Enzymatic methylation of inverted DNA repeats in the vicinity of the mouse beta-major globin gene

This paper examines the extent of enzymatic methylation in 5'-CCGG sequences of inverted repeats in DNA isolated from adult liver and bone marrow of DBA/2 mice, with special attention to the methylation of such sequences in the vicinity of the beta-major globin gene. Two thirds of inverted repeats contain 5'-AGCT and 5'-CCGG sequences, as found by a method based on the capability of inverted repeats of forming intramolecular duplexes under the conditions of "zero-time" reassociation. Methylation in internal cytosines of 5'-CCGG sequences of inverted DNA repeats differs between bone marrow and liver tissues. The beta-major globin gene was found in DNA covalently linked to inverted repeats. The enzymatic methylation of inverted repeats neighbouring the beta-major globin gene differs at HpaII recognition sites; the DNA of bone marrow tissue, in which this gene is expressed, is less methylated at such sites as compared to liver DNA.     

Repseek, a tool to retrieve approximate repeats from large DNA sequences

Chromosomes or other long DNA sequences contain many highly similar repeated sub-sequences. While there are efficient methods for detecting strict repeats or detecting already characterized repeats, there is no software available for detecting approximate repeats in large DNA sequences allowing for weighted substitutions and indels in a coherent statistical framework. Here, we present an implementation of a two-steps method (seed detection followed by their extension) that detects those approximate repeats. Our method is computationally efficient enough to handle large sequences and is flexible enough to account for influencing factors, such as sequence-composition biases both at the seed detection and alignment levels. AVAILABILITY: http://wwwabi.snv.jussieu.fr/public/RepSeek/     

Organization, structure, and polymorphisms of the human profilaggrin gene

Profilaggrin is a major protein component of the keratohyalin granules of mammalian epidermis. It is initially expressed as a large polyprotein precursor and is subsequently proteolytically processed into individual functional filaggrin molecules. We have isolated genomic DNA and cDNA clones encoding the 5'- and 3'-ends of the human gene and mRNA. The data reveal the presence of likely "CAT" and "TATA" sequences, an intron in the 5'-untranslated region, and several potential regulatory sequences. While all repeats are of the same length (972 bp, 324 amino acids), sequences display considerable variation (10-15%) between repeats on the same clone and between different clones. Most variations are attributable to single-base changes, but many also involve changes in charge. Thus, human filaggrin consists of a heterogeneous population of molecules of different sizes, charges, and sequences. However, amino acid sequences encoding the amino and carboxyl termini are more conserved, as are the 5' and 3' DNA sequences flanking the coding portions of the gene. The presence of unique restriction enzyme sites in these conserved flanking sequences has enabled calculations on the size of the full-length gene and the numbers of repeats in it: depending on the source of genomic DNA, the gene contains 10, 11, or 12 filaggrin repeats that segregate in kindred families by normal Mendelian genetic mechanisms. This means that the human profilaggrin gene system is also polymorphic with respect to size due to simple allelic differences between different individuals. The amino- and carboxyl-terminal sequences of profilaggrin contain partial or truncated repeats with unusual un-filaggrin-like sequences on the termini.(ABSTRACT TRUNCATED AT 250 WORDS)     

A novel method for determining linkage between DNA sequences: hybridization to paired probe arrays

Cooperative hybridization has been used to establish physical linkage between two loci on a DNA strand. Linkage was detected by hybridization to a new type of high-density oligonucleotide array. Each synthesis location on the array contains a mixture of two different probe sequences. Each of the two probes can hybridize independently to a different target sequence, but if the two target sequences are physically linked there is a cooperative increase in hybridization yield. The ability to create and control non-linear effects raises a host of possibilities for applications of oligonucleotide array hybridization. The method has been used to assign linkage in 50:50 mixtures of DNA containing single nucleotide polymorphisms (SNPs) separated by 17, 693, 1350 and 2038 bp and to reconstruct haplotypes. Other potential uses include increasing the specificity of hybridization in mutation detection and gene expression monitoring applications, determining SNP haplotypes, characterizing repetitive sequences, such as short tandem repeats, and aiding contig assembly in sequen-cing by hybridization.     

