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At least 811 records · Page 45Linked to original sources
PubMed2003

Immunoglobulin repertoire of B lymphocytes infiltrating breast medullary carcinoma.

Tumor specific peptides recognized by T lymphocytes infiltrating solid tumors, as well as the corresponding T cell receptor (TcR) repertoire usage, have been extensively investigated. By contrast, tumor infiltrating B cells and their immunoglobulin (Ig) repertoire have been studied only in a limited number of tumors. The objective of the present study was to determine, whether DNA sequence analysis of the expressed immunoglobulin variable regions of B cells that infiltrate breast cancer, could be used to reveal a potential specific tumor binding capacity of the antibodies. To answer this question, about 200 expressed Ig heavy (VH) and light chain variable gene (VL) regions were cloned, sequenced and comparatively analysed from a typical medullary beast carcinoma (MBC), where the massive B and plasma cell infiltration correlates with favourable prognosis despite of its high grade. The tumor infiltrating B cell Ig heavy and light chain sequences could be classified into clusters, families and subgroups, based on the identity level to germline, showing a pattern of oligoclonality. Some overrepresented clusters could be determined. In the course of a detailed analysis and search in Blastn database, a number of VH and VL sequences showed more than 99% homology to DNA sequences of Ig VH region, with proved tumor antigen binding capacity. Our data suggest, that potential tumor binder Ig VH and VL sequences might be selected using a detailed immunoglobulin variable region analysis. This new approach might have a benefit for further antibody engineering, as difficulties in search for tumor binders by phage library selection might be reduced and the time for selection shortened.

B-Lymphocytes↗
PubMed1997

Minisequencing: a specific tool for DNA analysis and diagnostics on oligonucleotide arrays.

We describe a method for multiplex detection of mutations in which the solid-phase minisequencing principle is applied to an oligonucleotide array format. The mutations are detected by extending immobilized primers that anneal to their template sequences immediately adjacent to the mutant nucleotide positions with single labeled dideoxynucleoside triphosphates using a DNA polymerase. The arrays were prepared by coupling one primer per mutation to be detected on a small glass area. Genomic fragments spanning nine disease mutations, which were selected as targets for the assay, were amplified in multiplex PCR reactions and used as templates for the minisequencing reactions on the primer array. The genotypes of homozygous and heterozygous genomic DNA samples were unequivocally defined at each analyzed nucleotide position by the highly specific primer extension reaction. In a comparison to hybridization with immobilized allele-specific probes in the same assay format, the power of discrimination between homozygous and heterozygous genotypes was one order of magnitude higher using the minisequencing method. Therefore, single-nucleotide primer extension is a promising principle for future high-throughput mutation detection and genotyping using high density DNA-chip technology.

DNA-Directed DNA Polymerase↗
PubMedDate not supplied

Species identification using genetic tools: the value of nuclear and mitochondrial gene sequences in whale conservation.

DNA sequence analysis is a powerful tool for identifying the source of samples thought to be derived from threatened or endangered species. Analysis of mitochondrial DNA (mtDNA) from retail whale meat markets has shown consistently that the expected baleen whale in these markets, the minke whale, makes up only about half the products analyzed. The other products are either unregulated small toothed whales like dolphins or are protected baleen whales such as humpback, Bryde's, fin, or blue whales. Independent verification of such mtDNA identifications requires analysis of nuclear genetic loci, but this is technically more difficult than standard mtDNA sequencing. In addition, evolution of species-specific sequences (i.e., fixation of sequence differences to produce reciprocally monophyletic gene trees) is slower in nuclear than in mitochondrial genes primarily because genetic drift is slower at nuclear loci. When will use of nuclear sequences allow forensic DNA identification? Comparison of neutral theories of coalescence of mitochondrial and nuclear loci suggests a simple rule of thumb. The "three-times rule" suggests that phylogenetic sorting at nuclear loci is likely to produce species-specific sequences when mitochondrial alleles are reciprocally monophyletic and the branches leading to the mtDNA sequences of a species are three times longer than the average difference observed within species. A preliminary test of the three-times rule, which depends on many assumptions about the species and genes involved, suggests that blue and fin whales should have species-specific sequences at most neutral nuclear loci, whereas humpback and fin whales should show species-specific sequences at fewer nuclear loci. Partial sequences of actin introns from these species confirm the predictions of the three-times rule and show that blue and fin whales are reciprocally monophyletic at this locus. These intron sequences are thus good tools for the identification of these species and will afford a chance to identify putative hybrid blue/fin whales thought to have entered the retail market after 1989.

