Ring Y chromosome: molecular characterization by DNA probes

A young male with a karyotype of 46,X,+ mar is described. Physical mapping of the marker chromosome by using Y-specific single-copy or moderately repeated DNA sequences as molecular probes showed that, in addition to the heterochromatic part of the Yq, a considerable portion of the euchromatin in both Yp and Yq had been lost. These findings suggest that the marker chromosome is a ring Y, for the generally accepted model of ring formation implies breakages in both chromosome arms. The clinical features of the patient correlated well with the phenotypic changes expected from the loss of genetic material from the Y.     

Breakage of the human Y-chromosome short arm between two blocks of tandemly repeated DNA sequences

Y-chromosomal rearrangements, a common cause of sex reversal in man, frequently occur between two blocks of repeated DNA. Both blocks are composed of 20-kb tandemly repeated Y-chromosome-specific DNA sequences. They are located in the proximal portion of the Y short arm on a NotI restriction fragment of approximately 5.3 Mb and on an MluI fragment of approximately 5.5 Mb. Chromosome breaks positioned between the two blocks were detected in two of three 46,XY females with deletions of Yp and in five of six 46,XX males positive for the repeat sequences. The rearranged NotI fragments in the 46,XX males were 4.4 Mb and the MluI fragments were 2.0 Mb in length. This indicates that breaks occur within a small region of Yp defined by the two blocks of specific repeated DNA sequences. The region between the two blocks thus appears to be a focus of structural lability in the human Y chromosome.     

The role of the sex-determining region of the Y chromosome (SRY) in the etiology of 46,XX true hermaphroditism

Summary The syndrome of 46,XX true hermaphroditism is a clinical condition in which both ovarian and testicular tissue are found in one individual. Both Mullerian and Wolffian structures are usually present, and external genitalia are often ambiguous. Two alternative mechanisms have been proposed to explain the development of testicular tissue in these subjects: (1) translocation of chromosomal material encoding the testicular determination factor (TDF) from the Y to the X chromosome or to an autosome, or (2) an autosomal dominant mutation that permits testicular determination in the absence of TDF. We have investigated five subjects with 46,XX true hermaphroditism. Four individuals had a normal 46,XX karyotype; one subject (307) had an apparent terminal deletion of the short arm of one X chromosome. Genomic DNA was isolated from these individuals and subjected to Southern blot analysis. Only subject 307 had Y chromosomal sequences that included the pseudoautosomal boundary, SRY (sex-determining region of Y), ZFY (Y gene encoding a zinc finger protein), and DXYS5 (an anonymous locus on the distal short arm of Y) but lacked sequences for DYZ5 (proximal short arm of Y) and for the long arm probes DYZ1 and DYZ2. The genomic DNA of the other four subjects lacked detectable Y chromosomal sequences when assayed either by Southern blotting or after polymerase chain reaction amplification. Our data demonstrate that 46,XX true hermaphroditism is a genetically heterogeneous condition, some subjects having TDF sequences but most not. The 46,XX subjects without SRY may have a mutation of an autosomal gene that permits testicular determination in the absence of TDF.     

Three cases of 45,X/46,XYnf mosaicism

Summary Three patients with 45,X/46,XYnf mosaicism were investigated by Southern hybridization using both X- and Y-specific DNa probes. Our patients seem to be hemizygous for the X chromosomal loci tested. Single-copy and low-copy repeated Y chromosomal sequences assigned to the short arm, centromere, and euchromatin of the long arm have been detected in our patients, suggesting the Y chromosomal origin of the marker chromosome both in male and female cases studied. Densitometry of autoradiographs revealed a double dose of Yp-specific fragments of the DXYS1 locus. None of the patients tested showed either the 3.4- or the 2.1-kb Hae III malespecific repeated DNa sequences. It seems likely that the Ynf is a pseudodicentric chromosome with duplication of Yp and euchromatic Yq sequences, the Yq heterochromatin being lost. Our findings indicate structural heterogeneity of the marker chromosome and in addition provide further information on the relative position of DNa sequences detectected by DNA probes 50f2, M1A, and pDP105.     

