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At least 1,495 records · Page 83Linked to original sources
PubMed1982

Analysis of foldback sequences and repetitive sequences in cloned segments of nuclear DNA from Physarum polycephalum.

The properties of DNA segments containing foldback elements were studied after their selection from a "random" recombinant library of Physarum polycephalum nuclear DNA sequences, cloned using the plasmid vector pBR322. Hybridisation of in vitro labelled recombinant plasmids to Southern blots of genomic restriction fragments demonstrated that each cloned segment contained repetitive elements located at several hundred sites in the genome. Two of the DNA clones generated hybridisation patterns which suggested that they contain repetitive elements with internal cleavage sites for the restriction endonuclease HaeIII. Cross-hybridisation of all combinations of the cloned sequences showed that most contain different arrangements of repetitive elements derived from different sequence families. The results are consistent with a model proposed previously on the basis of studies on total nuclear DNA, for the organisation of sequences closely associated with foldback elements in the Physarum genome.

Base Sequence↗
PubMed1984

Location of DNA sequences complementary to small nuclear repeat RNA (fr 3-RNA) in relation to DNA sequences coding for albumin and alpha-fetoprotein in rat liver cells.

Rat liver nuclei contain a 29-nucleotides-long RNA (fr 3-RNA) which is transcribed from middle repetitive DNA sequences. By Southern analysis of restriction fragments of rat albumin and alpha-fetoprotein genomic clones, DNA sequences complementary to this RNA were detected on a 4.6 kbp Eco RI fragment located 600 bp downstream from the termination exon of the albumin gene and on a 2 kbp Eco RI-HindIII fragment located 10 kbp downstream from the restriction fragment containing the alpha-fetoprotein site. No sequence complementary to this RNA was found either in the introns of exons of both genes or in the regions extending 7 kbp upstream from the first albumin exon and 10 kbp upstream of the first alpha-fetoprotein exon. We concluded that sequences complementary to fr 3-RNA are present at the 3'-end flanking regions of the rat albumin and alpha-fetoprotein gene complexes.

Animals↗
PubMed1999

Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

3' Untranslated Regions↗
PubMed1980

Sequence organization in nuclear deoxyribonucleic acid from Physarum polycephalum. Physical properties of foldback sequences.

An investigation was performed with the use of physical techniques, to determine the nature and organization of inverted repeat sequences in nuclear DNA fragments from Physarum polycephalum. From the average size of foldback duplexes (550 nucleotide pairs), and the foldback duplex yield as determined by treatment of DNA with S1 deoxyribonuclease followed by hydroxyapatite chromatography, it is estimated that there are at least 25000 foldback sequences in the Physarum genome. Foldback DNA molecules exhibit properties intermediate between single-stranded DNA and native duplexes on elution from hydroxyapatite with a salt gradient. In addition, thermal-elution chromatography of foldback DNA from hydroxyapatite crystals shows that foldback duplexes are less stable than native DNA. These properties can be explained on the basis that inverted repeat sequences are mismatched when in the foldback configuration. The results of experiments in which the binding of foldback DNA molecules to hydroxyapatite was determined, by using fragments of different single-chain size, agree with previous studies indicating that inverted repeat sequences are located, on average, every 7000 residues throughout the Physarum genome. The inverted repeats are derived from both the repetitive and single-copy components in Physarum nuclear DNA.

Base Sequence↗
PubMed1981

Sequencing long DNA fragments cloned in bacteriophage M13 by using internal primers. The sequence analysis of a yeast DNA fragment containing a replication origin.

In the ;shotgun' procedure for sequencing DNA, DNA fragments are cloned into a phage M13 vector and sequenced by using a flanking primer. In a variation of this procedure a longer DNA sequence is cloned into M13, the two single-stranded recombinants identified and sequenced by using a set of internal primers prepared by exonuclease III digestion of restriction fragments.

Base Sequence↗
PubMed1997

Diversity of DNA sequences among Vibrio cholerae O1 and non-O1 isolates detected by whole-cell repetitive element sequence-based polymerase chain reaction.

Vibrio cholerae strains isolated from patient, food and environmental sources in Taiwan and reference V. cholerae strains were examined by repetitive element sequence-based PCR (rep-PCR). Specimens from broth cultures were used directly in the PCR mixture with three different primers. The PCR fingerprinting profiles of toxigenic 01 isolates were not only homogeneous with primers from enterobacterial repetitive intergenic consensus (ERIC) sequences, but also allowed the differentiation from non-toxigenic O1 and non-O1 strains. Toxigenic 01 strains were further differentiated into El Tor and classical biotypes with primers designed from ERIC-related sequences of V. cholerae. Primers from the other V. cholerae repetitive DNA sequences, VCR, separated toxigenic El Tor strains into six groups and a unique pattern was also obtained in 16 isolates from imported cases of cholera and imported seafood. The results indicated that rep-PCR can be used to identify and differentiate different toxigenic 01, non-toxigenic 01 and non-O1 V. cholerae isolates.

