Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences

Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically demonstrable sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)_n/(gaca)_m are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)₄ probe. The (GATA)₄ oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In contrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)₄. Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)₄ probe.by E.R. SchmidtDedicated to Professor Karl Sperling on the occasion of his 50th birthday     

Simple GA C T A repeats characterize the X chromosomal heterochromatin of Microtus agrestis,European field vole (Rodentia,Cricetidae)

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have identified two prominent satellite bands of 2.0 and 2.8 kb in length after HaeIII and HinfI restriction enzyme digestion of genomic DNA, respectively. These satellites are located on the heterochromatic long arm of the X chromosome as shown using Microtus x mouse somatic cell hybrids. By in-gel hybridization with oligonucleotide probes, the organization of the two satellites was studied: among the many copies of the simple tandem tetranucleotide repeat GATA are interspersed rare single GACA tetramers. One of the satellites also harbours related GGAT simple tandem repeats. In situ hybridizations with plasmid-carried or oligonucleotide GA _C ^T A probes show clustered silver grains on the long and short arm of the X chromosome. Interspersion of differently organized (GATA)_n elements is also demonstrable in the autosomal complement and on the Y chromosome. These results are discussed in the context of the evolution of vertebrate sex chromosomes in relation to heterochromatin and simple repetitive DNA sequences.     

Heterogeneities in the distribution of (GACA)n simple repeats in the karyotypes of primates and mouse

Summary Tandemly organized simple repetitive sequences are widespread in all eukaryotes. The organization of the simple tetrameric (GACA)_n sequences at chromosomal loci has been investigated using in situ hybridization with chemically pure oligonucleotide probes. Both biotin- and digoxigenin-attached (GACA)₄ probes reveal specific hybridization signals over the short arms of all acrocentric autosomes in man. In the other examined primates the NOR-bearing autosomes could be detected by in situ hybridization with (GACA)₄, and a major concentration of the GACA simple repeats could be observed on the Y chromosome in the gibbon and mouse; the hybridization site in the gibbon Y chromosome coincides particularly with the silver-stainable NOR. In the past, accumulations of (GACA)_n sequences were demonstrated mainly on vertebrate sex chromosomes. Therefore, the organization of GACA simple sequences is discussed in the context of their evolutionary potential accumulation and the possible linkage with the primate rDNA loci.     

Simple repetitive sequences are associated with differentiation of the sex chromosomes in the guppy fish

Summary Hybridization of restriction enzymedigested genomic guppy (Poecilia reticulata, Poeciliidae) DNA with the oligonucleotide probe (GACA)₄ revealed a male-specific simple tandem repeat locus, which defines the Y chromosome in outbred populations. The related (GATA)₄ probe identifies certain males with the red color phenotype. In contrast only in two out of eight laboratory guppy strains was the typical (GACA)₄ band observed. By specific staining of the constitutive heterochromatin one pair of chromosomes could also be identified as the sex chromosomes, confirming the XX/XY mechanism of sex determination. All males exhibit Y chromosomes with a large region of telomeric heterochromatin. Hybridization in situ with nonradioactively labeled oligonucleotide probes localized the (GACA)_n repeats to this heterochromatic portion. Together these results may be regarded as a recent paradigm for the differentiation of heteromorphic sex chromosomes from a pair of autosomes during the course of evolution. According to the fish model system, this may have happened in several independent consecutive steps.     

Y enriched and Y specific DNA sequences from the genome of the mediterranean fruit fly,Ceratitis capitata

DNA sequences that are enriched or specific to the genome of the male medfly, Ceratitis capitata, have been isolated using a differential hybridization approach. Twelve phage clones from a genomic library have been identified that consistently display more intense hybridization with a genomic DNA probe from males as opposed to one from females. Southern DNA blot analysis reveals that these recombinant clones contain at least one EcoRI fragment that is either specific to the male genome, or more highly represented in it, as compared with the female genome. These EcoRI fragments, when used as probes, all generate a similar pattern of intense multiple bands in genomic DNA of males. This suggests the presence of repetitive sequences that are at least partially homologous in these regions of the genome that are specific to or enriched in males. In situ hybridization to mitotic chromosomes confirms a Y chromosomal origin for the male specific repetitive sequences. Data on the genomic organization, representation and evolutionary conservation of these sequences that are specific to or enriched in males are presented. Studies of the genomic organization and representation of flanking sequences that are not male specific are presented as well.by R. Appels     

Abundance and polymorphism of simple repetitive DNA sequences in Bmssica napus L.

