WO1989002440A2

WO1989002440A2 - Y-specific dna hybridization probes and uses therefor

Info

Publication number: WO1989002440A2
Application number: PCT/US1988/003255
Authority: WO
Inventors: David C. Page
Original assignee: Whitehead Institute For Biomedical Research
Priority date: 1987-09-21
Filing date: 1988-09-21
Publication date: 1989-03-23
Also published as: WO1989002440A3

Abstract

Y-specific DNA which can be used as probes to establish unambiguously the presence or absence of regions of the normal Y chromosome in DNA from a subject is disclosed, as is a method for the use of such probes. The Y DNA sequences can be used to determine whether homologous sequences are present in an individual's genome or not; to detect the presence or absence of specific portions of the Y chromosome in genomic DNA; and/or to determine the location of genes of interest on the Y chromosome.

Description

Y-SPECIFIC DNA HYBRIDIZATION PROBES AND USES THEREFOR

Description

Technical Field This invention is in the field of genetics and in particular relates to the analysis of genomes for the presence or absence of Y-specific DNA, particularly the testis-determining factor.

Background in mammals, the primary sex-determining signal is the Y chromosome. Regardless of the number of X chromosomes per cell, mammalian embryos with a Y chromosome develop testes and those without a Y chromosome develop ovaries. XY, XXY, XXXY and XXXY embryos develop testes and XO, XX, XXX and XXXX embryos develop ovaries. The embryonic testes or ovaries establish a male or female hormonal environment, which determines the remainder of the sex phenotype, including the sex of the internal accessory organs and external genitalia. Wilson, J.D. et al., In: The Metabolic Basis of Inherited Disease (5th ed.), New York. Thus, the entire sex phenotype -- male or female -- is determined at the beginning by a gene or genes on the Y chromosome. This Y-borne gene or gene complex is referred to as the testis-determining factor (TDF).

Many structural anomalies of the human Y chromosome have been detected by light microscopy of stained mitotic chromosomes. Inferences as to the regional location of the testis determinant ( s ) on the human Y chromosome have been drawn from correlations of these abnormal karyotypes with the sex phenotypes. Buhler, E.M., Human Genetics, 55: 145-175 (1980); Davis, R.M., Journal of Medical

Genetics, 18: 161-195 (1981). 'Generally, assessment of chromosomal structural anomalies has made use of chromosome banding techniques, in which characteristic horizontal (density) differences (or bands) are detectable after the chromosomes have been treated (e.g., by staining with quinacrine or Giemsa; by controlled heat denaturation). Vogel, F. and A.G. Motulsky, Human Genetics: Problems and Approaches, Springer-Verlag, pp. 23-31 (1982). Normal males have a 46,XY karyotype; that is, there is a total of 46 chromosomes , including one X and one Y sex chromosome. Normal females are 46,XX, indicating a total of 46 chromosomes, including two X sex chromosomes. There are numerous anomalies associated with Y chromosome abnormalities. For example, XX males are sterile males with a 46,XX karyotype and testicular but no ovarian tissue; that is, the total number of chromosomes is 46 and both sex chromosomes are X chromosomes. About 1 in 20,000 males is an XX male.

In addition, there are human XY females with an apparently normal 46,XY karyotype. Such gonadal dysgenesis females have female external genitalia, uterus, fallopian tubes and streak ovaries. Some XY females have features of the Turner syndrome, which is a condition usually associated with a 45,X chromosomal constitution. Many of the 46,XY females with the Turner phenotype are mosaic and have a 45,X cell line. There are also very rare 45,X individuals who are sterile males with testes.

The precision of chromosome banding techniques is limited; as a result, there is often considerable uncertainty as to the structure of abnormal Y chromosome's. For example, it has been hypothesized that XX males carry a small, male-determining portion of the Y chromosome, which cannot be detected by conventional chromosome banding techniques. It has been shown, through the use of cloned Y sequences as DNA hybridization probes, that some XX males do have Y-specific DNA and that these males are heterogeneous with respect to the amount of Y DNA in their genes. Guellaen, G. et al., Nature, 307: 172-173 (1984); Page, D.C. et al., Nature, 315:224-226 (1985). At this time, the testis determinant (s) has not been characterized and it is still unclear whether TDF maps to the short arm (Yp), the centromeric region, or long arm (Yq) of the Y chromosome.

The testis determinant(s) is not the only gene on the Y chromosome. However, genetic analysis of the mammalian Y chromosome has long been impeded by its haploid state. Unlike the other nuclear chromosomes, the Y has little opportunity to recombine with a homologue, making genetic linkage studies of the Y chromosome difficult, if not impossible. This at least partially accounts for the relative dearth of genes mapped to the Y chromosome in the mouse or human. Attempts to establish the Y-linkage of certain traits have been inconclusive because of the difficulty of distinguishing true Y-linked inheritance from sex-limited expression. Nonetheless, there is evidence for a number of genes on the Y chromosome in addition to the male determinant (s). For example, a structural gene for the antigen 12E7 has been shown to occur on the human Y chromosome. A structural or regulatory locus for the H-Y antigen probably maps to the Y chromosome in both the mouse and human. Two genes have been assigned to the weakly fluorescent proximal portion of Yq (band

Yq11): a gene affecting spermatogenesis and a gene affecting height and tooth size. Their exact locations are unknown, however. At the present time, there is no satisfactory way of identifying and mapping genes of interest on the Y chromosome; of detecting anomalies of the Y chromosome or of correlating the presence or absence of specific regions of the chromosome with the determination of gonadal sex or with effects on these and other phenotypes.

Disclosure of the Invention

The invention described herein is based on the determination of the location on the normal Y chromosome of a large number of segments of human Y-chromosomal DNA; localization of the male (testis) determinant ( s) on the Y chromosome; and construction of a physical or deletion map of the normal human Y chromosome. Genes located on the Y chromosome play an essential role in human male sexual development and the presence of the Y chromosome usually correlates with testis development. Conversely, Y chromosome abnormalities (e.g., deletions, rearrangements) are often associated with defects, such as sexual dysfunction and mental retardation. Using presently available techniques, such as light microscopic analysis of stained chromosomes, it is not possible to make a definitive determination of the presence or absence of specific segments of the

Y chromosome.

The deletion map of the human Y chromosome is composed of intervals which are defined by the portions of the Y chromosome shown, through hybridization with Y-DNA probes, to be present or absent in individuals with abnormal karyotypes or, as judged by cytogenetics, to have a structurally abnormal Y chromosome. The individuals whose genomic DNAs were tested included those with cytogenetically visible deletions of the long arm of the

Y chromosome (Yq) and an apparently intact short arm of the Y chromosome (Yp) and those with cytogenetically detectable deletions of Yp and an apparently intact Yq. The hybridization results from these samples serve to orient the deletion map with respect to the long and the short arms of the chromosome. The map serves as the basis for selection and cloning of Y-specific DNA sequences which are the subject of the present invention, as are methods for their use in analysis of sex chromosomal material. The map has great utility in clinical diagnosis and in the evaluation of any postulated Y function. With the map in hand, it is possible to systematically characterize structurally abnormal Y chromosomes by DNA hybridization, even in cases where no abnormality can be detected by currently available techniques (e.g., chromosome banding). For example, the testis determining factor(s) (TDF) is mapped to the short arm of the Y chromosome (Yp). Analysis of naturally-occurring deletions of the human Y chromosome has shown that a small region of the short arm of the Y chromosome contains this sex-determining function. That is, the TDF has been shown to map to the short arm (Yp) of the Y chromosome. It has been demonstrated that the presence of the region of the human Y chromosome designated interval 1A in the deletion map contains DNA sequences which are necessary and sufficient to induce testicular differentiation of the bipotential gonad, in the absence of downstream mutations. Interval 1A is divided into 1A1 and 1A2 and at least some of the DNA sequences necessary to determine maleness are in 1A2. That is, Y-speσific DNA present in interval 1A has been shown, in the absence of downstream mutations, to be necessary and sufficient to determine maleness. Y-specific DNA present in 1A2 has been shown to be necessary to determine maleness. By chromosome walking, interval 1A2, which represents approximately l/500th of the human Y chromosome (i.e., approximately 135 kilobase (kb) pairs in length), has been cloned and shown to contain some or all of the TDF, or the sex-determining function. TDF is by definition that portion of Y chromosomal material which is necessary and sufficient for determining maleness. Interval 1A2 extends from a deletion endpoint in a female with a reciprocal Y;22 translocation (associated with a deletion) to a Y-chromosomal breakpoint in a male carrying a small portion of the Y chromosome. A 1.2 kb HindIII fragment from the 135 kb region is highly conserved across evolutionary time and detects Y-specific DNA restriction fragments in all mammals tested. The nucleotide sequence of this fragment has been determined. In addition, CG-rich islands have been shown to occur within the 135 kb region. One CG-rich island occurs in a 3.0 kb SalI fragment which is located to the left of the 1.2 kb HindIII fragment; a second CG-rich island occurs in contiguous 4.6 and 2.8 Hind III fragments located to the right of the 1.2 kb HindIII fragment. All or a portion of the cloned Y-specific sequences can be used, according to the method of the present invention, as DNA probes to establish unambiguously the presence or absence of regions of the normal Y chromosome in DNA from an individual. Using the deletion map of the normal Y chromosome, Y chromosomes can be systematically characterized by DNA hybridization, even in cases where chromosome banding has been unable to demonstrate an abnormality. The cloned Y-DNA sequences are useful as hybridization probes to analyze structural characteristics of the Y chromosome; to determine whether Y-specific DNA is present in an individual's genome or not; to detect the presence or absence of specific portions of the Y chromosome in genomic DNA, as well as to determine the location of those portions or of the missing sequences on the Y DNA; and to diagnose genetic disorders and their related effects.

In particular, one or more of the Y hybridization probes is used to detect and characterize Y chromosome anomalies in individuals; to determine the occurrence in samples taken from individuals of DNA sequences specific to the short arm (Yp), the centromeric region or the long arm (Yq) of the Y chromosome; to detect the region of the Y chromosome which is male-determining, as well as the region determining the occurrence of spermatogenesis; and to detect other regions of interest (e.g., the region of the chromosome which apparently predisposes individuals with gonadal dysgenesis to gonadal neoplasms or the region responsible for expression of H-Y antigen).

All or a portion of the 135 kb-pair fragment is used to determine if the TDF is present or absent in DNA from an individual. For example, the 1.2 kb HindIII fragment, which detects Y-specific DNA restriction fragments in all mammals tested, is used to determine whether the TDF is present in sample DNA. Further, it can be used to determine the presence or absence of the evolutionarily homologous sex-determining gene in nonmammalian vertebrates.

The nucleotide sequence of the fragment, or of other portions of the Y chromosome, can be used to synthesize hybridization probes useful in analyzing chromosomal DNAs from subjects. DNA prepared from blood or other tissue from a subject is analyzed by hybridization with one or more of the Y-specific probes and, as a result, it is possible to establish the presence or absence of specific regions of the Y chromosome which are of interest in, for example, a clinical, diagnostic or agricultural context. It is also possible, using the predicted amino acid sequence of the encoded peptide, to make antipeptide antibodies to all or a portion of the 1.2kb HindIII fragment.

Brief Description of the Drawings Figure 1 is the deletion map of the normal human Y chromosome, constructed on the basis of DNA hybridization studies.

Figure 2 is a schematic representation of chromosome walking and of the location of the testis determining factor (s) (TDF).

Figure 3 is a schematic restriction map of portions of the human Y chromosome for the enzymes HindIII, SalI and EcoRI.

Figure 4 is the nucleotide sequence of the highly conserved 1.2 kb HindIII fragment from the 135 kb region of the Y chromosome.

Figure 5 is the amino acid sequence of the highly conserved 1.3 kb HindIII fragment from the 135 kb region of the Y chromosome. Figure 6 is an autoradiogram showing results of hybridization of the 1.2 kb HindIII fragment from the 135 kb interval which contains TDF with EcoRI-digested DNAs from the mammals indicated. Figure 7 is a deletion map in which an inversion polymorphism is represented. Figure 8 is an autoradiogram showing results of hybridization of two labeled DNA probes to a gel transfer of TaqI-digested DNAs from a normal female (case 28); an XX male (case 12); two XYq(-)males (cases 25 and 26) and a normal male (case 29).

