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At least 1,099 records · Page 61Linked to original sources
PubMed1991

Cloning and characterization of ERG8, an essential gene of Saccharomyces cerevisiae that encodes phosphomevalonate kinase.

Saccharomyces cerevisiae strains that contain the ery8-1 mutation are temperature sensitive for growth due to a defect in phosphomevalonate kinase, an enzyme of isoprene and ergosterol biosynthesis. A plasmid bearing the yeast ERG8 gene was isolated from a YCp50 genomic library by functional complementation of the erg8-1 mutant strain. Genetic analysis demonstrated that integrated copies of an ERG8 plasmid mapped to the erg8 locus, confirming the identity of this clone. Southern analysis showed that ERG8 was a single-copy gene. Subcloning and DNA sequencing defined the functional ERG8 regulon as an 850-bp upstream region and an adjacent 1,272-bp open reading frame. The deduced 424-amino-acid ERG8 protein showed no homology to known proteins except within a putative ATP-binding domain present in many kinases. Disruption of the chromosomal ERG8 coding region by integration of URA3 or HIS3 marker fragments was lethal in haploid cells, indicating that this gene is essential. Expression of the ERG8 gene in S. cerevisiae from the galactose-inducible galactokinase (GAL1) promoter resulted in 1,000-fold-elevated levels of phosphomevalonate kinase enzyme activity. Overproduction of a soluble protein with the predicted 48-kDa size for phosphomevalonate kinase was also observed in the yeast cells.

Amino Acid Sequence↗
PubMed1993

Genomic cloning of the rat histamine H1 receptor.

A rat histamine H1 receptor gene which lacked introns was isolated from a rat genomic library using recently cloned bovine histamine H1 receptor cDNA [Yamashita et al., Proc. Natl. Acad. Sci. USA, 88, 11515-11519 (1991)]. The receptor protein deduced from this isolated gene was composed of 486 amino acids and showed characteristic properties of G protein-coupled receptors. At the 5'-flanking region of the receptor gene, we have located potential TATA box sequences and consensus sequences for the glucocorticoid response element and AP-2 element. After being subcloned into a mammalian expression vector, the isolated gene was transfected to C6 glioma cells. These cells showed significant binding toward [3H]mepyramine. The binding was inhibited by H1 antagonists or histamine. The mode of this binding was comparable to the binding of membranes derived from rat tissues toward [3H]mepyramine. Northern blot analysis detected a 3.0 kb nucleotide band for histamine H1 receptor mRNAs from rat brain and small intestine when these mRNAs were hybridized with the isolated rat H1 gene. The present results demonstrate the isolation of the rat histamine H1 receptor gene.

Amino Acid Sequence↗
PubMed1990

Characterization and nucleotide sequence of the gene encoding the human pyruvate dehydrogenase alpha-subunit.

Genomic clones encompassing the entire gene (PDH alpha) encoding the human pyruvate dehydrogenase alpha-subunit (PDH alpha) have been isolated by screening a leukocyte genomic library in the cloning vector, lambda EMBL4. The PDH alpha gene spans 17082 bp and is composed of eleven exons and ten introns. All intron/exon splice junctions follow the GT/AG rule. A total of seven Alu repeats were found in five introns. The entire nucleotide (nt) sequence of the PDH alpha gene has been determined and typical consensus promoter sequences in the 5'-flanking region were found. The results of primer extension analysis imply that the PDH alpha gene transcription start point (tsp) is a thymine residue 124 bp upstream from the ATG start codon in exon 1. The structural organization and the tsp were compared with the recent report [Maragos et al., J. Biol. Chem. 264 (1989) 12294-12298]. Analysis of the PDH alpha gene resolves existing discrepancies among four published sequences of PDH alpha cDNAs. A 93-bp sequence that was missing in our sequence of cultured foreskin fibroblast PDH alpha cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45] was identified in the gene as exon 6.

Amino Acid Sequence↗
PubMed1994

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.

Amino Acid Sequence↗
PubMed1994

NAD(+)-dependent D-2-hydroxyisocaproate dehydrogenase of Lactobacillus delbrueckii subsp. bulgaricus. Gene cloning and enzyme characterization.

