Sex-specific DNA in reptiles with temperature sex determination

Banded krait minor ("Bkm") satellite DNA, originating in the W-chromosome of the snake Bungarus fasciatus, has been found in the genome of diverse eukaryotic species including fruit fly, quail, and horse. Concentrations of Bkm have been found in the presumptive W-chromosome of snakes with isomorphic sex chromosomes and in the male-determining region of the Y-chromosome in mouse and man. We therefore asked whether Bkm-related DNA might be present in quantitative excess in DNA from males or females in two related species of sea turtle, Chelonia mydas, in which sex is determined by the temperature of the incubating egg, and Lepidochelys kempi, in which the critical sex-determining temperature has recently been described. Filter hybridization with the Bkm 2(8) probe revealed male-specific fragments in both species; female-specific fragments were also revealed in C. mydas. Sex-specific DNA sequences in temperature-sex-determined species such as Kemp's ridley and the green turtle were unexpected, but could be explained if there were an underlying genetic mode of sex determination in these animals, or alternatively, if temperature-influenced sex determination involved structural modifications in DNA adjacent to, or directly concerned with, the sex-determining genes. If these results are confirmed across a broader sample of sea turtles, the techniques described in this paper might be used routinely to identify gener in the young of these endangered animals, in which male and female are grossly indistinguishable.     

The organization of the evolutionarily conserved GATA/GACA repeats in the mouse genome

Simple repeated GATA and GACA sequences which were originally isolated from sex-specific snake satellite DNA have been found subsequently in all eukaryotes studied. The organization of these sequences within the mouse genome was investigated here by using synthetic oligonucleotide probes as a novel tool in comparison with conventional hybridization probes. Southern blot hybridization showed sex-specific patterns with both the (GATA)₄ and (GACA)₄ oligonucleotide probes, as previously described with conventional probes. The quantitative analysis of two mouse DNA phage libraries and of 25 isolated GATA-positive phage clones revealed intensive interspersion of GATA sequences with GACA, and with other repetitive and single-copy sequences. Ubiquitous interspersion and homogeneous genomic distribution of GATA and GACA sequences were confirmed by hybridization in situ of the oligonucleotide probes to metaphase chromosomes. The lengths of the GATA and GACA stretches were found to vary considerably in the individual phage clones. DNA inserts from 20 phages were assigned to autosomes and sex chromosomes and three genomic fragments were found to be confined to the Y chromosome. The organization of GATA and GACA sequences is discussed in the context of their evolutionary potential and possible conservation mechanisms.     

Bkm satellite DNA and ZFY in the coral reef fish Anthias squamipinnis.

We studied DNA from the protogynous sex-changing fish Anthias squamipinnis to evaluate the recent observation that male-specific bands are identified after hybridization with Bkm, a probe originating in the W chromosome of the snake Bungarus fasciatus. Sex-specific hybridization would imply modification of DNA structure during the sex-changing process. No sex-specific Bkm fragments were identified in our study, after digestion of DNA from 15 males and 11 adult females, despite the use of 12 different restriction enzymes. However, hybridization with Bkm did produce a distinct fingerprint pattern, similar to the fingerprint patterns described for other species after hybridization with GATA (GACA) type probes. In other experiments, the pDP1007 probe, which identifies the ZFY gene in the male-determining region of the human Y chromosome, generated identical hybridization patterns in DNA from males and females of A. squamipinnis and estimation of DNA mass by flow cytometry revealed identical genome sizes.     

Conserved repetitive DNA sequences (Bkm) in normal equine males and sex-reversed females detected by in situ hybridization

In situ hybridization with a cloned banded krait sex-specific repetitive DNA probe (Bkm) indicates a high concentration of Bkm sequences on the horse Y chromosome in both normal XY males and XY sex-reversed females. Lesser, but still significant, concentrations of Bkm sequences were mapped to horse chromosomes 3, 4, and 30.     

Microsatellite fingerprinting in the genus Phaseolus.