The organization of repetitive sequences in a cluster of rabbit β-like globin genes

Several complementary procedures were used to identify and characterize DNA sequences which are repeated within a 44 kilobase (kb) segment of rabbit chromosomal DNA containing four different rabbit β-like globin genes (β1–β4). Cross-hybridization between cloned DNAs from different regions of the gene cluster indicates the presence of a complex array of repeat sequences interspersed with the globin genes. We classified 20 different repeat sequences into five families whose members cross-hybridize. Electron microscopy was used to determine the location, size and relative orientations of many of the repeat sequences. Both direct and inverted repeats were identified, with sizes ranging from 140 to 1400 base pairs (bp). Each of the four closely linked globin genes is flanked by at least one pair of inverted repeats of 140–400 bp, and the entire set of four genes is flanked by an inverted repeat of 1400 bp. Two of the five repeat families contain repeat sequences of different sizes. We found that the smaller sequence elements can occur individually or in association with the larger repeat sequences, suggesting that the larger repeats may be composed of more than one smaller repeat sequence. The restriction fragments containing the intracluster repeats also contain sequences which are repeated many times in total rabbit genomic DNA, but it is not known whether the genomic and intracluster repeats are the same sequences. The results provide the first demonstration of the relationship between single-copy and repetitive DNA sequences in a large segment of chromosomal DNA containing a well characterized set of developmentally regulated genes.     

HomologMiner: looking for homologous genomic groups in whole genomes

MOTIVATION: Complex genomes contain numerous repeated sequences, and genomic duplication is believed to be a main evolutionary mechanism to obtain new functions. Several tools are available for de novo repeat sequence identification, and many approaches exist for clustering homologous protein sequences. We present an efficient new approach to identify and cluster homologous DNA sequences with high accuracy at the level of whole genomes, excluding low-complexity repeats, tandem repeats and annotated interspersed repeats. We also determine the boundaries of each group member so that it closely represents a biological unit, e.g. a complete gene, or a partial gene coding a protein domain. RESULTS: We developed a program called HomologMiner to identify homologous groups applicable to genome sequences that have been properly marked for low-complexity repeats and annotated interspersed repeats. We applied it to the whole genomes of human (hg17), macaque (rheMac2) and mouse (mm8). Groups obtained include gene families (e.g. olfactory receptor gene family, zinc finger families), unannotated interspersed repeats and additional homologous groups that resulted from recent segmental duplications. Our program incorporates several new methods: a new abstract definition of consistent duplicate units, a new criterion to remove moderately frequent tandem repeats, and new algorithmic techniques. We also provide preliminary analysis of the output on the three genomes mentioned above, and show several applications including identifying boundaries of tandem gene clusters and novel interspersed repeat families. AVAILABILITY: All programs and datasets are downloadable from www.bx.psu.edu/miller_lab.     

REPuter: the manifold applications of repeat analysis on a genomic scale

The repetitive structure of genomic DNA holds many secrets to be discovered. A systematic study of repetitive DNA on a genomic or inter-genomic scale requires extensive algorithmic support. The REPuter program described herein was designed to serve as a fundamental tool in such studies. Efficient and complete detection of various types of repeats is provided together with an evaluation of significance and interactive visualization. This article circumscribes the wide scope of repeat analysis using applications in five different areas of sequence analysis: checking fragment assemblies, searching for low copy repeats, finding unique sequences, comparing gene structures and mapping of cDNA/EST sequences.     

