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PubMed1996

Detection and purification of sequence-specific DNA binding protein.

A simple assay for identifying DNA-binding proteins is described that involves loading of protein fractions onto nitrocellulose membrane using a slot-blot apparatus, incubating with 32P-labeled DNA probe in buffer in the presence of excess of nonspecific E. coli DNA at room temperature, and washing with increasing concentration of NaCl (from 50 to 500 mM) to obtain optimum signal. A simple and rapid scheme of purification of a sex and tissue-specific DNA-binding protein, which binds specifically to the GATA repeats of Bkm (banded krait minor satellite DNA), designated as Bkm-binding protein (BBP), is also described. This requires only a DNA affinity column after the initial ammonium sulfate precipitation. The insert (545 bp) of the Drosophila clone 2(8) containing 66 copies of GATA repeats was used to prepare the sequence-specific DNA-Sepharose affinity column. The slot-blot-binding assay and the simple scheme of purification described here may be used for routine screening and purification of sequence-specific DNA-binding proteins in general.

Animals↗
PubMed2001

Length of uninterrupted repeats determines instability at the unstable mouse expanded simple tandem repeat family MMS10 derived from independent SINE B1 elements.

Mouse expanded simple tandem repeats (ESTRs) provide highly informative loci for analyzing spontaneous and induced germline mutation. We have conducted an extensive sequence database search and identified 17 new members of the highly unstable rodent-specific ESTR family called MMS10. This family has arisen by independent expansions of a common GGCAGA repeat unit from within a subset of both ancestral and modern SINE B1 elements during the course of mouse evolution. Analysis of the interspersion patterns of variant repeats along alleles of 20 of these MMS10 loci revealed two distinct classes of tandem arrays: one composed of uninterrupted GGCAGA repeats and the second with generally larger arrays interrupted by variant units. Surveys of allelic diversity at 11 representative members of these two classes of loci in various laboratory strains and BXD recombinant inbred lines revealed that the level of repeat instability was positively correlated with the length of uninterrupted repeats. Turnover processes at MMS10 loci, therefore, appear similar to the type of mechanism observed at human microsatellites. The MMS10 family thus provides a potentially useful murine model for studying dynamic mutation at simple tandem repeats.

Alleles↗
PubMed2004

Mutation rates in the complex microsatellite MYCL1 and related simple repeats in cultured human cells.

Microsatellite instability is a phenotype observed in tumors cells that have defects in DNA mismatch repair (MMR). Most markers used for detecting microsatellite instability are mono- and dinucleotide repeats, but one tetranucleotide repeat (MYCL1) has been reported to be useful for this purpose. The MYCL1 repeat is actually a complex repeat, made up of approximately 14 GAAA tetranucleotides plus various other GA-rich repeats. In order to determine the nature of the instability of the this sequence, we have used a frameshift-reversion assay in MMR-proficient and -deficient human cells to compare the mutation rates and the types of mutation of MYCL1 to those of the related simple repeats (GAAA)17, (GA)17, and (CA)17. We found that the complex repeat was the most stable of the repeats examined in cells deficient in MMR; the tetranucleotide was less stable, while the dinucleotides were the least stable. In MMR-proficient cells, the relative rates were reversed; the MYCL1 repeat was the least stable, the tetranucleotide was more stable, and the dinucleotides were the most stable. These results suggest that MYCL1 and the pure tetranucleotide have relatively low rates of errors during replication, but that the errors in these repeats are corrected less efficiently than those in the smaller repeats. Because of their high rate of instability in MMR-proficient cells, MYCL1 and other tetranucleotide repeats appear to lack specificity for detection of tumors with defective MMR.

Base Sequence↗
PubMed2006

ProtRepeatsDB: a database of amino acid repeats in genomes.

