobes were attached to Yp (the short arm of the X chromosome). hybridized). In these six cases, the male phenotype is due to the presence of Y interval 1A. It is explained in the present.

Submolar amounts of YDNA were also not detected in the fifth XO male. See example 4 Light.

D, XY female Studies of XY females confirm that TDF is present in Y-interval individuals. XY female , have degenerated ovaries and no testicular tissue, despite the presence of an X chromosome. Not possible. In most cases, the XX male has a relative with a similar disorder and the genetics are It is chain recessive (Swyer syndrome). In such cases, the defect is clearly in the X chromosome. above, and Y is presumed to be normal. However, there are sporadic cases of XY female and TDF. It is also possible that this is due to deletion. By DNA hybridization, nine cases Deletion of Y interval IA (and several adjacent intervals) was observed in 4 cases with sporadic iXY females. One female had a balanced Y:22 translocation. See Figure 1. Deletion is DN In the two XY females detected by A hybridization, the prometaphase ding studies revealed a small deletion in Yp. of recent splits At the limit, deletions in class 1, 2 and 6 females are similar to those in class 3, 4 and 6 females, respectively. and 2xxts Note. These findings indicate that XY This suggests that females and XX males are mutual products of similar abnormal X-Y exchanges. Ru.

The findings in the XY female were also 45. Turna most commonly occurs in females with X karyotype - Clarified the etiology of the syndrome. Turner in all 4 XY females in which the deletion was detected. Showed some signs of the syndrome (eg, pterygium, lymphedema). Deletion detected No symptoms were observed in 591 patients who were not treated. These findings are associated with Turner syndrome. Strongly suggests that the group is the result of monomyelism of genes common to the X and X chromosomes do. For example, in XY females with Swyer syndrome, a familial X-linked recessive inheritance Here, the This suggests a downstream mutation. Features of these XY swire female turners The absence of a normal X chromosome prevents the Turner phenotype, as does a second X chromosome. It shows what you can do.

However, other Yp deletions were revealed by DNA/1 iridization. The presence of the Turner trait in XY females suggests that Yp, which is deleted in these females, Part b: means essential for Turner blocking action.

In other words, Turner syndrome is caused by one or more types common to the X and X chromosomes. It was revealed that this is due to genetic monomy. One of these genes or Inspection of Y-specific DNA sequences determined that two or more species are located in the distal segment VC of Yp. As a result of the identification and determination of the region where the X chromosome resides, blood or other Elucidate the existence of sequences homologous to the selected sequences for injection capsules prepared from body tissues. It becomes possible to use the selected DNA sequence for analysis. chosen D　IJ　A sequence binds homologous Y-DNA by hybridization with it. It is used as a probe to detect its presence or absence.

For example, if it is not well characterized and its location on the normal X chromosome is known, Using one or two types of probes, each interval of the sex chromosomes of the individual suspected of abnormality is detected. determine whether there is a homologous sequence in the The presence or absence of specific fragments such as known genes can be determined.

The DNA used for analysis is, for example, peripheral leukocytes, cultured skin fibroblasts, or KBV. - obtained from transformed lymphoblasts and prepared by known methods; Ru. In one method, the nucleus is released by cell lysis. The nucleus is then exposed to dodecyl sulfur. Acid) Decomposes in IJum/proteinase. DNA was extracted with phenol and and purify by dialysis (Kunke'l, L, M, et al.: proc, Natl .

Acad, Sci, USA, 74: 1245-1249.1977: Preparation The resulting DNA was then subjected to restriction digestion, electrophoresis and transfer, followed by and has already been reported by Page & de1aChape11e. Hybridization with one or more Y-specific DNA probes according to the method (Page, D, O, & de la 0hapella: Am6. T, Human Genet, 36: 565-575.198 See 4. (This disclosure is incorporated herein by reference). For example, DNA is Can be digested with restriction endonuclease Taq or E(!OR). Fragments are separated by tq e.g. agarose del electrophoresis and Y-specific DNA Transfer to a membrane filter for hybridization with the sequence (probe).

Hybridization can be carried out by the Sudden plot method (5outh ern, ni, M,: J, Mo1. Biol, 9 F3: 503-51 7.1975).

For individuals suspected of having sex chromosome abnormalities based on cytogenetic analysis, When detecting and identifying DNA sequences along a chromosome, a set of probes is can be used. 1 For example, one set of probes has A probe is included for each interval found to exist. this pro By using a combination of Detection of the presence of regions that do not normally exist on sex chromosomes and the detection of regions on the chromosome in question It is possible to determine the location of the object. The 10 normal Y chromosomes shown in Figure 1 One set of probes that can be used to determine the presence of an interval is as follows.

IA DP307 [Hln of JO-13 (i sub p p at DI site) Cloned 0,9kb ) 11th III Y fragment I B p D P 132 Subcloned at H1nll1m site of pUO-16 3.5 kb Hlnd m Y 7 Ragment 2　DP61　prJc-8 ACC engineering and 1 (! oR engineering site) subclonin Googled p115 l, Q kb EcORI/'raq engineering Y fragment from the insert of 3A pDP125 pUO-130H1ndI [subcloned into I site It was. ! , 6kb Hlnd m Y 7 Ragment 3B 50f2 1 subcloned into the ECOR site of pBR327, 1 kb B2coH engineering Y fragment 4A　DP34　2,2 subcloned into the ECO 5 site of pDP322 kb ECOR engineering Y fragment 4B pDP97 ptrc-13 subcloned into ECo 5 parts Sumido 5.3 kb ECOR Y fragment from Y97 5 12 f 5,0 subcloned into the EcoR site of pBR322 kb EQOR Y Fragment 6 50f2 1, 1 subcloned into the ECOR site of pBR322 kb ECOR Efragment A: pY431-atnfF cloned into the pat site of ApBR522 s　Q-3'Kb　Hlnf　1y　7ragment Note: pDP307; pDPl 32: pDPl 25; pDP3 4 detect ZXW-specific and Y-specific single copy DNA sequences, respectively.

The PDP 34 is the same as the DXYS1.

A combination or analog of this probe with one probe for each interval of the Y chromosome Using a combination of It becomes possible to determine their location on the color body.

When testing key IJDklA for the presence of a particular region of interest, Of course, only some of the probes mentioned above can be used. For example, The subset of probes that generate the can be used to determine the existence of

3B 50f2 A subset of probes can also be used for detection.

As previously mentioned, DNA sequences can be used to detect the presence of TDF. use The probe is located on the normal Y chromosome and is essential for testicular development. It is one or more types of sequences that have been shown to be Especially in this case , the probe shall be a Y-specific DN A present in one person at the interval shown in Figure 1. I can do it. Probes of this type that can be used include pDr3 [17 (mentioned above). Ru.