Figure 9 is an autoradiogram showing results of hybridization of two labeled probes to gel transfers of TaqI-digested DNAs from a normal male (case 29); a normal female (case 28) and an XX male (case 10). Figure 10 is a representation of the genetic deletion map of the human Y chromosome.

Figure 11 is an autoradiogram showing results of hybridization of probe Y431-HinfA, which detects a Y-specific 2.1kb Hae III fragment, with Hae III-digested DNA from parents of a 45,X/46,XY male; a control female; and a 45,X/46,XY male.

Detailed Description of the Invention

Most of the Y chromosome, which is the only haploid human chromosome, does not participate in meiotic recombination. As a result, it is impossible to construct a genetic map of the Y chromosome on the basis of recombinational distances among markers. A wide variety of deletions of the Y chromosome do, however, occur naturally. Attempts have been made to infer the regional location of the testis determining factor(s) (TDF) on the human Y chromosome from correlations of these abnormal karyotypes with the sex phenotypes of individuals having the abnormal karyotypes. The precision of these chromosome-banding studies is very limited, however, and there is considerable uncertainty as to the structure of abnormal Y chromosomes. Prior to the work described herein, the location of TDF (i.e., of the portion(s) of the Y chromosome whose presence determines maleness) was unknown. In fact, until construction of the deletion map of the Y chromosome as described herein, it was not clear whether TDF maps to the short arm (Yp), the centromeric region or the long arm (Yq) of the Y chromosome. The deletion map is represented in Figure 1.

It has been determined that the TDF maps to the short arm (Yp) of the Y chromosome. Analysis of naturally-occurring deletions of the human Y chromosome has shown that this sex-determining function maps to a small region of the Y chromosome, represented in Figure 1 as interval 1A. The presence of this interval has been shown to be both necessary and sufficient for determining maleness in humans. Interval 1A is subdivided into intervals 1A1 and 1A2.

Chromosome walking of the portions of the Y chromosome extending from interval 1C through interval 1B and 1A2 and into interval 1A1 has been carried out. By chromosome walking, the interval designated 1A2 in Figure 1, which contains some or all of the TDF, has been cloned in its entirety. Analysis of DNA from an XX male carrying a small portion of the Y chromosome and DNA from a female carrying a portion of the Y chromosome (referred to as an X,t(Y;22) translocation female) has shown that a region which represents about 1/500th of the Y chromosome contains some or all of the TDF. In Figure 1, the XX male is represented by the top bar (i.e., that extending under headings 1A1 and 1A2) and the X,t(Y;22) female is represented by the bottom bar (i.e., that extending under all headings except 1A2 and 1B). The region which contains some or all of the TDF, designated 1A2 in Figure 1, is approximately 135 kilobase (kb) - pairs in size and extends from a deletion endpoint in the X,t(Y;22) translocation female to a Y-chromosomal breakpoint in the XX male who carries a small portion of the Y chromosome. This is represented in Figure 2 and Figure 3.

A HindIII fragment which is approximately 1.2 kb in size (the 1.2 kb HindIII fragment) of the 135 kb region (the 1A2 interval) shows a striking degree of evolutionary conservation (is highly conserved) and hybridizes to the Y chromosome (i.e., detects Y-specific restriction fragments) of all mammals tested. It has been shown to hybridize to Y chromosome DNA from gorilla, chimpanzee, orangutan, New World monkey, Old World monkey, mouse, rat, rabbit, goat, horse and cow (See Figure 6). It has also been shown to hybridize to homologous sequences in DNA from chicken. It is also highly likely that it detects similar DNA sequences in other nonmammalian vertebrates.

The nucleotide sequence of this approximately 1.2 kb open reading frame has been determined (Figures 4 and 5) and compared with previously determined protein sequences. In Figure 5, the predicted protein is aligned to show 13 repeats of 28 to 32 amino acid residues each. The numbers at the very bottom indicate their portion in the repeat unit. Asterisks denote the stop codons that bound the open reading frame. There is a repetitive structure in the putative Y protein which is remarkably similar to the cysteine- and histidinerich "zinc finger" domains that are found in frog TFIIIA (Miller, J. et al., EMBO J., 41: 1609-1614 (1985)), human transcription factor Spl (Kadonaga, J.T. et al., Cell, 51: 1079-1090 (1987)) among others. See Page, D.C. et al., Cell, 51: 1091-1104 (1987) and references cited therein. It has been postulated that, in each finger domain, a pair of cysteines and a pair of histidines are arranged about a central zinc ion. (Miller et al. supra). Such Cys-Cys/His-His finger domains bind to nucleic acids of TFIIIA and Spl in a sequence-specific manner (Sakonju S. et al., Cell, 23: 665-669 (1980); Kadonaga et al. 1987 supra). Metal-binding finger domains appear to be characteristic of a host of nucleic acid binding proteins and, by analogy, the presence of multiple Cys-Cys/His-His finger domains in the 1.2 kb HindIII fragment strongly suggests that it binds to DNA or RNA is a sequence-specific manner. Lysates of three recombinant lambda phages, which each carry a human genomic DNA insert, were deposited at the American Type Culture Collection on September 3, 1987. Each of the phages carries human genomic DNA from the 1.2 kb HindIII fragment: Lambda OX82 carries a human genomic DNA insert of about 19 kb in vector lambda 2010, which is a derivative of lambda 2001, and is deposited under ATCC No. 40367. Lambda OX95 carries a human genomic DNA insert of about 18 kb in vector lambda 2010 and is deposited under ATCC No. 40368. Lambda OX107 carries a human genomic DNA insert of about 17 kb in vector lambda 2010 and is deposited under ATCC No. 40369. The location of each of these phages is represented in Figures 2 and 3. The nucleotide sequence of the entire 1.2 kb HindIII fragment has been determined and is represented in Figure 4. Two fragments of interval 1A2, represented in

Figure 3, are CpG-rich islands. One, which is a 3.5 kb SalI fragment, is located to the left of the 1.2 kb HindIII fragment; a second CG-rich island occurs in contiguous 4.6 and 2.8 HindIII fragments located to the right of the 1.2 kb HindIII fragment. As represented in Figure 2, the highly conserved 1.2kb HindIII fragment occurs at a location designated approximately -70; the left-hand CG-rich island occurs at a location designated approximately -115; and the right-hand CG-rich island occurs at a location designated approximately -50.

Many vertebrate genes are known to be preceded by a region of DNA which is rich in the non-methylated dinucleotide CpG and contains clusters of sites for CpG-methylation sensitive restriction enzymes. Bird, A. P., Nature, 321:209-213 (1986); Estivill, X. et al., Nature, 326:840-845 (1987). These sequences, known as CpG-rich or HTF islands (HpaII tiny fragments), generally have a sequence length of approximately 500-2,000 bp. Many genes have been shown to have HTF-like sequences (i.e., CpG-rich islands) at the region of the gene where transcription begins. The sequence often includes the first exons and the upstream sequences 5' (and sometimes 3') to the associated coding gene. It has been predicted that most mammalian genes will be shown to be associated with CpG-rich islands and such islands can be used as one set of signals marking coding sequences. It is likely that the two CpG-rich islands identified (as well as others present in interval 1A, but as yet not pinpointed) are associated with regions of the Y chromosome which contain coding sequences. For example, the CpG-rich islands might be associated with exons of a single "maleness-determining" gene or, alternatively, each might be associated with one of several "maleness-determining" genes.

In the former case, in which the "maleness-determining" gene is an interrupted gene, the DNA of the gene is made up of exons (regions represented in the corresponding messenger RNA

(mRNA) which is translated to produce the encoded protein) and introns (regions not present in the messenger RNA). Although an RNA copy which accurately represents the genome sequence is initially produced, this precursor RNA cannot be used for protein production. Introns must first be removed and the exons joined or spliced, resulting in the functional mRNA which is translated. If this is the case, the two CpG-rich islands would be associated with one of the two (or possibly more) exons which are represented in the functional (spliced) mRNA. The present invention relates to Y-chromosomal DNA sequences which occur on the normal Y chromosome and can be used as hybridization probes in the analysis of DNA (i.e., sex chromosomes), prepared from blood or other tissue, for the occurrence of homologous DNA sequences. Through their use, the presence or absence of Y DNA (i.e., DNA which occurs in normal Y chromosomes) in an individual can be determined unambiguously and the location of DNA sequences of interest on the Y chromosome can be determined. Detection of homologous sequences on the X-chromosome can also be carried out through their use as probes.

There appears to be a region on the human X chromosome which corresponds to that encoding the zinc finger region of the Y-encoded protein. (Page, D.C. et al. supra). Sequences of 1A2 detect a highly conserved locus on the X and Y chromosome of a wide variety of placental mammals (Page, D.C. et al. 1987, supra; See Example VII)). The sequences of the two gene regions predict extremely similar products. The zinc finger domain constitutes the carboxy terminal portion of the X-encoded and of the Y-encoded protein. The sequence of the carboxy-terminal 400 amino acids of both has shown that the X-encoded and the Y-encoded proteins differ by only nine amino acids, eight of which are very conservative.

For example, using Y-specific DNA probes of the present invention, the occurrence of the malenessdetermining region, or TDF, of the Y chromosome, the centromere, and other regions of the sex chromosome which are of interest in a diagnostic or clinical context (e.g., the region thought to predispose to gonadal neoplasms in individuals with gonadal dysgenesis; the region which affects spermatogenesis, the region responsible for H-Y antigen expression) can be detected.

In particular, the probes are used to determine the presence or absence of the TDF on DNA from an individual. The 135 kb fragment, or a portion thereof, is used as a probe to detect, in sample DNA, homologous Y-specific sequences. The 1.2 kb HindIII fragment hybridizes to the Y chromosome of all mammals assessed and all or a portion of it is thus useful in detecting the occurrence of Y-specific DNA in humans and other mammals. Thus, it is useful, for example, in determining the sex of any mammal (human or nonhuman). This is done, for example, by analyzing DNA obtained from a small number of cells from an embryo. In addition, all or a portion of the 1.2 kb fragment can be used to determine whether homologous sequences are present in DNA from nonmammalian vertebrates, such as chicken and fish.

Such Y-specific probes are also used to analyze a set of chromosomes which appears cytogenetically normal. For example, a chromosome which appears, on the basis of cytogenetic analysis, to be a normal X chromosome, can be analyzed for the presence of homologous sequences, such as TDF. In addition, the Y-specific DNA probes can be used prenatally to assess sex chromosome structure; detection and identification of Y chromosome anomalies is thus possible.

The amino acid sequence predicted from the nucleotide sequence of the 1.2 kb HindIII fragment can be used to produce peptides which can, in turn, be used to produce antipeptide antibodies. For example, a peptide whose amino acid sequence corresponds to all or a portion of the predicted amino acid sequence can be synthesized mechanically or, using known genetic engineering techiques, can be produced by introducing the nucleotide sequence which encodes the desired peptide into a suitable host, in which the peptide will be produced; the resulting peptide is then isolated from the host, again using known techniques. Antibodies to the peptide (antipeptide antibodies), which can be polyclonal or monoclonal in nature, are then produced, using known techniques, by innoculation of the peptide into an appropriate animal (e.g., mouse, rabbit, etc.) and subsequent recovery from the animal of antipeptide antibodies or by hybridoma technology. Antipeptide antibodies so produced can be used, for example, in diagnosing conditions or which are the result of or are related to the presence of abnormal sex chromosomal material.

In as much as the X- and Y-chromosomal genes detected by the probes strongly cross-hybridize, a DNA probe for X or Y of any mammal is likely to detect similar genes in any other placental mammal. Therefore, antibodies are also likely to react to both X and Y peptides across species. The following is a description of 1) the construction and characterization of the deletion map of the Y chromosome constructed on the basis of hybridization studies of genomic DNA from normal individuals and individuals having sex chromosome anomalies; 2) mapping of the testis determining factor (TDF), or sex-determining function, to a small region of the Y chromosome; 3) cloning of the region of the Y chromosome which includes TDF; and 4) the use of Y-specific DNA as hybridization probes. I. A Deletion Map of DNA Sequences in the Y

Chromosome

A. Hybridization of DNAs from Normal Subjects and DNAs from Subjects Having Sex Chromosome Anomalies with Y-specific DNA Probes A physical or deletion map of the normal human Y chromosome was constructed by analysis and comparison of DNAs from normal subjects and subjects with anomalies of sex chromosomes. The map is represented in Figure 1.