Nucleotide sequence of the rat gamma-crystallin gene region and comparison with an orthologous human region

The sequences of a 51-kb region containing the cluster of five rat gamma-crystallin-coding genes (CRYG) and of a 7-kb region surrounding the sixth rat CRYG gene were determined. Approximately 78% of the total sequence represents intergenic DNA. We also sequenced 22 kb of DNA from the human CRYG gene cluster. All CRYG genes are associated with CpG-rich regions. The sequence similarity between the human and rat gene regions drops sharply (to 65%) in intronic and 3'-flanking regions but decreases only gradually in the 5'-flanking region. Highly conserved regions (greater than 80%) are found as far upstream as 1.5 kb. Overall intergenic distances are conserved. The human region contains much more repetitive DNA (24% vs. 10%) but less simple-sequence (sps) DNA (0.7% vs. 4%) than the rat region. Almost all repeats and spsDNA elements are located in the intergenic region. The location of repetitive and spsDNA differs between the orthologous regions and these elements were probably inserted after the evolutionary separation of rat and man. The Alu repeats in man and the B3 repeats in the rat are close copies of their respective consensus sequences and bordered by virtually perfect repeats. In contrast, the B1 and B2 repeats in the rat have diverged considerably from the consensus sequence and the surrounding direct repeats are usually imperfect. Thus the dispersion of the B1 and B2 repeats in the rat probably preceded that of the B3 repeats. Within the rat genomic region the spacing of Z-DNA elements is surprisingly regular, they are located about 12 kb apart. A search for putative matrix-associated regions suggests that the rat CRYG gene cluster is organized into two chromosomal domains.     

Target genes of microsatellite sequences in head and neck squamous cell carcinoma: Mononucleotide repeats are not detected

Microsatellite instability (MSI) is detected in a wide variety of tumors. It is thought that mismatch repair gene mutation or inactivation is the major cause of MSI. Microsatellite sequences are predominantly distributed in intergenic or intronic DNA. However, MSI is found in the exonic sequences of some genes, causing their inactivation. In this report, we searched GenBank for candidate genes containing potential MSI sequences in exonic regions. Twenty seven target genes were selected for MSI analysis. Instability was found in 70% of these genes (14/20) with head and neck squamous cell carcinoma (HNSCC). Interestingly, no instability was detected in mononucleotide repeats in genes or in intergenic sequences. We conclude that instability of mononucleotide repeats is a rare event in HNSCC. High MSI phenotype in young HNSCC patients is limited to noncoding regions only. MSI percentage in HNSCC tumor is closely related to the repeat type, repeat location and patient's age.     

Rapid detection of CA polymorphisms in cloned DNA: application to the 5'' region of the dystrophin gene.

To identify CA repeats in genomic sequences which had been previously subcloned into plasmids, we performed PCR using a (CA)n primer and a flanking vector primer on the genomic inserts. By incorporation of a restriction enzyme site into the (CA)n primer, we have been able to subclone the genomic DNA so that the sequence flanking the CA repeat is readily determined. Primers can then be designed to amplify across the CA repeat in patient DNA samples. Application of this technique to genomic DNAs surrounding the upstream "brain" promoter of the dystrophin gene has led to the discovery of four new CA repeats. Three of these repeats are highly polymorphic, with PICs ranging from .586 to .768. The location of these markers at the extreme 5' terminus of the dystrophin gene, together with their high degree of polymorphism and ease of assay, makes them ideal for linkage analysis in families with Duchenne muscular dystrophy.     

A hemizygous short tandem repeat polymorphism 3′ to the human phosphoglycerate kinase gene

The human phosphoglycerate kinase (PGK) gene is located within Xq11-Xq13, a region implicated in genitourinary diseases including: prostate cancer, androgen insensitivity, perineal hypospadias, and other genetic abnormalities. The PGK gene and the androgen receptor gene are in linkage disequilibrium. PGK has been mapped extensively for nuclease-sensitive sites, methylation sites, and flanking DNA sequences. A PGK-associated BstXI polymorphism has been used to determine clonality of neoplastic tissues. Using fluorescent PCR product analysis and DNA sequencing, we discovered that a short tandem repeat (STR) in the 3 flanking region of the PGK gene is polymorphic. Among 231 individuals, there were nine distinct alleles, including eight based on variations in the number of TATC repeats. The PGK STR demonstrated hemizygosity, consistent with its X-chromosomal location and with an absence of cross-hybridizing autosomal homologs. The polymorphic PGK STR shows promise for rapid investigation of neoplastic clonality, for personal identification, and for studies of inherited predisposition to urologic disorders.     