The DNA sequence used as a probe was derived from the Y-rich incoming phage leipsisi ( For example, National Laboratory Gene Li1)rary library available from Project) and added to the plasmid. This is the cloned sequence that was cloned. Also, synthetic NA sequences can be used. Ru. Is the DNA probe labeled (e.g., radiolabeled, biotinylated, etc.)? (chemical modifications such as must be qualified with

Compare the data obtained with this method with the deletion map of the normal Y chromosome shown in Figure 1. By doing so, the DNA sequence homologous to the Y-specific probe (TDF) is detected. It becomes possible to determine whether or not a sample exists in sample DNA.

■Use of probes that recognize other regions of interest on the Y chromosome As mentioned above, the deletion map allows evaluation of the underlying Y chromosome function. and For example, if the Y chromosome has the gene for 1 (-Y antigen (transplant antigen), the deletion map Using selected probes, the gene can be localized to the chromosomal deletion interval Become. H-M antigen is TDF and H-Y is a genetic determinant of gonadal sex and/or has been hypothesized to be a determinant of spermatogenesis. Found to play a role in spermatogenesis For example, a probe specific to the Y region where 1(-Y is present would be Can be used to diagnose fertility.

As mentioned above, XY (gonadal hypoplasia) females have a 46°XY karyotype, female external reproductive organs, It is sterile female with a uterus, trumpet tubes, and underdeveloped ovaries. In addition, tar There is an XY/XQ female with Ner syndrome. Such individuals have gonadoblastoma Often, the X chromosome, or some part of it, predisposes these individuals to gonadoblastoma. It has been presumed that this role plays a role in making it easier to understand. The deletion map shows the injection capsule of such an individual. It can be used to characterize the body and detect abnormalities that are clearly responsible for this predisposition. Wear. Once the anomaly has been identified, spaced, and located, Homologous sequences are determined for the prepared DNA (e.g. from blood or other tissue). DNA probes can be created and used to investigate.

The X and X chromosomes have been considered partially homologous.

Using DNA hybridization probes, Page et al. Discovered a long single-copy DNA homology between chromosomes, tn, DXYSl. .

Restricting a 4.5 kb segment of single copy DNA from the Star Detum 2 library Hybridization by sudden transfer of human DNA digested with enzyme Taq tion. This probe is compatible with 11.12 and 15 kb long Taq engineering limit frames. clarified the component.

Among over 100 unrelated individuals, all males were found in one of the other fragments. In addition, a 15 kb fragment was shown. Some females have both 11 and 12 kb fragments are shown, the others have one fragment length of 11 or 12 kb. Ta. There were no i5 kb fragments. These results are 15 The kbT aq e fragment is derived from the X chromosome, while the 11 and 12k The b fragment suggests an X-linked allele, fs (Fagθ, D , (!, etc.: Research after Pr0C6 has confirmed these speculations 1111. Ta. High Resolution Image Download MS PowerPoint Slide of a 4.5 kb probe on TaqI-digested DNA from 48 individuals from a single family. Hybridization resulted in a 15 kb fragment of X-linked inheritance and 11 and 1 X-linked inheritance of a 2 kb fragment was demonstrated. starfish tooth hybrid thin From the cell system (with partial complementarity of human chromosomes) to Taq-engineered DNA Hybridization of the probe revealed a 15 kb allele on the human X chromosome. Human) Separation of b alleles on chromosomes 11 and 12 was observed on the X chromosome. child This is the first demonstration of single-copy sequence homology between X and X chromosomes in clear mammals. Met. This homology site between human x and X chromosomes was named DXYSl. (Fagθ et al.: Recombinant DNA Applied ions to Human piseaee.

caskey, C, & White, R, eds. c’o1a Spring 　Harl)or.

1983).

Deletion map studies using random Y-DNA loci revealed that nearly half of Yp, ca. 7.000 kb shows extremely high homology with Xq16-q21 (on the long chain). be suggested. these! -1 In this specification, it is referred to as the DXYSI-like locus. Yp interval I All 22 loci given A, IB, 2, 3A and 4AK positions are in this It seems to be the origin of the buttocks. DXYS 1 is the X-Y homologous gene of this class It is the most thoroughly researched of all time.

All of the DXYS1 loci were located at five different intervals on Yp. If it is the result of a single rearrangement from X at some point in the past several million years of human evolution. (as research suggests), homologous loci cluster closely together on the X chromosome; Expected to be in the same order. DXYSi's legacy, if not all. Most of the loci are found in - individuals with a known X chromosome segment or humans - By Southern hybridization using rtum DNA from hybrid cells found in the vicinity of xq13-q12, as shown by physical mapping. Ru.

This physical map of DXYSI-like loci on This can be supplemented by gene linkage maps. 8 X-linked at the DXYS1-like locus The characteristics of RFLP have been clarified. Several such intrafamilial X-linked RFLPs Tracing the inheritance of these Y homologous sites on the X chromosome simultaneously reveals genetic linkage You can create a map.

X-linked studies (i.e., linkage mapping these DXYSi-like loci to each other) ) map loci simultaneously within the larger association of the X chromosome and, in fact, A fine structure physical/genetic linkage map has been created near xq13 to q21.

Genetic mapping of these X-linked RF'LPs at the DXYSI-like locus Significantly increases its usefulness in the study of X-linked traits. For example, more than 50 people worldwide The above laboratories are currently working on choroidal deficiency, Charcot-Marie-Tooth disease and other We use DXYS1's X-linked RFLP to study linked diseases. DXY Sl also appears to be closely related to ectodermal dysplasia.

Pseudoautosomal loci on the X and X chromosomes Xp-Yp pairing during male meiosis is long For a long time, homology between these regions and even evidence of recombination has been considered. . Two loci provide concrete evidence for this previously hypothesized region of approximately 120 It was identified during the analysis of several Y-DNA loci. Both loci are X and X stained It is common in the body and often exhibits X-Y combination odor as a normal event during male meiosis.

These were located at distal X and distal Yp of the physical map, respectively.

Due to X-Y recombination, restriction polymorphisms (RFLPs) at these loci are strictly It does not show closely sex-linked inheritance and is inherited like an autosomal disorder, resulting in a pseudoautosomal disorder. It is called. One of the pseudoautosomal prosos is a close relative that exhibits a very high degree of RFLP. Detect family arrangement. Most of these polymorphisms can be detected using many restriction enzymes. , so it seems not to be due to base pair substitution. Homologous restriction fragments are Not only the length but also the number varies depending on the individual. However, family research suggests that This suggests that the probe detects a single locus. Within the family, Recognizing the collection of specific autoradiographic bands as constructing a gene I can do it. This R'F'LP does not show sex-linked inheritance, but the genetic locus is in 5 itu hybridization and de nodont-human hybrids. Deletion mapping maps the distal short arms of X and Y to T. These and others The pseudoautosomal locus probably maps distal to the short arm suprainterval 1A of the Y chromosome. cormorant.