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD(+)-dependent D-2-hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333-residue protein closely related to D-2-hydroxyisocaproate (i.e. 2-hydroxy-4-methyl-pentanoate) dehydrogenase (D-HO-HxoDH) of Lactobacillus casei and other NAD(+)-dependent D-lactate dehydrogenases (D-LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two-step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2-oxoacid substrates except those having a branched beta-carbon.

Alcohol Oxidoreductases↗
PubMed1990

Comparison of 5' region sequences of human and rabbit phosphofructokinase genes.

A phosphofructokinase gene was screened and cloned from a human genomic library prepared in the lambda EMBL4 phage vector. DNA sequencing shows that the first exon of this human phosphofructokinase gene is identical in length and highly homologous in sequence to that of a rabbit phosphofructokinase gene. Two amino acid replacements are indicated, an Arg----Lys and a Val----Ile at positions 9 and 13, respectively. Eleven base substitutions, 8 of them silent, are identified. Surprisingly, at ten of these sites, complete bias for A's and T's in the human gene and C's and G's in the rabbit gene are seen. Strong conservation is also observed in the 5' untranslated region and for the first 15 base pairs in the intron. All the nine variant nucleotides in these regions are, again, A's and T's in the human gene and G's and C's in the rabbit gene. The unit evolutionary period of change between the first exon of rabbit and human phosphofructokinase genes is estimated as 2.3 million years at silent sites and 15.6 million years at replacement sites.

Amino Acid Sequence↗
PubMed2001

A novel early estrogen-regulated gene gec1 encodes a protein related to GABARAP.

We have isolated, in guinea-pig endometrial cells, an estrogen-induced 1.8 kb RNA called gec1. Screening of a guinea-pig genomic library led to identification of gec1 gene consisting of 4 exons and 3 introns. Exon 1 contains the 5'UTR and the ATG initiation codon. A guinea-pig gec1 cDNA was obtained by 5'-RACE. The 351 bp coding sequence shares 76.8% identity with that of the human GABARAP 924 bp cDNA while UTRs of the two cDNAs differ. A gec1 probe from the 3'UTR revealed a 1.9 kb mRNA in human tissues and a human GEC1 cDNA was isolated from placenta. Its coding sequence shares 93 and 79% identity with that of guinea-pig gec1 and human GABARAP, respectively. The human and guinea-pig GEC1 proteins have 100% identity. GEC1 and GABARAP proteins have 87% identity and N terminus featuring a tubulin binding motif. Thus, estrogen-regulated gec1 is a new gene which could encode a microtubule-associated protein.

3' Untranslated Regions↗
PubMed1995

Gene structure and chromosomal localization of the human HSD11K gene encoding the kidney (type 2) isozyme of 11 beta-hydroxysteroid dehydrogenase.

11 beta-Hydroxysteroid dehydrogenase (11 beta HSD) converts glucocorticoids to inactive products and is thus thought to confer specificity for aldosterone on the type I mineralocorticoid receptor in the kidney. Recent studies indicate the presence of at least two isozymes of 11 beta HSD. In vitro, the NAD(+)-dependent kidney (type 2) isozyme catalyzes 11 beta-dehydrogenase but not reductase reactions, whereas the NADP(+)-dependent liver (type 1) isozyme catalyzes both reactions. We have now characterized the human gene encoding kidney 11 beta HSD (HSD11K). A bacteriophage P1 clone was isolated after screening a human genomic library by hybridization with sheep HSD11K cDNA. The gene consists of 5 exons spread over 6 kb. The nucleotide binding domain lies in the first and the second exon, and the catalytic domain in the fourth exon. The 5' flanking sequences and first exon are GC-rich (80%), suggesting that the gene may be transcriptionally regulated by factors that recognize GC-rich sequences. Fluorescence in situ hybridization of metaphase chromosomes with a positive P1 clone localized the gene to chromosome 16q22. In contrast, the HSD11L (liver isozyme) gene is located on chromosome 1 and contains 6 exons; the coding sequences of these genes are only 21% identical. HSD11K is expressed at high levels in the placenta and kidney of midgestation human fetuses and at lower levels in lung and testes. Different transcriptional start sites are utilized in kidney and placenta. These data should be applicable to genetic analysis of the syndrome of apparent mineralocorticoid excess, which may represent a deficiency of 11 beta HSD.