Sequences from a family of bovine Y-chromosomal repeats

A 307-bp Sau3AI fragment previously cloned by deletion enrichment from the bovine Y chromosome was used to isolate a larger lambda EMBL3A genomic cattle clone. The whole 13-kb insert did not give a sex-specific pattern of hybridization to Southern blots of cattle DNA. Subclones from this phage, however, did show that this fragment had a Y-chromosomal origin. It was estimated that at least 40% of the cattle Y chromosome is composed of repeated sequences related to those within these subcloned fragments. Sequences within these subclones are male-specific or male-enriched also in sheep, goats, and deer. Comparison of cattle and sheep homologues of these sequences reveals that much greater amplification and rearrangement have occurred on the cattle Y chromosome than on the sheep Y. The apparent insertion of sequences into cattle Y-specific sequences relative to the sheep homologues suggests possible mechanisms for the evolution of the artiodactyl Y chromosome.     

The location of highly repetitious DNA in the somatic chromosomes of Drosophila melanogaster

In situ hybridization of Drosophila melanogaster somatic chromosomes has been used to demonstrate the near exact correspondence between the location of highly repetitious DNA and classically defined constitutive heterochromatin. The Y chromosome, in particular, is heavily labeled even by cRNA transcribed from female (XX) DNA templates (i.e., DNA from female Drosophila with 2 Xs and 2 sets of autosomes). This observation confirms earlier reports that the Y chromosome contains repeated DNA sequences that are shared by other chromosomes. In grain counting experiments the Y chromosome shows significantly heavier label than any other chromosome when hybridized with cRNA from XY DNA templates (i.e., DNA from male Drosophila with 1 X and 1 Y plus 2 sets of autosomes). However, the preferential labeling of the Y is abolished if the cRNA is derived from XX DNA. We interpret these results as indicating the presence of a class of Y chromosome specific repeated DNA in D. melanogaster. The relative inefficiency of the X chromosome in binding cRNA from XY and XYY DNA templates, coupled with its ability to bind XX derived cRNA, may also indicate the presence of an X chromosome specific repeated DNA.     

Characteristics of the distribution of DNA repetitive sequences in the sex chromosomes of 4 species of rodent

Four rodent species with very large heterochromatic regions on the sex chromosomes have been studied using in situ DNA/DNA hybridization techniques. Repetitious DNA fractions were obtained at C0t 0-0.01. Heterochromatic regions of X and X chromosomes of Cricetulus barabensis and Phodopus sungorus, and the heterochromatic long arm of the Y chromosome of Mesocricetus auratus do not contain disproportionately high amounts of repeated DNA sequences. Heterochromatic regions on sex chromosomes of Microtus subarvalis contain high amounts of repeated DNA sequences. Additional heterochromatic autosomal arms, a heterochromatic arm of the X chromosome, and a short arm of the Y chromosome of Mesocricetus auratus contain high amounts of repeated DNA sequences too.     

Y chromosomal DNA of Drosophila hydei

Six recombinant DNA clones are described, which are derived from the Y chromosome of Drosophila hydei. They reveal characteristic features of Y chromosomal DNA sequences. Three of the cloned inserts are Y-specific and are members of the same family of repeated sequences associated with the lampbrush loop-forming fertility gene "nooses" in the short arm of the Y chromosome. The other three cloned sequences are members of three different families of repeated sequences, but display a small amount of homology to one another and to the family of the nooses sequences. These three cloned sequences are found preferentially in the Y chromosome, but also in other chromosomal positions. The Y chromosomal copies are located in the short arm of the Y chromosome. The other copies are found in autosomal kinetochore-associated heterochromatin or, for one of the cloned sequences, in one band of the giant chromosome 4, in addition to the kinetochore heterochromatin.     

