A tandemly repetitive centromeric DNA sequence of the fish Hoplias malabaricus (Characiformes : Erythrinidae) is derived from 5S rDNA

A substantial fraction of the eukaryotic genome consists of repetitive DNA sequences that include satellites, minisatellites, microsatellites, and transposable elements. Although extensively studied for the past three decades, the molecular forces that generate, propagate and maintain repetitive DNAs in the genomes are still discussed. To further understand the dynamics and the mechanisms of evolution of repetitive DNAs in vertebrate genome, we searched for repetitive sequences in the genome of the fish species Hoplias malabaricus. A satellite sequence, named 5SHindIII-DNA, which has a conspicuous similarity with 5S rRNA genes and spacers was identified. FISH experiments showed that the 5S rRNA bona fide gene repeats were clustered in the interstitial position of two chromosome pairs of H. malabaricus, while the satellite 5SHindIII-DNA sequences were clustered in the centromeric position in nine chromosome pairs of the species. The presence of the 5SHindIII-DNA sequences in the centromeres of several chromosomes indicates that this satellite family probably escaped from the selective pressure that maintains the structure and organization of the 5S rDNA repeats and become disperse into the genome. Although it is not feasible to explain how this sequence has been maintained in the centromeric regions, it is possible to hypothesize that it may be involved in some structural or functional role of the centromere organization.

Comparative chromosome mapping of repetitive sequences. Implications for genomic evolution in the fish, Hoplias malabaricus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A LINE2 repetitive DNA sequence from the cichlid fish, Oreochromis niloticus: sequence analysis and chromosomal distribution

We report the cloning and characterization of a long interspersed nucleotide element (LINE) fi-om a cichlid fish, Oreochromis niloticus, and show the distribution of this element, called CiLINE2 for cichlid LINE2, in the chromosomes of this species. The identification of an open reading frame in CiLINE2 with amino acid sequence similarity to reverse transcriptases encoded by LINE-like elements in Caenorhabditis elegans, Platemys spixii, Schistosoma mansoni, Gallus gallus (CRI), Drosophila melanogaster (I factor), and Homo sapiens (LINE2), as well as the structure of the element, suggest it is a member of this family of non-long terminal repeat-containing retrotransposons. Search of a DNA sequence database identified sequences similar to CiLINE2 in four other fish species (Haplotaxodon microlepis, Oreochromis mossambicus, Pseudotropheus zebra, and Fugu rubripes). Southern blot hybridization experiments revealed the presence of sequences similar to CiLINE2 in all Tilapiini species analyzed from the genera Oreochromis, Tilapia, and Sarotherodon, and gave an estimated copy number of about 5500 for the haploid genome of O. niloticus. Fluorescent in situ hybridization showed that CiLINE2 sequences were organized in small clusters dispersed over all chromosomes of O. niloticus, with a higher concentration near chromosome ends. Furthermore the long arm of chromosome 1 was strikingly enriched with this sequence. The distribution of LINE2-related elements might underlie the difference in chromosome banding patterns observed between cold-blooded vertebrates and mammals.

Chromosomal Mapping of Repetitive DNA and Cytochrome C Oxidase I Sequence Analysis Reveal Differentiation among Sympatric Samples of Astyanax fasciatus (Characiformes, Characidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Origin and distribution of AT-rich repetitive DNA families in Triatoma infestans (Heteroptera)

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Characterization of repetitive DNA in the Mycoplasma genitalium genome: possible role in the generation of antigenic variation.

We have characterized a family of repetitive DNA elements with homology to the MgPa cellular adhesion operon of Mycoplasma genitalium, a bacterium that has the smallest known genome of any free-living organism. One element, 2272 bp in length and flanked by DNA with no homology to MgPa, was completely sequenced. At least four others were partially sequenced. The complete element is a composite of six regions. Five of these regions show sequence similarity with nonadjacent segments of genes of the MgPa operon. The sixth region, located near the center of the element, is an A+T-rich sequence that has only been found in this repeat family. Open reading frames are present within the five individual regions showing sequence homology to MgPa and the adjacent open reading frame 3 (ORF3) gene. However, termination codons are found between adjacent regions of homology to the MgPa operon and in the A+T-rich sequence. Thus, these repetitive elements do not appear to be directly expressible protein coding sequences. The sequence of one region from five different repetitive elements was compared with the homologous region of the MgPa gene from the type strain G37 and four newly isolated M. genitalium strains. Recombination between repetitive elements of strain G37 and the MgPa operon can explain the majority of polymorphisms within our partial sequences of the MgPa genes of the new isolates. Therefore, we propose that the repetitive elements of M. genitalium provide a reservoir of sequence that contributes to antigenic variation in proteins of the MgPa cellular adhesion operon.

Mutations in the MSH3 gene preferentially lead to deletions within tracts of simple repetitive DNA in Saccharomyces cerevisiae.

