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At least 649 records · Page 36Linked to original sources

PubMed1997

Molecular genetic characterization of alternatively spliced CD44 transcripts in human stomach carcinoma.

CD44 is a member of cell surface glycoproteins which are involved in cell-matrix adhesion and tumor metastasis. Certain types of tumors express complex CD44 isoforms generated by alternative splicing of 2v-10v exons, and their expression appears to promote metastasis of tumor cells. Using a nested RT-PCR, we analyzed expression of CD44 variants in 26 stomach carcinoma, 21 matched normal tissues, and 2 carcinoma cell lines. We observed frequent and complex patterns of CD44 variant expression in tumor tissues. While exons 6v and 7v expression was detected in most normal and tumor tissues, exon 9v was most rarely detected. Exon 5v showed a significantly frequent expression in carcinoma, suggesting that its expression might contribute to the malignant progression. While exon 9v was frequently observed in diffuse-type tumors, the other 8 variant exons including 6v showed more frequent expression in intestinal-type tumors. Exons 9v and 10v were predominantly expressed in advanced tumor tissues and exon 8v was expressed more frequently in tumors of lymph node metastasis. We believe that series with a longer follow-up now need to be tested to clarify the association between CD44 splice variant expression and distant metastasis or long-term prognosis.

Adult↗

PubMed1992

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Amino Acid Sequence↗

PubMed1990

Molecular cloning, genetic characterization and DNA sequence analysis of the recM region of Bacillus subtilis.

In Bacillus subtilis the recM gene, whose product is associated with DNA repair and recombination, has been located between the dnaX and rrnA genes. The recM gene has been cloned and analyzed. Analysis of the nucleotide sequence (3.741-kilobase) around recM revealed five open reading frames (orf). We have assigned recM and dnaX to two of this orf, given the gene order dnaX-orf107-recM-orf74-orf87. The organization of genes of the dnaX-orf107-recM region resembles the organization of genes in the dnaX-orf12-recR region of the Escherichia coli chromosome. Proteins of 24.2 and 17.0 kDa would result from translation of the wild type and in vitro truncated recM genes, and radioactive bands of proteins of molecular weights of 24.5 and 17.0 kDa were detected by the use of the T7promoter-expression system. The RecM protein contains a potential zinc finger domain for nucleic acid binding and a putative nucleotide binding sequence that is present in many proteins that bind and hydrolyze ATP. Strains, in which the recM gene has been insertionally inactivated, were generated and show a phenotype essentially the same as previously described recM mutants.

Adenosine Triphosphate↗

PubMed2002

Biochemical and genetic characterization of a monomeric storage protein (T1) with an unusually high molecular weight in Triticum tauschii.

The protein named T1, present in Triticum tauschii, was previously characterized as a high-molecular-weight (HMW) glutenin subunit with a molecular size similar to that of the y-type glutenin subunit-10 of Triticum aestivum. This protein was present along with other HMW glutenin subunits named 2(t) and T2, and was considered as part of the same allele at the Glu-D (t) 1locus of T. tauschii. This paper describes a re-evaluation of this protein, involving analyses of a collection of 173 accessions of T. tauschii, by SDS-PAGE of glutenin subunits after the extraction of monomeric protein. No accessions were found containing the three HMW glutenin subunits. On the other hand, 17 lines with HMW glutenin subunits having electrophoretic mobilities similar to subunits 2(t) and T2 were identified. The absence of T1 protein in these gel patterns has shown that protein T1 is not a component of the polymeric protein. Rather, the T1 protein is an omega-gliadin with an unusually high-molecular-weight. This conclusion is based on acidic polyacrylamide gel electrophoresis (A-PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and two-dimensional gel electrophoresis (A-PAGE+ SDS-PAGE), together with analysis of its N-terminal amino-acids sequence. The inheritance of omega-gliadin T1 was studied through analyses of gliadins and HMW glutenins in 106 F(2)grains of a cross between synthetic wheat, L/18913, and the wheat cv Egret. HMW glutenin subunits and gliadins derived from T. tauschii ( Glu-D (t) 1 and Gli-D (t) 1) segregated as alleles of the Glu-D1 and Gli-D1loci of bread wheat. A new locus encoding the omega-gliadin T1 was identified and named Gli-DT1. The genetic distance between this new locus and those of endosperm proteins encoded at the 1D chromosome were calculated. The Gli-DT1 locus is located on the short arm of chromosome 1D and the map distance between this locus and the Gli-D1 and Glu-D1 loci was calculated as 13.18 cM and 40.20 cM, respectively.

