Nucleotide sequence analysis of a mouse Y chromosomal DNA fragment containing Bkm and LINE elements

The strong suppression of crossing-over between the X and Y chromosomes permits rapid accumulation of repetitive sequences in the Y chromosome. To gain insight into the mechanism responsible for the sequence amplification, it is essential to characterize Y chromosomal repetitive sequences at the molecular level. Here, we report the entire nucleotide sequence (3,902bp) of AC11, a mouse sequence that is repeated 300 times in the Y chromosome. AC11 is AT rich (32.8% GC), and contains many short poly(A) sequences. In addition, it has Bkm and LINE sequences as well as a Y chromosome-specific sequence. The Bkm sequence consists of typical (GATA) and (GACA) repeating units, whereas the LINE sequence deviates considerably from other mouse LINE sequences (71–76% identity) and may be considered atypical. The Y chromosome-specific region seems to be unique and does not identify similar sequences in the GenBank library. The information obtained from the nucleotide sequence should form the foundation to study the evolutionary processes through which AC11-related sequences have accumulated in the mouse Y chromosome.     

Tissue specific methylation of human Y chromosomal DNA sequences

This report describes two moderately repetitive human Y chromosomal DNA sequences isolated from a flow sorted Y chromosonal library. These sequences are present in XY male and XY female DNAs but absent in XX male and XX female DNAs. Genomic Southern blot analysis against DNAs isolated from different tissues showed tissue specific DNA methylation patterns. In contrast to the 2.1 kb Hae III repeats which are hypomethylated in sperm DNA, the moderately repetitive sequences used in this study are highly methylated in sperm, less methylated in blood and brain and least methylated in placental DNA.     

Globally dispersed Y chromosomal haplotypes in wild and domestic sheep

To date, investigations of genetic diversity and the origins of domestication in sheep have utilised autosomal microsatellites and variation in the mitochondrial genome. We present the first analysis of both domestic and wild sheep using genetic markers residing on the ovine Y chromosome. Analysis of a single nucleotide polymorphism (oY1) in the SRY promoter region revealed that allele A-oY1 was present in all wild bighorn sheep (Ovis canadensis), two subspecies of thinhorn sheep (Ovis dalli), European Mouflon (Ovis musimon) and the Barbary (Ammontragis lervia). A-oY1 also had the highest frequency (71.4%) within 458 domestic sheep drawn from 65 breeds sampled from Africa, Asia, Australia, the Caribbean, Europe, the Middle East and Central Asia. Sequence analysis of a second locus, microsatellite SRYM18, revealed a compound repeat array displaying fixed differences, which identified bighorn and thinhorn sheep as distinct from the European Mouflon and domestic animals. Combined genotypic data identified 11 male-specific haplotypes that represented at least two separate lineages. Investigation of the geographical distribution of each haplotype revealed that one (H6) was both very common and widespread in the global sample of domestic breeds. The remaining haplotypes each displayed more restricted and informative distributions. For example, H5 was likely founded following the domestication of European breeds and was used to trace the recent transportation of animals to both the Caribbean and Australia. A high rate of Y chromosomal dispersal appears to have taken place during the development of domestic sheep as only 12.9% of the total observed variation was partitioned between major geographical regions.     

Restriction fragment length polymorphism of Azospirillum strains

Abstract: Total DNA of various Azospirillum strains representing different species was digested with restriction enzymes, Southern blotted and hybridized with four A. brasilense probes. Pairwise comparison of the conserved hybridization fragments was applied to calculate sequence divergence and to group the strains using the unweighted pair group method. The resulting dendrogram grouped the strains according to the known species indicating that the analysis of the restriction fragment length polymorphism is an useful tool for characterizing Azospirillum isolates.     

Restriction fragment length polymorphism (RFLP) of wild perennial relatives of soybean

Summary Total DNA from callus tissue of 28 accessions representing seven wild perennial Glycine species was compared using recombinant genomic probes derived from G. max, the soybean. Using two probes, we show that this molecular approach both confirms and extends the model for the taxonomic relationships between the species derived from morphological and cytogenetic data, and that it provides clear evidence that RFLP analysis of genomic sequences has the potential for revealing the derivation of the member species of the wild perennial Glycine taxon. Although, in this preliminary report, the sample size for each species is small, it is clear that the greatest between-accession variation occurs in G. tabacina (B₂B₂) and G. clandestine (A₁A₁), suggesting that these may be the taxa from which further speciation occurred in the subgenus.     

