Y-encoded, species-specific DNA in mice: evidence that the Y chromosome exists in two polymorphic forms in inbred strains

We have investigated the structure of the murine Y chromosome by first developing a novel method for specifically cloning Y-encoded DNA and then generating a library enriched for Y-specific DNA sequences. Three randomly chosen Y DNA clones were studied and found to share several interesting properties: all three are members of small Y-specific multisequence families; all three are mouse-specific; and all three probes detect Y-encoded restriction fragments that are polymorphic. Examination of polymorphic Y chromosome restriction fragments in male DNA from nine different inbred strains suggests that only two polymorphic forms of Y chromosomal DNA exist among inbred strains of mice.     

Linkage studies of polymorphic, repeated DNA sequences in centromeric regions of human chromosomes.

The DNA at human centromeric regions was characterized by using a repetitive sequence, 308, which localizes in situ exclusively to centromeres of all chromosomes. We previously noted that this sequence is enriched on chromosome 6 and has chromosome-specific organization on 6, 3, 7, 14, X, and Y. In addition to this basic organization, sequences homologous to 308 are polymorphic among normal individuals. The variants are transmitted in a Mendelian manner within a family. To determine the chromosome origin of the variants, we studied their linkage to markers of various chromosomes. Linkage analysis of one pedigree segregating two polymorphisms shows that the 2.6-kilobase (kb) BamHI and 2.6-kb TaqI fragments are linked to each other and to the HLA loci on chromosome 6. Data from another family shows that 2.8-kb TaqI, 4.0-kb TaqI, and 1.3-kb BamHI polymorphic fragments are linked and are probably near the Fy locus on chromosome 1. By dot blot analysis, we determined that the relative amount of these sequences in the genome is not measurably different between unrelated individuals. Thus, the polymorphisms represent changes in homologous 308 sequences on specific chromosomes and can be used as chromosome-specific markers. Linkage studies using polymorphisms of repeated sequences will be most useful within a kindred, especially from an inbred population, because polymorphic repeats of the same restriction size may be heterogeneous in origin.     

Molecular characterization of a bovine Y-specific DNA sequence conserved in taurine and zebu breeds.

The identification of new bovine male-specific DNA sequences is of great interest because the bovine Y chromosome remains poorly characterized in terms of physical and genetic maps. Since taurine and zebu Y chromosomes are structurally different, the identification of Y-specific sequences present in both sub-species is particularly important: these sequences are of evolutionary significance and can be broadly used for embryo sexing. In this work, we initially used the random amplified polymorphic DNA (RAPD) technique to search for male-specific sequences present as monomorphic markers in genomic DNA from zebu and taurine bulls. A male-specific RAPD band was found to be present and highly conserved in both sub-species, as demonstrated by Southern blotting, fluorescent in situ hybridization (FISH) and DNA sequencing. In a previous work, a pair of primers derived from this marker was successfully used in taurine and zebu embryo sexing.     

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.     

Excessive variation in Y chromosomal DNA in Rumex acetosa (Polygonaceae)

Rumex acetosa is one of the few angiosperms that possesses sex chromosomes. The same types of abundant repetitive sequences cover both heterochromatic Y chromosomes present in males. The aim of this study was to investigate genetic variation in paternally inherited Y chromosomal DNA and in maternally inherited cpDNA, and to find out whether the examined genomic regions are suited to a phylogeographic study in R. acetosa. DNA sequence polymorphisms present in the 850-bp heterochromatic segment on the Y chromosomes were compared to variation in the 409-bp long chloroplast section (trnL- trnF spacer) in R. acetosa originating from several European locations and from the Altai mountains in Russia. A great amount of genetic variation was detected within the Y chromosomal region while only four chloroplast genotypes were detected. Although the chloroplast haplotypes possessed some geographic pattern, no clear phylogeographic pattern was detected based on the variable Y chromosomes. The mean Y chromosomal nucleotide diversity among all samples equaled 6.6 %, and the mean proportion of polymorphic sites per individual equaled 8.2 % among SNP sites and 1.7 % among all sites investigated. The high number of substitutions detected in the Y chromosomal DNA shows that this heterochromatic sequence has a high mutation rate. The diversity pattern indicates that gene flow via pollen is extensive and it blurs any geographical pattern in the Y chromosomal variation. The high number of repeats and uncertainty concerning the extent of recombination between the two Y chromosomes impair the usability of the Y chromosomal segment for phylogeographic or population genetic studies.     