11-beta-Hydroxysteroid Dehydrogenases↗
PubMed1992

Isolation of 2 novel RFLP markers and their localization at 2q35 by microdissection and subsequent enzymatic amplification.

We previously constructed a chromosome 2q-specific genomic library and isolated a number of microclones. In the present study, we first analyzed with Southern hybridization whether any of the microclones represent restriction fragment length polymorphisms (RFLPs), and then tried to map RFLP markers physically, using the recently developed chromosome microdissection/enzymatic amplification method. Of 13 clones analyzed, two were RFLP markers; a clone, pM2C83, showed a four-allele MspI RFLP, and the other, pM2C8, a two-allele RsaI RFLP. In order to assign the two polymorphic markers, two chromosomal segments, 2q32-q35 and 2q35-qter, on the chromosome 2 from a karyotypically normal person were microdissected, and the DNA from each segment was amplified with the polymerase chain reaction (PCR) using marker sequence-specific primers. With this method, both of the clones were assigned to 2q35. These two RFLP markers must be useful for linkage analysis of genetic diseases whose loci are at around 2q35.

Base Sequence↗
PubMed1991

Development of epidemiological method for the Helicobacter pylori by polymerase chain reaction.

The polymerase chain reaction was used to develop a method for the detection of Helicobacter pylori, a causative agent of gastritis, as well as for the elucidation of its mode of transmission. A genomic library of Helicobacter pylori DNA in Escherichia coli JM109 was constructed by cloning Hind III-digested DNA fragments into plasmid vector pUC18. The nucleotide sequences from seven recombinant clones were determined and five sets of oligonucleotide primers were synthesized on the basis of the sequences from five clones (B4, B9, B10, C15 and I22). The PCR amplifications with these primers were performed using DNA samples from five strains of Helicobacter pylori, two Campylobacter spp. and eleven species of enteric bacteria. Amplifications of the target DNA fragments in all of 5 strains of Helicobacter pylori were observed from the PCR with primers derived from clone B4, B9, C15 and I22. When the specificity was checked with the DNA samples from 13 other bacteria as template DNA for the PCR, specific amplification that produced the correct size of the target DNA of Helicobacter pylori was shown only in the PCR with primers derived from clone B9 and C15. The detection limit in the PCR amplification, determined by the heat-lysis method, was 500 cells of Helicobacter pylori.

Base Sequence↗
PubMed1999

Identification of microsatellite sequences in Vitis riparia and their applicability for genotyping of different Vitis species.

A Vitis riparia genomic library was screened for the presence of (GA)n simple sequence repeats (SSR) and 18 primer pairs yielding amplification products of the expected size were designed. Heterologous amplification with the primer pairs in related species (V. rupestris, V. berlandieri, V. labrusca, V. cinerea, V. aestivalis, V. vinifera, and interspecific hybrids) was successful in most primer-species combinations. Therefore, the new markers are applicable to the genotyping of a range of Vitis species. Variations in the SSR flanking sequence were detected between and within the species. The degree of polymorphism and performance of the markers were determined in up to 120 individuals of V. vinifera. Four of fifteen alleles per locus were detected and expected heterozygosity ranged between 0.37 and 0.88. Null alleles were shown to be present at two loci by a lack of heterozygous individuals and by transmission of the null alleles in a controlled cross. Regular Mendelian inheritance is indicated for all but one loci by a preliminary segregation analysis in 36 offspring. Thirteen of the markers were found suitable for the genotyping of grapevines (V. vinifera).

Base Sequence↗
PubMed1994

[Characterization of leptospiral DNA by southern hybridization serovar lai recombinant probe pCX7].