Long single between human x and Y chromosomes: +i-DNA homology section Characteristics of six types of Y-linked RFLPs, including those present in DXYSl, have been revealed. Santa. In most unrelated American white males, this 15kb protein is found on the Y chromosome. There is tandem duplication of homologous sequences. This is true of most Oriental and proto-Aust The same goes for Syrians. In other words, from the X that caused the DXYS1 homology itself Similar to the translocation to Y, this Y-linked RFLP appears to have occurred before the formation of the human species. be exposed.

These Y-linked RFLPs also contribute to the refinement of the deletion map in Figure 1 and the fact that the map It is also useful for determining whether it is actually an image shape. In other words, the period shown in Figure 1 Are there two types of Y chromosomes in the entire population that differ by distance, especially inversions of 3B and 4A? I think this will help you decide whether or not to do so. For example, the map shown in Figure 1 Class 4XX male or Class 2XY female are omitted, but in both cases, Y staining Three breakpoints are required on the color body. The order of intervals 3B and 4A is reversed If the inversion polymorphism is The idea that Note XXi has the final part of Yp is completely consistent. Class 3XXJ1m A and class 2XY females have a Y chromosome with a 3A-3B-, 4A-4B sequence. Class 4xx males and class 2XY females can be explained as arising from 3A The -4A-3B-4B sequence can be explained as originating from any Y chromosome.

The present invention will now be explained in further detail by means of the following examples, which are in no way intended. The present invention is not limited in taste either.

30711247a47z13d1151611t, +1JIXo Wml@ Ll:LIOγX/+Ld*R L to L61a Xd*1Tcp 1.4 LCL570 Xd@1Yqs 2 -4.

KL XYq-a*I Ma e NOe * MDkDLL9ZXIQ・m- KamekakuguchielIQI+11QMO01NOOFχ19φmal 15 - Kosha -yD Shame? ? '・115　X；1-1 LCL644 XY (Ti re Z, a *0u40Qalq:IN@ko--- -1101107'? -140XYp-fur am 47415221m CIL　n　　1m’　＊　Contest 1a-mxoy0247s47xHdIlffI! 11t7TA13rJ: 1 ttmoIIomorrrtmemtoosuot+tottomot+mouth...+ The dispute between the dispute and the dispute between Russia and Russia ◆ moi + ow participation KO + Φ ◆ and H mouth ◆ ◆ Hammer 01 10110*! B 5 (RZ 5arz SZd SZd 1150Xr (f gar'H50127l-7a NIAX 12f 5usolz I=@4 9111M　3.42-1121711-Y Normal individual and abnormal cutting die or thin DNAYY-specific DNA probe from individuals with genetically inherited sex chromosome abnormalities hybridized with bu.

The presence of 120 fl Y-DNA loci in the DNA obtained from 77 cases. was tested. Most of the individuals tested were xXj$a, xo andromastoid, or XY Are female or female, or have a structurally abnormal Y chromosome as determined by cytogenetics. there was.

The 1 patients studied are listed in Table 1, and the phenotype and pseudotype of each individual are shown. research 1 Most individuals are sterile and/or have small testicles and are removed during medical evaluation. The type was decided.

DNA1 release and rle transfer hybridization DNA is derived from peripheral leukocytes, cultured antisera fibroblasts, or EBV plasma lymphocytes. From blast cells, known methods (Kunkrl, L, M, et al.: Proc, Natl , Acad, Sci, UEIA, Mun: 1245-1249, 1977; Vergnaud, G, et al.: Br, MedJ, 289 Near 3-76.1 984). Restriction digestion, electrophoresis, transfer and DNA Hybridization was carried out as described above and one column (Pa6e, D, O, & dela 0hapelle: 1nuWWuutjLltlu,, 56 −j6b ~ b/! :+, 1 984).

Each hybridization probe was tested under low or high stringency conditions as described below. used in either. Low decision: Strict conditions mean hybridization 4 The final wash was carried out at 2°C at 68°C in zxssa or at 55°C in 0.1xssa. This is actually 5 things. High stringency conditions mean hybridization at 47°C and/or Or the final wash is carried out at 68°C in QAXBSO.

Use DNA hybridization probes with the exception of probes pDP34 and Pl. All probes described below, except for (described above) is a plasmid subclone (B18hOp, Q, et al.: J, MOl, B101. , 173:403-417.1984;B15 hop, (HoE, etc.: Hature, 305: 831-832.198 3) For multiple probes, the name of the homologous DNA segment or locus Human Gene M with the prefix (for example, DXYS5.DYZl) Received assignment of appingcon’rerencevn (5kolnick , M, H, et al.: Report of the committee on human gene mapping by recombinant DNA technique, Oytogsnet, cell, Genθt, 1-4 :210-273.1984).

Probes 47a and 47z have homologous sequences located high on the X2 and Y chromosomes (DXY 1743, 1985). Under high austerity conditions 47a detected a Y-specific Taq protein 4.3 kb fragment, and 47z detected a Y%-specific Taq fragment. q Engineering 3 kl) Detect fragments. These sites of X-Y homology are It is also detected by the lobe 470 (Guellaen, G., et al.: Na ture, 307:172-173.1984). 47a, 470 and 47Z is a subclone from the same cosmid.

Probe i3d targets a highly homologous sequence (DXYS7) on the X and X chromosomes. It has been revealed that it can be detected by BO:r,, 4: 1739-1743.1985n. Under conditions of high austerity, 1 3d detects a Y-specific 7 kb Taq effect.

Profoop 115 contains the highly homologous sequence CDIYS8 on the X and X chromosomes. Detect (Geldworth, D, Hoka: EMBo J,, 4: 1739-1743.1985).

Under high stringency conditions, 115 detects a 2.1 kb Y-specific Taq fragment do.

Probe 52d detects multiple loci on the X chromosome as well as one locus on the X chromosome (B15hop, C!, etc.: J, Mo1.ntol,, 173: 403-317.1984).

Under low stringency conditions 52d is 7 kb (restriction fragment 52d/A), 1.2k b (52d/B) and 1. Qkb (52d10. Dub that is not separated on the external surface Let]'s Y! Detection on different ECOR fragments. Corresponding Y-specific Ta qF2 segment is 9kb (52d10: unseparated doublet), 5 kb (52d/A) and 3kb (52d/B).

Probe 50f2 reveals multiple Y-specific and autosomal loci . Under low austerity conditions, 50 f2 is 10 kb (50 f2/A), 7.5 kb ( 50f2/B).

6kb (50f210), 4.5kb (50f2/OJ: and 1.7kb (50 f2/E) Y-specific BcoRI 7 label is detected. Corresponding Y special Different Taq Efragmen)t! 9kl) (50f2/E), 8kl) (50f2 /D).

3.5kb (50f2/A or 50f2/B) and 3kb (unsplit) No doublet, 50f210 J:hi5D f2/AmoL<ha50f 2/Bf) equivalent to Izurekani) (() uellan, G, etc.: Na ture, 307:172-173.1984>.

