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At least 721 records · Page 40Linked to original sources
PubMed1993

Sequence variation in the noncoding region of human papillomavirus type 16 detected by single-strand conformation polymorphism analysis.

Human papillomavirus (HPV) type 16 variants were found by single-strand conformation polymorphism (SSCP) analysis of the noncoding region of the viral genome. Two sets of primers were used to analyze a 1018 bp region spanning nucleotides 7109-222. Twelve SSCP patterns were demonstrated among HPV 16 DNAs purified from 48 anal specimens from 24 homosexual men. Seven patterns were detected among 10 HPV 16 isolates from cervical carcinomas. In two pairs of sex partners, identical variants were recognized in each partner of the pair. Infection with two variants of HPV 16 from 2 specimens was observed in 1 of 21 subjects for whom there were multiple samples over time. DNA sequence analysis of 7 isolates confirmed that the SSCP technique showed polymorphisms only in fragments that had base substitutions and that all of these base substitutions resulted in detectable shifts in fragment mobility.

Anal Canal↗
PubMed2003

Characterization of a PttRPS18 promoter active in the vascular cambium region of hybrid aspen.

By screening 273 hybrid aspen plants transformed with a luciferase-based promoter trap T-DNA vector, one plant was found in which the reporter gene (luxF2) was activated only in cells of the cambial region, i.e., vascular cambium, phloem and differentiating xylem. Southern blot analysis revealed that this plant denoted #24 had a single T-DNA insert. The chromosomal regions flanking the T-DNA were cloned by plasmid rescue. A 757 bp DNA fragment, originating from the rescued plasmid and covering the genomic region immediately upstream from the right-border sequence of the T-DNA, was used as a probe to isolate the corresponding chromosomal region from a wild-type hybrid aspen genomic library. A hybrid aspen small ribosomal protein gene, PttRPS18, was then isolated. By screening a wt cambial region-specific cDNA library, two cDNA clones encoding a putative 152 amino acid PttRPS18 protein were isolated. Comparison of the DNA sequence immediately flanking the T-DNA insert in #24 with the corresponding wild-type sequence showed that only a minor deletion occurred during the T-DNA integration. Northern analysis revealed that the PttRPS18 gene was expressed mainly in the cambial region. By RT-PCR and DNA sequencing analysis, the exact structures of the PttRPS18 and luxF2 transcripts were determined. Finally, the hybrid aspen PttRPS18 promoter was fused to the uidA reporter gene and transformed into hybrid aspen plants. Histochemical analysis of GUS activity showed that the PttRPS18 promoter was expressed in the cambial region in the same manner as the luciferase reporter gene in the initial screening.

Amino Acid Sequence↗
PubMed1994

Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein.

Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an initiator of the replication by inducing a single-stranded cut at the origin of replication (ori). The exact location of the cut induced by the A protein in vivo is determined in this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (iii) primer extensions. It is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, in a region devoid of secondary structures. The A gene has been cloned into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but it cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.

Amino Acid Sequence↗
PubMed2001

The Arabidopsis PBS1 resistance gene encodes a member of a novel protein kinase subfamily.

Specific recognition of Pseudomonas syringae strains that express the avirulence gene avrPphB requires two genes in Arabidopsis, RPS5 and PBS1. Previous work has shown that RPS5 encodes a member of the nucleotide binding site-leucine rich repeat class of plant disease resistance genes. Here we report that PBS1 encodes a putative serine-threonine kinase. Southern blot analysis revealed that the pbs1-1 allele contained a deletion of the 3' end of the PBS1 open reading frame. DNA sequence analysis of the pbs1-2 allele showed it to be a missense mutation that caused a glycine to arginine substitution in the activation segment of PBS1, a region known to regulate substrate binding and catalytic activity in many protein kinases. The identity of PBS1 was confirmed using both transient transformation and stable transformation of mutant pbs1 plants. Comparison of the predicted PBS1 amino acid sequence with other plant protein kinases revealed that PBS1 belongs to a distinct subfamily of protein kinases that contains no other members of known function. The Pto kinase of tomato, which is required for specific resistance to P. syringae strains expressing avrPto, did not fall in the same subfamily as PBS1 and is only 42% identical in the kinase domain. These data suggest that PBS1 and Pto may fulfil different functions in the recognition of pathogen avirulence proteins. We discuss several possible models for the roles of PBS1 and RPS5 in AvrPphB recognition.