The genetic variability of the genus Phaseolus was investigated by nonradioactive DNA fingerprinting. The simple repetitive sequences (GATA)4, (GACA)4, (CAC)5, and (CA)8 were used as probes to differentiate 18 species comprised of 90 genotypes. (GATA)4, (CAC)5, and (CA)8 could be detected in the genome of nearly all species, while the (GACA)4 motif occurred only in 13 species. Almost all fragments that hybridized with (GACA)4 also hybridized with (GATA)4. All but two cultivars of Phaseolus vulgaris, P. lunatus, P. acutifolius, and P. polyanthus showed specific banding patterns with (GATA)4. The other repetitive motifs revealed only limited or no intraspecific variation. In P. vulgaris, two group-specific patterns were found with (GATA)4, giving further evidence for a Middle American and an Andean origin of the P. vulgaris genotypes. The high intraspecific pattern variation that was revealed with (GATA)4 in the predominantly self-pollinating species P. vulgaris and P. lunatus can probably be explained by there being at least two primary centres of domestication and, hence, genetic diversification. In cross-pollinating species (e.g., P. coccineus), the observed intraspecific variation was, surprisingly, rather low. The present study shows that DNA fingerprinting with microsatellites successfully distinguishes among gene pools, cultivars, and, in some cases, among individuals.     

Genetic variation in parthenogenetic Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca, L. unisexualis) analyzed by DNA fingerprinting

Multilocus DNA fingerprinting has been used to study the variability of some mini- and microsatellite sequences in parthenogenetic species of Caucasian rock lizards of the genus Lacerta (L. dahli, L. armeniaca and L. unisexualis). We demonstrate that these clonally reproducing lizards possess species-specific DNA fingerprints with a low degree of intra- and interpopulation variation. Mean indices of similarity obtained using M13 DNA, (GACA)4 and (TCC)50 as probes were 0.962 and 0.966 in L. dahli and L. armeniaca, respectively. The mean index of similarity obtained using M 13 and GATA probes in L. unisexualis was estimated to be 0.95. However, despite the high degree of band-sharing, variable DNA fragments were revealed in all populations with the microsatellite probes. An particularly high level of variability was observed for (TCC)n microsatellites in populations of L. unisexualis. In fact TCC-derived DNA fingerprints were close to being individual-specific, with a mean index of similarity of 0.824. Fingerprint analysis of parthenogenetic families of L. armeniaca showed that all maternal fragments were inherited together by the progeny, and no differences in fingerprint patterns were observed. On the other hand, while identical DNA fingerprints were obtained from L. unisexualis families with M13 and (GATA)4 probes, use of the (TCC)50 probe revealed remarkable intrafamily variation in this species. It is assumed that the genetic heterogeneity observed in parthenogenetic populations may be explained, at least in part, by the existence of genetically unstable microsatellite loci. Our data serve to illustrate processes of spontaneous mutagenesis and the initial stages of clonal differentiation in natural populations of the lizard species studied.     

Genome-wide analysis of Bkm sequences (GATA repeats): predominant association with sex chromosomes and potential role in higher order chromatin organization and function

MOTIVATION: Bkm (Banded krait minor) satellite DNA sequences (GATA repeats) have been shown to be associated with the sex determining chromosomes of various eukaryotes and have been implicated in the evolution and differentiation of sex chromosomes in snakes. The objective of the study is to analyze the GATA repeats of human genome specifically, the Y-chromosome, and other model organisms to understand the possible function and potential role in higher order chromatin organization. RESULTS: Our extensive analysis of GATA repeats in the prokaryotic and eukaryotic genomes, which have been completely sequenced so far, has revealed that GATA repeats are absent in prokaryotes and have been gradually accumulated in higher organisms during the course of evolution. In human, the Y-chromosome has the highest GATA repeat density, which predominantly exists in the Yq centromeric region. Generally, occurrence of repeats in the genomes decreases steadily as the length of the repeat increases. In contrast, we report, that the occurrence of GATA repeats increases as the length of the repeat increases from six tandem repeats onwards and peaks at (GATA)(10-12). This has not been observed with any other simple repeat. Distribution of (GATA)(10-12) along the chromosome and their close proximity to Matrix Associated Regions (GATA-MAR) suggests that it may be demarking chromatin domains for a coordinated expression of genes residing in these domains.     