The recombinant probe pCX7, derived from genomic library of serovar lai strain 017, was applied for the characterization of DNA from 20 strains of leptospires in different genus species, sero-groups and serovars by southern hybridization. The probe, specific to partial species L. interrogans, did not hybridized with DNA of L. biflexa strain Patoc I and L. illini strain 3055, neither with DNA of serovars javanica, manhao 2 and ranarum which had shown very weak virulence, but hybridized with DNA of the other 15 strains of L. interrogans in 8 serogroups. It was easy to distinguish the banding patterns, since the pCX7 probe hybridized with a limited number of DNA fragments. Different leptospires showed different banding patterns. A few differences between strain 017 (strain of strong virulence) and strain 601 (strain of weak virulence) of serovar Lai were observed although their hybridization banding patterns were very similar. Differences in hybridization signals in combination with the banding pattern appear to provide good characteristics for identification of leptospiral serogroups and serovars, even strains. The result indicated that southern hybridization with the probe pCX7 might provide good tools for identification of leptospira.

Blotting, Southern↗
PubMed1999

Evaluating a putative bottleneck in a population of bowhead whales from patterns of microsatellite diversity and genetic disequilibria.

A size-selected Balaena mysticetus genomic library was screened for clones containing simple sequence repeat, or microsatellite, loci. A total of 11 novel loci was identified. These loci were combined with a set of 9 published loci, for a total of 20 markers, and were scored across a sample of 108 bowhead whales from the Bering-Chukchi-Beaufort Seas population of bowhead whales. Genetic variability was measured in terms of polymorphism information content values and unbiased heterozygosity. From the latter, estimates of long-term effective population size were obtained. In addition, gametic phase disequilibrium among loci was investigated. Moderate to high levels of polymorphism were found overall, and the long-term effective size estimates were large relative to total population size. Tests of heterozygosity excess (Cornuet and Luikart 1996) and allele frequency distribution (Luikart et al. 1998) indicated that the possibility of a recent genetic bottleneck in the Bering-Chukchi-Beaufort Seas population of bowhead whales is highly unlikely. However, the fact that five loci displayed a statistically significant heterozygote deficiency remains to be explained.

Animals↗
PubMed1991

Cloning and tissue expression of a rat histamine H2-receptor gene.

We have screened a rat genomic library using DNA probes derived from the sequence of a recently cloned canine histamine H2 receptor (Gantz et al., Proc. Natl. Acad. Sci. USA 1991, 88, 429-433). An intronless gene, encoding a 358 amino acid protein displaying all major features of G protein-coupled receptors and a 82% overall homology with the canine histamine H2 receptor, was isolated. Northern blot analysis, performed with a probe derived from the rat sequence, revealed a single approximately 6.0 kb transcript in various rat tissues. In the brain, this transcript is highly expressed in brainstem and, to a lesser extent, cerebral cortex, striatum, hippocampus and hypothalamus. These localizations are consistent with the distribution of the H2 receptor in this species. Among peripheral tissues, the stomach highly expresses the transcript which could not be detected in various other organs. All these features, together with the expression of the cloned gene in mammalian cells (Traiffort et al., submitted), are consistent with the idea that a single rat H2 receptor is encoded by the cloned gene.

Amino Acid Sequence↗
PubMed1994

Characterization of IS1110, a highly mobile genetic element from Mycobacterium avium.

A highly mobile insertion sequence designated IS1110 was detected in Mycobacterium avium strain LR541 following an observed increase in size of the plasmid pLR20. Genomic libraries of M. avium strains carrying either parental pLR20 or the modified plasmid (pLR20') were constructed and the sequence of the relevant clones was determined to characterize the insertion sequence and the target region. IS1110 is a 1457 bp element lacking terminal inverted repeats, and is related to IS900 (from Mycobacterium paratuberculosis), IS901 and IS902 (from M. avium) and to IS116 (from Streptomyces clavuligerus). LR541 carries several copies of IS1110. Individual colonies from the same plate show differences in Southern blot patterns when tested with an IS1110-derived probe; the ability to detect transposition events in random colonies, without any selection pressure, indicates an exceptionally high degree of mobility, which will be invaluable for transposon mutagenesis. Analyses of M. avium isolates from human, veterinary, and environmental sources showed that IS1110-hybridizing sequences are present in some M. avium isolates but they were not detected in strains of other mycobacterial species. The polymorphism exhibited in M. avium isolates suggests that this element may be useful for molecular epidemiological studies of M. avium infections.