Probe 118 detects multiple Y-specific restriction fragments. The existence or non-existence of In this study, the identified fragments were four types of Taq I fragments, 7kl ) (11B/A), 6kb (1187B), 5kb (1i 810) and 1 kb (118/D) and 14 (Guellan, G., et al.: Natu re, 307: 172-173.1984).

Probe pDP 34 is a highly homologous sequence CDXYS1 on the X and X chromosomes. (Page, D, O6, etc.: Proc, Natl, Acad , Elci+σ8A, 7ヱ: 2352~2356°~123.1984) . Under high stringency conditions, pDP34 generates a 15 kb YW heterologous Taq effect. Detect.

Probe 64a7 detects homologous sequences on Y and autosomes (()uel lan, G, et al.: Nature, 3 Q 7: 172-173.19 84). Under low stringency conditions, 64a7 contains a 4.5 kb Y-specific Taq effragment. Detect the event.

Probe 12f detects autosomes and multiple sequences on X and one sequence on Y. Know (B15hop, a, others+, JoMol, Biol, 17 3: 403-317 + 1984).

Under high stringency conditions, 12f can be linked to two or three Y-specific Taq molecules or ECOR. Detects efragment. The sample was an 8kb Y-specific Taq fragment ( The presence or absence of a 5kb ECOR fragment) was evaluated.

Under low stringency conditions, probe 49f detects a large number of Y-specific fragments ( B15hop, C, et al.: rlMol-Biol,, 173: 40 3-317.1984>. The sample is most strongly... Ipridized Y% different and old 7 fragments (2,0 and i, 8kb Taq construction or 2.8kl) Only EcoR engineering) was evaluated.

The DYZ1 repeat is a mixture of several cloned 3.4 kb Hae I [I repeat components It was used as a probe along with objects. The DYZI repeat is 3.4 kb under high stringency conditions. Y-specific ECOR effect or several discrete Y less than 0.6 kb It was detected as a specific Taq fragment.

The DYZ2 repeat consists of probe P1, a 2.1 kb H1ndlII repeat component. Subfragment (vergnaua, G, et al.: Br.

Mea, J, +289:73-76.1984) or probe pY 431-HlnfA (Bernheim, A, et al.: prOc.

Natl, Acad, Sci, [7SA, dan 0: 7571-7575°19 83). DYZ2 repeats are EC;OR and T under high stringency conditions. It was characterized as a high molecular weight Y-specific smear in aq-engineered digestion. Furthermore, Ta In the q-digestion, discrete Y-specific fragments were observed.

When DNA from a subject is hybridized with a Y-specific DNA probe, You can easily understand the result by referring to the figure. Figures 2 and 3 show typical Orton geogsim from hybridization experiments. Tani Zorobe used A clear Y-specific band could be recorded. 2nd various probes An example is shown in FIG. These probes are described in further detail in column 1. It will be revealed.

Figure 2 (left) shows autosomal bands, some of which are polymorphic, and multiple Y-specific bands. A probe (49f) for detecting a band gap is shown. Figure 2 (right side) shows the test Probe that detects multiple autosomal and X chromosome bands present in all individuals (12f) is shown. To reliably record the Y-specific 8kb Taq fragment Both were possible.

Figure 3 shows two proteins that detect car-copy sequences on the X and Y chromosomes, respectively. Showing robe. These are normal (46,XY) males! , normal (46,XX) female Hybridize to r-transfer of Taq-digested DNA from and XX males. tion. It is clear from the figure that 51c% each probe is an X chromosome fragment. and detect Y-specific fragments. Y-specific flag for probe 47a In the case of probe 47, it is 4.3 kb.

The tested individuals were recorded for the presence or absence of the restriction 7 fragment observed in normal males. However, restriction fragments observed in normal females were not recorded. Therefore, it was assumed that the restriction fragment was derived from the Y chromosome. detected The restriction fragments are summarized in Table 1 above. Some probes (e.g. 50f2 1118 and 52d), a large number of y% heterologous fragments were detected. This way For the probe, the presence or absence of specific Y-specific fragments shown in Table 1 was determined. Recorded for each individual. As shown in Table 1, Y-specific for XX and XX males No DNA sequence was detected, indicating Y(→XXXX male).

Other individuals are XX male (Y(→XXXX male).

xYq(→male) and t(Y;15) female are included.

Each of these individuals has one or more Yf¥! jExistence of different restriction fusigment presence was detected. However, among these individuals, all of the Y sequences tested There were no cases where this was detected. In studies of normal male controls and fathers of cases, Y-configuration All of the columns were present. Without exception, Taq or P detected in these individuals The COR restriction fragment was the same size as that seen in normal males. child The same was true for the fathers tested.

From this, we conclude that each of these cases represents a portion, but not the entire Y chromosome. Be it.

Normal control and case iC with chromosomal abnormality (using the described DNA probe) Y% heterogeneity in normal Y chromosome based on comparison of Y% heterogeneity fragments detected. A deletion map of target DNA sequences was constructed.

The obtained t′Lf data indicates that in each of these columns, a single contiguous portion of the Y chromosome This is consistent with the idea that there is a That is, the Y present in each patient tested The Y-specific sequences are arranged in such a way that they are disassembled without being cut in the middle of a single sequence.

In each individual with a cytogenetically detected abnormality, the results of chromosome banding studies The results are consistent with the existence of a single continuous segment of the Y chromosome. This kind of research It is not definitive, but is accurate enough to rule out the complex rearrangements of the Y chromosome. Hana (ゝ.

However, the pattern of Y-specific DNA sequences present in the tested individuals is It enables the creation of a consistent deletion map of the Y chromosome. This map shows the tested Y Each specific restriction fragment maps to one of the ten deletion gaps shown in the figure. Belong to. FIG. 1a is a deletion map showing the inversion polymorphism as described above.

In this map, the order of intervals 3B and 4A is reversed.

Previous experimental results showed that many and) However, it was suggested that the amount of Y chromosome material present is not constant (Gu ella*, G.

is crab 1 Jature, 307: 172-173.1984: Pag e, D, C0 are crab Nature, 315: 224-226゜19 85). In this study, this was the middle solution for 12 of the 19 XXjsfq tested. Two Y-specific DNA sequences were detected and expanded. All columns, observed Y-specific The length of the restriction fragment is identical to that of a normal female. 12 cases of Y (→XX XX Males can be classified into five groups according to the number of Y-specific fragments present. . The DNA sequences present in the first four classes increase in a stacking manner (see Figure 1). ). Class 1 It also does not have fl (defined as interval 1A in Figure 1). Class 1Bxxia is the same Y It has a specific fragment and another fragment (1B). class 2 Xx males have these same Y-specific fragments in addition to 161 other 7 lagomers. (defined as interval 2).

