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.     

DNA fingerprinting in reptiles: Bkm hybridization patterns in Crocodilia and Chelonia

The simple tetranucleotide repeat GATA (GACA) occurs in all eukaryotes so far studied. In many species, the arrangement of these sequences varies considerably among individuals. Thus GATA (GACA) type probes produce DNA fingerprints when hybridized with restricted DNA from different individuals within a species. Banded krait minor (Bkm) satellite DNA (related to sequences originally recovered from the W chromosome of the banded krait and consisting essentially of a series of GATA repeats) is found in a wide spectrum of vertebrates and invertebrates. We used the Bkm 2(8) clone to evaluate the occurrence of this satellite in DNA from five species of Crocodilia and six species of Chelonia, including the sea turtles Chelonia mydas and Lepidochelys kempi. Well-resolved DNA fingerprints were obtained. Among the crocodilians, fewer restriction fragments were generated and fewer of the fragments were polymorphic, than among the chelonians, consistent with the view that the crocodilians are less divergent within species. The Bkm 2(8) clone can accordingly be used to advantage in individual, familial, and population studies, and perhaps in the evaluation of taxonomic relationships in these animals. This is of potential value in endangered species such as C. mydas and L. kempi.     

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.     

Sequence organization of repetitive sequences enriched in small polydisperse circular DNAs from HeLa cells

A total of 36 clones were randomly selected from a recombinant DNA library of small polydisperse circular DNA (spcDNA) molecules from HeLa cells and were shown to contain repetitive sequences of different reiteration frequencies that ranged from several hundred to several hundred thousand per genome. Sequencing of representative clones revealed tandem repeats of alphoid (alpha) satellite DNA, clustered repeats of the Alu family, KpnI family sequences, tandem repeats of an alpha satellite DNA specific to the X chromosome (alpha X), and A + T-rich segments carrying short stretches of poly(A) or poly(T). DNA rearrangement was frequently found in the repetitive sequences enriched in these spcDNA clones. Short regions of homology that were patchy and inverted were often found, especially at the novel joint where spcDNA sequences are circularized. The presence of these inverted repeats suggests that HeLa spcDNAs are formed by a mechanism that involves looping out of the spcDNA region and joining of the flanking DNA by illegitimate recombination.     

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.     

Semiautomated clone verification by real-time PCR using molecular beacons

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.     

Repeat arrays in cellular DNA related to the Epstein-Barr virus IR3 repeat.

We isolated clones and determined the sequence of portions of mouse and human cellular DNA which cross-hybridize strongly with the IR3 repetitive region of Epstein-Barr virus. The sequences were found to be tandem arrays of a simple sequence based on the triplet GGA, very similar to the IR3 repeat. The cellular repeats have distinct differences from the viral repeat region, however, and their sequences do not appear capable of being translated into a purely glycine-plus-alanine protein domain like the portion of the Epstein-Barr nuclear antigen coded by IR3. Although the relationship between IR3 and the cellular repeats is left unclear, the cellular repeats have many interesting features. The tandem arrays are about 1 to several kilobases long, much shorter than satellite tandem repeats and larger than other interspersed, tandem repeats. Each of the repeats is a distinct variation, perhaps diverged from a common sequence, (GGA)n. This family is present in the genomes of all species tested and appears to be a ubiquitous feature of all higher eucaryotic genomes.     

Characterization of a 249-bp tandemly repetitive, satellite-like repeat in the translated portion of Balbiani ring c of Chironomus thummi.

A major part of Balbiani ring (BR)c DNA of Chironomus thummi consists of tandem 249-bp repeats which appear to be transcribed and translated into a polypeptide of very unusual composition. Whereas these 2549-bp repeats are evident by Southern blotting, sequence analysis reveals a finer tandemly repetitive substructure: more than half of the 249-bp repeat length consists of tandem 24-bp subrepeats , and these in turn may have been generated from even shorter sequences. Comparisons with partial BRb and BR1 sequences reveal that this hierarchically repetitive sequence structure is typical of BR genes. It resembles the structure of some satellite sequences, suggesting that mechanisms leading to satellite DNA evolution may also operate in the evolution of structural genes.     

Complex telomere-associated repeat units in members of the genus Chironomus evolve from sequences similar to simple telomeric repeats.

The dipteran Chironomus tentans has complex tandemly repeated 350-bp DNA sequences at or near the chromosome ends. As in Drosophila melanogaster, short simple repeats with cytosines and guanines in different strands have never been observed. We were therefore interested in learning whether the Chironomus repeats could have evolved from simple sequence telomeric DNA, which might suggest that they constitute a functional equivalent. We screened for repeat units with evolutionarily ancient features within the tandem arrays and recovered two clones with a less-evolved structure. Sequence analysis reveals that the present-day 350-bp unit probably evolved from a simpler 165-bp unit through the acquisition of transposed sequences. The 165-bp unit contains DNA with a highly biased distribution of cytosine and guanine between the two strands, although with the ratios inverted in two minor parts of the repeat. It is largely built up of short degenerate subrepeats for which most of the sequence can be reconstructed. The consensus for the subrepeat sequence is similar to the simple telomeric repeat sequences of several kinds of eukaryotes. We propose that the present-day unit has evolved from telomeric, simple sequence, asymmetric DNA from which it has retained some original sequence features and possibly functions.     

The 5S genes of Drosophila melanogaster.

