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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↗
PubMed1996

Evolution of the gonosomal heterochromatin of Microtus agrestis: rapid amplification of a large, multimeric, repeat unit containing a 3.0-kb (GATA)11-positive, middle repetitive element.

The sex chromosomes of Microtus agrestis are extremely large due to the accumulation of constitutive heterochromatin. We have cloned and characterized a 2,999-bp (GATA)n-positive sequence, following HaeIII digestion, that is confined to the noncentromeric heterochromatin of the X chromosome. The cloned element exhibits an accumulation of certain oligomers, which are scattered throughout its entire length, and several copies of Chi-related sequence motifs, which are thought to be implicated in recombination. The latter might have been responsible for the extensive amplification of homologous genomic elements. The sequence has been amplified to a copy number of 1-2 x 10(4) within the genome of M. agrestis. In contrast to many satellite DNAs, which are thought to be an inevitable constituent of constitutive heterochromatin, the sequence exhibits a tissue-specific methylation pattern and is organized, not as a simple tandem array, but as a component of an extremely large, multimeric, higher-order repeat unit with a length of over 20 kb. This higher-order repeat accounts for at least 15-30% of the gonosomal heterochromatin in M. agrestis. Sequences homologous to pMAHAE2 are abundant in the genomes of all Microtus species. The copy number varies from approximately 100 per diploid genome in M. arvalis, M. oeconomus, and M. cabrerae to approximately 500 per diploid genome in M. guentheri and up to 1-2 x 10(4) in M. agrestis. Our molecular data indicate that the sequences of the pMAHAE2 family probably arose during the evolution of the common ancestor of Microtus and have subsequently been amplified extensively in the X chromosomes of M. agrestis in the phylogenetically very short period of less than 1 million years.

Animals↗
PubMed1990

Centromeres of budding and fission yeasts.

Centromeres of the budding yeast Saccharomyces cerevisiae are structurally relatively simple, are specified by only about 125 base pairs of DNA, and contain no repeated DNA sequences. The centromere regions in the fission yeast Schizosaccharomyces pombe span many kilobase pairs of DNA and contain repeated DNA sequences that appear to be necessary for full centromere function. A portion of the repeated sequences is organized into a large inverted repeated structure in the centromere region of each S. pombe chromosome. Fission yeast provides an excellent model system for studying the role of repeated DNA sequences in centromere function.

Base Sequence↗
PubMed2005

Cloning and sequence analysis of the MAT-B (MAT-2) genes from the three Dutch elm disease pathogens, Ophiostoma ulmi, O. novo-ulmi, and O. himal-ulmi.

There were two successive pandemics of Dutch Elm Disease (DED) in Europe, parts of Asia and North America in the last century, caused by two ascomycete fungal species, Ophiostoma ulmi and O. novo-ulmi. A third DED species, O. himal-ulmi, was later discovered in the Himalayas. For each of these three species, we now report on the cloning and analysis of a 2.2 kb sequence containing the coding region and 5' and 3' flanking sequences of the mating type B (MAT-B) gene, which is involved in the control of sexual compatibility. The amino acid sequence of the single protein encoded by the gene for each species contained a conserved DNA-binding motif called the high mobility group (HMG) box which showed significant sequence similarity to corresponding sequences in many ascomycete MAT-2 genes. Phylogenetic trees constructed from the MAT-B (renamed MAT-2) nucleotide and derived amino acid sequences showed distinct clades corresponding to the three Ophiostoma species and a clear separation of the O. novo-ulmi clade into the two subspecies americana and novo-ulmi. The 3' flanking regions have been shown to contain variable numbers of repeated oligonucleotide sequences, the number of which is species-specific and readily distinguished by a simple PCR assay.

Amino Acid Motifs↗
PubMed2004

Telomere instability detected in sporadic colon cancers, some showing mutations in a mismatch repair gene.