Individuals were tested for the presence or absence of as many as 155 Y-DNA loci by hybridization to Southern transfers of restriction-digested genomic DNAs. Southern, E.M., Journal of Molecular Biology, 98:503-517 (1975). The majority of individuals tested were XX males, XO males (also referred to as 45X or X,t(Y;aut) males) or XY females, or, as judged by cytogenetics, had a structurally abnormal Y chromosome. DNAs from normal male (46,XY) subjects and normal female (46,XX) subjects were also analyzed. Many of the Y-DNA sequences used as hybridization probes were derived from a library made from flow-sorted Y chromosomes obtained from the National Laboratory Gene Library Project (Los Alamos). That is, the library consisted of a lambda phage (i.e., Charon 21A) into which fragments of Y chromosomal DNA (obtained by complete digestion of the Y DNA) had been cloned. Analysis of randomly selected Y-DNA-containing clones resulted in definition of deletion intervals on the chromosome. DNA sequences of interest were removed from the lambda phage and recloned into a plasmid (e.g., pUC8 or pUC13). Other DNA sequences used as probes are described by Page et al., Nature. 311: 119-123 (1984), by Bishop et al., Journal of Molecular Biology, 173: 403-417 (1984); and also in Nature, 303:831-832 (1983), the teachings of which are hereby incorporated by reference.

DNAs to be tested were prepared from peripheral leukocytes, cultured skin fibroblasts or EBV-transformed lymphoblasts, according to published methods. Kunkel L. M. et al., Proceedings of the National Academy of Sciences, U.S.A., 74: 1245-1249 (1977); Vergnaud, G. et al., British Medical Journal, 289: 73-76 (1984). They were then digested with the restriction endonucleases TaqI or EcoRI and hybridized, as indicated above, using the method described by Southern, with selected radiolabeled DNA probes which detect Y-specific restriction fragments. B. Construction of the Deletion Map of the Y

Chromosome Through Comparison of DNAs from Normal Subjects and Subjects of Abnormal Karyotype or Having A Structurally

Abnormal Y Chromosome

DNA studies of individuals having an abnormal karyotype or a structurally abnormal Y chromosome demonstrated that 70 of those tested carry part, but not all, of the Y chromosome. That is, in each of these 70 individuals, some, but not all, of the Y-specific restriction fragments which are invariably present in normal (46,XY) males were detected, using the method of Southern and the selected radiolabeled Y-specific DNA probes.

Only approximately one half of the Y deletions had been detected by cytogenetics; every Y deletion detected by cytogenetics was also revealed by DNA hybridization. The variety of patterns of Y-DNA loci occurring in these individuals are, as a group, most simply explained by the deletion map of the Y chromosome shown in Figure 1. This map accounts for each case on the basis of one Y breakpoint in all but two of the individuals. That is, the deletion map of Figure 1 reconciles the hybridization data with the presence of a single, contiguous portion of the Y chromosome in all but two of the cases.

The map has 20 deletion intervals (designated 1A1 through 7). Each of the intervals represented in Figure 1 is defined by the portion of the Y chromosome which is present or absent in a given individual or class of individuals or the difference (in the portion of the Y chromosome present) between two individuals or two classes of individuals.

For example, interval 1A, which includes 1A1 and 1A2, is that portion of the Y chromosome present in an XX male who carries only three of the Y-specific fragments for which testing was done; each of those fragments derives from or detects interval 1A and is represented in Figure 1 by a solid dot above the interval. The portion of the Y chromosome present in this XX male is represented in Figure 1 by the top bar.

Interval 1A is comprised of two smaller intervals, designated 1A1 and 1A2. As shown in Figure 1, interval 1A1 is present in both the XX male and the X,t(Y;22) female. Interval 1A2, however, is present in the XX male, who carries this small region of the Y chromosome, but is absent from the X,t(Y;22) female. Interval 1A2 is approximately 135 kb in length and contains at least one fragment (a 1.2 kb HindIII fragment) which hybridizes to the Y chromosome of all mammals tested, to DNA from chicken and is likely to hybridize to homologous DNA sequences from other vertebrates. Interval 1A2 also contains at least 2 CpG-rich islands. It is possible that the CpG-rich islands are associated with one (interrupted) "maleness-determining" gene or multiple "maleness-determining" genes.

It is possible to orient the deletion map with respect to Yp and Yq as a result of data obtained from hybridization studies of individuals with σytogenetically detectable deletions of Yq and an apparently intact Yp (XYq-males) and individuals with cytogenetically detectable deletions of Yp and an apparently intact Yq (XYp-females). The centromere is assigned to Interval 4B on the basis of two observations. First, 4B is the only interval present in all deleted but independently segregating Y chromosomes. Second, it is known that a particular Y-specific repeated sequence localizes to the centromere by in situ hybridization. Wolfe, J. et al., Journal of Molecular Biology, 182:477-485

(1985). This repeated sequence has now been mapped, using Y-specific DNA probes, to interval 4B. II. Mapping of the Testis-Determining Factor (TDF) The testis-determining factor (TDF), or sex-determining function, of the human Y chromosome was mapped to a small region of the Y chromosome by analysis of naturally-occurring deletions of the chromosome.

DNA from an XX male who had been shown to carry a small portion of the Y chromosome was analyzed, as described in Example 1, through hybridization with Y-specific DNA probes. This individual had been shown to carry the small portion of the Y chromosome present in the interval designated 1A, which includes 1A1 and 1A2. DNA from an X,t(Y;22) female with a reciprocal X,t(Y;22) translocation associated with a deletion was analyzed, also as detailed in Example 1.

Comparison of these results with DNA sequences present in the normal human Y chromosome demonstrated that in the XX male, Y DNA is present from both 1A1 and 1A2 (Figures 1 and 2) and that in the X,t(Y;22) female, Y DNA is present from 1A1 but not from 1A2. That is, there is a deletion in the X,t(Y;22) female of Y DNA in interval 1A2. Thus, this makes it clear that the XX male, who appeared to have two X chromosomes (based on cytogenetic assessment) in fact carries Y DNA in interval 1A, which includes intervals 1A1 and 1A2. The X,t(Y;22) female, who appeared to have all of the Y chromosome (based on cytogenetic assessment) actually has a deletion covering intervals 1A2 and IB. The presence of Y DNA from both interval 1A1 and interval 1A2 in the XX male and not in the X,t(Y;22) female (who has DNA from interval 1A1 but not from 1A2) is proof that Y-specific DNA whose presence is necessary, although possibly not sufficient, for determination of maleness is that whose location is indicated/represented by interval 1A2. It is also evident that Y-specific DNA- present in interval 1A (the sum of 1A1 plus 1A2) is necessary and sufficient for determination of maleness, in the absence of downstream mutations.

As described below, this DNA present on the Y chromosome in interval 1A2 represents approximately 1/500th of the Y chromosome and is approximately 135 kb-pairs in length. All or a portion of this 135-kb fragment is necessary for the determination of maleness but may need to be present in conjunction with the Y-specific DNA of interval 1A1; that is, although Y-specific DNA of interval 1A2 is necessary for determining maleness, it is not clear whether, alone, it is sufficient for determining maleness. III. Cloning of the Region of the Y Chromosome Which

Includes TDF

The region of the Y chromosome which includes at least a portion of the TDF has been cloned in its entirety. This was carried out, using conventional chromosome walking techniques and a lambda phage library containing Y DNA fragments. Genomic DNA was obtained from the human lymphoid cell line Oxen. Sirota, L. et al., Clinical Genetics, 19: 87-93 (1981). The Oxen cell line contains 49 chromosomes, which include a complete set of autosomes, one X chromosome and four Y chromosomes. That is, contiguous overlapping chromosomal segments were identified.

Chromosome walking is represented in Figure 2 and Figure 3. It makes use of an initial recombinant phage, containing a small segment of DNA which is a single-copy element or sequence in the genome. In walking the Y chromosome, the first probe used was pDP307, which is a 0.9 kb HindIII Y fragment subcloned into the HindIII site of pUC-13. This initial probe was used to screen the recombinant lambda phage library and resulted in isolation of phage lambda BER1, a lambda phage clone which contains the neighboring fragment of Y chromosome DNA; that is, the isolated lambda phage clone contains sequences which partially overlap the sequence of pDP307 and which partially extend beyond the pDP307 sequence.

In all, 49 recombinant lambda phages with overlapping inserts of the human Y chromosome were isolated from genomic libraries. Chromosome walking was carried out as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York 1982. The isolated lambda phage clone lambda BER1 was then used to rescreen libraries and produced clones BER27 and OX18. Figure 2 represents the order in which the clones were produced. Figure 3 is a schematic restriction map which resulted from 15 screening steps, each making use of the clone isolated in the previous screening step to identify a clone having partial overlap with the "screening" probe and, in addition, neighboring sequences of the Y chromosome.

As a result of this walking procedure, a region of the Y chromosome approximately 230kb in size has been cloned. In particular, the entire 135 kb region of the Y chromosome which includes at least part of TDF has been cloned. This region, represented. in Figure 1 as interval 1A2, has been shown to include a 1.2. kb HindIII fragment which hybridizes to Y chromosome DNA of all mammals tested. The nucleotide sequence of this fragment has been determined and is represented in Figure 4. This is explained in detail in Example 6.

In addition, the 135 kb 1A2 interval has been shown to contain at least two CpG-rich islands. Their locations are represented in Figure 3. As explained previously, CpG-rich islands are generally associated with a gene or a gene segment. Thus, in the case of the Y chromosome, it appears that the CpG-rich islands, as well as others present in the Y-specific DNA obtained from the XX male assessed, are associated with one or more "malenessdetermining" genes. IV. Use of Y-Specific DNA Hybridization Probes to

Detect and Identify Homologous DNA Sequences of

Sex Chromosomes

Thus, as a result of the detection and identification of Y-specific DNA sequences and their regional localization on the Y chromosome, selected DNA sequences are available for the analysis of sex chromosomes prepared from blood or other body tissues for the occurrence of sequences homologous to the sequences selected. The selected DNA sequences are used as probes which detect the presence or absence of the homologous Y-DNA through hybridization.

For example, one or more probes which are well characterized and of known location on the normal Y chromosome are used to determine the presence or absence of homologous sequences in each of the intervals of the sex chromosomes of individuals having suspected or presumed anomalies or to determine the occurrence of specific fragments, such as the TDF or the gene known to affect spermatogenesis.

DNA from, for example, peripheral leukocytes, cultured skin fibroblasts or EBV-transformed lymphoblasts, prepared according to published methods, can be analyzed. In one method of preparation, nuclei are first released by cell lysis in isotonic solution. Nuclei are then lysed in sodium dodecyl sulfate/proteinase K. DNA is purified by phenol extraction and dialysis. Kunkel, L.M. et al., Proceedings of the National Academy of Sciences, U.S.A., 74: 1245-1249 (1977); Vergnaud, G. et al., British Medical Journal, 289: 73-79 (1984). The prepared DNA is subsequently subjected to restriction digestion, electrophoresis and transfer. it is then hybridized with one or more Y-specific DNA probes, according to methods previously described by Page and de la Chapelle. See, Page, D.C. and A. de la Chapelle, American Journal of Human Genetics, 36: 565-575 (1984), the teachings of which are incorporated by reference. The DNA can be digested, for example, with the restriction endonucleases TaqI or EcoRI. The resulting fragments are separated, for example, by agarose gel electrophoresis and transferred to membrane filters for hybridization to the Y-specific DNA sequences (probes). Hybridization can be carried out by Southern blot technique. Southern, E.M., Journal of Molecular Biology, 98:503-517 (1975).