Actins↗
PubMed1999

Isolation and characterization of indene bioconversion genes from Rhodococcus strain I24.

Rhodococcus strain 124 is able to convert indene into indandiol via the actions of at least two dioxygenase systems and a putative monooxygenase system. We have identified a cosmid clone from 124 genomic DNA that is able to confer the ability to convert indene to indandiol upon Rhodococcus erythropolis SQ1, a strain that normally can not convert or metabolize indene. HPLC analysis reveals that the transformed SQ1 strain produces cis-(1R,2S)-indandiol, suggesting that the cosmid clone encodes a naphthalenetype dioxygenase. DNA sequence analysis of a portion of this clone confirmed the presence of genes for the dioxygenase as well as genes encoding a dehydrogenase and putative aldolase. These genes will be useful for manipulating indene bioconversion in Rhodococcus strain 124.

Amino Acid Sequence↗
PubMedDate not supplied

p53 is not mutated in hepatocellular carcinomas from Alaska Natives.

Hepatocellular carcinoma is common among Alaska Natives. The known risk factor in this population is hepatitis B viral infection; fungal toxins, including aflatoxin B1, have not been detected in foodstuffs. In this series of 14 patients (including 4 siblings and 2 second cousins), 3 patients were less than 12 years old at diagnosis of hepatocellular carcinoma, 8 patients were 13-24 years old, and 3 patients were more than 60 years old. Since p53 mutations occur in 29% of hepatocellular carcinomas worldwide, we tested the tumors for p53 mutations and serum samples for anti-p53 antibodies. Serum samples from these 14 patients did not contain detectable levels of anti-p53 antibodies. Loss of heterozygosity within the p53 locus was not detected in any of 9 informative cases. Immunohistochemical analysis for p53 protein accumulation was negative in all of 11 tumors. DNA sequence analysis of 12 tumor samples showed no evidence of p53 mutation in the highly conserved regions included in exons 5-8. These data, combined with one case from a previous report, indicate a mutation frequency of 0 of 13, which differs significantly from the worldwide frequency of 29% (chi 2 3.9; P = 0.048). These results indicate that liver carcinogenesis among Alaska Natives occurs independently of a traditional p53 pathway. The familial clustering and early onset in this population strongly suggest an inherited genetic predisposition to develop liver cancer. Germline mutations in a tumor suppressor or a cancer susceptibility gene are likely. Future studies of these samples should include investigations of candidate suppressor or susceptibility genes which map to chromosomal regions commonly deleted in liver cancers.

Adolescent↗
PubMed1999

[Tumour suppressor p53 gene sequence mutations in pleomorphic adenoma of salivary gland].

OBJECTIVE: To detect p53 exon 7 and 8 mutation and to study genesis and biological characteristics of pleomorphic adenoma of salivary galand. METHODS: Polymerase chain reaction- single-strand conformational polymorphism (PCR-SSCP) and DNA sequencing analysis were used. RESULTS: PCR-SSCP analysis showed that in 11 patients with positive SSCP, 7 showed abnormal exon 7 and 3 abnormal exon 8. Comparison of SSCP results with DNA sequencing showed that patients all patients with p53 exon 7 and 8 mutations were SSCP positive. Exon 7 mutation pattern was point mutation(T-->C). Exon 8 mutation pattern included base substitution (point mutation) (C-->A) and frame-shiftmutation (base insertion and base loss). CONCLUSIONS: Polycodon and poly-pattern mutations appeared in p53 exon 7 and 8. Pleomorphic adenoma of salivary galand was similar to malignant tumor in p53 gene mutation. Poly-codon mutation could occur in the same case.

Adenoma, Pleomorphic↗
PubMed1996

A periodic tryptophan protein 2 gene homologue (PWP2H) in the candidate region of progressive myoclonus epilepsy on 21q22.3.

The Unverricht-Lundborg type of progressive myoclonus epilepsy (EPM1) has been mapped to human chromosome 21q22.3, and the candidate region has been narrowed to a region of less than 300 kb. We now report the isolation of a novel gene, PWP2H, from this EPM1 candidate region. The putative protein encoded by this gene is a novel member of the beta transducin protein superfamily with high homology to the yeast periodic tryptophan protein 2 (PWP2). PWP2H cDNA was isolated from a 14 week human trisomy 21 fetal brain cDNA library by using a modification of a direct cDNA selection method. PFGE analysis showed that PWP2H is located proximal to D21S25 and distal to TMEM1. Northern analysis of the 3.1-kb PWP2H cDNA revealed that a 3.3-kb major transcript is ubiquitously expressed in human adult tissues. DNA sequence analysis reveals a complete coding region of 2,610 bp as well as 5'- and 3'-UTR. The structure of the putative PWP2H protein contains 6 WD40 repeats, 2 acidic regions and one leucine zipper domain, suggesting that the protein may form specific protein complexes in which the WD40 repeats and the leucine zipper represent protein-protein binding sites. In conclusion, the map location and the homology to a gene family involved in a large variety of biological processes including signal transduction and development make PWP2H an intriguing candidate for EPM1 as well as for APECED (autoimmune polyglandular disease) and HPE1 (holoprosencephaly-1) that also map in this region of chromosome 21.