Alleles↗
PubMed2005

Prevalence of myocilin and optineurin sequence variants in German normal tension glaucoma patients.

PURPOSE: To determine the prevalence of optineurin (OPTN) and myocilin (MYOC) sequence variants in a cohort of German patients with normal tension glaucoma. METHODS: All coding exons of the OPTN and MYOC genes were amplified by PCR from genomic DNA and subjected to direct DNA sequencing. Analysis of sequence variants in controls was done by denaturing high performance liquid chromatography (DHPLC) and restriction fragment length polymorphism (RFLP) analysis. RESULTS: Sequence variants were identified by DNA sequencing in 10 of 112 cases. The OPTN sequence variant M98K was found in seven patients. In addition, two novel sequence variants (A336G and A377T) in the OPTN gene were identified that were not present in a control group. Mutation screening also identified two potentially pathogenic MYOC mutations (T293K and A445V). CONCLUSIONS: The findings in the current study provide further evidence that MYOC and OPTN gene variants are rare causes of NTG.

Adolescent↗
PubMed1983

Isolation and characterization of an alpha-tubulin gene from Leishmania enriettii.

An alpha-tubulin gene of Leishmania enriettii has been identified in genomic Southern blots by hybridization with a heterologous alpha-tubulin gene from Drosophila melanogaster. A clone containing this gene has been isolated from a plasmid library of size-selected L. enriettii DNA. It was identified by hybridization with the D. melanogaster tubulin gene. The cloned DNA fragment was characterized by restriction analysis and partial DNA sequence analysis. The cloned DNA fragment is 2 kb in length, bounded by Pst I sites, and appears to contain the entire coding region of the alpha-tubulin gene.

Animals↗
PubMed2001

Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides.

Antimicrobial peptides (APs) are important components of the innate defenses of animals, plants, and microorganisms. However, some bacterial pathogens are resistant to the action of APs. For example, Proteus mirabilis is highly resistant to the action of APs, such as polymyxin B (PM), protegrin, and the synthetic protegrin analog IB-367. To better understand this resistance, a transposon mutagenesis approach was used to generate P. mirabilis mutants sensitive to APs. Four unique PM-sensitive mutants of P. mirabilis were identified (these mutants were >2 to >128 times more sensitive than the wild type). Two of these mutants were also sensitive to IB-367 (16 and 128 times more sensitive than the wild type). Lipopolysaccharide (LPS) profiles of the PM- and protegrin-sensitive mutants demonstrated marked differences in both the lipid A and O-antigen regions, while the PM-sensitive mutants appeared to have alterations of either lipid A or O antigen. Matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis of the wild-type and PM-sensitive mutant lipid A showed species with one or two aminoarabinose groups, while lipid A from the PM- and protegrin-sensitive mutants was devoid of aminoarabinose. When the mutants were streaked on an agar-containing medium, the swarming motility of the PM- and protegrin-sensitive mutants was completely inhibited and the swarming motility of the mutants sensitive to only PM was markedly decreased. DNA sequence analysis of the mutagenized loci revealed similarities to an O-acetyltransferase (PM and protegrin sensitive) and ATP synthase and sap loci (PM sensitive). These data further support the role of LPS modifications as an elaborate mechanism in the resistance of certain bacterial species to APs and suggest that LPS surface charge alterations may play a role in P. mirabilis swarming motility.

Amino Acid Sequence↗
PubMed1989

Specific-primer-directed DNA sequencing using automated fluorescence detection.

Automated fluorescence-based DNA sequence analysis offers the possibility to undertake very large scale sequencing projects. Directed strategies, such as the specific-primer-directed sequencing approach ('gene walking'), should prove useful in such projects. Described herein is a study involving the use of this approach in conjunction with automated fluorescence detection on a commercial instrument (ABI 370A DNA sequencer). This includes procedures for the rapid chemical synthesis and purification of labeled primers, the design of primer sequences that are compatible with the commercial analysis software, and automated DNA sequence analysis using such primers. A set of four fluorophore-labeled primers can be reliably synthesized in a twenty-four hour period, and greater than 300 nucleotides of analyzed new sequence obtained using this set in an additional twenty-four hours. Scale-up of these procedures to take advantage of the full capabilities of the sequencer is, at present, too slow and costly to be suitable for routine sequencing, and therefore the use of specific-primers is best suited to the closure of gaps in extended sequence produced using random cloning and sequencing strategies.