If the occurrence and identification of DNA sequences along the sex chromosome is to be carried out in an individual who is suspected, on the basis of cytogenetic analysis, to have a sex chromosome anomaly, a set of probes can be used. For example, the set of probes can include a probe for each of the intervals shown to occur on the normal Y chromosome. Use of this set of probes makes it possible to detect the absence of regions normally present in the Y chromosome; to detect the presence of regions not present in the normal sex chromosome; and to determine the location on the chromosome of regions of interest. One set of probes which can be used to determine the presence of the intervals of the normal Y chromosome as represented in Figure 1 is as follows:

Interval Detected Probe Characteristics 1A1 pDP1035 1.3Kb EcoRI Y fragment subcloned into EcoRI site of Bluescript

(Stratagene)

1A2 pDP1007 1.2Kb HindIII Y fragment subcloned into HindIII site of pUC-13

1B pDP307 0.9Kb HindIII Y fragment subcloned into HindIII site of pUC-13

1C 1.5Kb EcoRI fragment purified from lambda

BER38

1D pDP522b 2.0Kb BamHI Y fragment subcloned into BamHI site of pUC-12

1E pDP132 3.5Kb HindIII Y fragment -subcloned into HindIII site of pUC-13

2A pDP61 1.0Kb EcoRI/TaqI Y fragment from insert of pll5, subcloned into AccI and EcoRI sites of pUC-8 2B 4Kb HindIII fragment purified from lambda Y302 2C p8j 5Kb EcoRI fragment cloned into EcoRI site of pBR322 3A 3Kb HindIII fragment purified from lambda Y434 3B 4.3Kb HindIII fragment purified from lambda Y125 3C 50f2 1.1Kb EcoRI Y fragment subcloned into EcoRI site of pBR322

4A pDP34 2.2Kb EcoRI Y fragment subcloned into EcoRI site of pDP322 4B pDP97 5.3Kb EcoRI Y fragment from cosmid Y97 subcloned into EcoRI site of pUC-13 5 12f 5.0Kb EcoRI Y fragment subcloned into EcoRI site of pBR322

6A S232 7.0Kb EcoRI fragment subcloned into EcoRI site of plasmid 6B pDP527 2.4Kb HindIII Y fragment subcloned into HindIII site of pUC-13 6C 50f2 1.1Kb EcoRI fragment subcloned into EcoRI site of pBR322 6D 50f2 1.1Kb EcoRI fragment subcloned into EcoRI site of pBR322 7 pY431-HinfA 0.8Kb HinfI Y fragment cloned into PstI site of pBR322 Note: Probes for intervals 1B through 3B and for 4A each detect X-specific and Y-specific single copy DNA sequences; pDP34 is the same as DXYS1.

Use of this set of probes, or an analogous set having one probe for each interval of the Y chromosome, makes it possible to assess the occurrence of each of the regions along the Y chromosome DNA, as well as to determine their location on the chromosome. A subset of these probes can, of course, be used to assay prepared DNA for the occurrence of regions of particular interest. For example, a subset having the probes listed below can be used to determine the presence of DNA which occurs on the short arm of the normal Y chromosome.

Interval

Detected Probe

1A1 PDP1035 1A2 pDP1007 1B pDP307 1C 1.5Kb EcoRI fragment purified from lambda BER38 1D pDP522b 1E pDP132

2A pDP61

2B 4Kb HindIII fragment purified from lambda Y302 2C 5Kb EcoRI fragment cloned into EcoRI site of pBR322 3A 3Kb HindIII fragment purified from lambda Y434

3B 4.3Kb HindIII fragment purified from lambda Y125 3C 50f2 4A pDP34

4B pDP97 Similarly, a subset of probes can be used to detect the occurrence of DNA shown to occur on the long arm ofthe, normal Y chromosome. The presence or absence of the TDF in chromosome DNA from an individual is determined by using one or more DNA probes which hybridize to one or more sequences located on the normal Y chromosome and necessary for development of testes. For example, in this case, the probe can be Y-specific DNA present in interval 1A as represented in Figure 1. One such probe which can be used is pDP1035 (described above). In another embodiment of the present invention, DNA which hybridizes to DNA present in interval 1A2 of the normal Y chromosome is used as the probe or probes. In this case, a particularly useful probe is all or a portion of pDP1007, the 1.2 kb HindIII fragment which has been shown to be highly conserved in mammals and hybridizes to the Y chromosome of all mammals tested. (D.C. Page et al. 1987, supra). This probe is useful for determining the occurrence of the TDF in human and nonhuman sample DNA. In addition, the "maleness-determining" gene or genes, or a portion thereof, is useful for this purpose. The probe or probes which hybridize to sequences in interval 1A2 can be used separately or in combination (e.g., all or a portion of the 1.2 kb HindIII fragment, alone or in combination with all or a portion of the "maleness-determining" gene(s)).

The DNA sequences used as probes can be cloned sequences derived from a Y-enriched lambda phage library (such as that available from the National Laboratory Gene Library Project) and subcloned into plasmids or DNA sequences obtained, as described above, through chromosome walking, and subcloned into plasmids. Alternatively, synthetic DNA sequences can be used. The sequence of such DNA probes is determined by reference to the nucleotide sequence of normal Y chromosome DNA, such as the nucleotide sequence, represented in Figures 4 and 5, of the highly conserved 1.2 kb fragment. The DNA probes must be labelled (e.g., radioactively, by chemical modification such as biotinylation) or otherwise modified in such a way that in a sample they can be detected, identified and/or quantitated.

Comparison of data obtained by using a set of probes which detect two or more intervals on the normal Y chromosome with the deletion map of the normal Y chromosome represented in Figure 1 makes it possible to determine whether the DNA sequences homologous to the Y-specific probes are present in the sample DNA. Use of all or a portion of the 1.2 kb HindIII fragment of interval 1A2 as a probe can be used to show whether TDF is present in a sample or not.

V. Use of Y-Specific DNA Hybridization Probes to Determine the Genetic Basis for Sex Reversal Syndrome

There are several sex reversal syndromes in which it has been impossible to determine the genetic basis or explanation, using traditionally available methods (e.g. , chromosome banding studies). For example, there are individuals who have a 46,XX karyotype and are males; individuals who have a 46,XX karyotype and are hermaphrodites; individuals who have a 45,X karyotype and are males (designated XO males); and individuals who have an XY karyotype and are females.

Using Y-DNA specific probes and methods of detecting homologous DNA sequences according to the present invention, the genetic basis for these apparent anomalies is determined. A. XX Males

For example, XX males are individuals who are sterile but are otherwise phenotypically males; their karyotype is 46,XX. That is, they have no Y chromosome and apparently (as judged by cytogenetics) have the chromosomes of a normal female. However, by definition, the gonads of XX males are comprised exclusively of testicular elements. Testes in such individuals might be due to the presence of male-determining chromosomal DNA which cannot be detected by traditional methods. Y- specific DNA probes were used to determine the occurrence of homologous sequences (i.e., Y-specific sequences) in genomic DNA of 46,XX males. By hybridizing as many as 155 different Y-DNA probes to Southern transfers, Y-specific sequences were detected in the genomes of 46,XX males. Based on the Y-specific sequences present, the XX males are divided into 10 classes: Y(-) XX males, in whom no Y DNA sequences were detected, and nine classes of Y(+) XX males, in whom Y DNA was detected. The nine classes of Y(+)XX males are represented in Figure 1 by the first nine bars. The Y-specific DNA sequences present in the Y(+) XX males are those in intervals 1A1-4A of Figure 1. Based on the hybridization studies using Y-specific DNA probes, these are assigned to Yp, the short arm of the Y chromosome. The studies showed that intervals 1A1 and 1A2 were common to all Y(+) XX males, as well as to all XYq(-) males tested. As a result, if it is assumed that XX males have testes because of the presence of Y-derived TDF, TDF maps to at least one of these intervals. This assignment of TDF to Yp is in agreement with most karyotype-sex phenotype correlations.

Y-specific DNA sequences have also been detected in one 47,XXX male tested using the probes and methods described herein. The portion of the Y chromosome present is similar, if not identical, to that present in the males represented in Figure 1 by the eighth bar from the top.

As mentioned, in one class of XX males (designated Y(-)), no Y DNA sequences have been detected to date, either through chromosome banding or through hybridization using Y-specific DNA probes. It is possible that Y(-) XX males carry portions of the Y chromosome even smaller than those found in the Class 1A Y(+) male. Alternatively, Y(-) XX maleness may be the result of an autosomal or X-chromosomal mutation.

B. XX Hermaphrodites

XX hermaphrodites have a 46,XX karyotype and gonads which contain testicular as well as ovarian elements. Using Y-specific DNA probes, no Y-DNA sequences were detected in the genomes of three XX hermaphrodites tested. As with the Y(-) XX males, it is not known whether XX hermaphroditism is due to the presence of portions of the Y chromosome smaller than that in the Class 1A Y(+) male (who has DNA present in interval 1A only) or possibly to an autosomal or X-chromosomal mutation.

C. Males Having a 45,X Karyotype "XO males" are sterile but otherwise phenotypic males with a 45,X karyotype. As judged by chromosome banding, they carry no part of the Y chromosome. XO males were examined using the battery of Y-DNA probes described above. DNA hybridization studies using these probes showed one XO male to be a low grade mosaic with an XY sex chromosome constitution in less than 3% of fibroblasts. This mosaicism (later confirmed by karyotyping of many hundreds of cells) was detected and quantitated using probes detecting Y-specific repeated sequences. This analysis is described in detail in Example 4. Maleness is likely due to the XY cell line.

As judged by DNA hybridization, three other XO males carry intervals 1A through 4B of the Y chromosome (like Class 1 XYq(-) males). This is shown in Figure 1, in which the XO males are referred to as X translocation Y autosome males (X,t(Y;auto)). That is, although banding studies by expert cytogeneticists had not detected any Y material, DNA hybridization showed that these three "XO" males actually carry the entire short arm and centromere of the Y chromosome in all or at least most of their cells. In all three of these cases, in situ hybridization (i.e., hybridization of a radiolabelled DNA probe with metaphase stage chromosomes) and retrospective cytogenetics offer strong evidence that Y material has been translocated to an autosome, even though no Y DNA had been demonstrated by pure cytogenetics. In these three cases, the male phenotype is explained by the presence of Y DNA in interval 1A. See Example 4. D. XY Females

Studies of XY females confirm the mapping of TDF to Y interval 1A. XY females have degenerate ovaries and no testicular tissue, despite the presence of a Y chromosome. In most cases, XY females have similarly affected relatives, the inheritance being X-linked or autosomal recessive (Swyer syndrome). In such cases, the defect is clearly on the X chromosome or on an autosome, and the Y is presumably intact. However, it seemed possible that some sporadic cases of XY femaleness might be due to deletions of TDF. By DNA hybridization, deletions in part or all of Y interval 1A (and variable numbers of adjoining intervals) were detected in seven of 32 sporadic XY females and in one female with a "balanced" Y;22 translocation. See Figure 1. In two of the XY females in whom deletions were detected by DNA hybridization, prometaphase banding studies had revealed small deletions in Yp. The deletions in the various classes of XY females correspond roughly to the portions of the Y chromosome present in various classes of XX males. These findings suggest that XY females and XX males are reciprocal products of similar, aberrant X-Y exchanges.

DNA from the one (X,t(Y;22)) female with a "balanced" Y;22 translocation (associated with a deletion) was analyzed and compared with that from the XX male, described above, who had been shown to carry the small portion of the Y chromosome present in interval 1A. This provided further proof that the sex-determining function or TDF is present in interval 1A and, as described below, served as the basis of mapping this function to a smaller region, designated 1A2 , of the Y chromosome.

Findings in XY females also shed light on the etiology of Turner syndrome, which most commonly occurs in females with a 45,X karyotype. All seven of the XY females in whom deletions were detected had some signs of Turner syndrome (e.g., neck webbing, lymphedema); the 25 in whom deletions were not detected had no signs. These findings strongly suggest that Turner syndrome is the result of monosomy for a gene or genes common to the X and Y chromosomes. For example, in XY females with Swyer syndrome, the X-linked recessive inheritance in familial cases suggests mutation in an X-chromosomal gene functioning in the pathway of gonadal differentiation, perhaps "downstream" of the Y-encoded TDF. The absence of Turner stigmata in these XY Swyer females argues that a normal Y chromosome is--like a second X chromosome--capable of preventing the Turner phenotype. However, the presence o.f Turner stigmata in the other XY females--those with Yp deletions documented by DNA hybridization--demonstrates that the portion of Yp deleted in those females is required for the

"Turner-blocking" effect. Turner syndrome has thus been shown to be due to monosomy for one or more genes common to the X and the Y chromosome. One or more of these genes maps to the distal segment of Yp. It should be possible to map the Turner locus to a specific deletion interval on the Y chromosome and it appears to map to 1A1. VI. Use of Probes Recognizing Other Regions of

Interest on the Y Chromosome

As indicated previously, any postulated Y chromosome function can be evaluated using the deletion map of Figure 1. For example, if the Y chromosome carries a gene for H-Y antigen (a transplantation antigen), using the deletion map and selected probes, it is possible to map the gene to a deletion interval on the chromosome. It has been hypothesized that H-Y antigen is TDF and that H-Y is a genetic determinant of gonadal sex and/or a determinant of spermatogenesis. It has now been determined that H-Y is not TDF; H-Y has been excluded as a determinant of gonadal sex. If H-Y is found to have a role in spermatogenesis, for example, a probe specific to the Y region to which H-Y maps can be produced for use in diagnosis of male infertility.