Journal Article↗

PubMed2006

Genetic characterization and molecular mapping of Hessian fly resistance genes derived from Aegilops tauschii in synthetic wheat.

Two synthetic hexaploid wheat lines (xAegilotriticum spp., 2n = 6x = 42, genomes AABBDD), SW8 and SW34, developed from the crosses of the durum wheat cultivar Langdon (Triticum turgidum L. var. durum, 2n = 4x = 28, genomes AABB) with two Aegilops tauschii Cosson accessions (2n = 2x = 14, genome DD), were determined to carry Hessian fly [Mayetiola destructor (Say)] resistance genes derived from the Ae. tauschii parents. SW8 was resistant to the Hessian fly biotype Great Plains (GP) and strain vH13 (virulent to H13). SW34 was resistant to biotype GP, but susceptible to strain vH13. Allelism tests indicated that resistance genes in SW8 and SW34 may be allelic to H26 and H13 or correspond to paralogs at both loci, respectively. H26 and H13 were localized to chromosome 4D and 6D, respectively, in previous studies. Molecular mapping in the present study, however, assigned the H26 locus to chromosome 3D rather than 4D. On the other hand, mapping of the resistance gene in SW34 verified the previous assignment of the H13 locus to chromosome 6D. Linkage analysis and physical mapping positioned the H26 locus to the chromosomal deletion bin 3DL3-0.81-1.00. A linkage map for each of these two resistance genes was constructed using simple sequence repeat (SSR) and target region amplification polymorphism (TRAP) markers.

Alleles↗

PubMed1993

Cloning and genetic characterization of a calcium- and phospholipid-binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor-1 gamma.

We have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor-1 gamma (EF-1 gamma) first identified in the brine shrimp Artemia salina. The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included. The calculated M(r) of Cam1p (47,092 Da) is in close agreement with that of EF-1 gamma (M(r) = 49,200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles. Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non-essential. Attempts to elicit a translational phenotype have been unsuccessful. Since EF-1 gamma participates in the regulation of a GTP-binding protein (EF-1 alpha), double mutants with cam1 disruptions and various mutant alleles of known GTP-binding proteins were constructed and examined. No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4. The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP-dependent process is discussed.

Amino Acid Sequence↗

PubMed2001

Genetic characterization of the Klebsiella pneumoniae waa gene cluster, involved in core lipopolysaccharide biosynthesis.

A recombinant cosmid containing genes involved in Klebsiella pneumoniae C3 core lipopolysaccharide biosynthesis was identified by its ability to confer bacteriocin 28b resistance to Escherichia coli K-12. The recombinant cosmid contains 12 genes, the whole waa gene cluster, flanked by kbl and coaD genes, as was found in E. coli K-12. PCR amplification analysis showed that this cluster is conserved in representative K. pneumoniae strains. Partial nucleotide sequence determination showed that the same genes and gene order are found in K. pneumoniae subsp. ozaenae, for which the core chemical structure is known. Complementation analysis of known waa mutants from E. coli K-12 and/or Salmonella enterica led to the identification of genes involved in biosynthesis of the inner core backbone that are shared by these three members of the Enterobacteriaceae. K. pneumoniae orf10 mutants showed a two-log-fold reduction in a mice virulence assay and a strong decrease in capsule amount. Analysis of a constructed K. pneumoniae waaE deletion mutant suggests that the WaaE protein is involved in the transfer of the branch beta-D-Glc to the O-4 position of L-glycero-D-manno-heptose I, a feature shared by K. pneumoniae, Proteus mirabilis, and Yersinia enterocolitica.

Animals↗

PubMed2005

Genetic characterization of asymmetric somatic hybrids between Bupleurum scorzonerifolium Willd and Triticum aestivum L.: potential application to the study of the wheat genome.