Bacterial Typing Techniques↗
PubMed1974

Topology of repeated sequences: relationship of nuclear RNA to the repeated sequences of the main and satellite DNA in mouse plasmocytoma cells.

The topology of repeated sequences in mouse plasmocytoma DNA was studied by high-resolution CsCl density gradient centrifugation and heterogeneous nuclear RNA.DNA hybridization. Satellite region DNA of plasmocytoma cells contains additional components and hybridizes specifically with entire heterogeneous nuclear RNA molecules. A linkage is demonstrated between the A+T-rich satellite sequences and those hybridizing with heterogeneous nuclear RNA. Heavy DNA also hybridizes specifically with heterogeneous nuclear RNA molecules that show sequence similarity to heterogeneous nuclear RNA hybridized to satellite DNA. These results could suggest that part of satellite DNA became heavier after integration of some other DNA species, which could belong to a virus or to immunoglobulin repetitive genes. Dispersed, highly repetitive, short nucleotide sequences could constitute recognition sites for such a process.

Animals↗
PubMed1979

Parvovirus RNA transcripts containing sequences not present in mature mRNA: a method for isolation of putative mRNA precursor sequences.

We report here a method of RNA preparation that may enrich for precursor RNA sequences and the results of an investigation of adeno-associated virus (AAV) RNA transcription that used this method. Whole cells were lysed with detergent and high salt and separated into supernatant and pellet (crude chromatin) fractions. These fractions were then separately deproteinized by proteolytic digestion and phenol extractions. DNA was removed from the preparation by two cycles of pancreatic DNase digestion and phenol extraction. Hybridization analyses of the RNA obtained from AAV/adenovirus-infected KB (human) cells revealed some AAV-specific RNA sequences that were not present in the mature 20S mRNA. These additional sequences were contained in AAV RNA molecules present in the pellet fraction, whereas the 20S AAV mRNA accumulated in the supernatant. A species of AAV-specific RNA (about 22S), which was associated only with the pellet fraction and was labeled only after a short pulse, appeared to have a kinetic relationship with the more stable cytoplasmic 20S mRNA. These putative AAV mRNA "precursors" and precursor sequences were not observed previously when conventional methods were used to obtain RNA from either whole cells or isolated nuclei.

Base Sequence↗
PubMed1980

Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo.

The control region of a sea urchin H2A histone gene may be functionally dissected into at least three DNA segments, which we have termed modulator, selector, and initiator elements. While the initiator and in particular the selector containing the T-A-T-A-A-A-T-A sequence are specificity elements that dictate the generation of faithful 5' ends to H2A mRNA, the modulators control the rate at which these specificity elements operate [Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. USA 77, 1432-1436]. By functional tests of in vitro mutated histone DNA in the Xenopus oocyte we have now discovered that the segment E of the A+T-rich spacer DNA lying at a considerable distance upstream of the conservative T-A-T-A-A-A-T-A sequence is a strong modulator element of H2A gene transcription. Deletion of this element creates a 15- to 20-fold H2A-specific down mutation. Segment E by itself cannot elicit initiation of transcription except in coordination with the prelude sequence of the H2A gene. The nucleotide sequence of the relevant spacer element showing modulator activity has been determined and found to contain a pattern of T and A runs as well as a series of inverted repeats. Additional pre-H2A spacer mutants, including a spacer inversion mutant, have been constructed in vitro, that, when injected into the oocyte nucleus, modulate the expression of the H2A gene by an overall factor as large as 100. Other factors controlling promoter activity are discussed.

Animals↗
PubMed1985

mRNA precursor splicing in vivo: sequence requirements determined by deletion analysis of an intervening sequence.

To define the extent of intervening sequence required for splicing higher eukaryotic mRNA precursors in vivo, we constructed deletions within the second intervening sequence of the human G gamma-globin gene that progressively approach the donor or acceptor splice sites. Most of the intervening sequence can be deleted with no effect on splicing. At the donor splice site, 6 bases of intervening sequence are sufficient for accurate and efficient splicing. At the acceptor splice site, 20 bases are sufficient for accurate and efficient splicing, and 16 bases are sufficient for accurate splicing but at a reduced level. However, 15 bases are insufficient for splicing at a significant level. The effect of deletions ending near the acceptor splice site is independent of whether an A-G dinucleotide is introduced into the acceptor splice site region by the deletion.