Analysis of the non‐recombining Y chromosome defines polymorphisms in domestic pig breeds: ancestral bases identified by comparative sequencing

Sequences from 20 amplicons representing nine different loci and 11369bp from the short arm of the pig Y chromosome were compared using pools of DNA from different European and Chinese breeds. A total of 33 polymorphic sites were identified, including five indels and 28 single nucleotide polymorphisms (SNPs). Three high frequency SNPs within the coding regions of SRY were further analysed across 889 males representing 25 European and 25 Asian breeds or Lines, plus a European Line of Meishan. Two haplotypes seen to be associated with ‘European’ or ‘Chinese’ origin in the initial SNP discovery phase were found to be the most common in their respective groups of breeds in a more detailed genotyping study. Two further SRY haplotypes are relatively rare. One was found exclusively within Tamworth, at low frequency in Retinto, and in three Chinese breeds (Huai, Sahwutou and Xiaomeishan). The other uncommon haplotype is found exclusively in Bamajiang, two further Chinese breeds (Hangjiang Black and Longling) and two European rare breeds (Mangalica and Linderödssvin), but appears based on comparison with other suids to represent an ancestral sequence.     

Restriction fragment length polymorphisms in satellite DNA distinguish chromosomal races of the white-footed mouse Peromyscus leucopus

We describe a polymorphism revealed by a high-copy-number tandem repeat which serves to distinguish most individuals sampled (96%) from two chromosomal races of Peromyscus leucopus. Classical morphology, allozymes, mtDNA, and rDNA have all failed to provide fixed markers which separate these two chromosomal races. Data from P. leucopus further documents the utility of DNA polymorphisms to establish the natal origin (DNA ‘zipcodes’) of populations or individuals.     

Restriction fragment length polymorphism markers in relation to quantitative characters

Summary A simple consideration of the potential of restriction fragment length polymorphism mapping, as a method of analysing the inheritance of quantitative characters, suggests that there are severe limitations to the utility of this approach.     

The sex-determining region Y (SRY) gene is mapped to p12-p13 of the Y chromosome in pig (Sus scrofa domestica) by in situ hybridization

The sex-determining region Y is a gene located in the distal portion of the short arm of human (SRY) and mouse (Sry) Y chromosomes and considered to be the best candidate for the testis determining factor (TDF/Tdy). The gene is believed to be the key factor in sex differentiation in mammals and is conserved across mammalian species. We report herein that the SRY/Sry gene has been assigned to pi 2-p13 on the short arm of the Y chromosome in pig by in situ hybridization. The result confirms interspecies conservation of this chromosomal segment in the evolution of mammalian chromosomes, and suggests further use of this gene probe in genomic studies in other mammals. The assignment of the Sry gene is the second physical gene mapping data available for the Y chromosome in pigs. Such data can be used in the effort of constructing the pig gene map and for further establishment of a comparison of sex chromosome morphology in different mammalian species concerning sex-specific and pseudoautosomal regions.     

Cloning of Y derived DNA sequences of bovine origin inEscherichia coli

We have constructed a partial library of Y chromosome derived DNA sequences of bovine origin inEscherichia coli. That, the recombinants arc Y derived and Y specific was ascertained by differential colony hybridization using male and female DNA probes. Out of 1000 recombinants analysed, 17 were found to be Y derived as well as Y specific and were of repetitive nature. Restriction analysis revealed that most of them had short DNA inserts.     

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.     

Restriction fragment length polymorphism detected by cDNA and genomic DNA clones in Stylosanthes

A DNA isolation method suitable for genomic library construction and RFLP analyses of the forage legume Stylosanthes was developed. Probes isolated using this method were used to investigate the feasibility of constructing RFLP-based genetic maps in this genus. Two hundred and seventy-one PstI genomic DNA and 134 cDNA clones were analysed against four Stylosanthes accessions, including two tetraploids and two diploids, with the use of two restriction enzymes, DraI and HindIII. The proportion of clones which detected single-copy sequences from the PstI genomic library was higher than that from the cDNA library, but the percentage of clones which detected low-copy sequences was doubled in the latter. There was no significant difference in the level of RFLPs detected by gDNA and cDNA probes, although the level of polymorphism was lower in the diploids. A large proportion of RFLPs seemed to have resulted from mutation/base substitution events, and this was especially the case in diploids.     