Amino Acid Sequence↗
PubMed2004

An evaluation of 18S rDNA approaches for the study of fungal diversity in grassland soils.

Fungal community structure and diversity in two types of agricultural grassland soil were investigated by amplified 18S ribosomal DNA restriction analysis (ARDRA) and 18S ribosomal DNA sequence analysis. These two grassland sites represent a species-rich old hay meadow and an agriculturally improved site with low floristic diversity. Two primer sets were used in combination to amplify approximately 550 bp of rDNA from three major fungal groups, the zygomycetes, basidiomycetes, and ascomycetes, and clone libraries were created for each site. 18S ARDRA was used to analyze 170 rDNA clones, and three diversity indices were calculated. A small-scale culturing analysis was also carried out and the most common isolates analyzed using ARDRA and sequence analysis. The soil fungal community revealed by the rDNA approaches was significantly different from that produced by this limited culture-based analysis. Twenty-eight soil-derived clones were sequenced, and many represented fungal taxa rarely reported in culture-based studies. The PCR-based techniques detected differences in diversity between the two fungal communities and changes in patterns of dominance that paralleled higher plant diversity. The results suggest that 18S rDNA-based approaches are a useful tool for initial screening of fungal communities, and that they represent a more comprehensive picture of the community than plate culturing.

Base Sequence↗
PubMed2004

Census of the bacterial community of the gypsy moth larval midgut by using culturing and culture-independent methods.

Little is known about bacteria associated with Lepidoptera, the large group of mostly phytophagous insects comprising the moths and butterflies. We inventoried the larval midgut bacteria of a polyphagous foliivore, the gypsy moth (Lymantria dispar L.), whose gut is highly alkaline, by using traditional culturing and culture-independent methods. We also examined the effects of diet on microbial composition. Analysis of individual third-instar larvae revealed a high degree of similarity of microbial composition among insects fed on the same diet. DNA sequence analysis indicated that most of the PCR-amplified 16S rRNA genes belong to the gamma-Proteobacteria and low G+C gram-positive divisions and that the cultured members represented more than half of the phylotypes identified. Less frequently detected taxa included members of the alpha-Proteobacterium, Actinobacterium, and Cytophaga/Flexibacter/Bacteroides divisions. The 16S rRNA gene sequences from 7 of the 15 cultured organisms and 8 of the 9 sequences identified by PCR amplification diverged from previously reported bacterial sequences. The microbial composition of midguts differed substantially among larvae feeding on a sterilized artificial diet, aspen, larch, white oak, or willow. 16S rRNA analysis of cultured isolates indicated that an Enterococcus species and culture-independent analysis indicated that an Entbacter sp. were both present in all larvae, regardless of the feeding substrate; the sequences of these two phylotypes varied less than 1% among individual insects. These results provide the first comprehensive description of the microbial diversity of a lepidopteran midgut and demonstrate that the plant species in the diet influences the composition of the gut bacterial community.

Animals↗
PubMed1999

[Sub-cloning and preliminary sequence analysis of the gene encoding of a cefoperazone hydrolyzing beta-lactamase isolated from Escherichia coli].

Escherichia coli HX88108, which is resistant to cefoperazone(CPZ), was isolated from a severely infected patient. We studied genetical basis of beta-lactamase produced in E. coli HX88108 by pFL25, one of the recombinant plasmid of pFC. Largescale pFL25 plasmid was extracted, purified, and cleaved with restriction endonuclease EcoR I, Sal I, Pvu I, then subcloned into vector pUC19 as 1.9 kb, 0.9 kb, 0.65 kb fragments respectively. Recomminant plasmids were selected by alpha-complementation and determined by restriction endonuclease analysis. DNA sequencing was performed by the dideoxy polymerase chain termination method. Partial nucleotide sequence(1-78 nucleotide position) of the gene was found to be highly homologous (97%) with the gene coding for TEM-52 extended spectrum beta-lactamase of K. pneumonise, suggesting that the beta-lactamase coded by the cefoperazone resistant gene might be derived from TEM-type beta-lactamase.