Molecular Characterization of Ribosomal Genes on the Ybb- Chromosome of DROSOPHILA MELANOGASTER

The question of whether the Ybb- chromosome contains ribosomal genes has been examined by using Southern blot analysis and comparing rDNA hybridization patterns for X/X and X/Ybb- DNA. The results demonstrate that the Ybb- chromosome contains sequences that hybridize to an rDNA probe under stringent conditions. Differential hybridization of some of these sequences with DNAs corresponding to different regions of a complete ribosomal gene repeat provides evidence that some of the genes on the Ybb- chromosome are type 2 repeats. Because data obtained by other workers suggest that type 2 repeats are transcribed only to low levels, these repeats may be classed as "nonfunctional". A further finding is that the ribosomal genes on the Ybb- chromosome do not undergo multiple rounds of DNA replication during polytenization of X/Ybb- cells.     

Telomeric site position heterogeneity in macronuclear DNA of Paramecium primaurelia.

In Paramecium primaurelia, the macronuclear gene encoding the G surface protein is located near a telomere. In this study, multiple copies of this telomere have been isolated and the subtelomeric and telomeric regions of some of them have been sequenced. The telomeric sequences consist of tandem repeats of the hexanucleotides C4A2 or C3A3. We show that the location where these repeats are added, which we call the telomeric site, is variable within a 0.6-0.8-kb region. These results are discussed in relation with the formation of macronuclear DNA.     

FORRepeats: detects repeats on entire chromosomes and between genomes

MOTIVATION: As more and more whole genomes are available, there is a need for new methods to compare large sequences and transfer biological knowledge from annotated genomes to related new ones. BLAST is not suitable to compare multimegabase DNA sequences. MegaBLAST is designed to compare closely related large sequences. Some tools to detect repeats in large sequences have already been developed such as MUMmer or REPuter. They also have time or space restrictions. Moreover, in terms of applications, REPuter only computes repeats and MUMmer works better with related genomes. RESULTS: We present a heuristic method, named FORRepeats, which is based on a novel data structure called factor oracle. In the first step it detects exact repeats in large sequences. Then, in the second step, it computes approximate repeats and performs pairwise comparison. We compared its computational characteristics with BLAST and REPuter. Results demonstrate that it is fast and space economical. We show FORRepeats ability to perform intra-genomic comparison and to detect repeated DNA sequences in the complete genome of the model plant Arabidopsis thaliana.     

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.     

Differential distribution of simple sequence repeats in eukaryotic genome sequences.

Complete chromosome/genome sequences available from humans, Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana, and Saccharomyces cerevisiae were analyzed for the occurrence of mono-, di-, tri-, and tetranucleotide repeats. In all of the genomes studied, dinucleotide repeat stretches tended to be longer than other repeats. Additionally, tetranucleotide repeats in humans and trinucleotide repeats in Drosophila also seemed to be longer. Although the trends for different repeats are similar between different chromosomes within a genome, the density of repeats may vary between different chromosomes of the same species. The abundance or rarity of various di- and trinucleotide repeats in different genomes cannot be explained by nucleotide composition of a sequence or potential of repeated motifs to form alternative DNA structures. This suggests that in addition to nucleotide composition of repeat motifs, characteristic DNA replication/repair/recombination machinery might play an important role in the genesis of repeats. Moreover, analysis of complete genome coding DNA sequences of Drosophila, C. elegans, and yeast indicated that expansions of codon repeats corresponding to small hydrophilic amino acids are tolerated more, while strong selection pressures probably eliminate codon repeats encoding hydrophobic and basic amino acids. The locations and sequences of all of the repeat loci detected in genome sequences and coding DNA sequences are available at http://www.ncl-india.org/ssr and could be useful for further studies.