Autoanalysis↗
PubMed1997

Amplification of CFTR exon 9 sequences to multiple locations in the human genome.

Cloning and characterization of the cystic fibrosis transmembrane conductance regulator (CFTR) gene led to the identification and isolation of cDNA and genomic sequences that cross-hybridized to the first nucleotide binding fold of CFTR. DNA sequence analysis of these clones showed that the cross-hybridizing sequences corresponded to CFTR exon 9 and its flanking introns, juxtapositioned with two segments of LINE1 sequences. The CFTR sequence appeared to have been transcribed from the opposite direction of the gene, reversely transcribed, and co-integrated with the L1 sequences into a chromosome location distinct from that of the CFTR locus. Based on hybridization intensity and complexity of the restriction fragments, it was estimated that there were at least 10 copies of the "amplified" CFTR exon 9 sequences in the human genome. Furthermore, when DNA segments adjacent to the insertion site were used in genomic DNA blot hybridization analysis, multiple copies were also detected. The overall similarity between these CFTR exon 9-related sequences suggested that they were derived from a single retrotransposition event and subsequent sequence amplification. The amplification unit appeared to be greater than 30 kb. Physical mapping studies including in situ hybridization to human metaphase chromosomes showed that multiple copies of these amplified sequences (with and without the CFTR exon 9 insertion) were dispersed throughout the genome. These findings provide insight into the structure and evolution of the human genome.

Animals↗
PubMed1999

p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma: p53 mutations in primary tumor and matched lymph node metastases.

In order to define the diagnostic value of p53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma (HNSCC), we investigated p53 mutations in primary tumors (PT) and matched lymph node metastases (LNM); the underlying question being whether differentiation between metastatic disease of a known PT or (a metastasis of) a synchronous or metachronous second tumor is possible by means of p53 sequencing-based mutation analysis. In 15 PT, the p53 status was analyzed, following RNA isolation, cDNA synthesis and polymerase chain reaction amplification, by direct sequencing full-length mRNA. Mutations thus found were confirmed by DNA sequencing analysis of the corresponding exon in the PT. When RNA isolation was defective, DNA sequencing analysis of exons 1 through 11 was performed. In the matched LNM, DNA analysis of the corresponding exon was performed to prove the presence of the same p53 mutation. In the event of small clones not detectable by direct sequencing, an oligo ligation assay was developed to detect a specific mutation. The presence of germline mutations was excluded by DNA sequencing analysis of the corresponding exon of peripheral blood leucocytes. In 14 PT (94%), a mutation was identified. In one PT, no p53 mutation could be identified either after full-length mRNA sequencing or after sequencing exons 1 through 11. In all cases of PT and matched LNM, the mutations proved to be identical. We conclude that p53 mutations develop in carcinogenesis before metastases occur and are maintained during metastasis. Consequently, p53 may serve as a clonal marker not susceptible to change during tumor metastasis. This merits further exploration of the application of p53 mutation analysis in differentiating between metastatic disease from a known PT versus a metastasis of another second PT.

Adult↗
PubMed2004

Genomic organization, expression, and comparative analysis of noncoding region of the rat Ndrg4 gene.