Organization and chromosomal specificity of autosomal homologs of human Y chromosome repeated DNA

The human Y chromosome contains a group of repeated DNA elements, identified as 3.4-kilobase pair (kb) fragments in Hae III digests of male genomic DNA, which contain both Y-specific and non-Y-specific sequences. We have used these 3.4-kb Hae III Y fragments to explore the organizational properties and chromosomal distribution of the autosomal homologs of the non-Y-specific (NYS) 3.4-kb Hae III Y elements. Three distinct organizations, termed domains, have been identified and shown to have major concentrations on separate chromosomes. We have established that domain K is located on chromosome 15 and domain D on chromosome 16 and suggested that domain R is on chromosome 1. Our findings suggest that each domain is composed of a tandemly arrayed cluster of a regularly repeating unit containing two sets of repeated sequences: one that is homologous to the NYS 3.4-kb Hae III Y sequences and one that does not cross-react with the 3.4-kb Hae III Y repeats. Thus, these autosomal repeated DNA domains, like their Y chromosome counterparts, consist of a complex mixture of repeated DNA elements interspersed among each other in ways that lead to defined periodicities. Although each of the three identified autosomal domains cross-reacts with 3.4-kb Hae III Y fragments purified from genomic DNA, the length periodicities and sequence content of the autosomal domains are chromosome specific. The organizational properties and chromosomal distribution of these NYS 3.4-kb Hae III homologs seem inconsistent with stochastic mechanisms of sequence diffusion between chromosomes.     

Cosmid clones derived from both euchromatic and heterochromatic regions of the human Y chromosome.

Clones containing sequences derived from the human Y chromosome have been isolated from cosmid libraries of a human-mouse hybrid cell line. These libraries were constructed in the new expression vectors Homer V and Homer VI. The collection of cosmids isolated is enriched for unique sequence DNA and only a few of the cosmids contain the tandemly repeated sequences which constitute a major portion of the Y chromosome. Three cosmids have been studied in detail. One cosmid shows extensive homology over at least 20 kb with the long arm of the X chromosome; this homology is outside the predicted homology region required for sex chromosome pairing. The other two clones contain unique sequences specific to the Y chromosome and both map to the heterochromatic region of the Y chromosome long arm.     

The identification of a repeated DNA sequence involved in the karyotype polymorphism of the human Y chromosome

We show that individual men are polymorphic for the amount of two different repeated DNA sequences. The amount of one of these sequences is proportional to the length of the brightly fluorescent heterochromatin on the Y chromosome. There are no detectable alterations in sequence between polymorphic individuals. Female DNA contains sequences complementary to those found on the Y, but at a much reduced level.     

Y-190, a DNA probe for the sensitive detection of Y-derived marker chromosomes and mosaicism

Summary A DNA probe (Y-190) is described that specifically hybridizes with repeated DNA sequences in the short arm of the human Y chromosome. The suitability of Y-190 to detect Y-derived DNA is shown in two patients with a 45,X/46,X+marker earyotype and in a third patient previously described as having a 45,X karyotype.     

The majority of the marker chromosomes in Japanese patients with stigmata of turner syndrome are derived from Y chromosomes

Summary DNA analyses of 41 individuals with stigmata of Turner syndrome and a 45,X/46,X+mar or 46,X+mar karyotype were carried out. Southern-blot analysis employing 17 Y-specific probes was used to determine whether the marker chromosome was Y-chromosomal in origin. Of the 41 DNA samples from these patients, 23 contained detectable Y-chromosomal DNA. Points of chromosome breakage were distributed over the entire length of the Y long arm. Three individuals, who carry different portions of the Y chromosome, had developed gonadoblastoma. GBY (the gonadoblastoma locus on the Y chromosome) is mapped proximal to DYS132, midway between the 13 Yq loci that we have studied. We also used a polymerase chain reaction technique that could detect 7 loci over the length of the Y chromosome. This technique may be useful for the rapid assessment of marker chromosomes, especially for evaluating the risk of gonadoblastoma.     