BACKGROUND: Genome wide and cross species comparisons of amino acid repeats is an intriguing problem in biology mainly due to the highly polymorphic nature and diverse functions of amino acid repeats. Innate protein repeats constitute vital functional and structural regions in proteins. Repeats are of great consequence in evolution of proteins, as evident from analysis of repeats in different organisms. In the post genomic era, availability of protein sequences encoded in different genomes provides a unique opportunity to perform large scale comparative studies of amino acid repeats. ProtRepeatsDB http://bioinfo.icgeb.res.in/repeats/ is a relational database of perfect and mismatch repeats, access to which is designed as a resource and collection of tools for detection and cross species comparisons of different types of amino acid repeats. DESCRIPTION: ProtRepeatsDB (v1.2) consists of perfect as well as mismatch amino acid repeats in the protein sequences of 141 organisms, the genomes of which are now available. The web interface of ProtRepeatsDB consists of different tools to perform repeat s; based on protein IDs, organism name, repeat sequences, and keywords as in FASTA headers, size, frequency, gene ontology (GO) annotation IDs and regular expressions (REGEXP) describing repeats. These tools also allow formulation of a variety of simple, complex and logical queries to facilitate mining and large-scale cross-species comparisons of amino acid repeats. In addition to this, the database also contains sequence analysis tools to determine repeats in user input sequences. CONCLUSION: ProtRepeatsDB is a multi-organism database of different types of amino acid repeats present in proteins. It integrates useful tools to perform genome wide queries for rapid screening and identification of amino acid repeats and facilitates comparative and evolutionary studies of the repeats. The database is useful for identification of species or organism specific repeat markers, interspecies variations and polymorphism.

Amino Acid Sequence↗
PubMed2003

Noncoding plastid trnT-trnF sequences reveal a well resolved phylogeny of basal angiosperms.

Recent contributions from DNA sequences have revolutionized our concept of systematic relationships in angiosperms. However, parts of the angiosperm tree remain unclear. Previous studies have been based on coding or rDNA regions of relatively conserved genes. A phylogeny for basal angiosperms based on noncoding, fast-evolving sequences of the chloroplast genome region trnT-trnF is presented. The recognition of simple direct repeats allowed a robust alignment. Mutational hot spots appear to be confined to certain sectors, as in two stem-loop regions of the trnL intron secondary structure. Our highly resolved and well-supported phylogeny depicts the New Caledonian Amborella as the sister to all other angiosperms, followed by Nymphaeaceae and an Austrobaileya-Illicium-Schisandra clade. Ceratophyllum is substantiated as a close relative of monocots, as is a monophyletic eumagnoliid clade consisting of Piperales plus Winterales sister to Laurales plus Magnoliales. Possible reasons for the striking congruence between the trnT-trnF based phylogeny and phylogenies generated from combined multi-gene, multi-genome data are discussed.

Base Sequence↗
PubMed1987

Slipped-strand mispairing: a major mechanism for DNA sequence evolution.

Simple repetitive DNA sequences are a widespread and abundant feature of genomic DNA. The following several features characterize such sequences: (1) they typically consist of a variety of repeated motifs of 1-10 bases--but may include much larger repeats as well; (2) larger repeat units often include shorter ones within them; (3) long polypyrimidine and poly-CA tracts are often found; and (4) tandem arrangements of closely related motifs are often found. We propose that slipped-strand mispairing events, in concert with unequal crossing-over, can readily account for all of these features. The frequent occurrence of long tandem repeats of particular motifs (polypyrimidine and poly-CA tracts) appears to result from nonrandom patterns of nucleotide substitution. We argue that the intrahelical process of slipped-strand mispairing is much more likely to be the major factor in the initial expansion of short repeated motifs and that, after initial expansion, simple tandem repeats may be predisposed to further expansion by unequal crossing-over or other interhelical events because of their propensity to mispair. Evidence is presented that single-base repeats (the shortest possible motifs) are represented by longer runs in mammalian introns than would be expected on a random basis, supporting the idea that SSM may be a ubiquitous force in the evolution of the eukaryotic genome. Simple repetitive sequences may therefore represent a natural ground state of DNA unselected for coding functions.

Base Composition↗
PubMed1995

The unusual telomeres of Drosophila.

The telomeres of most eukaryotes contain short, simple repeats that are highly conserved. Drosophila, on the other hand, does not have such sequences, but carries at the ends of its chromosomes one or more LINE-like retrotransposable elements. Instead of elongation by telomerase, incomplete DNA replication at the termini of Drosophila chromosomes is counterbalanced by transposition of these elements at high frequency specifically to the termini. These transposable elements are not responsible for distinguishing telomeric ends in Drosophila from broken chromosome ends; the structure performing this function is not yet known. Proximal to the terminal array of transposable elements are regions of tandem repeats that are structurally, and probably functionally, analogous to the subterminal regions in other eukaryotes.

Animals↗
PubMedDate not supplied

Do mutant woodchuck hepatitis viruses play a role in hepatocellular carcinogenesis?