Rat Ndrg4 is a member of the NDRG gene family and has been suggested to relate to brain development. The structure of the rat Ndrg4 gene was studied to understand the mechanism for the expression of multiple forms of Ndrg4 protein, which were revealed in the brain. Subcloning and DNA sequencing analysis of a bacterial artificial chromosome (BAC) clone, together with analysis of a transcriptional start site by a cap-site hunting, indicated that the Ndrg4 gene spans about 39 kilobases (kb) and consists of 19 exons, in which the first and second exons were first found in rat. An alternative promoter usage at different transcriptional start sites may produce three types of messages, Ndrg4-A, Ndrg4-B, and Ndrg4-C, and there is a variant that lacks exon 18 for each type of transcript. Thereby, Ndrg4-A1, Ndrg4-A2, Ndrg4-B1, Ndrg4-B2, Ndrg4-C1, and Ndrg4-C2 were identified to be expressed. These six variants might explain the heterogeneity of the Ndrg4 protein in the brain. The variants without exon 18 were revealed in the embryonic and early postnatal brains while those with exon 18 were detected in the maturing and adult brains. Radiation hybrid mapping suggests that the rat Ndrg4 gene is located on chromosome 19 at 90.6 centirays (cR) from the top. Comparison of the noncoding sequence of the rat Ndrg4 gene to those of the orthologous mouse and human genes suggests that the AP-1 binding site is a candidate regulatory element.

Alternative Splicing↗
PubMed1991

Primary structures of ribosomal proteins from the archaebacterium Halobacterium marismortui and the eubacterium Bacillus stearothermophilus.

Approximately 40 ribosomal proteins from each Halobacterium marismortui and Bacillus stearothermophilus have been sequenced either by direct protein sequence analysis or by DNA sequence analysis of the appropriate genes. The comparison of the amino acid sequences from the archaebacterium H marismortui with the available ribosomal proteins from the eubacterial and eukaryotic kingdoms revealed four different groups of proteins: 24 proteins are related to both eubacterial as well as eukaryotic proteins. Eleven proteins are exclusively related to eukaryotic counterparts. For three proteins only eubacterial relatives-and for another three proteins no counterpart-could be found. The similarities of the halobacterial ribosomal proteins are in general somewhat higher to their eukaryotic than to their eubacterial counterparts. The comparison of B stearothermophilus proteins with their E coli homologues showed that the proteins evolved at different rates. Some proteins are highly conserved with 64-76% identity, others are poorly conserved with only 25-34% identical amino acid residues.

Amino Acid Sequence↗
PubMed1998

Tandem repeat structure of the duplicated Y-chromosomal STR locus DYS385 and frequency studies in the German and three Asian populations.

The Y-chromosomal short tandem repeat (STR) locus DYS385 can be typed using PCR amplification and separation of the resulting polymorphic fragments by non-denaturing high resolution polyacrylamide gel electrophoresis followed by silver staining. The PCR primers amplify a duplicated repeat sequence on the Y chromosome revealing a two-band pattern in male individuals. To determine the internal repeat structure as a basis for a consensus nomenclature, DNA sequence analysis was carried out after subcloning of PCR-amplified fragments revealing the uniform 4-bp repeat structure 'GAAA'. The shortest allele observed consisted of 10 repeat units thus providing the basis for the designation 'allele 10'. Except for isolated point mutations, no systematic differences could be observed either in the repeat sequence or in the flanking regions between the two fragments of a given individual. Thus it was not possible to discriminate between the two loci of the DYS385 system. Four population samples of German (n = 146), Chinese (n = 100), Japanese (n = 100), and Thai (n = 95) origin were studied. In the four groups, alleles 10 to 24 could be observed and genotype frequencies differed significantly. In Germans only one common genotype was present (11-14; 33.8% frequency). In the Asian populations, the frequencies were more evenly distributed with the 13-13 genotype (9%) in Chinese, the 13-17 genotype (14%) in Japanese and the 14-18 genotype (7%) in Thai being the most common. Overall, 69 different genotypes were found, of these 36 were observed in Germans, 36 in Chinese, 33 in Japanese and 44 in Thai. No mutations were detected in 62 father-son pairs. Thus DYS385 is a highly polymorphic STR system with population-specific genotype distributions.

Asia, Southeastern↗
PubMed1997

Childhood hepatocellular adenoma in familial adenomatous polyposis: mutations in adenomatous polyposis coli gene and p53.