Heterochromatin and microsatellites detection in karyotypes of four sea turtle species: Interspecific chromosomal differences

The wide variation in size and content of eukaryotic genomes is mainly attributed to the accumulation of repetitive DNA sequences, like microsatellites, which are tandemly repeated DNA sequences. Sea turtles share a diploid number (2n) of 56, however recent molecular cytogenetic data have shown that karyotype conservatism is not a rule in the group. In this study, the heterochromatin distribution and the chromosomal location of microsatellites (CA)_n, (GA)_n, (CAG)_n, (GATA)_n, (GAA)_n, (CGC)_n and (GACA)_n in Chelonia mydas, Caretta caretta, Eretmochelys imbricata and Lepidochelys olivacea were comparatively investigated. The obtained data showed that just the (CA)_n, (GA)_n, (CAG)_n and (GATA)_n microsatellites were located on sea turtle chromosomes, preferentially in heterochromatic regions of the microchromosomes (mc). Variations in the location of heterochromatin and microsatellites sites, especially in some pericentromeric regions of macrochromosomes, corroborate to proposal of centromere repositioning occurrence in Cheloniidae species. Furthermore, the results obtained with the location of microsatellites corroborate with the temperature sex determination mechanism proposal and the absence of heteromorphic sex chromosomes in sea turtles. The findings are useful for understanding part of the karyotypic diversification observed in sea turtles, especially those that explain the diversification of Carettini from Chelonini species.     

Bkm sequences are polymorphic in humans and are clustered in pericentric regions of various acrocentric chromosomes including the Y

Summary Probes of uncloned Bkm satellite DNA and a Drosophila clone 2(8), consisting mainly of GATA repeasts related to a major sequence component in Bkm, have been used to probe Southern blots of human male and female DNAs obtained from a Caucasian and an Australian aboriginal population and to human chromosomes in situ. Hybridization was observed to a distinct and an indistint series of bands against a smeared background. The same distinct bands are identified in the DNA samples with both probes, but are most readily detected using the uncloned Bkm probe. Most restriction bands are common to both populations and some are polymorphic. However, certain bands appear to be characteristic of the Australian aboriginal samples. There are no distinct sex-linked patterns. However all of the small acrocentric human chromosomes, including the Y chromosome show hybridization to uncloned Bkm in situ.     

Genomic variation in parthenogenetic lizard Darevskia armeniaca: evidence from DNA fingerprinting data

Microsatellites, or short tandem repeats, are abundant across genomes of most organisms. It is evident that the most straightforward and conclusive way of studying mutations in microsatellite-containing loci is to use clonally transmitted genomes or DNA sequences inherited in multigeneration pedigrees. At present, little is known about the origin of genetic variation in species that lack effective genetic recombination. DNA fingerprinting in 43 families of the parthenogenetic lizard species Darevskia armeniaca (131 siblings), using (GACA)(4), (GGCA)(4), (GATA)(4), and (CAC)(5) probes, revealed mutant fingerprints in siblings that differed from their mothers in several restriction DNA fragments. In some cases, the mutant fingerprints detected in siblings were also found in population samples. The mutation rate for new restriction fragment length estimated by using multilocus probes varied from 0.8 x 10(-2) to 4.9 x 10(-2) per band/per sibling. Probably, the most variations detected as restriction fragment length polymorphism have germ-line origin, but somatic changes of (CAC)(n) fingerprints in adult lizards were also observed. These results provide new evidence of existing unstable regions in genomes of parthenogenetic vertebrate animals, which provide genetic variation in unisexual populations.     

Banded krait minor satellite (Bkm) contains sex and species-specific repetitive DNA

A novel class of repetitive DNA was isolated from a Bkm DNA library by exclusion hybridization. This sequence was mapped to the short arm of the W chromosome of banded krait, Bungarus fasciatus. Southern blot hybridization showed that these sequences are sex and species specific. Sequence analysis of a 206 bp long clone, BR87, revealed the presence of a tandem array of two internal repeat units of 18–19 bp alternating with each other with a gap of 1,2 or 3 nucleotides. To our knowledge, this is the first report of an exclusively W chromosome-and species-specific repeat isolated from any reptile. The functional significance of this sequence based on its organisation is discussed.     