Although hepadnaviruses are implicated in the aetiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence, DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection, in the serum of woodchucks with hepatocellular carcinoma, of mutant woodchuck hepatitis viruses that are strikingly similar to the rearranged genomes found previously as integrated sequences in cellular DNA. Of the four mutants studied, two had large inverted duplications, one a 219 nucleotide direct duplication, and one a 219 nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at topoisomerase I cleavage sites. Thus, it is possible that rearrangement of the hepadnavirus genome precedes integration of viral DNA into cellular DNA and that mutant genomes that are preferentially integrated into cellular DNA have an aetiological role in hepatocarcinogenesis.

Animals↗
PubMed1993

Mutant woodchuck hepatitis virus genomes from virions resemble rearranged hepadnaviral integrants in hepatocellular carcinoma.

Although hepadnaviruses are implicated in the etiology of hepatocellular carcinoma, the pathogenic mechanisms involved remain uncertain. Clonally propagated integrations of hepadnaviral DNA into cellular DNA can be demonstrated in most virally induced hepatocellular carcinomas. Integration occurs at random sites in cellular DNA, but the highly preferred sites in viral DNA are adjacent to the directly repeated sequence DR1, less often DR2, or in the cohesive overlap region. Integrants invariably contain simple deletions or complex rearrangements that have been thought to occur after integration. We report here the detection of mutant woodchuck hepatitis virus (WHV) genomes cloned from virions in serum that are strikingly similar to the rearranged hepadnaviral genomes found previously as integrated sequences in cellular DNA. Of 102 cloned genomes studied, 2 had large inverted duplications, 1 a 219-nucleotide direct duplication, and 1 a 219-nucleotide deletion. Virus-virus DNA junctions occurred either adjacent to DR1 or DR2 or in the cohesive overlap region at preferred topoisomerase I cleavage sites. Since these sites are located in the single-stranded regions of the genome, cleavage by topoisomerase I would produce linear molecules that would be expected to be highly recombinogenic since this enzyme, possessing nicking and ligating activities, would remain covalently attached. Sucrose density gradient centrifugation coupled with polymerase chain reaction studies confirmed that the mutant WHV DNA forms resided in virions and did not represent free viral DNA released from infected cells or were unlikely to be an artifact of the cloning process. Thus, the finding in virions of mutant WHV DNA similar to WHV DNA integrated into cellular DNA suggests that the processes of mutation and integration are linked in some instances. Furthermore, the mutant genomes that are preferentially integrated into cellular DNA may have an etiologic role in hepatocarcinogenesis.

Animals↗
PubMed1997

Binding of cationic alpha-helical peptides to plasmid DNA and their gene transfer abilities into cells.

Polycationic reagents such as cationic lipids and poly-L-lysine are widely used for gene transfer into cells in vitro and show promise as vectors for in vivo gene therapy applications as nonviral gene transfer techniques. We have developed a novel transfection method using cationic amphiphilic alpha-helical oligopeptides with repeated sequences. Oligopeptide has the advantages of being easily designed and modified because of its simple structure. In this study, we synthesized five kinds of peptides of which the total chain length and the width of the hydrophobic region were changed. The binding of the peptides to plasmid DNA was evaluated by agarose gel electrophoresis. It was found that the long and/or hydrophobic peptides can strongly bind to the DNA. The formation of large aggregates with a 0.5-5-microm diameter, which consisted of the long peptides and the DNA, was observed by electron microscopy. The transfection abilities of the peptides were determined by the expression of luciferase from its cDNA in COS-7 cells. The long peptides showed high transfection abilities. As a result, it could be said that the transfection ability of these peptides was parallel to their ability to form aggregates with DNA. Furthermore, the transfection ability was increased by the addition of chloroquine in the transfection procedure. This result indicated that the internalization of the peptide-DNA aggregates would be mediated by the endocytosis pathway.

Animals↗
PubMed1998

Dissociable processes for learning the surface structure and abstract structure of sensorimotor sequences.

A sensorimotor sequence may contain information structure at several different levels. In this study, we investigated the hypothesis that two dissociable processes are required for the learning of surface structure and abstract structure, respectively, of sensorimotor sequences. Surface structure is the simple serial order of the sequence elements, whereas abstract structure is defined by relationships between repeating sequence elements. Thus, sequences ABCBAC and DEFEDF have different surface structures but share a common abstract structure, 123213, and are therefore isomorphic. Our simulations of sequence learning performance in serial reaction time (SRT) tasks demonstrated that (1) an existing model of the primate fronto-striatal system is capable of learning surface structure but fails to learn abstract structure, which requires an additional capability, (2) surface and abstract structure can be learned independently by these independent processes, and (3) only abstract structure transfers to isomorphic sequences. We tested these predictions in human subjects. For a sequence with predictable surface and abstract structure, subjects in either explicit or implicit conditions learn the surface structure, but only explicit subjects learn and transfer the abstract structure. For sequences with only abstract structure, learning and transfer of this structure occurs only in the explicit group. These results are parallel to those from the simulations and support our dissociable process hypothesis. Based on the synthesis of the current simulation and empirical results with our previous neuropsychological findings, we propose a neuro-physiological basis for these dissociable processes: Surface structure can be learned by processes that operate under implicit conditions and rely on the fronto-striatal system, whereas learning abstract structure requires a more explicit activation of dissociable processes that rely on a distributed network that includes the left anterior cortex.