Summary The Brassica napus genome has been investigated by DNA fingerprinting with six synthetic oligonucleotide probes complementary to simple repetitive sequences, namely (GATA)₄, (GACA)₄, (GGAT)₄, (CA)₈, (CT)₈ and (GTG)₅. While all sequence motifs were found to be present in the B. napus genome, their organization and abundance varied considerably. Among the investigated probes, (GATA)₄ revealed the highest level of intraspecific polymorphism and distinguishes not only between cultivars but even between different individuals belonging to the same cultivar. In contrast, (GTG)₅, (GACA)₄ and (GGAT)₄ produced relatively homogeneous fingerprint patterns throughout different cultivars, while hybridization to (CT)₈ and (CA)₈ resulted in only a few weak bands superimposed on a smear. The isoschizomeric pair Hpa II and Msp I revealed the presence of methylated cytosines in the vicinity of (GATA)_m repeats. The applicability of simple repetitive sequence polymorphisms as molecular markers for Brassica species is discussed.     

GATA repeats in the genome of Asellus aquaticus (Crustacea,Isopoda)

A 500 bp fragment of Drosophila genomic DNA containing 37 copies of the tetranucleotide GATA was used to probe, by Southern DNA blotting and in situ hybridization, two natural populations of the isopod crustacean Asellus aquaticus collected from the Sarno and Tiber rivers. This species does not have a recognizable sex chromosome pair. In a number of males from the Sarno population chromomycin A₃ staining reveals a heteromorphic chromosome pair. The heterochromosome has two blocks of heterochromatin. After digestion of genomic DNA with six restriction endonucleases and hybridization with the GATA probe, the two populations exhibit different fragment length patterns. No sex-linked pattern was observed in either population. In situ hybridization to chromosomes of males and females from the Sarno population does not reveal any sex-specific pattern of labelling and indicates a scattered distribution of GATA sequences on most chromosomes with some areas of preferential concentration. The heterochromatic arcas of the male heterochromosome are not labelled.by E.R. Schmidt     

Clustered GATA repeats (Bkm sequences) on the human Y chromosome

Summary Sixty eight individual clones of a human Y chromosome cosmid library were screened for the presence of GATA. repeats, the major component of Bkm-related DNA sequences. Nine cosmid clones were found to cross-hybridize. The sequence organization of the repetitive base quadruplet GATA was analyzed using synthetic oligonucleotide probes. Subclones of GATA-positive cosmid clones were used for chromosomal localization of the Y-derived DNA sequences thus revealing male-specificity or male-female homology.     

The chromosomal organization of simple sequence repeats in wheat and rye genomes

The physical distribution of ten simple-sequence repeated DNA motifs (SSRs) was studied on chromosomes of bread wheat, rye and hexaploid triticale. Oligomers with repeated di-, tri- or tetra-nucleotide motifs were used as probes for fluorescence in situ hybridization to root-tip metaphase and anther pachytene chromosomes. All motifs showed dispersed hybridization signals of varying strengths on all chromosomes. In addition, the motifs (AG)₁₂, (CAT)₅, (AAG)₅, (GCC)₅ and, in particular, (GACA)₄ hybridized strongly to pericentromeric and multiple intercalary sites on the B genome chromosomes and on chromosome 4A of wheat, giving diagnostic patterns that resembled N-banding. In rye, all chromosomes showed strong hybridization of (GACA)₄ at many intercalary sites that did not correspond to any other known banding pattern, but allowed identification of all R genome chromosome arms. Overall, SSR hybridization signals were found in related chromosome positions independently of the motif used and showed remarkably similar distribution patterns in wheat and rye, indicating the special role of SSRs in chromosome organization as a possible ancient genomic component of the tribe Triticeae (Gramineae). Received: 13 February 1998; in revised form: 18 August 1998 / Accepted: 18 August 1998     

Random amplification of polymorphic DNA with conserved sequences reveals genome-specific monomorphic amplicons: Implications in clad identification

The enzymatic amplification of genomic DNA with an arbitrary primer generates informative band profile useful for genome analysis. We used a set of synthetic oligodeoxyribonucleotide primers OAT15.2 (GACA)_3.75, OAT18. 2 (GACA)_4.5, OAT24.2 (GACA)₆, OAT36 (GACA)₉, comprising 4–9 consecutive units of GACA repeat, O33.15 (CACCTCTCCACCTGCC) and 033.6 (CCTCCAGCCCTCCTCCAGCCCT) for RAPD reactions of genomic DNA from different sources. The GACA based oligos of 15 and 18 base residues generated discernible genome specific amplicons whereas primers larger than 18 bases revealed smeary signals. The other oligos O33.15 and O33.6 also generated genome specific amplicons with more bands compared with those obtained from OAT15.2 or OAT18.2. The presence of OAT15.1 (GATA)_3.75 and OAT15.2 (GACA)_3.75 sequences in different genomes were ascertained by independent dot-blot hybridization prior to using them for RAPD reactions. The RAPD amplicons generated by evolutionarily conserved primer(s) or sequences shared by many species may be useful for clad identification in controversial systematics, comparative genome analysis, and for establishing the phylogenetic status of an organism.     