Amino Acid Sequence↗
PubMed1994

Cloning and nucleotide sequence analysis of the Lactobacillus delbrueckii ssp. lactis DSM7290 cysteine aminopeptidase gene pepC.

A genomic library of Lactobacillus delbrueckii ssp. lactis DSM7290 in the low copy number vector pLG339, was screened for the presence of peptidase genes. Using the chromogenic substrate gly-ala-beta-naphthylamide, which is not a substrate for any of the recently cloned peptidases of DSM7290, and the multiple peptidase deficient Escherichia coli strain CM89, allowed the isolation of clones, which contained the equivalent hydrolytic activity. To identify genes encoding the conserved catalytic active site of cysteine proteases, partial nucleotide sequencing with a degenerate oligonucleotide was performed on recombinant plasmids isolated from such clones. This allowed to identify two out of nine clones to carry the Lactobacillus pepC gene. A total of 2026 nucleotides were determined, and sequence analysis revealed a gene with strong homology to the recently cloned Lb. helveticus (73.2%) and Lactococcus lactis (51.03%) pepC genes, and the derived protein showed homology with the active site of a large number of cysteine proteases. The predicted open reading frame consists of 449 codons, coding for a protein of 50,909 Da. The enzyme is functional and extremely overexpressed in E. coli.

Amino Acid Sequence↗
PubMed1996

Increased unsaturated fatty acid production associated with a suppressor of the fabA6(Ts) mutation in Escherichia coli.

Plasmids that corrected the temperature-sensitive unsaturated fatty acid auxotrophy of strain M6 [fabA6 (Ts)] were isolated from an Escherichia coli genomic library. Subcloning and physical mapping localized the new gene (called sfa for suppressor of fabA) at 1,070 kb on the E. coli chromosome. DNA sequencing revealed the presence of a 227-bp open reading frame which directed the synthesis of a peptide of approximately 8 kDa, which correlated with the correction of the fabA6(Ts) phenotype. However, the sfa gene was an allele-specific suppressor since plasmids harboring the sfa gene corrected the growth phenotype of fabA6(Ts) mutants but did not correct the growth of fabA2(Ts) or fabB15(Ts) unsaturated fatty acid auxotrophs. Overexpression of the sfa gene in fabA6(Ts) mutants restored unsaturated fatty acid content at 42 degrees C, and overexpression in wild-type cells resulted in a substantial increase in the unsaturated fatty acid content of the membrane. Thus, the suppression of the fabA6(Ts) mutation by sfa was attributed to its ability to increase the biosynthesis of unsaturated fatty acids.

Alleles↗
PubMed1991

Cloning and functional characterization of a novel mas-related gene, modulating intracellular angiotensin II actions.

The mas oncogene codes for a GTP binding protein-coupled receptor that determines a physiological response to angiotensin when expressed in Xenopus laevis oocytes or in the neuronal cell line NG115-401L. However, another gene, rat thoracic aorta gene, structurally related to mas, is devoid of any functional similarity with the angiotensin receptor(s). The relationships between the mas-related proteins and the angiotensin receptors were investigated by identifying and characterizing new members of the mas gene family. A new mas-related gene (mrg) was cloned in a human genomic library at low stringency using the mas cDNA as probe. Mrg codes for a seven-hydrophobic-segment receptor that is 35% identical to the mas product and 29% identical to the rat thoracic aorta gene product. Mrg mRNA was not detected in several rat and human adult tissues that normally express the angiotensin II (AII) receptor, and transfections of COS and CHO cells with the mrg gene did not modify the number of AII binding sites. These results indicate that mrg and the human AII receptor genes are not identical. However, injection of mrg mRNA into Xenopus oocytes markedly increased the electrophysiological response to angiotensin peptides, indicating some functional similarities with the mas product. The reduction of the response after defolliculation of the oocyte, together with the full agonist effect of Sar1IIe8AII and the partial agonist effect of Sar1Ala8AII, seem to indicate that mrg interacts with the signaling pathways of the endogenous Xenopus angiotensin receptor to potentiate the response to AII.

Amino Acid Sequence↗