Human telomeres are essential for genome stability and are composed of long simple tandem repeat arrays (STRs), comprising the consensus TTAGGG repeat interspersed, at the proximal end, with sequence-variant repeats. While the dynamics of telomere attrition through incomplete replication has been studied extensively, the effects on telomeres of error-prone DNA repair processes, known to affect other STRs, are poorly understood. We have compared the TTAGGG and sequence-variant interspersion patterns in the proximal 720 bp of telomeres in colon cancer and normal DNA samples. The frequency of telomere mutations was 5.8% per allele in a randomly collected panel of sporadic colon cancers, showing that telomere mutations occur in vivo. The mutation frequency rose to 18.6% per allele in sporadic tumours that exhibit instability at the polyA tract in the TGFbetaRII gene and to 35% per allele in tumours with somatic mutations in the hMSH2 gene. The majority of the characterized mutations resulted in the loss of one or a few repeats. If the mutation spectrum and frequency described here is reiterated in the rest of the array, there is the potential for extensive telomere destabilization especially in mismatch repair-defective cells. This may in turn lead to a greater requirement for telomere length maintenance earlier in tumourigenesis.

Base Pair Mismatch↗
PubMed1988

Developmentally controlled telomere addition in wild-type and mutant paramecia.

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.

Animals↗
PubMed1992

Fast one-step procedure for the detection of nucleic acids in situ by primer-induced sequence-specific labeling with fluorescein-12-dUTP.

We provide fast, simple, one-step procedures for sequence-specific detection of nucleic acids in situ. Tandem repeat sequences in DNA are stained within 30 min, and mRNA is stained within 2 h. The procedures are based on the incorporation of the newly available fluorescein-labeled dUTP into DNA synthesized in situ by primed in situ labeling, with denatured fragments of cloned DNA or oligonucleotides as primers. The extreme speed and simplicity of the reaction make it attractive for automatization in routine laboratory procedures and opens up new diagnostic possibilities.

DNA↗
PubMed2006

Activation mapping as a percentage of local excitation: fMRI stability within scans, between scans and across field strengths.

Functional magnetic resonance imaging (fMRI) does not typically yield highly reproducible maps of brain activation. Maps can vary significantly even with constant scanning parameters and consistent task performance conditions (Liu et al., Magn. Reson. Med., 2004, 52:751-760). Reproducibility is even more of a problem when comparing fMRI signal magnitude and spatial extent of activation across scans involving different task performance levels, scan durations, pulse sequences or magnetic field strengths. In this report, the consistency of fMRI was reexamined by considering the relative spatial and temporal distribution of fMRI blood oxygen level dependent (BOLD) activation signals separately from the absolute magnitude of the activation signal in each brain area. Subjects repeatedly performed the same simple motor task but under a variety of imaging conditions, using both spiral and standard echo-planar pulse sequences and at 1.5- and 4.0-T magnetic field strengths. The results demonstrate that the absolute amplitude of BOLD statistical activation signals varied significantly across time and scanning conditions, but the relative spatial pattern of BOLD activation was highly reproducible across all conditions. Analysis of realistic simulated fMRI data sets indicates that stability of relative activation patterns could provide a useful tool for assessing the accuracy of fMRI maps.

Adult↗
PubMed2002

Simple sequences are rare in the Protein Data Bank.

A simple sequence is abundant in the proteins that have been sequenced to date. But unusual protein features, such as a simple sequence, are not present in the same high frequency within structural databases. A subset of these simple sequences, a group with a highly repetitive nature has been shown to be abundant in eukaryotes but not in prokaryotes. In this study, an examination of the eukaryotic proteins in the Protein Data Bank (PDB) has revealed a large deficiency of low complexity, highly repetitive protein repeats. Through simulated databases of similar samples of eukaryotic proteins taken from the National Center for Biotechnology Information (NCBI) database, it is shown that the PDB contains a significantly less highly repetitive, simple sequence than artificial databases of similar composition randomly derived from NCBI. When the structural data for those few PDB sequences that did contain a highly repetitive simple sequence is examined in detail, it is found that in most cases the tertiary structure is unknown for the regions consisting of a simple sequence. This lack of a simple sequence both in the PDB database and in the structural information suggests that this type of simple sequence may produce disordered structures that make structural characterization difficult.

Amino Acid Sequence↗
PubMed1994

The analysis of simple repeat loci as applied in evolutionary and behavioral sciences.

This chapter describes several aspects of tandemly organized, simple repetitive DNA sequences and their usefulness for genetic relationship analyses. After introducing the structure, the evolution and the biological meaning of such target sequences in a particularly well-studied gene, we discuss oligonucleotide probes for generating individual specific multilocus banding patterns. Thus, oligonucleotide fingerprinting allows to approach novel problems in behavioral sciences. Here, we use a passerine bird, the great tit (Parus major) as an example. Finally, genomic fingerprinting is compared to sensitive amplification methods requiring less DNA. Advantages and shortcomings of these techniques need to be evaluated in the context of the biological question(s) asked and, above all, the quality and quantity of the starting material.