RAPD markers encoding retrotransposable elements are linked to the male sex in Cannabis sativa L.

Male-associated DNA sequences were analyzed in Cannabis sativa L. (hemp), a dioecious plant with heteromorphic sex chromosomes. DNA was isolated from male and female plants and subjected to random amplified polymorphic DNA analysis. Of 120 primers, 17 yielded 400 to 1500-bp fragments detectable in male, but not female, plants. These fragments were cloned and used as probes in gel-blot analysis of genomic DNA. When male and female DNA was hybridized with 2 of these male-specific fragments, MADC(male-associated DNA sequences in C. sativa)3 and MADC4, particularly intense bands specific to male plants were detected in addition to bands common to both sexes. The MADC3 and MADC4 sequences were shown to encode gag/pol polyproteins of copia-like retrotransposons. Fluorescence in situ hybridization with MADC3 and MADC4 as probes revealed a number of intense signals on the Y chromosome as well as dispersed signals on all chromosomes. The gel-blot analysis and fluorescence in situ hybridization results presented here support the hypothesis that accumulation of retrotransposable elements on the Y chromosome might be 1 cause of heteromorphism of sex chromosomes.     

Mouse minor satellite DNA genetically maps to the centromere and is physically linked to the proximal telomere.

As an adjunct to attempts to define functionally important sequences at human centromeres, we have undertaken a long-range physical analysis of these regions in the mouse. Mouse centromeres are usually situated very close to the chromosome ends and are closely associated with minor satellite sequences on the basis of cytological observations. Using pulsed-field gel electrophoresis we find that this satellite DNA is arranged as tandem arrays, predominantly uninterrupted by nonsatellite sequences. These arrays can be released largely intact by digestion with a range of enzymes that generally cleave frequently in non-satellite DNA. The restriction fragments carrying these arrays are polymorphic in size between inbred strains and provide direct markers for mouse centromeres. To illustrate the possible use of these polymorphic markers we have mapped a 1.3-Mb PvuII variant in a set of RI strains to the centromere of Chromosome 7. The minor satellite arrays are very close to the centromeric telomere and physical linkage with terminal repeat sequences can readily be detected, placing many minor satellite arrays on terminal restriction fragments smaller than 1 Mb. The apparent lack of any sizable amount of nonsatellite DNA between the minor satellite and the terminal repeat arrays indicates that many mouse chromosomes are truly telocentric.     

Co-amplification of tail-to-tail copies of MuRVY and IAPE retroviral genomes on the Mus musculus Y Chromosome

We have isolated a clone from a C57BL/6 genomic library that contains both part of the Y Chromosome-specific 8.7 kbp MuRVY genome (Hutchinson and Eicher, J. Virol. 63, 4043, 1989) and a full-length 8.3 kbp Intracisternal A Particle genome (IAPE-Y), in a tail-to-tail organization. Although IAPs are encoded by a disperse multigene family (∼1000 copies per haploid genome), we present evidence that a significant proportion of the IAP-related sequences are present on the Y Chromosome (Chr) and that a >25 kbp genomic sequence, which contains the two proviral genomes, has been amplified on the Y Chr. Two discrete amplified families of MuRVY retroviral genomes distinguishable by a polymorphic restriction site were detected, suggestive that amplification occurred in incremental stages. The presence of MuRVY-related DNA sequences, but absence of IAPE-Y-related DNA sequences in Mus spretus suggests that the IAPE-Y retrotransposition event occurred after the evolutionary divergence of the lineages leading to Mus musculus and Mus spretus, and that the amplification of MuRVY occurred independently in the two lineages. Received: 28 July 1995 / Accepted: 1 September 1995     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Characterization of a human Y chromosome Pvu II repeated sequence