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross- hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.      

Anchored simple-sequence repeats as primers to generate species-specific DNA markers in Lolium and Festuca grasses

Simple-sequence repeats (SSRs) comprising three tetranucleotide repeat sequences with two-base ’anchors’, namely 5′-(AGAC)₄GC, 5′-AC(GACA)₄ and 5′-(GACA)₄GT, were used in PCR reactions as primers to develop inter-SSR DNA fingerprints of the outbreeding grass species Lolium multiflorum, L. perenne, Festuca pratensis and F. arundinacea. Each species was represented by DNA samples from 3 to 6 varieties. In all four species distinctive species-specific DNA profiles were produced that were common across a number of varieties despite their diverse origin. While the fingerprints of the two ryegrasses, L. multiflorum and L. perenne, were the most similar, a number of inter-SSR DNA markers were generated that enabled them to be distinguished from each other. Some slight variations were found between varieties, which provided putative variety-specific markers for cultivar identification. In addition, variations in the DNA profiles of the genotypes of L. multiflorum and F. pratensis were examined, and the results showed that variety-specific fingerprints are integrated patterns made up from the profiles of individual genotypes. Amongst the primers used, AC(GACA)₄ generated the best distinction between Lolium and Festuca individuals and provides an effective new tool for genome identification. A number of species-discriminating sequences, ranging in size between 550 bp and 1,600 bp, were cloned: three clones for F. pratensis, one clone for L. multiflorum and one clone for F. arundinacea. A F. pratensis fragment pFp 78H582 was sequenced. Southern hybridization confirmed the presence of this fragment in F. arundinacea (which contains one genome of F. pratensis), but no homology was found with L. multiflorum. However, a F. arundinacea clone amplified with (GACA)₄GT, pFa 104H1350, was found to be unique to the F. arundinacea genome. Received: 23 June 1999 / Accepted: 27 August 1999     

Molecular genetics of sex determination in channel catfish: studies on SRY, ZFY, Bkm, and human telomeric repeats.

In amniotes, the banded krait minor (Bkm) minisatellite (GATA), the human telometric sequence (TTAGGG)7, and the Y-specific genes, ZFY and SRY, are associated with a particular sex. These sequences were studied in the channel catfish, Ictalurus punctatus. However, none was sex-specific in catfish; homologs of each were present in males and females. Our data suggest that components of mammalian sex-determining systems may be widespread and shared among the vertebrates in general. Whether those components are involved in sex determination in lower vertebrates or merely represent evolutionary precursors of sex-determining factors in amniotes remains to be determined.     

Hypervariable Bkm DNA Loci in a Moth, Ephestia kuehniella : Does Transposition Cause Restriction Fragment Length Polymorphism?

Bkm sequences, originally isolated from snake satellite DNA, are a component of eukaryote genomes with a preferential location on sex chromosomes. In the Ephestia genome, owing to the presence of only a few Bkm-positive BamHI restriction fragments and to extensive restriction fragment length polymorphisms between and within inbred strains, a genetic crossbreeding analysis was feasible. No sex linkage of Bkm was detected. Instead—depending on the strain—two or three autosomal Bkm DNA loci were identified. All three loci were located on different chromosomes. Fragment length and transmission of fragments was stable in some crosses. In others, changes in fragment length or loss of the Bkm component were observed, probably depending on the source strain of the fragment. The anomalous genetic behaviour is best accounted for by the assumption that Bkm sequences are included in mobile genetic elements.     

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.     

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.     

A Y-chromosomal DNA fragment is conserved in human and chimpanzee.

A human male-specific Y-chromosomal DNA fragment (lambda YH2D6) has been isolated. By deletion-mapping analysis, 2D6 has been localized to the euchromatic portion of the long arm (Yq11) of the human Y chromosome. Among great apes, this fragment was found to be conserved in male chimpanzee but was lacking in male gorilla and male orangutan. No homologous fragments were detected in females of orangutan, gorilla, chimpanzee, or human. Nucleotide sequence analysis indicated the presence of partial-Alu-elements and of sequences similar to the GATA repeats of the snake Bkm sequence.