As mentioned, XY (gonadal dysgenesis) females are sterile females with a 46,XY karyotype, female external genitalia, uterus, Fallopian tubes and "streak" ovaries. In addition, there are XY/XO females, who have Turner syndrome. Such individuals often have gonadoblastoma and it has been postulated that the Y chromosome, or some portion of it, has a role in predisposing such individuals to gonadoblastoma.

Gonadoblastomas are neoplasms defined histologically by the occurrence of both germ cells and sex cord elements (cells resembling immature Sertoli and granulosa cells) within well circumscribed nests. Stromal elements resembling Leydig or lutein cells are usually present. Gonadoblastomas are so named because 1) they recapitulate gonadal development (the primitive sex cords and stroma) to a degree seen in no other tumor and 2) they arise in markedly abnormal gonads, often not recognizable as either testis or ovary. Such gonads, often lacking the usual architecture of either an ovary or a testis and usually devoid of normal germ cells, are frequently described as being "dysgenetic." Germinomas and other more malignant cancers can arise within gonadoblastomas. Virtually all gonadoblastomas arise within such dysgenetic gonads.

There appears to be a gene on the Y chromosome that strongly predisposes dysgenetic gonads to develop gonadoblastomas. It appears that this postulated GBY gene (GonadoBlastoma locus on Y chromosome) has a physiologic function in normal males. GBY may, for example, function in or prior to spermatogenesis in normal testes.

Y-DNA hybridization analysis of individuals with gonadoblastoma and partial deletions of the Y chromosome suggest that GBY maps to the region that includes deletion intervals 4B through 7, (i.e., this evidence suggests it is located near the centromere or on the long arm of the Y chromosome). Gonadoblastomas occur almost exclusively in a very select population--individuals with dysgenetic gonads and a Y chromosome. Among individuals with dysgenetic gonads who are otherwise phenotypically female, gonadoblastoma occurs in a substantial fraction of those with a 46,XY or mosaic 45,X/46,XY karyotype; it rarely occurs in those with a 45,X or 46,XX karyotype. The tumor also occurs in females with gonadal dysgenesis and a deleted or otherwise structurally abnormal Y chromosome in some or all cells. Gonadoblastomas are sometimes present in individuals with marked sexual ambiguity, a Y chromosome, and abnormal, abdominal or inguinal testes. Scully, R.E., Cancer, 25:1340-1356 (1970). Only a very small fraction of gonadoblastomas develop in the apparent absence of Y-chromosomal material; in these cases, the presence of Y chromatin has been excluded by conventional cytogenetic (chromosome banding) analysis, not DNA hybridization. if gonadal dysgenesis alone predisposes to gonadoblastoma, then the tumor should occur at comparable rates in gonadal dysgenesis females with and without Y chromosomal material, which is decidedly not the case. If pleiotropic X-linked or autosomal mutations cause, by independent mechanisms, gonadal dysgenesis and gonadoblastoma, then the tumor should largely be limited to individuals carrying such mutations. On the contrary, gonadoblastoma is a frequent occurrence in females without such Mendelian mutations, in whom 1) gonadal dysgenesis is due to mosaicism for a Y-bearing cell line or in whom 2) gonadal dysgenesis (and sex reversal) is due to the presence of a Y chromosome lacking the male-determining region. The presence of GBY is not a sufficient condition for the development of gonadoblastoma. First, oncogenic manifestation of GBY requires a markedly abnormal gonad. Second, the combination of GBY and a dysgenetic gonad constitutes a strong but not absolute predisposition to gonadoblastoma. Some individuals with an intact Y chromosome and gonadal dysgenesis do not develop gonadoblastoma, and gonadoblastomas often appear to be of focal origin within dysgenetic gonads. Thus, additional, unrecognized factors or events may be required for the development of the tumor. it is possible that, in the context of dysgenetic gonads, GBY acts as an oncogene and that GBY has a physiologic function in normal males, likely in the testes, perhaps in or prior to spermatogenesis. It is reasonable to suppose that, while GBY induces gonadoblastomas in dysgenetic gonads, it functions in or prior to spermatogenesis in normal testes.

Since gonadoblastoma can occur in the presence of partially deleted Y chromosomes, it should be possible to define the small portion of the Y in which the GBY locus is found. Most if not all individuals with gonadoblastoma should carry, in at least some cells, the segment of the Y chromosome that contains GBY. Conversely, the absence of the GBY locus should prevent gonadoblastoma, even in the presence of other portions of the Y chromosome.

The deletion map represented in Figure 1 provides a framework for examining the role of the Y chromosome in various biological functions. Several issues arise in attempting to deletion map GBY. First, the phenotype must be identified accurately. The consistent application of defined histological criteria in diagnosing the presence of gonadoblastoma is of critical importance. Scully, R.E., Cancer, 25:1340-1356 (1970). Second, the presence of Y chromatin merely predisposes; gonadoblastomas will not develop in all individuals with dysgenetic gonads and GBY. Third, the tumors can first manifest themselves over a wide range of patient ages. The gonads of individuals at risk are frequently removed (and examined histologically) in childhood. Consequently, in attempting to map GBY, deleted-Y cases with gonadoblastoma can be interpreted with certainty, while cases without gonadoblastoma provide information of more limited usefulness. The limited evidence presently available suggests that, if there is a single GBY locus on the Y chromosome, it maps near the centromere or on the long arm. A female with gonadal dysgenesis, bilateral gonadoblastoma, and a 46,XY karyotype was found to carry DNA sequences specific to intervals 3 and 4B through 7 of the Y chromosome. Her Y chromosome was deleted for intervals 1, 2, and 4A, which can be excluded as sites for GBY. DNA hybridization findings in a second female with gonadal dysgenesis, bilateral gonadoblastoma, and a 46,XY karyotype suggest that she lacks interval 3 (as well as other intervals on Yp). Together, the findings in these two XY females suggest that. GBY maps to the region defined by intervals 4B through 7, which includes the centromere and all of the long arm (Figure 1). Since TDF, the testis-determining factor, maps to interval 1, GBY and TDF cannot be one and the same. The role of the Y chromosome in gonadoblastoma is likely independent of its role in gonadal sex determination.

The deletion map can be used to characterize sex chromosomes in such individuals and detect the abnormality apparently responsible for this predisposition. Once that abnormality has been identified and the interval(s) characterized and localized, DNA probes can be produced and used to assess prepared DNA (e.g., from blood or other tissues) from subjects for the occurrence of homologous sequences. VII. Homologous DNA Sequences on the Human X and Y Chromosomes DXYS1 and similar loci. It has long been thought that the mammalian X and Y chromosomes are partially homologous. Using DNA hybridization probes, Page et al. discovered DXYS1, a site of extensive single-copy DNA homology between the human x and Y chromosomes. A 4.5-kb segment of singlecopy DNA from a human genomic library was hybridized to Southern transfers of human DNAs digested with the restriction enzyme TaqI. This probe revealed TagI restriction fragments 11, 12 and 15 kb long. Among more than 100 unrelated individuals, all males exhibited the 15-kb fragment in addition to one of the other fragments. Some females displayed both the 11- and 12-kb fragments, while others had only the 11- or the 12-kb fragment, and none had the 15-kb fragment. These results suggested that the 15-kb TagI fragment derived from the Y chromosome, while the 11- and 12-kb fragments were X-linked alleles. Page, D.C. et al., Proceedings of the National Academy of Sciences, USA, 79: 5352-5356 (1982).

Subsequent studies confirmed these inferences. Hybridization of the 4.5-kb probe to TaqI-digested

DNAs from 48 members of a single family demonstrated Y-linked inheritance of the 15-kb fragment and X-linked inheritance of the 11- and 12-kb fragments. Hybridization of this probe to TagI-digested DNAs from human-rodent hybrid cell lines (which have partial complements of human chromosomes) showed segregation of the 15-kb allele with the human Y chromosome and segregation of the 11- and 12-kb alleles with human X chromosomes. This was the first demonstration of single-copy sequence homology between the X and Y chromosomes of any mammal. This site of homology between the human X and Y chromosomes has been designated DXYS1. Page et al., in: Recombinant DNA Applications to Human Disease (Caskey C. and White R., eds.) Cold Spring Harbor (1983)).

Deletion mapping studies using random Y-DNA loci suggest that as much as half of Yp is highly homologous to Xq13-q21 (on the long arm); these are referred to here as DXYS1-like loci. All loci mapping to Yp intervals 1B-3B and 4A appear to have this origin. DXYS1 is the most thoroughly studied member of this class of X-Y homologous loci.

If all of the DXYS1 loci mapped to the 10 different intervals on Yp are the result of a single transposition from the X at some time in the last few million years of human evolution (as studies suggest), then one might expect the homologous loci to occur tightly clustered, and in the same order, on the X chromosome. Most, if not all of these, DXYS1-like loci are found in the vicinity of Xq13- q21, as shown by physical mapping by Southern hybridization, using genomic DNAs from individuals or human-rodent hybrid cells carrying known portions of the X chromosome.

This physical map of DXYS1-like loci on the X can be complemented by a genetic linkage map among such loci. Eight X-linked RFLPs at DXYS1-like loci have been characterized. By simultaneously tracing the inheritance of several such X-linked RFLPs infamilies, it is possible to construct a genetic linkage map among these Y-homologous sites on the X chromosome.

X-linkage studies (i.e., to linkage map these DXYS1-like loci with respect to each other) simultaneously map the loci within the larger context of the X chromosome, in effect, generating a "finestructure" physical/genetic linkage map in the vicinity of Xq13-q21.

Genetic mapping of these X-linked RFLPs at DXYS1-like loci tremendously enhances their usefulness in studies of X-linked traits. For example, more than 50 laboratories around the world are already using the X-linked RFLP at DXYS1 to investigate choroideremia, Charcot-Marie-Tooth disease and other X-linked diseases, such as X-linked cleft palate and anhidrotic ectodermal dysplasia. Pseudoautosomal Loci on the X and Y Chromosomes Xp-Yp pairing during male meiosis has long been taken as evidence of homology and even recombination between those regions. Two loci that provide concrete evidence of this previously hypothetical region have been identified in the course of analyzing about 155 Y-DNA loci. Both loci are common to the X and Y chromosomes and exhibit frequent X-Y recombination as a normal event during male meiosis. They have been physically mapped to distal Xp and distal Yp, respectively. Because of X-Y recombination, restriction polymorphisms (RFLP) at these loci do not show strictly sex-linked inheritance, but instead are inherited as though autosomal; hence the term "pseudoautosomal." One of the pseudoautosomal probes detects a closely related family of sequences displaying a very high degree of RFLP. Most of this polymorphism can be detected using a number of restriction enzymes, so it is likely not due to base-pair substitutions. Not only the lengths but the numbers of homologous restriction fragments vary from individual to individual. Nonetheless, family studies suggest that this probe is detecting a single genetic locus. Within a family, it is possible to recognize a particular collection of autoradiographic bands as constituting an allele. Although this RFLP does not show sex-linked inheritance, the locus has been mapped to the distal short arms of the X and Y by in situ hybridization and deletion mapping in rodent-human hybrids. These and other pseudoautosomal loci probably map distal to Interval 1A1 on the short arm of the Y chromosome. The 1.2 kb HindIII fragment described above detects highly conserved DNA sequences on both the X and the Y chromosome. The DNA sequences detected by the 1.2 kb HindIII fragment on the Y chromosome are in interval 1A2; in the X chromosome, they occur at Xp21 to Xp223. This is not pseudoautosomal in nature and, thus, this is the first evidence of Xp-Yp homology that is not pseudoautosomal.

The protein sequence of the zinc finger domain of this X-chromosome protein has been determined and has been compared to the corresponding Y-chromosome protein of the approximately 400 amino acids, only 9 of the positions differ between X and Y (2.2%). VIII. Identification of Y-linked RFLPs Six Y-linked RFLPs have been characterized, including one occurring at DXYS1, a site of extensive single-copy DNA homology between the human X and Y chromosomes. In most unrelated American white males, the Y chromosome carries a tandem duplication of 15 kb of this X-homologous sequence. The same is true of most Oriental and aboriginal Australian males. Thus, like the X-to-Y transposition that gave rise to the DXYS1 homology itself, this Y-linked RFLP appears to have arisen prior to the formation of the human races.