Computer Simulation↗
PubMed1996

Hebbian learning of context in recurrent neural networks.

Single electrode recording in the inferotemporal cortex of monkeys during delayed visual memory tasks provide evidence for attractor dynamics in the observed region. The persistent elevated delay activities could be internal representations of features of the learned visual stimuli shown to the monkey during training. When uncorrelated stimuli are presented during training in a fixed sequence, these experiments display significant correlations between the internal representations. Recently a simple model of attractor neural network has reproduced quantitatively the measured correlations. An underlying assumption of the model is that the synaptic matrix formed during the training phase contains in its efficacies information about the contiguity of persistent stimuli in the training sequence. We present here a simple unsupervised learning dynamics that produces such a synaptic matrix if sequences of stimuli are repeatedly presented to the network at fixed order. The resulting matrix is then shown to convert temporal correlations during training into spatial correlations between attractors. The scenario is that, in the presence of selective delay activity, at the presentation of each stimulus, the activity distribution in the neural assembly contain information of both the current stimulus and the previous one (carried by the attractor). Thus the recurrent synaptic matrix can code not only for each of the stimuli presented to the network but also for their context. We combine the idea that for learning to be effective, synaptic modification should be stochastic, with the fact that attractors provide learnable information about two consecutive stimuli. We calculate explicitly the probability distribution of synaptic efficacies as a function of training protocol, that is, the order in which stimuli are presented to the network. We then solve for the dynamics of a network composed of integrate-and-fire excitatory and inhibitory neurons with a matrix of synaptic collaterals resulting from the learning dynamics. The network has a stable spontaneous activity, and stable delay activity develops after a critical learning stage. The availability of a learning dynamics makes possible a number of experimental predictions for the dependence of the delay activity distributions and the correlations between them, on the learning stage and the learning protocol. In particular it makes specific predictions for pair-associates delay experiments.

Animals↗
PubMed1985

D1 protein of Drosophila melanogaster. Purification and AT-DNA binding properties.

D1 protein of Drosophila melanogaster is a sequence-specific DNA-binding protein which recognizes AT-rich DNA sequences. AT-rich DNA sequences in eukaryotic organisms are distributed in two characteristic ways: flanking transcriptional units and in constitutive heterochromatin. D1 could play a role in regulation of gene expression and in geographical localization of DNA sequences within the nucleus. D1 has been partially purified by ion exchange chromatography. DNA-binding activity was investigated by nucleoprotein gel electrophoresis, using end-labeled restriction fragments varying in AT sequence content. D1 binds most tightly to the satellite sequence -AATAT-, with intermediate strength to the complex satellite (359-base pair repeat) and another AT-rich (68% AT) mixed sequence DNA, and least to the simple satellite sequence -AAGAG-.

Adenine↗
PubMed2006

Detection of a large genomic deletion in the pancreatic secretory trypsin inhibitor (SPINK1) gene.

Mutations and polymorphisms in the SPINK1 gene, which encodes trypsin's physiological inhibitor, pancreatic secretory trypsin inhibitor, have been found to be associated with chronic pancreatitis. However, to date, all currently reported SPINK1 variants are either single-nucleotide substitutions or microinsertions/deletions. It is possible that large genomic rearrangements at this locus may underlie certain cases of chronic pancreatitis. However, such events, if indeed they exist, may have been overlooked by conventional PCR-based techniques. Here we attempted to screen all four exons as well as the promoter region of the SPINK1 gene for large genomic deletions by means of quantitative high-performance liquid chromatography analysis. Of the 47 pancreatitis families (not carrying any known PRSS1, SPINK1 and CFTR variants/mutations after screening the coding regions by our previously established denaturing high-performance liquid chromatography methods), one family was suggested to carry a large genomic deletion in the SPINK1 gene. The aberrant chromosomal junction was encapsulated by long-range PCR and the breakpoints were determined by direct sequencing of the rearranged fragment. A 2-bp short direct repeat was present at the deletion breakpoints; this simple deletion (c.1-320_c.55+961del1336 bp) can thus in principle be explained by replication slippage. Identification of this lesion has not only expanded the SPINK1 mutational spectrum but also served to identify a novel mutational mechanism causing chronic pancreatitis.