Restriction fragment polymorphism in mitochondrial DNA of Cryptococcus neoformans

The restriction patterns of mitochondrial DNA from 20 isolates of the two varieties of Cryptococcus neoformans were compared. The patterns exhibited extensive heterogeneity among the isolates regardless of their serotype or varietal status. Hybridizations with cloned fragments of the conserved cytochrome oxidase gene from Saccharomyces cerevisiae exhibited at least seven patterns among the 20 isolates. There were, however, similarities in the restriction patterns among isolates within the same serotype that were not shared by isolates of other serotypes. Intra-varietal similarities were observed in the restriction patterns among the isolates of C. neoformans var. neoformans which were not present in the restriction patterns among the isolates of C. neoformans var. gattii. Hybridization of some cloned mitochondrial DNA fragments to total DNA digests of various isolates revealed polymorphic as well as variety-specific patterns of homology. These findings agree with the antigenic heterogeneity among the isolates and support the current taxonomic classification of C. neoformans into two varieties.     

A conserved motif of vertebrate Y RNAs essential for chromosomal DNA replication

Noncoding Y RNAs are required for the reconstitution of chromosomal DNA replication in late G1 phase template nuclei in a human cell-free system. Y RNA genes are present in all vertebrates and in some isolated nonvertebrates, but the conservation of Y RNA function and key determinants for its function are unknown. Here, we identify a determinant of Y RNA function in DNA replication, which is conserved throughout vertebrate evolution. Vertebrate Y RNAs are able to reconstitute chromosomal DNA replication in the human cell-free DNA replication system, but nonvertebrate Y RNAs are not. A conserved nucleotide sequence motif in the double-stranded stem of vertebrate Y RNAs correlates with Y RNA function. A functional screen of human Y1 RNA mutants identified this conserved motif as an essential determinant for reconstituting DNA replication in vitro. Double-stranded RNA oligonucleotides comprising this RNA motif are sufficient to reconstitute DNA replication, but corresponding DNA or random sequence RNA oligonucleotides are not. In intact cells, wild-type hY1 or the conserved RNA duplex can rescue an inhibition of DNA replication after RNA interference against hY3 RNA. Therefore, we have identified a new RNA motif that is conserved in vertebrate Y RNA evolution, and essential and sufficient for Y RNA function in human chromosomal DNA replication.     

Two DNA sequences specific for the canine Y chromosome

Data are presented on the characterization of two nucleotide sequences found exclusively in the DNA of male dogs. In polymerase chain reactions (PCRs) of canine genomic DNA with a decanucleotide primer of arbitrary sequence (OP-W17), two nucleotide segments (650 and 990 bp) were amplified only from male samples, whereas a number of other fragments between 400 and 2500 bp in size were amplified from both male and female samples. The two male-specific segments were cloned and sequenced, and terminal 24mer oligonucleotide primer pairs were synthesized. PCR with these specific primer pairs resulted in amplification of the two male-specific sequences only from DNA samples of 34 male dogs; no product was amplified from 42 samples of females. A segment of the SRY gene previously localized on the Y chromosome could be amplified in DNA samples that had the two new sequences. Eco RI digested genomic male DNA when hybridized with the 650 bp or the 990 bp sequences, resulted in a single band for each on Southern analysis; DNA from females did not yield any bands. Comparisons between the two new sequences and the SRY gene segment revealed no homologies. We concluded that the two new sequences are specific for the canine Y chromosome and do not contain the short characterized segment of the SRY gene.     