Animals↗
PubMed2006

Quantitative analysis of chimerism using a short tandem repeat method on a fluorescent automated DNA sequencer.

Stem cell transplantation (SCT) is the treatment of choice for a number of malignant and nonmalignant diseases. Monitoring of SC engraftment or microchimerism (MC) is important for diagnosis of relapse, rejection or graft vs. host disease (GVHD). The goal of this study was to develop a sensitive and relatively simple method for MC lineage analysis using the Visible Genetics fluorescence automated sequencer. Sensitivity of the method was studied by polymerase chain reaction (PCR) amplification of informative short tandem repeats (STR) using donor/recipient DNA mixtures as the templates and DNA extracted from donor and recipient CD3+, CD19+ and CD15+ cells mixed at various ratios. Semi-quantitative analysis was performed using the Visible Genetics software and percent of donor specific signal was calculated. The sensitivity of this method varied from 0.8% to 6.2% for both DNA and cellular MC in CD3+, CD19+ and CD15+ subsets. Regression analysis revealed linearity (r = 0.94) between the number of donor cells in the mixture and intensity of MC fluorescent signal. These data indicate that the Visible Genetics polyacrylamide gel sequencer can be successfully used for MC analysis in SC recipients providing a relatively high level of sensitivity.

Antigens, CD↗
PubMed1999

Use of a quantitative structure-property relationship to design larger model proteins that fold rapidly.

A quantitative structure-property relationship (QSPR) was used to design model protein sequences that fold repeatedly and relatively rapidly to stable target structures. The specific model was a 125-residue heteropolymer chain subject to Monte Carlo dynamics on a simple cubic lattice. The QSPR was derived from an analysis of a database of 200 sequences by a statistical method that uses a genetic algorithm to select the sequence attributes that are most important for folding and a neural network to determine the corresponding functional dependence of folding ability on the chosen attributes. The QSPR depends on the number of anti-parallel sheet contacts, the energy gap between the native state and quasi-continuous part of the spectrum and the total energy of the contacts between surface residues. Two Monte Carlo procedures were used in series to optimize both the target structures and the sequences. We generated 20 fully optimized sequences and 60 partially optimized control sequences and tested each for its ability to fold in dynamic MC simulations. Although sequences in which either the number of anti-parallel sheet contacts or the energy of the surface residues is non-optimal are capable of folding almost as well as fully optimized ones, sequences in which only the energy gap is optimized fold markedly more slowly. Implications of the results for the design of proteins are discussed.

Databases, Factual↗
PubMed2000

Elongation of tandem repetitive DNA by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis at a hairpin-coil transitional state: a model of amplification of a primordial simple DNA sequence.

DNA is replicated by DNA polymerase semiconservatively in many organisms. Accordingly, the replicated DNA does not become larger than the original DNA (template DNA), implying that replicative synthesis by DNA polymerase alone cannot explain the diversification of primordial simple DNA. We demonstrate that a single-stranded tandem repetitive oligodeoxyribonucleic acid (oligoDNA) composed of a palindromic or quasi-palindromic motif sequence and 25-50% GC content is elongated in vitro to more than 20,000 bases at 70-74 degrees C by the DNA polymerase of the hyperthermophilic archaeon Thermococcus litoralis without a bimolecular primer-template complex. The efficiency of elongation decreased when the palindromic structure of the oligoDNA was destroyed or when the GC content of the oligoDNA was outside the range of 25-50%. The thermal melting transition profile of the oligoDNA, as observed by ultraviolet spectroscopy, exhibited a biphasic curve, reflecting a duplex-hairpin transition at 31-40 degrees C and a hairpin-coil transition at 70-77 degrees C. The optimal reaction temperature for the elongation, for instance, of oligoDNA (AGATATCT)(6) (72 degrees C) was very close to its hairpin-coil transition melting temperature (70.4 degrees C), but was markedly higher than the temperature at which duplex oligoDNA can exist stably (<35.9 degrees C). These results suggest that a hairpin-based "intramolecular primer-template structure" is formed transiently in the oligoDNA, and it is elongated by the DNA polymerase to long DNA through repeated cycles of folding and melting of the hairpin structure. We discuss the implication of this phenomenon, "hairpin elongation", from the standpoint of potential amplification of simple DNA sequences during the evolution of the genome.

Base Composition↗