Class 6xx males have all Y% heterologous fragments in class 2 plus 8 more (defined as interval 3).

The Y chromosome deletion map was mapped to the arm region (Yp and and Yq) and centromeres are also possible.

Five males with microscopically visible deletion of the long arm of Y (the short arm is normal in appearance); One female case with translocation of q-heterochromatin to chromosome 15 was particularly affected by Y-specific regulation. The presence or absence of limited fragments was recorded. Sterile 46°xYq-males are DYZl and and DYZ2 repeats as well as certain single copy Y sequences. These 46° xYq-masculinities can be divided into two classes depending on the number of single-copy Y sequences they lack. Wear. One example (OHMol B) is probe 12f (specific for interval 5 in Figure 1). 9 (therefore, the presence of the detected 8 kb Taq fragment Q4. EIL; LL92). Normal male tgl with non-fluorescent Y chromosome t <aHIA007) is specific for DYZl and DYZ2 repeat c interval 7) only case OHM 01 due to the presence of several single-copy sequences (specific to interval B) Different from 8. Conversely, Yq with the quinacrine-emitting distal portion translocated to chromosome 15 46. xx.

der(15), t(y:15)(q12.pii) Females are DYZl and DY Z2 repeats (interval 7), but the Y-specific single copy sequences listed in Table 1 (interval 1 ~6) I don't have it. However, this female has one other Y-specific single female. A column was detected.

These Y9 deletions assign intervals 5, 6, and 7 to the long arm of the Y chromosome. (Figure 1).

In other words, the above deletion serves to establish a deletion map regarding the long and short arms of the Y chromosome. stand. These Y, chromosomes are simple deletions (e.g. not transposition products) Assuming that, all have a Y centromere.

On the other hand, the Y chromosome was not detected cytogenetically (→XXXX male @ original body) It's hard to imagine it being a motsu. The Y centromere is found in interval 4B. Interval 4B is XYq( → All male sex exists, but xxt! & Notes are all absent (Figure 1).

Interval 4 is also the Y gene detected with probe: [)DP 34 (DXYSl) Contains loci. This locus was identified by inθitu hybridization as Y p (short arm of the Y chromosome). Therefore, interval 4 is one part of the short arm of the Y chromosome. It is thought to include the centromere as well as the centromere. Interval 4A was assigned to Yp. Of course , intervals 1 to 3 are completely in the Yp circle. These conclusions indicate that the Yq-chromosome It is based on the assumption that the rearrangement in is a simple deletion that affects only Yq. .

The morphology of each Yq (→ chromosome is compatible with such a deletion, but based on cytogenetic studies In the end, complex rearrangements cannot be eliminated. If this is reversed or transposed In the unlikely event that the above is involved, the above conclusion will need to be revised.

Y (+) x The Y-specific DNA sequence present in males was assigned to Yp. These are the sequences in intervals 1-3. Class 4Y (→XX'ts Note also indicates the arrangement of 4 people at intervals. do. All y (+) XXjs notes (and XYq- male also have It has a distance of 1A. XX males have testes due to the presence of the male sex-determining part of the Y chromosome Then, the male determiner will be located at the interval IAK. This male determiner Yp The assignment to is consistent with most punch-out phenotype correlations (Davis, R.M. : J, Med, Genet, 13: 161-1 75.1 981) .

The work described herein has provided considerable information on the organization of DNA sequences within the Y chromosome. give. Under the hybridization conditions used, probe 50f2°52 d and 118 detect multiple Y-specific restriction fragments. These pros The fragments detected in each strip are all clustered within a single interval. isn't it.

In class 6xX males, bands 52 dlo and 52 d/B are equal in strength. stomach. In a normal female (or XYq-male), 52d10 is stronger than 52d/B. Ba 52 dlo is one copy found in interval 3 and one in interval 4 if It is concluded that this is at least a doublet consisting of two or more copies.

Probe 118 detects many Y-specific Taq fragments. record securely Of the four possible Y-specific fragments, six (118/A, B, O, 1 interval 6) , one (118/D) is assigned to interval B.

By the way, the probes 50f2 and 52d are spaced at intervals of 3 (on Yp), 4 and 52d, respectively. and 6 (Y, top). Probe 118 is located at intervals 3 and 6 (and Detects arrays of minutes and more). These Yp-Yq homologies are an evolutionary process This can be considered to be the result of one or more duplications of parts of the Y chromosome in .

A group of highly homologous DNA sequences has also been found on the mouse Y chromosome (L amar, T2. E, & Pa1+ner, E,: Ce1l, 37: 171-177, 1984: B15hop, c.

E. et al.: Nature, 315 Near 0-72.1985).

Normal female, xxts, two rows of XYq-male and Ta9 engineer from normal male Hybridization of two labeled probes to the del transfer of digested DNA I turned it on.

Figure 2 is an autoradiogram showing the results of these hybridizations. Ru. On the left side of Figure 2, “2p labeled blow-)” A9f is added to Del Transfer. The results of hybridization are shown below. 49f is at the end of these individuals. Detect the two autosomal bands present. 49f also tested normal female and part of the XYq-male of the two cases shown in Table 1 above (Example 26/CHJJOO The 2,0 kb y-specific fragment in 7) is also detected. 49f is further Several types of Y-specific fragments can be detected. XY, (→male 26 (OHM 0 07) with normal females (see Table 1), it is clear that these flags Hens are plesiomorphic in size and presence. These cases and Table 1 Non-polymorphic note Y% different for the other individuals listed 2. Record the presence or absence of Okb fragments (indicated by the arrow in the figure on the left). Probe 12f uses the same TaqIr transfer. hybridized with. 12f is the majority that exists in all of these individuals detect autosomal and X chromosome fragments. Furthermore, 12f is several types of Y Detect specific fragments. Individuals are recorded for the presence or absence of 8kb fragments. (as shown by the arrow on the right side of Figure 2).

Example 3 Hybridization of probes 47a and 47z to xx male NA Two types of 32pi zolobes, 47a and 472, were isolated from normal females, normal females and Deltrans of Taq digested DNA from X7s Note 10 (OONlol) Hybridized with fur. Figure 3 shows these hybridizations. This is an autoradiogram showing the results of the analysis. Hybridization with 47a The results are shown in the left panel of Figure 3. 47a is an 8kb X chromosome fragment and 3 Y% heterologous fragment of kb was detected (indicated by arrow "f" in Figure 6). The hybridization results are shown in the figure on the right. 47z is 1.5 kb and 2. Detecting a 1 kb X chromosome fragment and a 4.3 kb X chromosome fragment (indicated by the arrow in Figure 3).

Regarding the presence or absence of this 3kb fragment and 4.3kl) fragment, The body was recorded. The result is an extremely rare 45.5% as discussed above in Table 1 above. x pieces The body is sterile and male with testes. The genetic basis of masculinization is chromosome banding cannot be determined using research studies.