Mapping Simple Repeated DNA Sequences in Heterochromatin of Drosophila Melanogaster

Heterochromatin in Drosophila has unusual genetic, cytological and molecular properties. Highly repeated DNA sequences (satellites) are the principal component of heterochromatin. Using probes from cloned satellites, we have constructed a chromosome map of 10 highly repeated, simple DNA sequences in heterochromatin of mitotic chromosomes of Drosophila melanogaster. Despite extensive sequence homology among some satellites, chromosomal locations could be distinguished by stringent in situ hybridizations for each satellite. Only two of the localizations previously determined using gradient-purified bulk satellite probes are correct. Eight new satellite localizations are presented, providing a megabase-level chromosome map of one-quarter of the genome. Five major satellites each exhibit a multichromosome distribution, and five minor satellites hybridize to single sites on the Y chromosome. Satellites closely related in sequence are often located near one another on the same chromosome. About 80% of Y chromosome DNA is composed of nine simple repeated sequences, in particular (AAGAC)(n) (8 Mb), (AAGAG)(n) (7 Mb) and (AATAT)(n) (6 Mb). Similarly, more than 70% of the DNA in chromosome 2 heterochromatin is composed of five simple repeated sequences. We have also generated a high resolution map of satellites in chromosome 2 heterochromatin, using a series of translocation chromosomes whose breakpoints in heterochromatin were ordered by N-banding. Finally, staining and banding patterns of heterochromatic regions are correlated with the locations of specific repeated DNA sequences. The basis for the cytochemical heterogeneity in banding appears to depend exclusively on the different satellite DNAs present in heterochromatin.     

Organization and heterogeneity of sequences within a repeating unit of human Y chromosome deoxyribonucleic acid.

Fragments of 3.4 kilobases (kb) are released from DNA of human males, but not DNA of human females, by cleavage with restriction endonucleases HaeIII, EcoRI, or EcoRII. Most, if not all, reiterated DNA which is specific for the Y chromosome (it-Y DNA) is present within these male-specific 3.4-kb molecules. Although such 3.4-kb molecules are themselves localized to the Y chromosome, this is not true for all sequences within them. At least two distinguishable types of reiterated sequences are found within each 3.4-kb molecule. One type consists of at least two families which are highly reiterated and are not confined to the Y chromosome. The other type is composed of an estimated minimum of 39 families, each moderately reiterated and localized to the Y chromosome. Y-specific and non-Y-specific sequences are interspersed with one another in the same 3.4-kb molecule. In the average 3.4-kb molecule, three 800 nucleotide lengths of Y-specific sequences alternate with four 250 nucleotide lengths of non-Y-specific sequences. Since the total number of families of Y-specific sequences, calculated on the basis of reiteration frequency and total abundance in a male genome, greatly exceeds the number of Y -specific sequences present in a single 3.4-kb molecule, it necessarily follows that the population of these 3.4-kb molecules is heterogeneous.     

A Repeated Segment on the Mouse Y Chromosome Is Composed of Retroviral-Related, Y-Enriched and Y-Specific Sequences

We report the isolation and characterization of two recombinant clones containing DNA derived from the Y chromosome of the C57BL/10 inbred mouse strain. Both clones were isolated from a lambda phage library derived from a partial EcoRI digest of C57BL/10 male DNA using the murine retrovirus M720. Characterization of these clones showed they were derived from a repeated segment present on the C57BL/10J Y chromosome that contains sequences found elsewhere in the genome. In addition, one clone contained a sequence, designated YB10, that is unique to the Y chromosome and present in approximately 500 copies on the C57BL/10J Y chromosome. Analysis of Southern blots containing DNAs prepared from females and males of representative species from four subgenera of Mus probed with pYB10 and the 3'LTR from one of the Y-associated retroviruses (MuRVY) revealed that, with the exception of a single fragment observed in both female and male DNA of Mus saxicola, hybridization to pYB10 was observed only to male DNA of the species Mus spretus, Mus hortulanus, Mus musculus, Mus domesticus and Mus abbotti. In addition, the pattern and intensity of hybridization to YB10 and the MuRVY-LTR indicated that sequence of divergence was followed by amplification of Y chromosome sequences containing YB10 and MuRVY. The divergence and amplification occurred separately in each of the ancestral lineages leading to M. spretus, M. hortulanus, M. abbotti, M. musculus and M. domesticus. We suggest that acquisition and amplification of DNA sequences by the mammalian Y chromosome has contributed to its evolution and may imply that the mammalian Y chromosome is evolving at a faster rate than the rest of the genome.     