The human Y chromosome carries numerous copies of a tandemly repeated Pvu II sequence, 2.4 kb long. These sequences are specific to humans, and are present in a much smaller amount in the DNA of females. They are localized on the long arm of the Y chromosome. We have compared this sequence with the Hae III 2.1 kb Y-specific repeated sequence, already described.     

Detection of male and female fetal DNA in maternal plasma by multiplex fluorescent polymerase chain reaction amplification of short tandem repeats

The purpose of this study was to develop a fluorescent polymerase chain reaction (PCR) assay for the detection of circulating fetal DNA in maternal plasma. Maternal DNA extracted from plasma samples of pregnant women at term and newborn DNA isolated from cord blood were used to genotype 12 mother/child pairs at nine different polymorphic short tandem repeat loci. Multiplex fluorescent PCR was used to detect fetus-specific alleles in the corresponding maternal plasma samples. Fetus-specific alleles were found in all maternal plasma samples studied. Using these polymorphic repeat sequences, every mother/child pair was informative in at least four of nine loci. Paternally inherited fetal alleles were detected in 84% of informative short tandem repeats. This approach may have implications for non-invasive prenatal diagnosis. Compared with other fetal DNA detection systems that use fetus-derived Y sequences to detect only male fetal DNA in maternal plasma, our proposed technique can be applied to both female and male fetuses.     

Characterization of a low copy repetitive element S232 involved in the generation of frequent deletions of the distal short arm of the human X chromosome.

There are several copies of related sequences on the distal short arm of the human X chromosome and the proximal long arm of the Y chromosome which were originally detected by cross hybridization with a genomic DNA clone, CRI-S232. Recombination between two S232-like sequences flanking the steroid sulfatase locus has been shown to cause frequent deletions in the X chromosome short arm, resulting in steroid sulfatase deficiency. We now report the characterization of several S232-like sequences. Restriction mapping and sequence analysis show that each S232 unit contains 5 kb of unique sequence in addition to two elements, RU1 and RU2, composed of a variable number of tandem repeats. RU1 consists of 30 bp repeating units and its length shows minimal variation between individuals. The RU2 elements in the hypervariable S232 loci on the X chromosome consist of repeating sequences which are highly asymmetric, with about 90% purines and no C's on one strand. The X-derived RU2 elements range from 0.6 kb to over 23 kb among different individuals, accounting entirely for the observed polymorphism at the S232 loci. Although the repeating units of the RU2 elements in the nonpolymorphic S232 loci on the Y chromosome share high sequence homology with those on the X chromosome, they exhibit much higher intrarepeat sequence variation. S232 homologous sequences are found in great apes, old world and new world monkeys. In chimpanzees and gorillas the S232-like sequences are polymorphic in length.     

豚鼠基因组26个多态性微卫星标记的筛选

目的筛选豚鼠基因组的多态性微卫星标记,为豚鼠遗传质量控制及基因定位等工作奠定基础。方法采用磁珠富集法和豚鼠基因组数据库筛选法获取微卫星位点序列,通过分析和初步筛选,挑选部分候选位点,根据其序列设计引物,对5种不同来源的豚鼠基因组DNA标本进行PCR扩增,以期获得多态性分子标记。结果本实验采用磁珠富集法共获得微卫星序列304个,设计引物125对,最终获得多态性位点1个,暂未发现多态性的特异性位点17个;用数据库筛选法共获得微卫星序列292个,设计并合成相应引物178对,最终发现多态性位点25个,暂未发现多态性的特异性位点28个。结论本实验获得26个多态性微卫星标记,45个潜在的候选标记,为微卫星标记在豚鼠遗传质量监测及突变基因定位等工作的应用奠定了基础。     

DNA variation in a 5-Mb region of the X chromosome and estimates of sex-specific/type-specific mutation rates.