These Y-linked RFLPs also may be useful in the refinement of the deletion map of Figure 1 and in determining whether the map is, in fact, dimorphic. That is, it may be instrumental in determining whether, among the population, there are two Y chromosomes which differ by an inversion of the intervals represented in Figure 1, particularly intervals 3C and 4A. For example, the map shown in Figure 1 does not account for the Class 2 XY female (represented by the third bar from the bottom of Figure 1), in whom DNA from intervals 3C and 4B-7 is present; in either case, three breakpoints would be required on the Y chromosome. An inversion polymorphism in which the order of intervals 3C and 4A are reversed accounts for each of these cases via one breakpoint on the Y and is consistent with the idea that XX males have a terminal portion of Yp.

Such an inversion is represented in Figure 7. Class 3 XX males (those in whom DNA is present in intervals 1A1 through 3C) and Class 1 XY females (those in whom DNA is present in intervals 4A through 7) can be explained as arising from a Y chromosome having a 3B-3C-4A-4B sequence; the Class 2 XY female (in whom DNA in intervals 3C and 4B through 7 are present) can be explained as arising from a Y chromosome having a 3B-4A-3C-4B sequence. The present invention will be further illustrated by the following examples, which are not intended to be limiting in any way.

Example 1 Construction of Deletion Map of the Normal Y Chromosome Patients Studied

DNAs from normal individuals and individuals having abnormal karyotypes or cytogenetically determined sex chromosome anomalies were hybridized with Y-specific DNA probes. DNAs obtained from 77 individuals were tested for the presence of as many as 155 Y-DNA loci. The majority of individuals tested were XX males, XO males or XY females or had, as judged by cytogenetics, a structurally abnormal Y chromosome.

The patients studied are listed in Table 1, which lists the phenotype and karyotype of each individual. Most of the individuals studied were karyotyped during the course of a medical evaluation because of infertility and/or small testes.

DNA Extraction and Gel-Transfer Hybridization

DNA was prepared from peripheral leukocytes, cultured skin fibroblasts, or EBV-transformed lymphoblasts by published methods. Kunkel, L.M. et al., Proceedings of the National Academy of

Sciences, U.S.A., 74: 1245-1249 (1977); Vergnaud, G. et al., British Medical Journal, 289:73-76 (1984). Restriction digestion, electrophoresis, transfer and hybridization of DNA were performed as previously described. Page, D.C. and A. de la Chapelle, American Journal of Human Genetics, 36: 565-575 (1984). Each hybridization probe was used at either "reduced" or "high" stringency, as described below. "Reduced" stringency implies that hybridizations were carried out at 42ºC and that the final wash was in 2X SSC at 68ºC or in 0.1X SSC at 55ºC. "High" stringency implies that hybridizations were carried out at 47ºC and/or that the final wash was in 0.1X SSC at 68ºC. DNA Hybridization Probes Used

The probes used are described below. Many of them are plasmid subclones derived from a Y-enriched lambda phage library (see above). Bishop, C. et al., Journal of Molecular Biology, 173:403-417 (1984); Bishop, C.E. et al., Nature, 303:831-832 (1983). For many of the probes, the names of the homologous DNA segments or loci (e.g., DXYS5 , DYZ1) are designated as assigned at the Human Gene Mapping Conference VII. Skolnick, M.H. et al., Report of the Committee on human gene mapping by recombinant DNA techniques. Cytogenetics and Cell Genetics, 1-4:210-273 (1984). Probes 47a and 47z have been shown to detect highly homologous sequences on the X and Y chromosomes (DXYS5). Geldwerth, D. et al., EMBO Journal, 1739:1743 (1985). At high stringency, 47a detects a Y-specific TaqI fragment of 4.3 kb; 47z detects a Y- specific TaqI frament of 3 kb. These sites of X-Y homology are also detected by probe 47c. Guellaen, G. et al., Nature, 307: 172-173 (1984). 47a, 47c and 47z are subclones from the same cosmid. Probe 13d has been shown to detect highly homologous sequences on the X and Y chromosomes (DXYS7). Geldwerth, D. et al., EMBO Journal, 1739:1743 (1985). At high stringency, 13d detects a Y-specific TaqI fragment of 7 kb. Probe 115 detects highly homologous sequences on the X and Y chromosomes (DXYS8). Geldwerth, D. et al., EMBO Journal, 4:1739-1743 (1985). At high stringency, 115 detects a Y-specific TaqI fragment of 2.1 kb. Probe 52d detects multiple loci on the Y chromosome as well as one on the X. Bishop, C. et al., Journal of Molecular Biology, 173:403-417 (1984). At reduced stringency, 52d detects Y-specific EcoRI fragments of 7 kb (restriction fragment 52d/A), 1.2 kb (52d/B), and 1.0 kb (52d/C; apparently an unresolved doublet). The corresponding Y-specific TaqI fragments are 9 kb (52d/C; an unresolved doublet), 5 kb (52d/A), and 3 kb (52d/B). Probe 50f2 defines multiple Y-specific loci and an autosomal locus. At reduced stringency, 50f2 detects Y-specific EcoRI fragments of 10 kb (50f2/A), 7.5 kb (50f2/B), 6 kb (50f2/C), 4.5 kb (50f2/D) and 1.7 kb (50f2/E). The corresponding Y-specific TaqI fragments are 9 kb (50f2/E), 8 kb (50f2/D), 3.5 kb (50f2/A or 50f2/B), and 3 kb (an unresolved doublet, corresponding to 50f2/C and either 50f2/A or 50f2/B). Guellan, G. et al., Nature, 307:172-173 (1984).

Probe 118 detects numerous Y-specific restriction fragments. Four TaqI fragments whose presence or absence could be unambiguously determined were considered in this study: 7 kb (118/A), 6 kb (118/B), 5 kb (118/C), and 1 kb (118/D). Guellan, G. et al., Nature, 307: 172-173 (1984).

Probe pDP34 detects highly homologous sequences on the X and Y chromosomes (DXYS1). Page, D.C. et al., Proceedings of the National Academy of Science, U.S.A., 79:2352-2355 (1982); Page, D.C. et al., Nature, 311: 119-123 (1984). At high stringency, pDP34 detects a Y-specific TaqI fragment of 15 kb. Probe 64a7 detects homologous sequences on the Y and on an autosome. Guellaen, G. et al., Nature, 307: 172-173 (1984). At reduced stringency, 64a7 detects a Y-specific TaqI fragment of 4.5 kb.

Probe 12f detects sequences on autosomes and the X as well as on the Y. Bishop, C. et al.,

Journal of Molecular Biology, 173:403-417 (1984). At high stringency, 12f detects two or three Y-specific TaqI or EcoRI fragments. Samples were scored for the presence or absence of an 8-kb, Y-specific TaqI fragment (corresponding to a 5-kb EcoRI fragment). At reduced stringency, probe 49f detects numerous Y-specific fragments. Bishop, C. et al., Journal of Molecular Biology, 173:403-417 (1984). Samples were scored only for the most intensely hybridizing Y-specific fragments (2.0- and 1.8-kb TaqI, or 2.8-kb EcoRI).

DYZ1 repeats were probed with a mixture of several cloned 3.4-kb HaeIII repeated elements. DYZ1 repeats have been detected at high stringency as a Y-specific EcoRI fragment of 3.4 kb or as several discrete Y-specific TaqI fragments of less than 0.6 kb.

DYZ2 repeats were examined using probe P1, a subfragment of the 2.1-kb HaeIII repeated element, Vergnaud, G. et al., British Medical Journal, 289: 73-76 (1984), or probe pY431-HinfA; Vergnaud, G. et al., American Journal of Human Genetics, 38 : 109-124 (1986). DYZ2 repeats were characterized at high stringency as Y-specific smears of high molecular weight in both EcoRI and TaqI digests. In addition, discrete Y-specific fragments were observed in TaqI digests.

Probe pDP1035 is a 1.3Kb EcoRI Y fragment subcloned into the EcoRI site of Bluescript (Stratagene).

Probe pDP1007 is a 1.2Kb HindIII Y fragment subcloned into the HindIII site of pUC-13.

Probe pDP522b is a 2.0Kb BamHI Y fragment subcloned into the BamHI site of pUC-12. Probe pDP8j is a 5Kb EcoRI fragment cloned into the EcoRI site of pBR322.

Probe pDP527 is a 2.4Kb HindIII Y fragment subcloned into the HindIII site of pUC-13. The results of hybridization of DNAs from the subjects with Y-specific DNA probes are best described with reference to the figures. Figures 8 and 9 are autoradiograms from representative hybridization experiments. For each probe used it was possible to score for unambiguously Y-specific bands.

In Figure 8 (left panel) is shown a probe (49f) which detects autosomal as well as multiple Y-specific bands, some of which are polymorphic. In Figure 8 (right panel) is shown a probe (12f) which detects many autosomal and X-chromosomal bands which are present in all the individuals tested. It was also possible to score unambiguously for a Y-specific 8-kb TaqI fragment. In Figure 9 are shown two probes which each detect single-copy sequences on the X and Y chromosomes. They were hybridized to gel transfers of TaqI-digested DNAs from a normal (46,XY) male, a normal (46,XX) female and an XX male. As shown, each probe detects X-chromosomal fragments, as well as a Y-specific fragment. In the case of probe 47a, the Y-specific fragment is 3kb and in the case of probe 47, it is 4.3kb.

The individuals tested were scored for the presence or absence of restriction fragments observed in normal males but not in normal females, and therefore assumed to derive from the Y chromosome. The restriction fragments detected are summarized in Table 1. Some probes (e.g. 50f2, 118, and 52d) detect multiple Y-specific fragments. For such probes, individuals were scored for the presence or absence of the particular Y-specific fragments indicated in Table 1. As indicated in Table 1, no Y-specific DNA sequences were detected in XX hermaphrodites or in a few XX males who can be referred to as Y(-) XX males. The other individuals included XX males (referred to as Y(+) XX males); XYq- males; and a t(Y;15) female. In each of these individuals, the presence of one or more Y-specific restriction fragments was detected. However, in none of these individuals were all the Y sequences for which testing was done detected. All of the Y sequences were present in each of the normal male controls and, when tested, in the fathers of the cases. Without exception, the TaqI or EcoRI restriction fragments detected in the individuals were of the same size as those seen in normal males. This was also true in fathers of the cases who were tested. This leads to the conclusion that each of these cases contains a portion, but not all, of the Y chromosome.

Based on a comparison of the Y-specific fragments detected (using the DNA probes described) in normal subjects and in subjects having chromosome anomalies, a deletion map of the Y-specific DNA sequences in the normal Y chromosome was constructed (Figure 10). The intervals in which DNAs from subjects occur are shown in Table 2.

The data obtained are consistent with the idea that, in each of these cases, only a single contiguous portion of the Y chromosome is present. That is, the Y-specific sequences can be ordered so that, in each of the patients tested, with two exceptions, the Y sequences present are a single, uninterrupted cluster. In each of the persons with cytogenetically detected abnormalities the chromosome banding studies are consistent with the presence of a single, contiguous portion of the Y chromosome. Such studies are not definitive and are not sufficiently precise to exclude more complicated rearrangements of the Y chromosome.

However, the patterns of Y-specific DNA sequences present in the individuals tested make it possible to generate a consistent deletion map of the human Y chromosome. In this map, each of the Y-specific restriction fragments tested is assigned to one of 20 deletion intervals, as shown in Figure Figure 7, as mentioned above, is a deletion map in which an inversion polymorphism is represented; the order of intervals 3B and 4A are reversed in this map.

Earlier evidence suggested that many human XX males contain Y-specific DNA sequences but are heterogeneous with respect to the amount of Y-chromosomal material present. Guellaen, G. et al., Nature, 307:172-173 (1984); Page, D.C. et al., Nature, 315:224-226 (1985). The present work extends that evidence: Y-specific DNA sequences were detected in the genomes of 24 of 26 XX males tested. In all cases, the Y-specific restriction fragments observed are of the same lengths as in. normal males. The 24 Y(+) XX males can be divided into nine classes according to the number of Y-specific fragments present. The DNA sequences present in the nine classes comprise a nested series (see Figure 1). The Class 1A XX male carries three of the Y-specific restriction fragments for which testing was done (defining interval 1A in Figure 1). Class 1B XX males carry that same Y-specific fragment and two additional fragments (1B). It is also possible to orient the Y-chromosome deletion map with respect to the arms (Yp and Yq) and the centromere, as a result of hybridization data.