In this paper, we describe how Bupleurum scorzonerifolium/Triticum aestivum asymmetric somatic hybrids can be exploited to study the wheat genome. Protoplasts of B. scorzonerifolium Willd were irradiated with ultraviolet light (UV) and fused with protoplasts of common wheat (T. aestivum L.). All cell clones were similar in appearance to those of B. scorzonerifolium, while the regenerated plantlets were either intermediate or B. scorzonerifolium-like. Genotypic screening using isozymes showed that 39.3% of cell clones formed were hybrid. Some of the hybrid cell clones grew vigorously, and differentiated green leaves, shoots or plantlets. DNA marker analysis of the hybrids demonstrated that wheat DNA was integrated into the nuclear genomes of B. scorzonerifolium and in situ karyotyping cells revealed that a few wheat chromosome fragments had been introgressed into B. scorzonerifolium. The average wheat SSR retention frequency of the RH panel was 20.50%, but was only 6.67% in fusions with a non-irradiated donor. B. scorzonerifolium chromosomes and wheat SSR fragments in most asymmetric hybrid cell lines remained stable over a period of 2.5-3.5 years. We suggest the UV-induced asymmetric somatic hybrids between B. scorzonerifolium Willd and T. aestivum L. have the potential for use in the construction of an RH map of the wheat genome.

Bupleurum↗

PubMed1991

Use of a broad-host-range gyrA plasmid for genetic characterization of fluoroquinolone-resistant gram-negative bacteria.

The gyrA genotypes of ciprofloxacin-resistant clinical isolates of Escherichia coli (n = 3), Klebsiella pneumoniae (n = 4), Providencia stuartii (n = 2), Pseudomonas aeruginosa (n = 1), and Acinetobacter calcoaceticus (n = 1) were analyzed in a dominance test. This test is based on the dominance of a wild-type gyrA gene (gyrA+) over the quinolone resistance allele (gyrA) in a heterodiploid strain. Plasmid pBP515, developed to carry the gyrA+ gene of E. coli K-12 on a broad-host-range vector derived from pRSF1010, was used to obtain heterodiploid strains. Plasmid pBP515 encodes kanamycin and gentamicin resistance and is transferable via mobilization by a pRP1-derived helper plasmid (pRP1H) to strains of several gram-negative species. After the introduction of pBP515, single-cell MICs (as measured by reduction of the viable cell count) of ciprofloxacin and nalidixic acid decreased by 4- to greater than 8,000-fold for all strains tested, and 8 of the 11 strains regained ciprofloxacin susceptibilities similar to those of the respective wild types. The results indicate that (i) high-level fluoroquinolone resistance in clinical isolates of E. coli, K. pneumoniae, P. aeruginosa, and A. calcoaceticus can result from mutational alteration of the gyrA gene, and (ii) gyrA mutations are involved in high levels of fluoroquinolone resistance in P. stuartii. Additional mutations outside the gyrA locus may contribute to resistance in K. pneumoniae and P. stuartii.

Acinetobacter↗

PubMed1978

Biochemical and genetic characterization of kynurenine formamidase from Drosophila melanogaster.

The molecular weight forms of kynurenine formamidase were studied both gentically and biochemically. Formamidase I (native molecular weight 60,000) was purified using (NH4)2SO4 and pH fractionation, DEAE-cellulose chromatography at two different pH's, hydroxylapatite chromatography, and Sephadex G-100 gel filtration. Its subunit molecular weight, as determined by SDS gel electrophoresis, is 34,000, indicating that formamidase I is a dimer. Its Km is 1.87 X 10(-3) M. Its isoelectric point is pH 5.3. Its amino acid composition is reported. Formamidase II (native molecular weight 31,000) was partially purified using techniques similar to those above. Its Km is 2.31 X 10(-3) M. The response of formamidase activity to change in gene dosage was measured in segmental aneuploids generated in the second, third, and X chromosomes. Two separate chromosomal regions were identified which when present in extra dosage result in an elevation of the level of formamidase activity close to that predicted for the addition of a structural gene in a two-gene system. These tentative map positions were substantiated by demonstration that addition of one of the regions, 25A-27E, causes a 50% elevation in the relative amount of formamidase II. Addition of the other region, 91B-93F, causes a similar elevation in the relative amount of formamidase I. A model of the evolutionary origin of the two forms is presented, and the significance of these results to this model is discussed.

Amidohydrolases↗

PubMed2005

Genetic characterization of type 2 porcine circoviruses detected in Hungarian wild boars.