We describe a new approach for the study of human genome variation, based on our solid-phase fluorescence chemical mismatch-cleavage method. Multiplex screening rates >/=80 kb/36-lane gels are achieved, and accuracy of mismatch location is within +/-2 bp. The density of differences between DNA from any two humans is sufficiently low, and the estimate of their position is accurate enough, to avoid sequencing of most polymorphic sites when defining their allelic state. Furthermore, highly variable sequences, such as microsatellites, are distinguished easily, so that separate consideration can be given to loci that do and do not fit the definition of infinite mutation sites. We examined a 5-Mb region of Xq22 to define the haplotypes of 23 men (9 Europeans, 9 Ashkenazim, and 5 Pygmies) by reference to DNA from one Italian man. Fifty-eight 1.5-kb segments revealed 102 segregating sites. Seven of these are shared by all three groups, two by Pygmies and Europeans, two by Pygmies and Ashkenazim, and 19 by Ashkenazim and Europeans. Europeans are the least polymorphic, and Pygmies are the most polymorphic. Conserved allelic associations were recognizable within 40-kb DNA segments, and so was recombination in the longer intervals separating such segments. The men showed only three segregating sites in a 16.5-kb unique region of the Y chromosome. Divergence between X- and Y-chromosome sequences of humans and chimpanzees indicated higher male mutation rates for different types of mutations. These rates for the X chromosomes were very similar to those estimated for the X-linked factor IX gene in the U.K. population.     

Homologies between X and Y chromosomes detected by DNA probes: localisation and evolution.

We have isolated and characterized DNA probes that detect homologies between the X and Y chromosomes. Clone St25 is derived from the q13-q22 region of the X chromosome and recognizes a 98% homologous sequence on the Y chromosome. Y specific fragments were present in DNAs from 5 Yq-individuals and from 4 out of 7 XX males analysed. An X linked TaqI RFLP is detected with the St25 probe (33% heterozygosity) which should allow one to establish a linkage map including other polymorphic X-Y homologous sequences in this region and to compare it to a Y chromosome deletion map. Probe DXS31 located in Xp223-pter detects a 80% homologous sequence in the Y chromosome. The latter can be assigned to Yq11-qter outside the region which contains the Y specific satellite sequences. ACT1 and ACT2, the actin sequences present on the X and Y chromosomes respectively, have been cloned. No homology was detected between the X and Y derived fragments outside from the actin sequence. ACT2 and the Y specific sequence corresponding to DXS31 segregate together in a panel of Y chromosomes aberrations, and might be useful markers for the region important for spermatogenesis in Yq. Various primate species were analysed for the presence of sequences homologous to the three probes. Sequences detected by St25 and DXS31 are found only on the X chromosome in cercopithecoidae. The sequences which flank ACT2 detect in the same species autosomal fragments but no male specific fragments. It is suggested that the Y chromosome acquired genetic material from the X chromosome and from autosomes at various times during primate evolution.     

Multiple forms of male-specific simple repetitive sequences in the genusMus

Summary Previous reports indicate that in laboratory strains of mice, males are distinct from females in possession of repetitive DNA, notably devoid of Eco RI and Hae III sites and rich in the simple tetranucleotides GATA/GACA. We report here that such sequences originated in an ancestor common to laboratory mice,Mus hortulanus, M. spretus, and possibly alsoM. cookii. Interestingly, other male-specific satellite sequences were detected inM. caroli, M. cookii, M. saxicola, andM. minutoides. This novel satellite is also likely to be composed of simple repetitious sequences, but does not contain GATA and GACA. Thus, the Y chromosome appears to contain a disproportionately large amount of simple repetitious DNA. An attractive explanation for these results is that long tandem arrays of simple repeated sequences are generated at high frequency throughout the genome and that they are retained for a longer time on the Y chromosome due to the absence of homologous pairing at meiosis.     