No signature of Y chromosomal resemblance between possible descendants of the Cimbri in Denmark and Northern Italy

Two European populations are believed to be related to the ancient Germanic tribe Cimbri: one living in Northern Italy, the other living in Jutland, Denmark. The people called Cimbri are documented in the ancient Roman historical record. Arriving from the far north their movements can be tracked from successive battles with the Romans. The Cimbri finally entered Italy from the northeast and were defeated at Vercellae (present day Vercelli) in 101 BC by Gaius Marius and his professional legions. Classical sources from the first centuries AD relate the homeland of the Cimbri to the coasts around the Elb estuary (northern Germany) or specifically towards the north (Himmerland in northern Jutland). In the alpine parts of Veneto, northeast of the historical battlefield, local traditions dating back to late medieval time, identify a local population as Cimbri living in Terra dei Cimbri. They are considered the descendents of the Germanic combatants that fled the battlefield at Vercelli. As the defeated Cimbri that possibly fled to the mountains of Northern Italy most likely would have been male (warriors), the present study investigated the possible Y chromosomal diversity of the two present populations using microsatellite markers and single nucleotide polymorphisms. While Cimbri from Himmerland resembled their geographical neighbors from Denmark for the Y-chromosome markers, Cimbri from Italy were significantly differentiated both from Cimbri from Himmerland and from Danes. Therefore, we were not able to show any biological relationship for uniparentally transmitted markers.     

Age‐dependent DNA methylation patterns on the Y chromosome in elderly males

The Y chromosome, a sex chromosome that only exists in males, has been ignored in traditional epigenetic association studies for multiple reasons. However, sex differences in aging‐related phenotypes and mortality could suggest a critical role of the sex chromosomes in the aging process. We obtained blood‐based DNA methylation data on the Y chromosome for 624 men from four cohorts and performed a chromosome‐wide epigenetic association analysis to detect Y‐linked CpGs differentially methylated over age and cross‐validated the significant CpGs in the four cohorts. We identified 40–219 significant CpG sites (false discovery rate <0.05) with >82% of them hypermethylated with increasing age, which is in strong contrast to the patterns reported on the autosomal chromosomes. Comparing the rate of change in the Y‐linked DNA methylation across cohorts that represent different age intervals revealed a trend of acceleration in DNA methylation with increasing age. The age‐dependent DNA methylation patterns on the Y chromosome were further examined for their association with all‐cause mortality with results suggesting that the predominant pattern of age‐related hypermethylation on the Y chromosome is associated with reduced risk of death.     

Restriction fragment length polymorphism in the mitochondrial DNA of cloned cattle

The clonal origin of 4 Holstein bulls was determined by hybridization experiments with 2 different minisatellite probes, and all 4 animals showed identical genomic DNA fingerprints, hence confirming monozygosity. Extra-chromosomal differences were observed among these 4 Holstein bulls. Mitochondrial DNA restriction fragment length polymorphisms with restriction endonucleases Avall and Hhal sites were found, and these polymorphisms can be explained by the loss of a single site for each of these 2 enzymes. Since mitochondrial DNA are maternally transmitted, all 4 bulls would produce genetically equivalent spermatozoa and offspring. The combination of embryo cloning and specific cytoplasmic markers would provide an ideal system for the study of maternal cytoplasmic effects on quantitative traits.     

Age‐related DNA methylation on Y chromosome and their associations with total mortality among Chinese males

In view of the sex differences in aging‐related diseases, sex chromosomes may play a critical role during aging process. This study aimed to identify age‐related DNA methylation changes on Y chromosome (ChrY). A two‐stage study design was conducted in this study. The discovery stage contained 419 Chinese males, including 205 from the Wuhan‐Zhuhai cohort panel, 107 from the coke oven workers panel, and 107 from the Shiyan panel. The validation stage contained 587 Chinese males from the Dongfeng‐Tongji sub‐cohort. We used the Illumina HumanMethylation BeadChip to determine genome‐wide DNA methylation in peripheral blood of the study participants. The associations between age and methylation levels of ChrY CpGs were investigated by using linear regression models with adjustment for potential confounders. Further, associations of age‐related ChrY CpGs with all‐cause mortality were tested in the validation stage. We identified the significant associations of 41 ChrY CpGs with age at false discovery rate (FDR) <0.05 in the discovery stage, and 18 of them were validated in the validation stage (p < 0.05). Meta‐analysis of both stages confirmed the robust positive associations of 14 CpGs and negative associations of 4 CpGs with age (FDR<0.05). Among them, cg03441493 and cg17816615 were significantly associated with all‐cause mortality risk [HR(95% CI) = 1.37 (1.04, 1.79) and 0.70 (0.54, 0.93), respectively]. Our results highlighted the importance of ChrY CpGs on male aging.