In this example, X-linked restriction fragment long flame form a (RFLP) and/or Y-specific restriction fragments in rtum DNA from two rows of 45X males and their relatives. A DNA hybridization probe for detection will be described below.

subject 45 in two cases from two other families. X males and their dams and sire were tested. 45. X male Lymphocyte and skin fibroblast cell division studies revealed that both parents were The patient was cytogenetically normal. My father is 46.゛XY, mother is 46. It's XX Ta. We have repeatedly considered professional bands. Blood cultures between 1971 and 1979 tested four times and one fibroblast culture showed only 45,x mitosis. . Blood cultures in 1979 revealed several hundred mitotic and 1,000 interphase nuclei. We screened the X chromosome or Y chromatin using quinacrine fluorescence, but none Neither was recognized.

In 1971, 15/1000 cells of the oral mucosa showed fluorescent spots; The possibility of an X chromosome was considered. In 1977, approximately 5% of oral mucosal cells The Y-stained room appeared to be positive.

In a 1982 study of skin fibroblasts, the majority of cells were mitotic (179/186). is 45. x, but 5/186 cells were G-banded by trypsin. (GTG) and Q-banding (QFQ) by quinacrine fluorescence revealed 4 6. XY was recognized.

QFQ banding shows the bright fluorescent band Y912 and the same length as in the father's case. The length of the X chromosome was longer than the average length), indicating that the X chromosome was structurally normal. Sara 34/1000 of the interphase nuclei from the same culture were Y-stained, corresponding in size to an X chromosome. It has a chamber body. A frozen sample of the same fibroblast culture was thawed in 1984. , chromosome tests were repeated. During a total of 434 mitoses examined with quinacrine fluorescence, 5 cases were 46. One child has an XY karyotype, all others have less than 46 chromosomes, and the X chromosome is I was not able to admit. In addition, 8/1000 interphase nuclei gave birth to Y-chromosomal bodies. X staining The DNA used to detect body-specific DNA sequences was from these 1982 and 198 Prepared from 4 years of fibroblast culture.

patient 2 Both parents and older brother are cytogenetically normal, and the father and older brother are 46. XY, mother Parents are 46. It was XX.

The proband received 4 blood cultures (2 times) and skin fibroblast cultures (3 times). 5. Only X cells were detected. In addition, from blood culture in 1975, 1.00 Open phase nucleus of 0, nucleus of 1.000 from oral mucosal force, blood culture performed in 1979 1,000 mitoses and i,oo.

Screening of open phase nuclei with quinacrine fluorescence reveals Y chromatin-! or presence of X chromosome I checked the location, but couldn't find anything. In other words, this patient has an X chromosome. No results were obtained that would indicate its existence.

DNA research Restriction eradication of human starch DNA obtained from peripheral leukocytes or cultured skin serum fibroblasts synthesis, agarose electrophoresis-del transfer, and radiolabeled cloned DNA 1. Performing hybridization with Azorobe. DNA hybrid used X-linked restriction fragment length polymorphism (RFLP) and/or or Y-specific restriction fragments.

45 in 2 cases. To investigate the parental origin of a single X chromosome in x-masculinity, both families were classified into eight X-linked RFLPs. These X-linked RFLPs happened to be the probands of the X chromosome only if it is a living combination of alleles present in only one parent. It will give information about the origin. The probes used for this purpose are: It is a cage.

1) RO8 is X-linked, with Taq alleles of 3.0, 3.4 and 5.7k Detect b fragment.

2) D2 contains the 6.0 and 6.6 kb X-linked allelic pvun fragments. Detect.

3) L 1.28 is 10 and 12kb0) X-linked allele Taq efrag Detecting ment.

4) pDP34 contains 11 and 12;irb X-linked alleles Taq effragment Detection of Y-specific 15kb72 protein and Y-specific 15kb72 protein.

5) 19-2 is 4.4 and 12 kb (7, IX-linked allele Map effrag Detecting ment.

6) S21 is a 2.5 and 2.7 kb X-linked allele Taq fragment Detect.

7) 22-33 is the X-linked allele Taq efrag of 10 and i7kl) Detecting ment.

8) 46-±5 contains the 6 and 9'ibX-linked allele BglI [fragment Detect.

The results are shown in Table 2 below.

In both cases, it is clear that the proband's single X chromosome is of maternal origin. was made into Many Y% heterologous sequences for 45°X males and their relatives at 21 pH To test for the presence of A probe was used. The probes leached for this purpose are:

9) 47c is 3 and 4,3 kb o) y% different Taq I 77 segment Detect.

Y0) 115 is either 2.1 or 2.6 kb Y'#different Taq efrag Detecting ment.

11) 50f2 is ICOR engineered or L is Taq engineered to detect multiple Y-specific loci. know

12) ECOR 52d detects multiple Y-specific loci by Taq digestion do.

13) 1.8kbPet efrag produced from plasmid 71-71 by '1'R ment detects multiple Y-specific Taq fragments.

14) pY 431-HlnfA is a 2.1 kb highly repetitive Y-specific Hae Detect DI fragment.

15) px 3-4 is a 3.4 kb highly repetitive Y-specific gg aae m fl detect the component.

Probe 47 (!, 115.50f2.52’i, ppDP 34 and 7l -7A detects a single copy o)y-specific sequence. Three of these (50 f2.52d and 7l-7A) detect multiple Y-specific restriction fragments . Probe pY 431-HinfA and p'y3.4 are Y-specific repeat sequences Detect. The results are shown in Table 3 below.

Both families are normal at 46. XY males (father and siblings) compared to unrelated control males. The number of Y-specific sequences produced is shown. The mother does not exhibit these Y-specific sequences. stomach.

Any 45. No single copy Y-specific sequences tested were detected in X males either. won. In patient 1, probes pY431-HlnfA and py3.4 Highly homologous repeats, Y-specific, 2.1 kb and 3.4 kb Hae DI 7. A fragment was detected. However, these Y'l'T-heterorepeat sequences The abdomen of the column was significantly lower than that detected in patient 1's father or an unrelated control male. In order to stabilize the loss of these repeat sequences, high Hybridization Shiri 2.1 J: Hi 3.4 kbuae nt fragment The strength is determined by the amount of decrease in paternal DNA (10 times, 100 times, 1.000 times and and 10.000 times). A5+xtS 2, i kb Hae I [[band intensity decreased 100 times The intensity was somewhat higher than that observed for the amount of paternal DNA (Figure 2). This 45° A similar decrease in the intensity of the 3.4 kb Hasm band was observed in males. . Both the Y-specific 2.1 kb and 3.4 kb Hae m fragments are It is approximately 1-3% of the amount present in the father. In patient 2, these Y-specific repeat sequences were , if normal male DNA is reduced by io or ooo times ('f, that is, normal Y staining Even under conditions where it can be detected (even if the body is present in only one out of io, ooo cells), I couldn't find even a trace of it.