Porcine circoviruses (PCV) are present in pigs worldwide; they are grouped into two types: PCV1 comprising non-pathogenic viruses and PCV2 responsible for several clinical manifestations. Both types are frequently detected in domestic pigs, the prevalence and role of PCV in wild boars however, is not well studied. During the years 2002-2003 over 2000 organ samples of Hungarian wild boars were collected, grouped and samples from 307 different animals were tested by polymerase chain reaction for the presence of PCV. 35.5% of the wild boars were positive for one or both PCV types and PCV2 was detected in 20.5% of the animals. The PCV2 viruses were divided into 7 groups (WB-H1-7) based on sequencing data and genomes representing these groups were sequenced completely. The wild boar PCV2 groups were distributed evenly in the geographical region, regardless of the time and place of collection. The phylogenetic analysis of the PCV2 sequences of wild boar and domestic pig origin showed the possibility of an epidemiological link between wild boar and domestic pig infections. Interestingly, the complete nucleotide sequence of the viruses and the predicted amino acid sequence of the replication associated protein (ORF1) grouped the viruses similarly, whereas the capsid protein (ORF2) comparisons revealed different relations among the groups, suggesting the possibility of genomic recombination in PCV2.

Animals↗

PubMed1998

Genetic characterization of HIV type 1 from migrant workers in three South African gold mines.

The phylogenetic relationships between 44 HIV-1 isolates from 43 infected subjects employed by three adjacent South African gold mines were investigated. The patients were migrant workers originating from rural areas of South Africa and the neighboring countries of Lesotho, Botswana, Swaziland, and Mozambique. Proviral HIV-1 DNA was subtyped using a heteroduplex mobility assay (HMA) based on the 700-bp V3-V5 region of the env gene. DNA sequence analysis was used to confirm the subtype designation and to determine phylogenetic relationships between isolates. All 44 HIV-1 isolates were identified as env subtype C using both HMA and phylogenetic analysis. These isolates did not show a distinct phylogenetic relatedness based on the geographic origins of the migrant workers or show close homology to other subtype C sequences from southern Africa or India. However, five clusters of closely related sequences were identified, mainly involving miners of disparate geographic origins, suggesting possible epidemiological linkage in these few cases. The characteristic tetrapeptide sequence, GPGQ, at the tip of the V3 loop of subtype C viruses was conserved in the predicted amino acid sequences of most isolates. The heterogeneity of HIV-1 sequences among migrant workers in a mining cohort suggests multiple introductions of HIV-1 subtype C into this population that are not apparently linked to the geographic origins of the patients.

Adolescent↗

PubMed1990

Transformation of chloroplast ribosomal RNA genes in Chlamydomonas: molecular and genetic characterization of integration events.

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.

Chlamydomonas↗

PubMed1998

Biochemical and genetic characterization of the dominant positive element driving transcription ofthe yeast TBP-encoding gene, SPT15.

We previously demonstrated that a combination of both positive and negative cis -acting upstream elements control the transcription of the gene encoding TBP ( SPT15 ) in Saccharomyces cerevisiae . One of these elements found in that study, resident between 5' flanking sequences -147 and -128 , and termed PED (for positive element distal), was found to play an essential positive role in driving transcription of the gene encoding TBP. In this report, we map at nucleotide-level resolution, the critical residues which comprise PED, purify and sequence the protein that binds to it and determine that this PED binding factor is Abf1p, an abundant yeast protein previously broadly implicated in both gene regulation and DNA replication. In the case of the TBP-encoding gene, however, Abf1p works through the PED element which is a non-consensus binding site. Based upon the work of others, the PED-variant ABF1 site would be predicted to be a very poor binding site for this factor yet Abf1p binds PED and a consensus ABF1 site with comparable affinity. These results are discussed in light of the broader context of Abf1p-mediated gene regulation.

Base Sequence↗

PubMed1995

Genetic characterization of the Mutator system in maize: behavior and regulation of Mu transposons in a minimal line.

Most Mutator lines of maize harbor several different classes of Mu transposons, each of which may be present in high copy number. The regulatory element is also often found in high copy number, and it is this element's behavior that is presumed to cause the non-Mendelian inheritance of Mutator activity. Using a very simple Mutator line, we demonstrate tha MuDR-1, a regulator of the Mutator system, can functionally replace standard non-Mendelian Mutator activity and that MuDR-1 is associated with the loss of methylation of the termini of another Mu transposon. Further, we show that Mu transposons can transpose duplicatively, that reinsertion tends to be into unlinked sites, and that MuDR-1 frequently suffers deletions. Changes in chromosomal position and the mode of sexual transmission are shown to be associated with changes in the frequency of MuDR-1 duplication and with the activity of MuDR-1 as monitored by the excision frequency of a reporter transposon of the Mu family, Mu1. Our data are derived from a Minimal Mutator Line in which there are relatively few Mu transposons, including one MuDR-1 regulator and as few as one Mu1 reporter. The seemingly enigmatic results that have been obtained using more complicated Mu genotypes are reinterpreted using simple Mendelian principles. We have borrowed a gap-repair model from Drosophila biologists to explain both duplications and deletions of MuDR-1.