Eukaryotic genomes contain tracts of DNA in which a single base or a small number of bases are repeated (microsatellites). Mutations in the yeast DNA mismatch repair genes MSH2, PMS1, and MLH1 increase the frequency of mutations for normal DNA sequences and destabilize microsatellites. Mutations of human homologs of MSH2, PMS1, and MLH1 also cause microsatellite instability and result in certain types of cancer. We find that a mutation in the yeast gene MSH3 that does not substantially affect the rate of spontaneous mutations at several loci increases microsatellite instability about 40-fold, preferentially causing deletions. We suggest that MSH3 has different substrate specificities than the other mismatch repair proteins and that the human MSH3 homolog (MRP1) may be mutated in some tumors with microsatellite instability.

Repetitive DNA fragments as taxonomic markers for {\it Penaeus\/} sibling taxa (Decapoda: Dendrobranchiata: Penaeidae) from the southern terminus of the Florida peninsula, U.S.A.

Sympatric populations of P. brasiliensis and P. duorarum from Biscayne Bay, Florida, revealed species-specific satellite DNA organizational patterns with the restriction endonuclease EcoRI. The species-specific satellite DNA patterns can be explained as resulting from differential amplification/deletion events having altered monomer arrays after the divergence of these two species. Two discontinuous populations of P. duorarum (Biscayne Bay and Dry Tortugas) were found to exhibit distinct EcoRI satellite fragment patterns; BamHI repetitive fragments specific to the Dry Tortugas P. duorarum population were also detected. In addition, the evolutionary conservation of the Penaeus (Farfantepenaeus) satellites was investigated. The putative conservation of sequences related to one cloned P. duorarum satellite monomer unit suggests that the FTR satellite DNA family may not only be of use as a genome tag to distinguish between sibling and cryptic Penaeus species but may also serve as a probe to better understand decapod crustacean genome organization and evolution. ^

Combined mitochondrial and nuclear DNA sequences resolve the interrelations of the major Australasian marsupial radiations

Australasian marsupials include three major radiations, the insectivorous/carnivorous Dasyuromorphia, the omnivorous bandicoots (Peramelemorphia), and the largely herbivorous diprotodontians. Morphologists have generally considered the bandicoots and diprotodontians to be closely related, most prominently because they are both syndactylous (with the 2nd and 3rd pedal digits being fused). Molecular studies have been unable to confirm or reject this Syndactyla hypothesis. Here we present new mitochondrial (mt) genomes from a spiny bandicoot (Echymipera rufescens) and two dasyurids, a fat-tailed dunnart (Sminthopsis crassicaudata) and a northern quoll (Dasyurus hallucatus). By comparing trees derived from pairwise base-frequency differences between taxa with standard (absolute, uncorrected) distance trees, we infer that composition bias among mt protein-coding and RNA sequences is sufficient to mislead tree reconstruction. This can explain incongruence between trees obtained from mt and nuclear data sets. However, after excluding major sources of compositional heterogeneity, both the “reduced-bias” mt and nuclear data sets clearly favor a bandicoot plus dasyuromorphian association, as well as a grouping of kangaroos and possums (Phalangeriformes) among diprotodontians. Notably, alternatives to these groupings could only be confidently rejected by combining the mt and nuclear data. Elsewhere on the tree, Dromiciops appears to be sister to the monophyletic Australasian marsupials, whereas the placement of the marsupial mole (Notoryctes) remains problematic. More generally, we contend that it is desirable to combine mt genome and nuclear sequences for inferring vertebrate phylogeny, but as separately modeled process partitions. This strategy depends on detecting and excluding (or accounting for) major sources of nonhistorical signal, such as from compositional nonstationarity.

Restoration of native folding of single-stranded DNA sequences through reverse mutations: an indication of a new epigenetic mechanism

We used in vivo (biological), in silico (computational structure prediction), and in vitro (model sequence folding) analyses of single-stranded DNA sequences to show that nucleic acid folding conservation is the selective principle behind a high-frequency single-nucleotide reversion observed in a three-nucleotide mutated motif of the Maize streak virus replication associated protein (Rep) gene. In silico and in vitro studies showed that the three-nucleotide mutation adversely affected Rep nucleic acid folding, and that the single-nucleotide reversion [C(601)A] restored wild-type-like folding. In vivo support came from infecting maize with mutant viruses: those with Rep genes containing nucleotide changes predicted to restore a wild-type-like fold [A(601)/G(601)] preferentially accumulated over those predicted to fold differently [C(601)/T(601)], which frequently reverted to A(601) and displaced the original population. We propose that the selection of native nucleic acid folding is an epigenetic effect, which might have broad implications in the evolution of plants and their viruses.