However, these repeat HaQ III fragments are not unique. However, it is mainly present in distal Yq, and therefore an abnormal X chromosome lacking that region It can also be said that it is not useful for detecting the mosaic involved. DNA hybridization The grade of the structurally abnormal X chromosome in patient 2 was determined from the analysis alone. You can't argue with low mosaics. Similarly, the quinacrine-emitting distal portion of Yq Nor can such mosaicism be addressed using cytogenetic methods based on the detection of .

Case 1 has Turner features. Her gonads were removed when she was 4 years old, but her glands remained dense. The ovarian stroma consisted of a stroma, and no primitive follicles or testicular tissue was observed.

Dermal fibroblast cultures established from this patient used two different constant fjk methods. Repeated testing revealed borderline positivity for H-Y antigen.

Case 2 showed some features of Turner syndrome.

She had congenital lymphedema, primary amenorrhea, and developed gonadoblastoma. set of gonads Histological examination showed a gonadoblastoma and striae without primordial follicles. this patient Gonadal fibroblast cultures established from 2000 were positive for H-Y antigen.

Cytogenetic analysis included peripheral blood samples from cases 1 and 2, skin samples from case 1; Fibroblast culture from the specimen and the gonadal biopsy specimen of case 2 were carried out. .

Prometaphase cells are labeled with G-banding, R-banding, C-banding and Stained by Q-banding. Small deletion in the short arm of the X chromosome (46, X, d el(Y)(pll)] was identified in both patients. The deletion is slightly on the metaphase chromosome. was detected. Whether this deletion is intermediate or terminal and differs between the two patients. It was not possible to determine whether there were any. The long arm of the X chromosome in both patients was of normal size. The size encompassed an area of average size Q-dominant heterochromatin. each patient's father's The X chromosome was normal. No other abnormality of the cutting die was found in any patient, and the analysis Nor could they find any evidence of mosaicism in the tissues that had been used. In case 1, blood and 105 and 101 cells were calibrated for each skin sample and case 2. 120 cells for blood and 54 cells for bilateral gonad samples. and 58 cells were calibrated.

DNA research Blood samples and fibroblasts from both patients, the father of case 2, and normal male and female controls. DIJA was prepared from cell culture. This DNA is digested with restriction endonuclease Ta. q engineering (for probe pDP 34) or ECOR engineering (probe 50f2. 52d, 47b, 113. About p12f2 and p12f3] did. DNA fragments separated by agarose gel electrophoresis are passed through a membrane filter. and hybridization was performed using the Sudden plot method. Map to the X chromosome Plug the 7 types of 32p-labeled DNA probes into patient and control rzum DNA plots. (see 1 and Example 1 above). Probe pDP 34 (D Detect columns. Probes 50f2 and 52 (l on and on the X chromosome Hybridization is performed with the DNA sequences of both autosomes. probe 47 b detects X, Y and autosomal sequences. Probe 118 is exclusively Y-specific It is. Furthermore, the two probes p12f2 and p12f3 are on the long arm of the X chromosome. located above. The results of these analyzes can be summarized as follows.

1) Based on the hybridization of probe pDP 34, patient 1 is normal male. DXYS1, which shows a characteristic hybridization pattern, is deleted. patient 2 had 15kb17)Y¥i free bond (i.e. It was later revealed that Y-specific DNA homologous to DXYS1 was deleted. Ta.

2) Hybridization of probes 118, 52d, 50f2 and 47b From the results, patient 1 lacks 6 male-specific bands for all 4 probes. (confirmation of cytogenetic data showing deletion of part of the Y chromosome), patient 2 probe 50f2 and Y-specific band at probe 47b (the latter It was revealed that the band was the same as the missing band of 1.

3) Hive of probes p12f2 and p12f3 (present on the long arm of the Y chromosome) On redization, both patients showed a normal male hybridization pattern. Shi1ko.

Hybridization of probe pDP 34 to patient and control DNA The results were compared. Case 1 is characteristic of normal males with bands at 11 and 15 kb. DXYSl was deleted in this case. It was suggested that it is not. In case 2, however, the band at 15 kb Y-specific DNA homologous to DXYSl was deleted in this patient. was suggested. The father of the latter patient had normal X at 15 and 1'lkb, respectively. and Y-specific band. 32F labeled probe 1 i 8.52d, 50f2 and 47'b, normal female , normal male, father of case 1, case 2 and ECOR engineered D from case gAJ2 Hybridization to LJA was also detected. Case 1 is 4 A total of 6 males for species probe i 1 B, 52d, 50f2 and 47b It showed the deletion of specific bands. This means that case 1 has a deletion of part of the Y chromosome. This confirmed the cytogenetic data.

Probe 1 i 8, 52d and 50 f21! , compared to normal males, Example 1 showed several male-specific bands that were clearly not deleted. child These three types of probes exist in regions of the Y chromosome other than those deleted in case 1. It is suggested that the present sequence is recognized.

In case 2, probes 118 and 52 (no disappearance of the Y-specific band were observed for probes 118 and 52 (1). One thing that didn't happen. With probe 50f2, both the father of case 2 and case 2 had the polymorphism. It lacked a likely Y-specific band (indicated by a small arrow). P In Loeb 471), Case 2 is corrected by mentioning Case 2's father, which Case 1 lacks. It was revealed that the Y-specific band, which is present in permanent male species, is lacking. moreover Two types of probes, p12f2 and pl2f3, located on the long arm of the Y chromosome, In a study, patients 1 and 2 both showed normal male hybridization patterns. Indicated.

Table 4 shows hybridizations showing that cases 1 and 2 have different deletions. The data was compiled. The only band these patients commonly lack is the probe. Bu 47b and phase l? 5] It is a gender band. This probe is a 46,xx male patient. The most frequently encountered probes (470 Kl!4 in contact with It is likely that one or both of the deletions exist in the middle, and the deletion at position 47b It seems to be on either side.

Two rows of patients have different but overlapping deletions. This is Turner syndrome in both cases. Although it shows the characteristics of a group, it is thought to be able to explain that it differs in certain respects. Case 1 is short Case 2 has a normal body height. This is a height-regulating gene on the short arm of the Y chromosome. This indicates the presence of the gene, which is deleted in case 1 but is present in case 2. Exists. Furthermore, case 2 developed gonadal myeloma, but case 1 did not. . However, in case 1, the gonads were removed at an early stage. MY antigen is clearly case 2 The short arm of the Y chromosome, which is present only in the male sex and affects HY antigen expression independently of male differentiation The absence of another area is suggested. The independence of HY antigen and male differentiation has recently been reported in mice. I was told. 46. Whether the HY antigen is positive or negative in the XY gonadal hypoplasia column is not constant. do not have. It seems that there are several causes for this article (Page, D, O, et al.: Nature, 311: 119-123.1984).