Actin-like sequences are present on human X and Y chromosomes.

The human genome contains greater than 20 actin-related sequences, six of which at least are expressed as protein. We have shown by blot hybridization the presence of actin-like sequences on both the X and the Y chromosomes. These sequences can be detected in HindIII digests of genomic DNA, using as probe cDNA clones corresponding to human alpha skeletal actin or to a hamster (beta or gamma) cytoskeletal actin; they show more homology to the latter probe. The actin probes also detect a polymorphic DNA fragment showing autosomal inheritance with a frequency for the major allele of 0.55 in the population studied. The X-linked actin sequence has been assigned to a centromeric region between Xp11 and Xq11 by hybridization to DNAs from a panel of human-mouse hybrid cell lines, and thus lies outside the postulated region of homology between the X and Y chromosomes. The Y-linked actin sequence can serve as a marker to analyse anomalies of sex determination or of gametogenesis in man. It was found in all XY males studied but was absent from the genomic DNA of four unrelated 'XX male' subjects and two XX hermaphrodites. This shows that the region of chromosome Y which contains the actin sequence is not translocated onto the X chromosome (or onto autosomes) in these patients.     

A recent insertion of an alu element on the Y chromosome is a useful marker for human population studies

A member of the Alu family of repeated DNA elements has been identified on the long arm of the human Y chromosome, Yq11. This element, referred to as the Y Alu polymorphic (YAP) element, is present at a specific site on the Y chromosome in some humans and is absent in others. Phylogenetic comparisons with other Alu sequences reveal that the YAP element is a member of the polymorphic subfamily-3 (PSF-3), a previously undefined subfamily of Alu elements. The evolutionary relationships of PSF-3 to other Alu subfamilies support the hypothesis that recently inserted elements result from multiple source genes. The frequency of the YAP element is described in 340 individuals from 14 populations, and the data are combined with those from other populations. There is both significant heterogeneity among populations and a clear pattern in the frequencies of the insertion: sub-Saharan Africans have the highest frequencies, followed by northern Africans, Europeans, Oceanians, and Asians. An interesting exception is the relatively high frequency of the YAP element in Japanese. The greatest genetic distance is observed between the African and non-African populations. The YAP is especially useful for studying human population history from the perspective of male lineages.      

New, Male-Specific Microsatellite Markers from the Human Y Chromosome

Seven novel microsatellite markers have been developed from a new cosmid library constructed from flow-sorted human Y chromosomes. These microsatellites are tetranucleotide GATA repeats and are polymorphic among unrelated individuals. Five of the seven markers are male-specific, with no PCR product being generated from female DNA. One marker produces male-specific, polymorphic PCR products but occasionally produces a much larger, invariant product from female DNA. The remaining marker is polymorphic in both males and females with many shared alleles between the sexes. This report of six new, male-specific markers doubles the number of tetranucleotide markers that are currently available for the human Y chromosome. These new markers will be valuable where nonrecombining, gender-specific DNA markers are desired, including forensic investigations as well as studies of populations and their evolutionary histories.     

Isolation of recombinant bacteriophage containing male-specific mouse DNA

The mammalian Y chromosome is an isolated piece of genetic material that directs sexual determination and gametogenesis. Very little is understood about the mechanism whereby the Y chromosome carries out these functions. Also, there is a severe lack of genetic markers on this chromosome. In order to understand the structure and function of the Y chromosome at the level of its DNA sequences and to provide genetic markers, we are isolating clones of DNA whose sequences are found primarily in DNA from male mice. To this end, we have developed a procedure for the identification of such clones. Application of this screening procedure to a lambda library derived from mouse sperm DNA has yielded 12 distinct clones, part of whose sequences are present predominantly in male DNA. Besides this DNA, they also contain other sequences that are shared with female DNA. These clones are either derived from the Y chromosome or they represent autosomal sequences specifically amplified during male development.