Chromosome Mapping↗

PubMed1999

Genetic characterization of cytological region 77A-D harboring the presenilin gene of Drosophila melanogaster.

We performed a systematic lethal mutagenesis of the genomic region uncovered by Df(3L)rdgC-co2 (cytological interval 77A-D) to isolate mutations in the single known Presenilin (Psn) gene of Drosophila melanogaster. Because this segment of chromosome III has not been systematically characterized before, inter se complementation testing of newly recovered mutants was carried out. A total of 79 lethal mutations were isolated, representing at least 17 lethal complementation groups, including one corresponding to the Psn gene. Fine structure mapping of the genomic region surrounding the Psn transcription unit by transgenic rescue experiments allowed us to localize two of the essential loci together with Psn within an approximately 12-kb genomic DNA region. One of these loci, located 3' to Psn, encodes a Drosophila protein related to the yeast 60S ribosomal protein L10 precursor. We also determined which of the newly recovered lethal mutant groups correspond to previously isolated lethal P-element insertions, lethal inversion breakpoints, and lethal polo gene mutants. Point mutations were identified in all five recovered Psn alleles, one of which results in a single amino acid substitution G-E at a conserved residue in the C-terminal cytoplasmic tail of the protein, suggesting an important functional role for this C-terminal domain of Presenilin. In addition, some viable mutations were recovered in the screen, including new alleles of the clipped and inturned loci.

Animals↗

PubMed2006

Genetic characterization and expression of the novel fungal protease, EPg222 active in dry-cured meat products.

EPg222 protease is a novel extracellular enzyme produced by Penicillium chrysogenum (Pg222) isolated from dry-cured hams that has the potential for use over a broad range of applications in industries that produce dry-cured meat products. The gene encoding EPg222 protease has been identified. Peptide sequences of EPg222 were obtained by de novo sequencing of tryptic peptides using mass spectrometry. The corresponding gene was amplified by PCR using degenerated primers based on a combination of conserved serine protease-encoding sequences and reverse translation of the peptide sequences. EPg222 is encoded as a gene of 1,361 bp interrupted by two introns. The deduced amino acid sequence indicated that the enzyme is synthesized as a preproenzyme with a putative signal sequence of 19 amino acids (aa), a prosequence of 96 aa and a mature protein of 283 aa. A cDNA encoding EPg222 has been cloned and expressed as a functionally active enzyme in Pichia pastoris. The recombinant enzyme exhibits similar activities to the native enzyme against a wide range of protein substrates including muscle myofibrillar protein. The mature sequence contains conserved aa residues characteristic of those forming the catalytic triad of serine proteases (Asp42, His76 and Ser228) but notably the food enzyme exhibits specific aa substitutions in the immunoglobulin-E recognition regions that have been identified in protein homologues that are allergenic.

Amino Acid Sequence↗

PubMed2004

Molecular-genetic characterization of CMS-S restorer-of-fertility alleles identified in Mexican maize and teosinte.

Restorer-of-fertility (Rf) alleles for S-type cytoplasmic male sterility (CMS-S) are prevalent in Mexican races of maize and teosinte. Forty-five Rf alleles from 26 races of maize and 6 Rf alleles from different accessions of teosinte were found to be homozygous viable, consistent with the hypothesis that they are naturally occurring Rf alleles. Mapping and allelism studies were performed to assess the number of genes represented by these 51 alleles. Forty-two of the Rf alleles mapped to the long arm of chromosome 2 (2L), and 5 of these were further mapped to the whp1-rf3 region. The Rf3 restoring allele, found in some U.S. maize inbred lines, cosegregates with internal processing of CMS-S mitochondrial transcripts. Three of the 5 mapped Rf alleles were associated with a similar RNA processing event. Allelism or tight linkage was confirmed between Rf3 and 2 teosinte alleles (Rf K-69-6 and Rf 9477) and between Rf3 and the Cónico Norteño allele Rf C-N (GTO 22). The rf3 region of 2L potentially encodes a complex of linked rf genes. The prevalence of restoring alleles in this chromosomal region, among normal-cytoplasm accessions of Mexican maize and teosinte, supports the conclusion that these alleles have functions in normal mitochondrial gene expression that by chance allow them to restore male fertility in S cytoplasm.

Chromosome Mapping↗