As mentioned above, the use of DNA probes that are homologous regions on the short or long arm of the Y chromosome is , a small deletion in the short arm of the Y chromosome and an abnormality of the injected envelope 46. I solved the XYfi note. .

Although a small deletion on the short arm of the Y chromosome was detected, the probe used detected a deletion on the long arm. 1 item not detected. Although different, these deletions are clearly essential for male differentiation. The overlapping region (detected by probe 47kl) was included.

Additionally, the KHY antigen was clearly present only in patient 2, and is located in another region of the short arm of the Y chromosome. It was suggested that its absence affects Y antigen expression (independent of male differentiation).

The results are summarized in Table 4 below.

Gender Female Female MY antigen Gonads, streaks, Qin marks gonadoblastoma a: For each probe, (+-) indicates the presence of a Y-specific band, and (-) indicates the band. Indicates the absence of For DXYSI, the band was obtained by Taq 1 digestion of DNA; Other probes were generated using the Iteha DNO EcoR digestion.

b = This polymorphic pattern was also found in the father of case 2 and is a sequence that exists in the normal Y chromosome. DNA probe that detects DNA sequences homologous to the sequence and YDN in individuals The method of using the above DNA to evaluate the presence or absence of A is applicable to clinical and other medical fields. can be used to diagnose and evaluate many putative functions. For example, chromosomal abnormalities determining the genetic basis of phenotypic abnormalities, determining genetic disorders and their associated Those skilled in the art will be able to use the features described herein to diagnose the effects involved. Equivalents to the foot embodiment are easily recognized and more than routine experiments. It is clear that this can be confirmed by experiments that do not. Such an equivalent is It is a matter of course that it is included in the scope of the claims.

FIG.I FIG.lA 49f 12f XX XX XYQ-XYrXY XX XX xycr xyq-xycas a211225212121+2252@2947a 47z xy xx XX XY XX XX 1.5J-One verse! ! W--- 0 interval 29 2B+0 29 28 TOFIG, 5 Procedural amendment (Mutsu) November 16, 1985

Claims (1)

[Claims] (1) A single species that hybridizes to at least a portion of one of the Y chromosome intervals in Figure 1. separation DNA (2) Claim No. 1 that hybridizes to the Y chromosome interval in which the testis-determining factor exists. Isolated DNA according to claim 1 (3) The unit according to claim 2 having a detectable label separation DNA (4) Isolated DNA sequence that hybridizes to the short arm of the normal human Y chromosome (5) Isolated DNA sequence that hybridizes to the long arm of the normal human Y chromosome (6) Isolated DNA that hybridizes to the region of the normal human Y chromosome necessary for spermatogenesis A 7) The isolation according to claim 6, which hybridizes to the long arm of a normal human Y chromosome. DNA (8) Isolated DNA sequences that are Y-linked restriction fragment length polymorphisms (9) Isolated DN that detects pseudoautosomal loci common to the X and Y chromosomes A array (10) DN that hybridizes to a sequence homologous to a sequence present on the normal Y chromosome A method for detecting Y-specific DNA in human sex chromosomes improved in that it uses A ( 11) Is the DNA hybridizing to the D envelope A sequence homologous to the normal Y chromosome sequence? Probes for detecting human sex chromosome abnormalities (12) The number of probes in each interval in the deletion map of the normal Y chromosome shown in Figure 1 The DNA probe (13) according to claim 11, comprising at least one each ) A probe that detects the presence or absence of a DNA sequence homologous to the DNA sequence of a normal chromosome. Is the vector carrying a restriction fragment of a normal chromosome or an equivalent sequence? probe (14) with a 0.9 kb HindIII fragment of the Y chromosome or equivalent sequence. A vector that is a plasmid with a certain restriction fragment inserted into the HindIII site The probe according to claim 12, consisting of (15) with a 3.5 kb HindIII fragment of the Y chromosome or equivalent sequence. A vector that is a plasmid with a certain restriction fragment inserted into the HindIII site The poob according to claim 12 consisting of (16) 1.0 kb EcoRI/TaqI fragment of Y chromosome or equivalent sequence Plasmid with a restriction fragment inserted into the AccI and EcoRI sites of the program. Probe (17)Y according to claim 12, which is a vector consisting of Sumid. The restriction fragment, which is a 4.6 kb HindIII restriction fragment of the chromosome or equivalent sequence. A vector consisting of a plasmid inserted into the restriction fragment HindIII site. The probe according to claim 12 (18) (a) DNA present in the sample is available for hybridization (b) transfer the DNA in the sample to hybridization; DNA that hybridizes with the Y chromosome under suitable conditions to cause lysis. (c) DNA in the sample hybridizes with Y chromosome DNA. in a sample consisting of each step of measuring hybridization with a DNA sequence. Method for detecting the presence of Y-specific DNA (19) Hybridization with Y chromosome DNA 19. The method of claim 18, wherein the DNA sequence has a detectable label. (20) DNA sequences that hybridize with Y chromosome DNA can be radioactively or chemically The method according to claim 19, which is labeled as (21) (a) making the DNA in the sample available for hybridization; (b) Hybridize the DNA in (a) with the DNA in the sample and the DNA probe. Under conditions appropriate to the deletion, at least one of the deletion maps of the normal human Y chromosome (c) in the sample; From each step of detecting hybridization between DNA and DNA probe. A method for detecting the presence of a DNA sequence homologous to a sequence in a normal Y chromosome (22) The method according to claim 21, wherein the DNA probe is labeled. (23) DNA probes are radioactively, enzymatically, or chemically labeled. Method according to claim 22 (24) DNA from the individual was added to each of the deletion maps of the normal human Y chromosome shown in Figure 1. Consisting of a set of spaced DNA probes, the DNA sequence of the normal human Y chromosome and hybrid DHA hybridization probe, which is a probe made of soybean DNA (b) and one set of b ) Each method detects hybridization between DNA from an individual and a DNA probe. A method for detecting the presence of normal human Y chromosome DNA in an individual consisting of a process (25) DHA probes are PDP307, pDP132, PDP61, pDP1 Group consisting of 25,50f2, pDP34,12f and pY431-HinfA The method according to claim 24 selected from (26) (a) DNA from human sex chromosomes, DNA encoding testis-determining factors DNA hybridization probe, which is a sequence, and hybridization probe (b) bring the DNA from the sex chromosomes into contact with the DNA under suitable conditions for Each step of detecting hybridization with a hybridization probe Detection of the presence of DNA sequences encoding testis-determining factors in human sex chromosomes How to get out (27) A normal human Y chromosome, at least one of the Y chromosome intervals shown in Figure 1. The point of using a DNA hybridization probe that hybridizes to the Improved evaluation method for sex chromosome abnormalities (28) 0.9 kb Hind III fragment from normal Y chromosome DNA The human probe was subcloned into the HindIII site of pUC-13. DNA hybridization for detecting testis-determining factors in Y chromosome DNA Detailed description of Yonprobe invention