Base Sequence↗
PubMed1988

DNA base sequence changes and sequence specificity of bromodeoxyuridine-induced mutations in mammalian cells.

By using a shuttle vector system developed in our laboratory, we have carried out studies on the molecular mechanism by which 5-bromodeoxyuridine (BrdUrd) induces mutations in mammalian cells. The target for mutagenesis in these studies was the Escherichia coli gpt gene that was contained within a retroviral shuttle vector and integrated into chromosomal DNA in mouse A9 cells. Shuttle vector-transformed cells expressing the gpt gene were mutagenized with BrdUrd and cells with mutations in the gpt gene were selected. Shuttle vector sequences were recovered from the mutant cells, and the base sequence of the mutant gpt genes was determined. The great majority of the BrdUrd-induced mutations involving single-base changes were found to be G.C----A.T transitions. We have shown that mutagenesis by BrdUrd depends upon perturbation of deoxycytidine metabolism. Thus, the current results suggest that BrdUrd mutagenesis involves mispairing and misincorporation of BrdUrd opposite guanine in DNA, driven by nucleotide pool perturbation caused by BrdUrd and the resulting imbalanced supply of triphosphates available for DNA synthesis. The results also revealed a very high degree of sequence specificity for the BrdUrd mutagenesis. BrdUrd-induced G.C----A.T transitions occurred almost exclusively in sequences with two adjacent guanine residues. Furthermore, in approximately equal to 90% of the cases, the guanine residue involved in mutation was the one in the more 3' position.

Base Composition↗
PubMed2004

Sequence features of Oxytricha trifallax (class Spirotrichea) macronuclear telomeric and subtelomeric sequences.

We sequenced and analyzed the subtelomeric regions of 1356 macronuclear "nanochromosomes" of the spirotrichous ciliate Oxytricha trifallax. We show that the telomeres in this species have a length of 20 nt, with minor deviations; there is no correlation between telomere lengths at the two ends of the molecule. A search for open reading frames revealed that the 3' and 5' untranslated regions are short, with a median length of approximately 130 nt, and that surprisingly there are no detectable differences between sequences upstream and downstream of genes. Our results confirm a previously reported purine bias in the first approximately 80 nucleotides of the subtelomeric regions, but with this larger data set we curiously detected a 10 bp periodicity in the bias; we relate this finding to the possible regulatory and structural functions these regions must serve. Palindromic sequences in opposing subtelomeric regions, although present in most sequences, are not statistically significant.

Animals↗
PubMed1998

Comparative performance of high-density oligonucleotide sequencing and dideoxynucleotide sequencing of HIV type 1 pol from clinical samples.

The performance of the high-density oligonucleotide array methodology (GeneChip) in detecting drug resistance mutations in HIV-1 pol was compared with that of automated dideoxynucleotide sequencing (ABI) of clinical samples, viral stocks, and plasmid-derived NL4-3 clones. Sequences from 29 clinical samples (plasma RNA, n = 17; lymph node RNA, n = 5; lymph node DNA, n = 7) from 12 patients, from 6 viral stock RNA samples, and from 13 NL4-3 clones were generated by both methods. Editing was done independently by a different investigator for each method before comparing the sequences. In addition, NL4-3 wild type (WT) and mutants were mixed in varying concentrations and sequenced by both methods. Overall, a concordance of 99.1% was found for a total of 30,865 bases compared. The comparison of clinical samples (plasma RNA and lymph node RNA and DNA) showed a slightly lower match of base calls, 98.8% for 19,831 nucleotides compared (protease region, 99.5%, n = 8272; RT region, 98.3%, n = 11,316), than for viral stocks and NL4-3 clones (protease region, 99.8%; RT region, 99.5%). Artificial mixing experiments showed a bias toward calling wild-type bases by GeneChip. Discordant base calls are most likely due to differential detection of mixtures. The concordance between GeneChip and ABI was high and appeared dependent on the nature of the templates (directly amplified versus cloned) and the complexity of mixes.

Anti-HIV Agents↗
PubMed1994

Sequence Similarity Presenter: a tool for the graphic display of similarities of long sequences for use in presentations.

A new method for the presentation of alignments of long sequences is described. The degree of identity for the aligned sequences is averaged for sections of a fixed number of residues. The resulting values are converted to shades of gray, with white corresponding to lack of identity and black corresponding to perfect identity. A sequence alignment is represented as a bar filled with varying shades of gray. The display is compact and allows for a fast and intuitive recognition of the distribution of regions with a high similarity. It is well suited for the presentation of alignments of long sequences, e.g. of protein superfamilies, in plenary lectures. The method is implemented as a HyperCard stack for Apple Macintosh computers. Several options for the modification of the output are available (e.g. background reduction, size of the summation window, consideration of amino acid similarity, inclusion of graphic markers to indicate specific domains). The output is a PostScript file which can be printed, imported as EPS or processed further with Adobe Illustrator.