Anti-Bacterial Agents↗
PubMed1999

Germline mutations of E-cadherin gene in Korean familial gastric cancer patients.

Gastric cancer is the most common cancer in Korea. Germline mutations of the E-cadherin gene have recently been identified in familial gastric cancer patients. We screened five Korean familial gastric cancer patients to investigate germline mutations of the E-cadherin gene. These patients fulfilled the following criteria: presence of at least two gastric cancer patients within first-degree relatives and one patient diagnosed before the age of 50 years. Abnormal band patterns were found in exons 6 and 10 in two familial gastric cancer patients by polymerase chain reaction-single strand conformation polymorphism analysis (probands from the SNU-G2 and SNU-G1001 families, respectively). DNA sequencing analysis of the E-cadherin gene of these two patients revealed missense mutations in each exon. The SNU-G2 proband harbored a missense mutation from aspartic acid (GAT) to glycine (GGT) at codon 244 in exon 6 of the E-cadherin gene, and the SNU-G1001 proband had a missense mutation from valine (GTG) to alanine (GCG) at codon 487 in exon 10. The SNU-G2 proband was diagnosed with gastric cancer at the age of 38; three brothers and two sisters had died of gastric cancer under the age of 50, and their mother had died of gastric cancer at the age of 63. The SNU-G1001 proband was diagnosed with gastric cancer at the age of 42 and one brother had died of gastric cancer at the age of 49. In summary, we found germline mutations of the E-cadherin gene in two of five Korean familial gastric cancer patients screened.

Adenocarcinoma↗
PubMed1999

Analysis of homeobox gene HOXA10 mutations in cryptorchidism.

PURPOSE: Cryptorchidism is the most common congenital abnormality of the genitalia. However, its exact etiology remains to be defined. Homeobox (HOX) containing genes have a key role in the morphogenesis of segmental structures along the primary body axis, including the urogenital mesenchyma. In male mice with a targeted deletion of the HOXA10 gene cryptorchidism manifests in the absence of other major defects. Because to our knowledge this gene has never been examined for alterations in humans, we evaluated whether mutations of HOXA10 are associated with cryptorchidism in humans. MATERIALS AND METHODS: Genomic deoxyribonucleic acid (DNA) was extracted from human blood or tissue samples from 16 noncryptorchid control subjects and 45 cryptorchid boys. To screen for mutations exons 1 and 2 of the HOXA10 gene were amplified individually by polymerase chain reaction using 6 overlapping oligonucleotide primer pairs. Single strand conformational polymorphism (SSCP) analysis of the amplified radiolabeled DNA fragments was performed. Variant band shifts were detected due to abnormal migration of the denatured DNA fragment compared to controls, suggesting an alteration in the DNA sequence. Sequence analysis of these variant bands was then done to define any mutations. RESULTS: SSCP analysis revealed variants in 2 controls. Of the 45 samples from cryptorchid patients 30 had SSCP variants in exon 1. No variants were found in other regions of the gene. Sequence analysis revealed several DNA polymorphisms in exon 1 in controls and boys with cryptorchidism. Other nucleotide changes (point mutations) were noted only in exon 1 in the DNA of 5 cryptorchid patients, of whom 1 had a 24 nucleotide deletion. CONCLUSIONS: Our initial analysis of the HOXA10 gene in humans demonstrates that genetic alterations of this gene may be present in some boys with cryptorchidism. HOXA10 polymorphisms exist in normal control subjects as well as in cryptorchid patients. Further analysis of the function of the mutated protein will elucidate the role of this gene as a potential causative factor of testicular descent.

Child↗
PubMed2000

[Cloning and identification of a new telomeric-associated zinc finger protein cDNA].

In order to isolate novel genes related to early human embryo development and differentiation, a directional cDNA library was constructed from 3-week-old human embryo. Single-pass DNA sequence analysis was used to sequence 47 randomly picked low-abundance cDNA clones. This approach led us to select a clone, L30, showing significant homology with the telomeric-associated DNA and zinc finger protein genes. It is about 3.8 kb in length and contains an open reading frame of notable length within 5'-region, and a tailing signal of AAUAAA and poly (A+) with 39 A in 3'-region. The gene was transcribed in human embryo by Northern blot hybridization and assigned to human chromosome 12 by in situ hybridization.

Cloning, Molecular↗
PubMed2004

Essential residues in the chromate transporter ChrA of Pseudomonas aeruginosa.