Computational analysis of epigenetic information in human DNA sequences

Over the last few years, investigations of human epigenetic profiles have identified key elements of change to be Histone Modifications, stable and heritable DNA methylation and Chromatin remodeling. These factors determine gene expression levels and characterise conditions leading to disease. In order to extract information embedded in long DNA sequences, data mining and pattern recognition tools are widely used, but efforts have been limited to date with respect to analyzing epigenetic changes, and their role as catalysts in disease onset. Useful insight, however, can be gained by investigation of associated dinucleotide distributions. The focus of this paper is to explore specific dinucleotides frequencies across defined regions within the human genome, and to identify new patterns between epigenetic mechanisms and DNA content. Signal processing methods, including Fourier and Wavelet Transformations, are employed and principal results are reported.

DNA sequences and austral taxa indicate generic synonymy of Paratrichocladius Santos-Abreu with Cricotopus Wulp (Diptera: Chironomidae)

In the century since the description of the orthoclad genus Paratrichocladius Santos-Abreu (Diptera: Chironomidae), separation in any life stage from the cosmopolitan, diverse Cricotopus Wulp has been problematic. Molecular analysis reveals the presence of two species in Australia that conform in morphology to Paratrichocladius and which form a well-supported clade including Paratrichocladius micans (Kieffer) from Africa and a distinct southern African larva. This clade clusters with taxa allied with Cricotopus albitibia (Walker), in turn nested within all other sampled Australian Cricotopus. Relevant nodes strongly support Cricotopus as nonmonophyletic without inclusion of Paratrichocladius. We synonymize Paratrichocladius with Cricotopus syn.n, treating Paratrichocladius as a subgenus. Cricotopus (Paratrichocladius) australiensis Cranston sp.n. is described for Trichocladius pluriserialis Freeman from Australia, which is not the same species under that name in New Zealand. Cricotopus (Paratrichocladius) bifenestrus Cranston sp.n. from Australia is described, also in all life stages. The many new combinations, listed in an Appendix, include three replacement names for new secondary homonyms, namely: Cricotopus (Paratrichocladius) sinobicinctus Cranston & Krosch nom.n. for Paratrichocladius bicinctus Fu, Sæther & Wang, Cricotopus draysoni Cranston & Krosch nom.n. for Cricotopus brevicornis Drayson, Krosch & Cranston, and Cricotopus (Paratrichocladius) sikhotealinus Makarchenko & Makarchenko nom.n. for Cricotopus orientalis Kieffer. We conclude with comments on wider issues in the taxonomy of Paratrichocladius, especially concerning New Zealand species.

Polypurine/polypyrimidine sequences as cis-acting transcriptional regulators

Genome sequence information has generated increasing evidence for the claim that repetitive DNA sequences present within and around genes could play a important role in the regulation of gene expression. Polypurine/polypyrimidine sequences [poly(Pu/Py)] have been observed in the vicinity of promoters and within the transcribed regions of many genes. To understand whether such sequences influence the level of gene expression, we constructed several prokaryotic and eukaryotic expression vectors incorporating poly(Pu/Py) repeats both within and upstream of a reporter gene, lacZ (encoding β-galactosidase), and studied its expression in vivo. We find that, in contrast to the situation in Escherichia coli, the presence of poly(Pu/Py) sequences within the gene does not significantly inhibit gene expression in mammalian cells. On the other hand, the presence of such sequences upstream of lacZ leads to a several-fold reduction of gene expression in mammalian cells. Similar down-regulation was observed when a structural cassette containing poly(Pu/Py) sequences upstream of lacZ was integrated into yeast chromosome V. Sequence analysis of the nine totally sequenced yeast chromosomes shows that a large number of such sequences occur upstream of ORFs. On the basis of our experimental results and DNA sequence analysis, we propose that these sequences can function as cis-acting transcriptional regulators.

Recognition and alignment of homologous DNA sequences between minichromosomes and single-stranded DNA promoted by RecA protein

The incorporation of DNA into nucleosomes and higher-order forms of chromatin in vivo creates difficulties with respect to its accessibility for cellular functions such as transcription, replication, repair and recombination. To understand the role of chromatin structure in the process of homologous recombination, we have studied the interaction of nucleoprotein filaments, comprised of RecA protein and ssDNA, with minichromosomes. Using this paradigm, we have addressed how chromatin structure affects the search for homologous DNA sequences, and attempted to distinguish between two mutually exclusive models of DNA-DNA pairing mechanisms. Paradoxically, we found that the search for homologous sequences, as monitored by unwinding of homologous or heterologous duplex DNA, was facilitated by nucleosomes, with no discernible effect on homologous pairing. More importantly, unwinding of minichromosomes required the interaction of nucleoprotein filaments and led to the accumulation of circular duplex DNA sensitive to nuclease P1. Competition experiments indicated that chromatin templates and naked DNA served as equally efficient targets for homologous pairing. These and other findings suggest that nucleosomes do not impede but rather facilitate the search for homologous sequences and establish, in accordance with one proposed model, that unwinding of duplex DNA precedes alignment of homologous sequences at the level of chromatin. The potential application of this model to investigate the role of chromosomal proteins in the alignment of homologous sequences in the context of cellular recombination is considered.