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.      

Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes

Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-α) and fibroblast (HuIFN-β) interferon gene sequences. Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA. Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes. The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones. One HuIFN-α clone, 201, has a nucleotide sequence different from published HuIFN-α sequences. Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-α in Escherichia coli.     

On sex determination in the Turkish desert woodlouse Hemilepistus elongatus (Crustacea, Isopoda, Oniscidea): searching for sex chromosomes and for sex-specific differences in simple DNA repeats.

The karyotype of male and female Hemilepistus elongatus was investigated by means of C-banding. The diploid chromosome number in both sexes is 2n = 50. By scrutinizing general morphology and localization of the constitutive heterochromatin, no heteromorphic sex chromosomes were found. All chromosome pairs in males are well paired during diakinesis. Hybridization of genomic DNA with (GACA)4 and (GATA)4 oligonucleotides revealed no sex-specific patterns. Key words : karyotype, C-banding, sex determination, simple DNA-repeats, Isopoda.     

DNA fingerprinting in sugar beet (Beta vulgaris) — identification of double-haploid breeding lines

Summary The distribution and abundance of simple repetitive sequences complementary to the synthetic oligonucleotides (GACA)₄, (GATA)₄, (GTG)₅ and (CA)₈ in the genomes of several cultivars of Beta vulgaris and in the wild beet B. vulgaris ssp. maritima were investigated. Hybridization experiments revealed that all four motifs were present, though at different abundances, in the genomes of all of the investigated beet cultivars. Considerable intraspecific variation of the resulting DNA fingerprints was observed. The extent of polymorphism depends on the oligonucleotide probe. The most informative banding patterns were obtained with the (GATA)₄ probe hybridized to HinfI-, HaeIII-, or RsaI-restricted DNA, respectively. DNA fingerprinting with (GATA)₄ allowed a clear differentiation of double-haploid breeding lines (DH lines). We demonstrated that the application of oligonucleotide probes for DNA fingerprinting is a sensitive tool for genome diagnosis in cultivated beet.     

Organization of the micronuclear genome of oxytricha nova

In the hypotrichous ciliated protozoan Oxytricha nova, approximately 95% of the micronuclear genome, including all of the repetitive DNA and most of the unique sequence DNA, is eliminated during the formation of the macronuclear genome. We have examined the interspersion patterns of repetitive and unique and eliminated and retained sequences in the micronuclear genome by characterizing randomly selected clones of micronuclear DNA. Three major classes of clones have been defined: (1) those containing primarily unique, retained sequences; (2) those containing only unique, eliminated sequences; and (3) those containing only repetitive, eliminated sequences. Clones of type one and three document two aspects of organization observed previously: clustering of macronuclear destined sequences and the presence of a prevalent repetitive element. Clones of the second type demonstrate for the first time that eliminated unique sequence DNA occurs in long stretches uninterrupted by repetitive sequences. To further examine repetitive sequence interspersion, we characterized the repetitive sequence family that is present in 50% of the clones (class three above). A consensus map of this element was obtained by mapping approximately 80 phage clones and by hybridization to digests of micronuclear DNA. The repeat element is extremely large (approximately 24 kb) and is interspersed with both macronuclear destined sequences and eliminated unique sequences.     

DNA fingerprinting in rice using oligonucleotide probes specific for simple repetitive DNA sequences

In this report we describe the use of five oligonucleotide probes, namely (GATA)₄, (GACA)₄, (GGAT)₄, (GAA)₆ and (CAC)₅, to reveal highly polymorphic DNA regions in rice. With each of the oligonucleotide probes, the level of polymorphism was high enough to distinguish several rice genotypes. Moreover, individual plants of one cultivar showed the same cultivar-specific DNA fingerprint. The multilocus fingerprint patterns were somatically stable. Our study demonstrates that microsatellite-derived DNA fingerprints are ideally suited for the identification of rice genotypes. As the majority of the probes detected a high level of polymorphism, they can be very useful in monitoring and aiding gene introgression from wild rice into cultivars.     

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.