The chrA gene of Pseudomonas aeruginosa plasmid pUM505 encodes the hydrophobic protein ChrA, which confers resistance to chromate by the energy-dependent efflux of chromate ions. Chromate-sensitive mutants were isolated by in vivo random mutagenesis. Transport experiments with cell suspensions of selected mutants showed that 51CrO4(2-) extrusion was drastically lowered as compared to suspensions of the strain with the wild-type plasmid, confirming that the mutations affected a chromate efflux system. DNA sequence analysis showed that most point mutations affected amino acids clustered in the N-terminal half of ChrA, altering either cytoplasmic regions or transmembrane segments, and replaced residues moderately to highly conserved in ChrA homologs. PhoA and LacZ translational fusions were used to confirm the membrane topology at the N-terminal half of the ChrA protein.

Amino Acid Sequence↗
PubMed1998

Sequence analysis of DNA randomly amplified from the Saccharomyces cerevisiae genome.

Despite its widespread use, the molecular basis of random amplification is poorly understood. Here the basis of random amplification has been investigated by cloning and sequencing the products of a random amplification of polymorphic DNA (RAPD) amplification from Saccharomyces cerevisiae DNA. The genomic origin of the amplified products was determined by sequence comparison with the S. cerevisiae Genome Database (SGD). This allowed analysis of the degree of identity between the random primer and the primer binding sites on the genome. There was no relationship between RAPD size, GC content and relative abundance. The degree of matching between the primer and the primer binding sites increased towards the 3; end of the primer and decreased towards the 5; end. The maximum number of mismatches observed between primer and primer binding sites was never more than one between positions 1-7 of the primer. Nucleotide compositional biases were also observed upstream and downstream of the primer binding site with a marked preference for AT richness upstream of the primer binding sites and for a GC preference directly following the 3; end of the primer. These findings have important ramifications for primer design for multiplex, low stringency and degenerate polymerase chain reaction (PCR).

Base Sequence↗
PubMed2000

A polymerase chain reaction method for the amplification of full-length envelope genes of HIV-1 from DNA samples containing single molecules of HIV-1 provirus.

Polymerase chain reaction (PCR) amplification of full-length envelope genes from the human immunodeficiency virus type 1 (HIV-1) directly from uncultured clinical samples is difficult. This paper describes a comparative assessment of the performance of three thermostable polymerases in an HIV-1 full-length envelope gene PCR. The PCR method utilising Expand HiFi polymerase was successful when using DNA samples extracted from a variety of sources including blood, semen and various tissues. This method generated high and specific yields of product from samples containing as little as one copy of HIV-1 proviral DNA. The resulting PCR products were suitable for a variety of downstream analytical methods including DNA sequence analysis.

Base Sequence↗
PubMed2002

[Analysis of 16S rDNA sequences and DNA-DNA hybridization of moderately halophilic bacteria from Xinjiang region].

Based on the previous studies on numerical taxonomy and 16S rDNA PCR-RFLP analysis, the moderately halophilic bacteria isolated from Xinjiang Region constituted a new cluster, and the phylogenetic tree was constructed by comparing with the 16S rDNA sequences of the other moderately halophilic bacteria species. In the phylogenetic tree, most of the reference strains were clustered in a group, and the similarity values of 16S rDNA sequence were above 96%. However, AI-3, Alcanivorax borkumensis and Halobacillus litoralis were clustered in another group, and the similarity value of 16S rDNA sequences between AI-3 and Alcanivorax borkumensis was 96%, and that of 16S rDNA sequences between AI-3 and Halobacillus litoralis was 99%. The results indicated that AI-3 was different from the reference strains in phylogeny. The values of DNA homology in the new cluster were more than 70%, but the value between AI-3 and Halomonas elongata was less than 50%. Thus, the strain AI-3 possibly represent a new moderately halophilic bacteria species.

Base Sequence↗
PubMed1995

Clonal structure of invasive Streptococcus pyogenes in Northern Scotland.

We have used molecular techniques to characterize 51 group A streptococci from Scotland and 17 'serious disease' isolates from other countries, in order to establish the clonal structure of invasive Streptococcus pyogenes strains circulating between 1986 and 1993. Strains were grouped by restriction endonuclease analysis, pulsed field gel electrophoresis and ribotyping patterns, and were examined for the presence of alleles of the speA gene by polymerase chain reaction and DNA sequence analysis. Serious and fatal infections in Scotland were caused by several clones. One clone (9 of 51 strains) was M type 1 and possessed the speA gene allele 2. This was the clone previously identified as causing severe infection in the USA. Another clone (5 of 51 strains) was M type 3 and had speA gene allele 3. In view of the clear association of more than one clone with severe, invasive and fatal infections, horizontal gene exchange between genotypes merits further investigation.

Base Sequence↗