We have cloned embryonic Drosophila DNA using the poly (dA-DT) connector method (Lobban and Kaiser, 1973) and the ampicillin-resistant plasmid pSF2124 (So, Gill and Falkow, 1975) as a cloning vehicle. Two clones, containing hybrid plasmids with sequences complementary to a 5S RNA probe isolated from Drosophila tissue culture cells, were identified by the Grunstein and Hogness (1975) colony hybridization procedure. One hybrid plasmid has a Drosophila insert which is comprised solely of tandem repeats of the 5S gene plus spacer sequences. The other plasmid contains an insert which has about 20 tandem 5S repeat units plus an additional 4 kilobases of adjacent sequences. The size of the 5S repeat unit was determined by gel electrophoresis and was found to be approximately 375 base pairs. We present a restriction map of both plasmids, and a detailed map of of the5S repeat unit. The 5S repat unit shows slight length and sequence heterogeneity. We present evidence suggesting that the 5S genes in Drosophila melanogaster may be arranged in a single continuous cluster.     

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.     

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.     

Characterization of cloned human alphoid satellite with an unusual monomeric construction: evidence for enrichment in HeLa small polydisperse circular DNA.

A recombinant DNA plasmid library was constructed from HeLa cell extrachromosomal circular DNA and the sequence organization of one family of clones, which contain sequences enriched in HeLa small polydisperse circular (spc) DNA, was studied by restriction mapping and base sequence analysis. Restriction mapping revealed each clone to be composed solely of imperfect tandem repeats of ca. 170 bp. The entire DNA sequence of one clone was determined and found to be alphoid satellite with a variant monomeric construction.     

Base sequence studies of 300 nucleotide renatured repeated human DNA clones

A band of 300 nucleotide long duplex DNA is released by treating renatured repeated human DNA with the single strand-specific endonuclease S₁. Since many of the interspersed repeated sequences in human DNA are 300 nucleotides long, this band should be enriched in such repeats. We have determined the nucleotide sequences of 15 clones constructed from these 300 nucleotide S₁-resistant repeats. Ten of these cloned sequences are members of the Alu family of interspersed repeats. These ten sequences share a recognizable consensus sequence from which individual clones have an average divergence of 12.8%. The 300 nucleotide Alu family consensus sequence has a dimeric structure and was evidently formed from a head to tail duplication of an ancestral monomeric sequence. Three of the remaining clones are variations on a simple pentanucleotide sequence previously reported for human satellite III DNA. Two of the 15 clones have distinct and complex sequences and may represent other families of interspersed repeated sequences.     

A new rice repetitive DNA shows sequence homology to both 5S RNA and tRNA.

Moderately repetitive DNA sequences are found in the genomes of all eucaryotes that have been examined. We now report the discovery of a novel, transcribed, moderately repetitive DNA sequence in a higher plant which is different from any of the known repetitive DNA sequences from any organism. We isolated a rice cDNA clone which hybridizes to multiple bands on genomic blot analysis. The sequence of this 352 bp cDNA contains four regions of homology to the wheat phenylalanine tRNA, including the polymerase III-type promoter. Unexpectedly, two regions of the same 352 bp sequence also show homology to the wheat 5S RNA sequence. Using the cDNA as a probe, we have isolated six genomic clones which contain long tandem repeats of 355 bp sequence, and have sequenced nine repeat units. Our findings suggest that the rice repetitive sequence may be an amplified pseudogene with sequence homology to both 5S RNA and tRNA, but organized as long tandem repeats resembling 5S RNA genes. This is the first example showing homology between the sequences of a moderately repetitive DNA with unknown function and 5S RNA.     

Satellite DNA and speciation: A species specific satellite DNA of Drosophila guanchel

The heterochromatin of the chromosomes of Drosophila gunche consists mainly of a satellite DNA composed of multiple, tandemly arranged copies of a 290 b p basic sequence. Five clones containing one or two copies of the basic unit were sequenced. As expected from CsCl density centrifugation and AT specific staining of mitotic chromosomes the sequence is AT rich. The average nucleotid variability between the cloned sequences is 11.6%. In situ hybridization on the mitotic chromosomes revealed, that this satellite DNA is present in the centromeric regions of all chromosomes but the Y. The nucleotide variability between copies of different tandem clusters seems to be higher than between members of the same cluster. The copy number of the sequence in the haploid genome was estimated to be approximately 80000. The sequence is species specific and is not present in the genome of sibling species D. subobscura and D. madeiren-sis. The evolutionary origin of the satellite DNA and its possible role in species formation is discussed.     

Satellite DNA in the elm leaf beetle,Xanthogaleruca luteola (Coleoptera,Chrysomelidae): characterization,interpopulation analysis,and chromosome location

In this paper the satellite DNA (stDNA) of the phytophagous beetle Xanthogaleruca luteola is analyzed. It is organized in a tandem repeat of 149-bp-long monomers, has an AT content of 59%, and presents inverted internal repeats. Restriction analysis of the total DNA with methylation-sensitive enzymes suggests that this repetitive DNA is not methylated. Analysis of the electrophoretic mobility of stDNA on non-denaturing polyacrylamide gels showed that this stDNA is not curved. In situ hybridization with a biotinylated probe of the stDNA revealed a pericentromeric localization of these sequences in the majority of the meiotic bivalents. We have studied the stDNA of X. luteola from two populations with very distinct geographical origins. The sequence and phylogenetic analysis of monomers from these two populations showed that the repetitive element is conserved within the species. Putative gene conversion tracts are identified when the different monomers of the same population are compared. These results could indicate the existence of processes of homogenization that would extend these mutations to all the satellite repeats.