BACKGROUND & AIMS: Infantile and childhood liver tumors have been found in 0.42% of individuals with a germline mutation in the adenomatous polyposis coli (APC) gene. This study analyzed a hepatocellular adenoma of a 2-year-old child at risk for familial adenomatous polyposis to identify genetic alterations in hepatic tumors initiated by APC germline mutations. METHODS: Mutation screening was performed for the APC gene (protein truncation test and DNA sequence analysis), p53 gene (complementary DNA cloning and sequencing), and members of the Ras gene family (complementary DNA sequence analysis). RESULTS: Both the mother and child had a germinal CGA-->TGA transition at codon 1451 leading to an Arg1451Ter stop mutation in the APC gene. Loss of the wild-type APC allele as a second hit revealed hemizygosity of the inherited mutation in the tumor. Furthermore, a CGC-->CAC transition in the p53 gene of the adenoma resulted in an Arg-->His missense mutation in codon 175. No loss of heterozygosity was detected at the p53 locus. Ras gene mutations were not found. CONCLUSIONS: Biallelic inactivation of APC gene and p53 mutation are early events in hepatocellular tumorigenesis. Additional reports will confirm whether inherited APC gene mutations between codon 1444 and 1578 increase the risk for hepatic tumors.

Adenoma, Liver Cell↗
PubMed1991

Identification and characterization of novel human endogenous retroviral sequences prefentially expressed in undifferentiated embryonal carcinoma cells.

A novel endogenous retroviral sequence (ERV-9) has been isolated from a human embryonal carcinoma cDNA library by hybridization to a probe containing a recently described human repetitive element. DNA sequence analysis of the 4kb cDNA insert (pHE.1) revealed the presence of ORFs potentially coding for putative retrovirus-related gag, pol and env proteins. Northern blot and RNase protection experiments showed that RNA homologous to the pHE.1 insert is detected only in embryonal carcinoma cells as a 8 kb mRNA, and its expression is negatively regulated during retinoic acid induced differentiation of the human teratocarcinoma cell line NT2/D1. Using a pol specific probe we have isolated a genomic locus containing the ERV-9 sequences. Characterization by restriction enzyme analysis and DNA sequencing allowed us to define LTR-like sequences, that are composed by a complex array of subrepetitive elements. In addition we show that ERV-9 LTR sequences are capable to drive expression of linked CAT gene in a cell specific manner as LTR promoter activity has been detected only in NT2/D1 cells.

Amino Acid Sequence↗
PubMed1980

Deletions and DNA rearrangements within the transposable DNA element IS2. A model for the creation of palindromic DNA by DNA repair synthesis.

Three derivatives of mutant ga10P-308::IS2-I of Escherichia coli were characterized by DNA sequence analysis. Deletions and DNA sequence rearrangements were observed which apparently were initiated at short A-T rich inverted repeats within IS2. Two of the mutants carried newly synthesized DNA sequences which were inverted copies of already existing IS2 sequences. Thus long stretches with twofold symmetry were formed. It is discussed whether these inverted repeats were formed by DNA repair synthesis which was initiated at the A-T rich palindromes of IS2.

Base Sequence↗
PubMed1998

A software system for data analysis in automated DNA sequencing.

Software for gel image analysis and base-calling in fluorescence-based sequencing consisting of two primary programs, BaseFinder and GelImager, is described. BaseFinder is a framework for trace processing, analysis, and base-calling. BaseFinder is highly extensible, allowing the addition of trace analysis and processing modules without recompilation. Powerful scripting capabilities combined with modularity and multilane handling allow the user to customize BaseFinder to virtually any type of trace processing. We have developed an extensive set of data processing and analysis modules for use with the program in fluorescence-based sequencing. GelImager is a framework for gel image manipulation. It can be used for gel visualization, lane retracking, and as a front end to the Washington University Getlanes program. The programs were designed using a cross-platform development environment, currently allowing them to run in Windows NT, Windows 95, Openstep/Mach, and Rhapsody. Work is ongoing to deploy the software on additional platforms, including Solaris, Linux, and MacOS. This software has been thoroughly tested and debugged in the analysis of >2 million bp of raw sequence data from human chromosome 19 region q13. Overall sequencing accuracy was measured using a significant subset of these data, consisting of approximately 600 sequences, by comparing the individual shotgun sequences against the final assembled contigs. Also, results are reported from experiments that analyzed the accuracy of the software and two other well-known base-calling programs for sequencing the M13mp18 vector sequence. [The sequence data described in this paper have been submitted to the GenBank data library under accession no. AF025422]

Algorithms↗