Males with microscopically visible deletions of the long arm of the Y (and an apparently intact short arm) and females with a translocation of Yq heterochromatin to other chromosomes were among those scored for the presence or absence of Y-specific restriction fragments. The sterile 46,XYq- males lack the DYZ1 and DYZ2 repeated sequences as well as certain single-copy Y sequences. These sterile 46,XYq- males can be divided into three classes according to the number of single-copy Y sequences they lack. A normal male with a non-fluorescent Y chromosome lacks only the DYZ1 and DYZ2 repeats (specific for interval 7) and differs from a few of the other cases by the presence of several single-copy sequences (specific to interval 6). Conversely, a 46,XX,der(15), t(Y; 15) (q12;p11) female, (designated X,t(Yq; 15) in Figure 1) with the quinacrine-bright distal portion of Yq translocated to chromosome 15, carries the DYZ1 and DYZ2 repeats (interval 7), but none of the Y-specific single copy sequences listed in Table 1 (intervals 1 through 6).

These Yq deletions make it possible to assign intervals 5 through 7 to the long arm of the Y chromosome (Figure 1). They thus serve to orient the deletion map with respect to the long and short arms of the Y chromosome. Assuming that these Yq- chromosomes represent simple deletions (and not, for instance, translocation products), they all have the

Y centromere. On the other hand, it seems unlikely that the Y(+) XX males, in whom no Y chromosomal was detected cytogenically, have the Y centromere. The

Y centromere is found, then, in interval 4B, which is present in all the XYq(-) males but absent in all the XX males (Figure 1). Interval 4A contains the Y locus detected by probe pDP34 (DXYS1). This locus has been mapped to Yp (the short arm of the Y chromosome) by in situ hybridization. Page, D.C. et al., Nature, 311:119-123 (1984). It follows that intervals, 1 to 4A are entirely within Yp. These conclusions are based on the assumption that the rearrangement in the Yq- chromosomes is a simple deletion affecting Yq only. While the morphology of each Yq(-) chromosome is compatible with such a deletion, more complicated rearrangements cannot be excluded on the basis of cytogenetic studies. If events such as inversions or translocations were involved, the above conclusions might have been modified.

The Y-specific DNA sequences which are present in Y(+) XX males are those in intervals 1 through 4A, which have been assigned to Yp. All of the Y(+) XX males (and all of the XYq- males) have interval 1A in common. Assuming that XX males have testes because of the presence of a male-determining portion of the Y chromosome, that male determinant can be mapped to interval 1A. This assignment of the male determinant to Yp is in agreement with most karyotype-sex phenotype correlations. Davis, R.M., Journal of Medical Genetics, 18: 161-175 (1981). The work described herein provides considerable information as to the organization of DNA sequences within the Y chromosome. Under the hybridization conditions used, probes 50f2, 52d, and 118 detect multiple Y-specific restriction fragments. The fragments detected by each of those probes are not all clustered within single intervals.

In Class 3 XX males, bands 52d/C and 52d/B are of equal intensity; in normal males (or in the XYq-males), 52d/C is more intense than 52d/B. It is concluded that band 52d/C is at least a doublet, composed of one copy found in interval 3C and. one or more copies in interval 4B.

Probe 118 detects many Y-specific TaqI fragments; of the four Y-specific fragments which could be scored unambiguously, three (118/A,B,C) map to interval 3C, while one (118/D) maps to interval 6.

Thus, probes 50f2 and 52d each detect sequences in intervals 3C (on Yp), 4B, and 6 (on Yq); probe 118 detects sequences in intervals 3C and 6 (and probably elsewhere). These Yp-Yq homologies may be the result of one or more duplications of portions of the Y chromosome during evolution. Families of highly homologous DNA sequences have also been observed on the mouse Y chromosome. Lamar, E.E. and E. Palmer, Cell, 32:171-177 (1984); Bishop, C.E. et al., Nature, 315:70-72 (1985).

Example 2 Hybridization of Two DNA Probes

Detecting Yq(-)Specific Sequences to XX Male DNA and XYq(-) Male DNA Two labeled probes were hybridized to a gel transfer of Taql-digested DNAs from a normal female, an XX male, two XYq- males, and a normal male. Figure 7 is an autoradiogram showing results of these hybridizations. The left panel of Figure 8 shows the results of hybridization of ³²P-labeled probe 49f to the gel transfer. 49f detects two autosomal bands present in all of these individuals. 49f also detects a Y-specific 2.0-kb fragment in all normal males tested and in one of the two XYq- males shown in Table 1 above. 49f detects several additional Y-specific fragments; the size and presence of some of these is polymorphic, as can be seen in the comparison of XYq(-) male 26 (CHM007) with a normal male (see Table 1). These and the other individuals listed in Table 1 were scored for the presence or absence of the nonpolymorphic Y-specific 2.0-kb fragment (indicated in the left panel by an arrow). Probe 12f was hybridized to the same TaqI gel transfer. 12f detects many autosomal and X-chromosomal fragments present in all of these individuals. In addition, 12f detects several Y-specific fragments; individuals were scored for the presence or absence of the 8-kb fragment (indicated in the right panel of Figure 8 by an arrow).

Example 3 Hybridization of Probes 47a and 47z to DNA of an XX Male Two ³²P-labeled probes, 47a and 47z, were hybridized to gel transfers of TaqI-digested DNAs from a normal male, a normal female, and XX male 10 (CON 101). Figure 8 is an autoradiogram showing results of these hybridizations. Results of hybridization with 47a are shown in the left panel of

Figure 8. 47a detects an X-chromosomal fragment of 8 kb and Y-specific fragment of 3 kb (shown by an arrow in Figure 9). Results of hybridization with 47z are shown in the right panel. 47z detects X-chromosomal fragments of 1.5 kb and 2.1 kb and a Y-specific fragments of 4.3 kb (shown by an arrow in Figure 9). Individuals were scored for the presence or absence of the 3 kb fragment and the 4.3 kb fragment; results are shown in Table 1 (above).

Example 4 Use of Y-specific DNA Hybridization

Probes to Explain Maleness in Two 45,X Males As discussed above, very rare 45,X individuals are sterile males with testes. The genetic basis for their maleness cannot be determined using chromosome banding studies. In this example, the use of DNA hybridization probes to detect X-linked restriction fragment length polymorphisms (RFLP) and/or Y-specific restriction fragments in genomic DNAs from two 45,X males and their relatives is described.

Subjects

Two 45,X males from two families and their mothers and fathers were tested, as was the brother of one of the 45,X males. Cytogenetic Studies Patient 1 Both parents were cytogenetically normal, as shown by lymphocyte and skin fibroblast mitotic studies; the father was shown to be 46,XY and the mother to be 46,XX. The proband was studied repeatedly. Four blood cultures and one fibroblast culture tested between 1971 and 1979 showed 45,X mitoses only. Several hundred mitoses and 1000 interphase nuclei from the 1979 blood culture were screened by quinacrine fluorescence for the presence of a Y chromosome or a Y chromatin body, but neither was found. In 1971 a buccal mucosa smear had shown 15/1000 cells with a fluorescent spot judged at the time to be a possible Y chromosome. Again in 1977 some 5% of buccal mucosa cells appeared to be Y chromatinpositive. Skin fibroblast studies in 1982 showed that though most mitoses (179/186) were 45,X, 5/186 cells were clearly 46,XY as seen in G-banding by trypsin (GTG) and Q-banding by quinacrine fluorescence (QFQ). QFQ banding indicated that the Y chromosome was structurally normal with a brightly fluorescent band Yq12 and that it was the same length (longer than average) as the father's. Moreover, 34/1000 of interphase nuclei from the same culture has a Y-chromatin body corresponding in size to the Y chromosome. In 1984 a frozen aliquot of the same fibroblast culture was thawed and the chromosome studies repeated. Of a total of 434 mitoses studied by quinacrine fluorescence, 5 had the karyotype 46,XY while all others had less than 46 chromosomes and no Y chromosome. Moreover, 8/1000 interphase nuclei had a Y chromatin body. The DNAs used for the detection of Y chromosome-specific DNA sequences were prepared from these 1982 and 1984 fibroblast cultures.

Patient 2

Both parents and the brother were cytogenetically normal; the father and the brother were shown to be 46,XY and the mother to be 46,XX. Only 45,X cells were detected in the propositus, in blood cultures (on 2 occasions) and skin fibroblast cultures (on 3 occasions). In addition, 1000 interphase nuclei from the 1975 blood culture, 1000 nuclei from a buccal smear, and 100 mitoses and 1000 interphase nuclei from the blood culture made in 1979 were screened by quinacrine fluorescence for the presence of Y chromatin or a Y chromosome, but none was found. Thus, there was no indication of the presence of any Y chromosome in this patient. DNA Studies

Human genomic DNAs, prepared from peripheral leukocytes or cultured skin fibroblasts, were analyzed by restriction digestion, agarose electrophoresis, gel transfer, and hybridization with radiolabeled cloned DNA probes. The DNA hybridization probes used detect X-linked restriction fragment length polymorphisms (RFLP) and/or Y-specific restriction fragments.

To determine the parental origin of the single X chromosome in the two 45,X males, both families were typed for as many as eight X-linked RFLPs. These X-linked RFLPs provide information as to the parental origin of the X chromosome only when, by chance, the propositus is hemizygous for an allele present in only one parent. The probes used for this purpose are as follows:

1. RC8 detects X-linked, allelic Taq I fragments of 3.0, 3.4 and 5.7kb.

2. D2 detects X-linked, allelic Pvu II fragments of 6.0 and 6.6kb.

3. L1.28 detects X-linked, allelic Taq I fragments of 10 and 12kb.

4. pDP34 detects X-linked, allelic Taq I fragments of 11 and 12kb as well as a Y-specific 15kb fragment.

5. 19-2 detects X-linked, allelic Msp I fragments of 4.4 and 12kb.

6. S21 detects X-linked, allelic Taq I fragments of 2.5 and 2.7kb. 7. 22-33 detects X-linked, allelic Taq I fragments of 10 and 17kb. 8. 43-±5 detects X-linked, allelic Bg1 II fragments of 6 and 9kb.

The results are shown in Table 3.

In both cases, the propositus' single X chromosome was demonstrated to be maternal in origin.

Hybridization probes derived from the human Y chromosome were used to test the two 45,X males and their relatives for the presence of a number of Y-specific sequences. The probes used for this purpose are as follows: 9. 47c detects Y-specific Taq I fragments of 3 and

4.3kb. 10. 115 detects a Y-specific Taq I fragment of either 2.1 or 2.6kb.

11. 50f2 detects multiple Y-specific loci on Eco Rl or Taq I digests.

12. 52d detects multiple Y-specific loci on Eco Rl or Taq I digests.

13. A 1.8-kb PstI fragment purified from plasmid 71-7A detects multiple Y-specific Taq I fragments.

14. pY431-HinfA detects a highly repeated Y- specific Hae III fragment of 2.1kb.

15. pY3.4 detects a highly repeated Y-specific Hae III fragment of 3.4kb.

Probes 47c, 115, 50f2, 52dpDP34 and 71-7A detect single-copy, Y-specific sequences. Three of them (50f2, 52d and 71-7A) detect multiple Y-specific restriction fragments. Probes pY431-HinfA and pY3.4 detect Y-specific repeated sequences. The results are shown in Table 4.

In both families, the normal 46,XY males (the fathers and the brother) exhibit all of the Y- specific sequences found in unrelated control males. The mothers (normal 46,XX karyotypes) exhibit none of these Y-specific sequences.

None of the single-copy, Y-specific sequences tested for were detected in either of the 45, X males. In patient 1, the highly repeated, Y- specific 2.1kb and 3.4kb HaeIII fragments homologous to probes pY431-HinfA and pY3.4 were detected.

However, these Y-specific repeated sequences were present in greatly reduced amounts, in comparison with amounts detected in the father of patient 1 or in unrelated control males. To quantitate the reduction of these repeated sequences, the intensity of the hybridizing 2.1- and 3.4kb Hae III fragments in patient 1 was compared with the corresponding intensities obtaining using equal or reduced (10-fold, 100-fold, 1000-fold, and 10,000-fold) amounts of paternal DNA. The intensity of the 2.1-kb Hae III band in the 45,X male is somewhat greater than that observed with paternal DNA in 100-fold reduced amount (Figure 11). A similar reduction in the intensity of the 3.4kb Hae III band in this 45,X male was also observed. Both Y-specific 2.1kb and 3.4kb Hae III fragments are present in about 1 to 3% of the amount present in the father. No trace of these Y-specific repeated sequences was found in patient 2, even when conditions were used in which it is possible to detect the presence of normal male DNA in 10,000-fold reduced amount (i.e., the presence of a normal Y chromosome in as few as 1 in 10,000 cells.).