Algorithms↗
PubMed1995

DnaSP, DNA sequence polymorphism: an interactive program for estimating population genetics parameters from DNA sequence data.

DnaSP, DNA sequence polymorphism, is an interactive computer program for the analysis of DNA polymorphism from nucleotide sequence data. The program, addressed to molecular population geneticists, calculates several measures of DNA sequence variation within and between populations, linkage disequilibrium parameters and Tajima's D statistic. The program, which is written in Visual Basic v. 3.0 and runs on an IBM-compatible PC under Windows, can handle a large number of sequences of up to thousands of nucleotides each.

Animals↗
PubMed2001

A new approach to sequence comparison: normalized sequence alignment.

The Smith-Waterman algorithm for local sequence alignment is one of the most important techniques in computational molecular biology. This ingenious dynamic programming approach was designed to reveal the highly conserved fragments by discarding poorly conserved initial and terminal segments. However, the existing notion of local similarity has a serious flaw: it does not discard poorly conserved intermediate segments. The Smith-Waterman algorithm finds the local alignment with maximal score but it is unable to find local alignment with maximum degree of similarity (e.g. maximal percent of matches). Moreover, there is still no efficient algorithm that answers the following natural question: do two sequences share a (sufficiently long) fragment with more than 70% of similarity? As a result, the local alignment sometimes produces a mosaic of well-conserved fragments artificially connected by poorly-conserved or even unrelated fragments. This may lead to problems in comparison of long genomic sequences and comparative gene prediction as recently pointed out by Zhang et al. (Bioinformatics, 15, 1012-1019, 1999). In this paper we propose a new sequence comparison algorithm (normalized local alignment ) that reports the regions with maximum degree of similarity. The algorithm is based on fractional programming and its running time is O(n2log n). In practice, normalized local alignment is only 3-5 times slower than the standard Smith-Waterman algorithm.

Algorithms↗
PubMed2000

Sequence-based structural features between Kvlqt1 and Tapa1 on mouse chromosome 7F4/F5 corresponding to the Beckwith-Wiedemann syndrome region on human 11p15.5: long-stretches of unusually well conserved intronic sequences of kvlqt1 between mouse and human.

Mouse chromosome 7F4/F5 is a syntenic locus of human 11p15.5 in which many imprinted genes are clustered. Transmission of aberrant human 11p15.5 or duplicated 11p causes Beckwith-Wiedemann syndrome (BWS) depending on which parent the chromosome is derived from. To analyze a syntenic mouse locus corresponding to human 11p15.5, mouse BAC contigs were constructed between Nap2 and Tapa1, in which 390 kb was sequenced between Kvlqt1 and Tapa1. An unexpected finding was that of highly conserved intronic sequences of Kvlqt1 between mouse and human, and their homologies came up to at least 160 kb because the length of this gene extended to 350 kb, suggesting the possibility of some functional constraint due to transcriptional and/or post-transcriptional regulation of this region. Many expressed sequence tags (ESTs) were mapped on this locus. Three genes, Lit1 (Kvlqt1-AS), Mtr1 and Tssc4, were identified and characterized. Lit1 is an antisense-transcript of Kvlqt1 and paternally expressed and maternally methylated throughout the developmental stage. The position where Lit1 exists corresponded to a highly conserved region between mouse and human. This transcript extends at least 60 kb from downstream to upstream of exon 10 in Kvlqt1. Tssc4 and Mtr1 carried putative open reading frames but neither was imprinted. Further characterization of this locus based on the sequence comparison between mouse and human will contribute valuable information towards resolving the mechanism of the occurrence of BWS and the associated childhood tumor.

Alleles↗
PubMed1983

Unusual domains of human alphoid satellite DNA with contiguous non-satellite sequences: sequence analysis of a junction region.

The sequence organization of cloned segments of Human DNA carrying unusual domains of alphoid satellite was studied by restriction mapping, electron microscopy and base sequence analysis. In some cases restriction mapping revealed the absence of the typical 340 bp EcoR 1 dimer, although blot hybridizations showed the extensive presence of alphoid satellite. A variant monomeric construction was demonstrated by DNA sequencing. Furthermore, inverted repeats within these domains were detected by electron microscopy. In one case these were shown to be the result of interruptions in the satellite sequence by members of a family of repetitive, conserved elements.

Base Sequence↗