A human moderately repeated Y-specific DNA sequence is evolutionarily conserved in the Y chromosome of the great apes.

Evolutionary conservation of the human-derived moderately repeated Y-specific DNA sequence Y-190 (DYZ5) was investigated in the chimpanzee, orangutan, and gorilla. Southern blot analysis showed the presence of the sequence in the Y chromosome of all great apes. Pulsed-field gel electrophoresis and in situ hybridization revealed that the repeat is organized in one major block and confined to a small region of the Y chromosome of the three species. DYZ5 was assigned to the proximal short arm of the Y chromosome of the chimpanzee and orangutan and to the long arm of the Y chromosome of the gorilla. In light of its evolutionary conservation, DYZ5 may have an as yet undetermined structural function in the Y chromosome.     

Distribution and Evolution of Repeated Sequences in Genomes of Triatominae (Hemiptera-Reduviidae) Inferred from Genomic In Situ Hybridization

The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily.     

Sequence instability and functional inactivation of murine Y chromosomes can occur on a specific genetic background.

When the Y chromosome from Mus. poschiavinus (YPos) is backcrossed onto the C57BL/6J laboratory strain, testicular dysfunction occurs at high frequencies. When five different multicopy probes from the recombinationally suppressed region of the Y chromosome were used, genomic DNAs from sibling female progeny of C57BL/6J YPos males were found to contain YPos-specific sequences ranging from trace levels to levels consistent with an intact Y chromosome. Females with a high copy number of YPos-specific sequences had a karyotype of XYPos and were sterile. Females with trace levels of these sequences were XX and fertile. Repeated sequences in the testis-determining-region (Sxr) of inactive YPos chromosomes were unstable relative to sequences in non-Sxr regions. In contrast, the YPos chromosome was stable and functioned normally in other inbred laboratory strains such as 129/Sv. The frequency and extent of YPos chromosome instability increased with successive backcrosses from stable (129/Sv) to unstable (C57BL/6J) genetic backgrounds. Traces of YPos-specific sequences were first detected in N2 female offspring of F1 males. Therefore, sequences were deleted from YPos chromosomes in the F1 male germ line and were transmitted to N2 females; inactive YPos chromosomes (XYPos females) were first detected in the N3 generation. The mouse line being derived by backcrossing the YPos chromosome onto C57BL/6J inbred strains ended in the N7 generation, since all XYPos offspring were sterile. Even stable repeated sequences from the non-Sxr regions of their inactive YPos chromosomes were precisely rearranged in these N7 offspring at high frequencies. These data are consistent with hybrid dysgenesis in mammals.     

Distamycin A-DAPI banding of nonfluorescent Y (Ynf) chromosomes in 45,X/46,XYnf mosaicism

Summary The distamycin A-DAPI banding patterns of nonfluorescent, nonheterochromatic Y chromosomes (Y^nf) in two patients with 45,X/46,XY^nf mosaicism were investigated. In both cases moderately fluorescent bands were observed near the centromere and on the distal long arm of the Y^nf. These bands were similar to the centrometric band on normal Y chromosomes and support the hypothesis that the Y^nf is an isodicentric chromosome derived from the proximal portion of the Y chromosome.