However, these repeated Hae III fragments are located principally if not exclusively in distal Yq, and thus would be of little use in detecting mosaicism involving an abnormal Y chromosome lacking that region. The DNA hybridization studies alone, then, cannot argue against low-grade mosaicism for a structurally abnormal Y chromosome in patient 2. Similarly, cytogenetic methods based on detection of the quinacrine-bright distal portion of Yq cannot argue against such mosaicism.

Example 5 Detection of Small Deletions of the

Short Arm of the Y Chromosome in 46,XY Females

Subjects

Case 1 has Turner stigmata. Her gonads, which were removed at age 4, were streaks which consisted of dense ovarian stroma with no primordial follicles or testicular tissue.

Case 2 has several features of Turner syndrome. She has congenital lymphedema. She had primary amenorrhea and developed bilateral gonadoblastoma. Histological examination of the gonads showed gonadoblastoma and streaks with no primordial follicles. Cytogenetic Studies

Cytogenetic analysis was performed on peripheral blood samples from cases 1 and 2 and on fibroblast cultures from a small skin biopsy of case 1 and from a gonadal biopsy of case 2. Prometaphase cells were stained by G-banding, R-banding, C-banding and Q-banding. A small deletion of the short arm of the Y chromosome [46,X,del(Y) (p11)] was identified in both patients, as shown in Figure 6, where the Y chromosome of case 1 (1) and case 2 (2) are compared to a normal Y chromosome (3) after G- and Q-bandings. The deletions were barely detectable on metaphase chromosomes. It was not possible to determine whether the deletions were interstitial or terminal, or whether they differed in the two patients. The long arm of the Y chromosome of both patients appeared of normal size, including a Q-bright heterochromatic region of average size. The Y chromosome of the father of each patient was normal. No other karyotypic abnormalities were identified in either patient and no evidence of mosaicism was found in the tissues analyzed: in case 1, 105 cells and 101 cells were examined in the blood and skin samples, respectively, while in case 2, 155 cells were examined in the blood and 54 and 58 cells in bilateral gonadal samples, respectively. DNA Studies

DNA was prepared from blood samples and fibroblast cultures of both patients, the father of case 2 and normal control male and female individuals. The DNAs were digested to completion with the restriction endonucleases TaqI (for probe pDP34) or EcoRI (for probes 50f2, 52d, 47b, 118, p12f2 and p12f3). DNA fragments separated, on agarose gel electrophoresis were transferred to membrane filters for hybridization by Southern blot technique. Seven

32P-labeled DNA probes that have been mapped to the

Y chromosome were hybridized to patient and control genomic DNA blots. (See I above and Example 1) Probe pDP34 (DXYSl) detects homologous sequences on the short arm of the Y chromosome and on the long arm of the X chromosome. Probes 50f2 and 52d hybridize with DNA sequences both on the Y chromosome and on autosomes on the X chromosome. Probe 47b detects Y-, X- and autosomal sequences; probe 118 is exclusively Y-specific. Two additional probes, p12f2 and p12f3 are located on the long arm of the Y chromosome. Results of these analyses can be summarized as follows: 1. Hybridization of probe pDP34 showed

(Figure 8) that: patient 1 had a hybridization pattern characteristic of a normal male (DXYS1 was not deleted) and patient 2 was missing the 15kb Y-specific bond (i.e., Y-specific DNA homologous to DXYS1 was deleted). 2. Hybridization of probes 118, 52d, 50f2 and 47b showed that patient 1 was missing six of the male-specific bands with all four probes (confirming cytogenetic data indicating a deletion of a portion of the Y chromosome) and patient 2 had a missing

Y specific band with probe 50f2 and probe 47b (the latter identical to that missing in patient 1). 3. Hybridization with probes p12f2 and p12f3 (located on the long arm of the Y chromosome) showed normal male hybridization patterns for both patients. Results of the hybridization of probe pDP34 to patient and control DNAs were compared. Case 1 showed a hybridization pattern characteristic of a normal male with bands at 11 and 15 kb, indicating that DXYS1 was not deleted in this case. Case 2, however, was missing the band at 15 kb, indicating that the Y-specific DNA homologous to DXYS1 had been deleted in that patient. The father of the latter patient showed normal Y- and X-specific bands at 15 and 12 kb, respectively. The hybridization of 32P-labeled probes 118,

52d, 50f2 and 47b to EcoR1 digested DNA's from normal female, normal male, case 1, the father of case 2 and case 2 was also assessed. Case 1 showed deletions of a total of six of the male-specific bands with all four probes 118, 52d, 50f2 and 47b. This confirmed the cytogenetic data which indicated that case 1 had a deletion of a portion of the Y chromosome. Probes 118, 52d and 50f2 showed a missing Y-specific band identical to the one missing in case 1 but present in normal males, including the father of case 2. Studies with two additional probes, p12f2 and p12f3, which are located on the long arm of the Y chromosome showed normal male hybridization patterns for both patients 1 and 2. Table 4 summarizes the hybridization data which indicate that cases 1 and 2 have different deletions. The only band these patients are missing in common is homologous to probe 47b. This probe is adjacent to probe 47c, which was most often present in patients who are 46,XX males. One or both of the deletions described here are likely to be interstitial, extending on either side of the location of 47b. The two patients have different but overlapping deletions which could explain that while both patients have features of Turner syndrome, they differ in some respects. Case 1 has short stature, while case 2 is of normal height: this may indicate the presence of genes controlling height on the short arm of the Y chromosome, which are deleted in case 1 but not in case 2. In addition, case 2 but not case 1 developed gonadoblastoma; however the gonads were removed at an early age in case 1. Thus, use of the DNA probes described, which are homologous to regions on the short and the long arms of the Y chromosome, demonstrated that the sex chromosomal anomaly, the 46,XY female, resulted from small deletions of the short arm of the Y chromosome. That is, small deletions of the short arm of the Y chromosome were detected; no deletions on the long arm were detected with the probes used. The deletions, although different, included a common overlapping region (detected by probe 47b) apparently essential for male differentiation. The results are summarized in Table 5.

Industrial Applicability

DNA probes which detect DNA sequences homologous to those occurring in the normal Y chromosome can be used in assessing the presence of Y-specific DNA in a clinical or other medical context for diagnosis and evaluation of many postulated functions and in an agricultural context for DNA-sexing of animals. For example, they can be used for the assessment of the presence or absence of the testis determining factor(s), the analysis of suspected chromosomal abnormalities, the determination of the genetic basis of phenotypic abnormalities and the diagnosis of genetic disorders and their related effects.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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Claims

1. Isolated DNA which hybridizes to at least a portion of one of the Y chromosomal intervals represented in Figure 1.

2. Isolated DNA of Claim 1 which hybridizes to Y chromosomal DNA necessary and sufficient, in the absence of downstream mutations, for determining maleness in a mammal.

3. Isolated DNA of Claim 2 which has a detectable label.

4. Isolated DNA of Claim 2 which hybridizes to Y chromosomal interval 1A.

5. Isolated DNA of Claim 4 which has a detectable label.

6. Isolated DNA of Claim 4 which hybridizes to Y chromosomal interval 1A2.

7. Isolated DNA which hybridizes to a selected interval of the short arm of the normal human Y chromosome.

8. Isolated DNA which hybridizes to a selected interval of the long arm of the normal human Y chromosome.

9. Isolated DNA which hybridizes to an interval of the normal human Y chromosome required for spermatogenesis.

10. Isolated DNA of Claim 9 which hybridizes to the long arm of the normal human Y chromosome.

11. Isolated DNA which is a Y-linked restriction fragment length polymorphism.

12. Isolated DNA which detects pseudoautosomal loci common to the X chromosome and the Y chromosome.

13. Isolated DNA having a nucleotide sequence of all or a portion of the nucleotide sequence of Figure 4.

14. Isolated DNA present in lysate of lambda phage OX82, as deposited at the American Type Culture

Collection under deposit number 40367.

15. Isolated DNA present in lysate of lambda phage OX95, as deposited at the American Type Culture Collection under deposit number 40368.

16. Isolated DNA present in lysate of lambda phage OX107, as deposited at the American Type Culture Collection under deposit number 40369.

17. Isolated DNA which hybridizes to chromosomal DNA from humans, gorilla, chimpanzee, orangutan, New World monkey, Old World monkey, mouse, rat, rabbit, goat, horse, cow and chicken.

18. Isolated DNA of Claim 17 which hybridizes to Y chromosome DNA from humans, gorilla, chimpanzee, orangutan, New World monkey, Old World monkey, mouse, rat, rabbit, goat, horse, and cow.

19. A DNA probe for the detection of human sex chromosomal anomalies, the probe comprising DNA which hybridizes to DNA sequence homologous to an interval of the normal Y chromosome designated in Figure 1.

20. A probe which detects the presence or absence of DNA sequences homologous to DNA sequences of the normal human Y chromosome, said probe comprising a vector having therein a restriction fragment of the normal human Y chromosome, or equivalent sequences.

21. A probe of Claim 20 comprising a plasmid having inserted at its HindIII site a restriction fragment which is an 0.9Kb HindIII DNA fragment of the Y chromosome, or equivalent sequences.

22. A probe of Claim 20 comprising a plasmid having inserted at its HindIII site a restriction fragment which is a 3.5Kb HindIII DNA fragment of Y chromosome DNA present in interval 1A2 of Figure 1, or equivalent sequences.

23. A probe of Claim 20 comprising a plasmid having inserted at its HindIII site a restriction fragment which is a 1.2 kb HindIII fragment of Y chromosome DNA present in interval 1A2 of Figure 1, or equivalent DNA sequences.

24. A probe of Claim 20 comprising a plasmid having inserted at its AccI and EcoRI sites a restriction fragment which is a 1.0Kb

EcoRI/TaqI fragment of the Y chromosome or equivalent sequences.

25. A probe of Claim 20 comprising a plasmid having inserted at its HindIII site a restriction fragment which is a 4.6Kb HindIII restriction fragment of the Y chromosome or equivalent sequences.

26. A method of detecting in a sample the presence of DNA sequences homologous to those in the normal Y chromosome, comprising the steps of: a. rendering DNA in the sample available for hybridization; b. contacting DNA in (a) with DNA probes which hybridize with DNA from at least one interval in the deletion map of the normal human Y chromosome represented in Figure 1, under conditions appropriate for hybridization of DNA in the sample with the DNA probes; and c. detecting the hybridization of DNA in the sample with the DNA probes.

27. A method of Claim 26 in which the DNA probes are labelled.

28. A method of Claim 27 in which the DNA probes are radioactively, enzymatically or chemically labelled.

29. A method of detecting the presence in human sex chromosomes of DNA sequences necessary and sufficient, in the absence of downstream mutations, for determining maleness in a human, comprising the steps of: a. contacting DNA from human sex chromosomes with DNA hybridization probes for all or a portion of interval 1A in the deletion map of the normal human Y chromosome represented in Figure 1, under conditions suitable for hybridization to occur; and b. detecting hybridization of the DNA from sex chromosomes with the DNA hybridization probes.

30. A method of diagnosing Turner's Syndrome in an individual, comprising: a. preparing a sample from the individual, so as to render DNA present in the sample available for hybridization; and -90¬

b. contacting the sample with at least one probe which is a DNA sequence which hybridizes with DNA, present in the distal segment of the short arm of the normal Y chromosome, whose absence has been shown to be specifically associated with the occurrence of Turner's Syndrome.

31. A method of Claim 30 wherein the probe is at least one DNA sequence which hybridizes with DNA from interval 1A1 of the deletion map of Figure 1.

32. Essentially pure polypeptide encoded by all or a portion of the nucleotide sequence of Figure 4.

33. An antibody reactive with a polypeptide of Claim 32.

34. An antibody reactive with a polypeptide encoded by a nucleotide sequence complementary to the nucleotide sequence of DNA which hybridizes to all or a portion of interval 1A of the normal Y Chromosome designated in Figure 1.

35. An antibody of Claim 34 in which the portion of interval 1A is interval 1A2.