Base Sequence↗
PubMed1998

Precise sequence complementarity between yeast chromosome ends and two classes of just-subtelomeric sequences.

The terminal regions (last 20 kb) of Saccharomyces cerevisiae chromosomes universally contain blocks of precise sequence similarity to other chromosome terminal regions. The left and right terminal regions are distinct in the sense that the sequence similarities between them are reverse complements. Direct sequence similarity occurs between the left terminal regions and also between the right terminal regions, but not between any left ends and right ends. With minor exceptions the relationships range from 80% to 100% match within blocks. The regions of similarity are composites of familiar and unfamiliar repeated sequences as well as what could be considered "single-copy" (or better "two-copy") sequences. All terminal regions were compared with all other chromosomes, forward and reverse complement, and 768 comparisons are diagrammed. It appears there has been an extensive history of sequence exchange or copying between terminal regions. The subtelomeric sequences fall into two classes. Seventeen of the chromosome ends terminate with the Y' repeat, while 15 end with the 800-nt "X2" repeats just adjacent to the telomerase simple repeats. The just-subterminal repeats are very similar to each other except that chromosome 1 right end is more divergent.

Base Sequence↗
PubMed1983

Sequence specific cleavage of African green monkey alpha-satellite DNA by micrococcal nuclease.

The sequence specificity of micrococcal nuclease complicates its use in experiments addressed to the still controversial issue of nucleosome phasing. In the case of alpha-satellite DNA containing chromatin from African green monkey (AGM) cells cleavage by micrococcal nuclease in the nucleus was reported to occur predominantly at only one location around position 126 of the satellite repeat unit (Musich et al. (1982) Proc. Natl. Acad. Sci. USA 79, 118-122). DNA control experiments conducted in the same study indicated the presence of many preferential cleavage sites for micrococcal nuclease on the 172 bp long alpha-satellite repeat unit. This difference was taken as evidence for a direct and simple phase relationship between the alpha-satellite DNA sequence and the position of the nucleosomes on the DNA. We have quantitatively analyzed the digestion products of the protein-free satellite monomer with micrococcal nuclease and found that 50% of all cuts occur at positions 123 and 132, 5% at position 79, and to a level of 1-3% at about 20 other positions. We also digested high molecular weight alpha-satellite DNA from AGM nuclei with micrococcal nuclease. Again cleavage occurred mostly at positions 123 and 132 of the satellite repeat unit. Thus digestion of free DNA yields results very similar to those reported by Musich et al. for the digestion of chromatin. Therefore no conclusions on a possible phase relationship can be drawn from the chromatin digestion experiments.

Animals↗
PubMed1989

Mouse U3-RNA-processed pseudogenes are nonrandomly integrated into genomic DNA. Implications for the process of retrogene formation.

We have characterized three mouse U3-RNA-processed pseudogenes. Together with previous analyses of mouse functional genes and of rat genes and pseudogenes, these data provide some insights into the processes of U3 retrogene formation during the evolution of rodents. All the mouse retrogenes correspond to a full-length U3B-coding sequence with a 3'-poly(A) tail and are precisely flanked by a pair of direct repeats, in agreement with formation through an RNA intermediate followed by insertion at staggered nicks in the genome. All the rodents U3 retrogenes identified so far are produced from a U3B-RNA form, with two of them (one for each rodent) formed from a 3'-elongated U3-RNA precursor, pointing to the particular susceptibility of RNA precursors forms to serve as templates for retrogene formation. Rodent full-length U3 retrogenes are not inserted randomly in the genome but are systematically found in a context of simple sequences, prone to slipped-strand mispairings and likely to favour the appearance of single-stranded DNA. Moreover their flanking repeats share not only the same size (15 bp) but also common sequence features (which extend to vicinal upstream nucleotides) suggesting that common mechanisms, specific to this class of retrogenes, have been involved in their formation. For the first time, a model accounting for the generation of full-length cDNA copies from nonpolyadenylated RNA templates is proposed. Rodent U3 retrogenes appear to be of rather recent origin (posterior to the mouse/rat divergence) and some of them could have undergone subsequent genetic exchanges with the functional genes.

Animals↗