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At least 955 records · Page 53Linked to original sources
PubMed1999

The pimB gene of Mycobacterium tuberculosis encodes a mannosyltransferase involved in lipoarabinomannan biosynthesis.

The biosynthesis of lipoarabinomannan (LAM), a key mycobacterial lipoglycan that has been implicated in numerous immunoregulatory functions, was examined utilizing D-mannosamine (ManN) as a tool to identify mannosyltransferase genes involved in LAM synthesis. Cell-free reactions utilizing cellular membranes of mycobacteria as the enzyme source indicated that ManN inhibited the synthesis of phosphatidylinositol mannosides, early precursors to LAM. A selection strategy was devised to screen a Mycobacterium tuberculosis genomic library in Mycobacterium smegmatis for clones conferring conditional resistance to ManN, with the rationale that overexpression of the gene(s) encoding a target of ManN would impart a ManN-resistant phenotype under these conditions. This strategy led to the identification of pimB, whose deduced amino acid sequence shows similarity to mannosyltransferases and other glycosyltransferases. Partially purified recombinant PimB protein from Escherichia coli or membranes from M. smegmatis overexpressing the pimB gene were used in cell-free assays to show that PimB catalyzes the formation of triacylphosphatidylinositol dimannoside from GDP-mannose and triacylphosphatidylinositol monomannoside.

Bacterial Proteins↗
PubMed2000

Mapping of four simple sequence repeat (SSR) markers on rat chromosome 4.

We previously reported that several markers on rat chromosome (Chr) 4 cosegregated with the occurrence of cerebral stroke and brain edema in stroke-prone spontaneously hypertensive rats (SHRSP). To obtain insights into the positional candidate genes for stroke susceptibility in this region, we mapped four genes, Taurine transporter (Tau), tumor necrosis factor receptor (Tnfr), GABA transporter (Gat1) and glucose transporter-3 (Glut3) genes, using newly developed simple sequence repeat (SSR) markers on rat Chr 4. We isolated the SSRs for the genes either by screening a rat genomic library or by searching the GenBank database. By linkage analysis using two sets of backcrosses, Gat1 and Tnfr were mapped in the region associated with stroke, while Taut was located distant from the region. The Glut3 locus was also assigned to rat Chr 4 using a rat x mouse hybrid clone panel. These results indicated that the Tnfr, Gat1 and Glut3 genes were good positional candidates for the stroke susceptibility in SHRSP, suggesting that further evaluation of these genes by functional studies could prove useful.

Animals↗
PubMed1995

The gene encoding human 2-oxoglutarate dehydrogenase: structural organization and mapping to chromosome 7p13-p14.

Genomic clones covering the entire sequence of the gene encoding human 2-oxoglutarate dehydrogenase (OGDH) were isolated by screening leukocyte and placenta genomic libraries with radio- and digoxigenin (DIG)-labeled human OGDH cDNAs. The human OGDH gene contains 22 exons spanning approx. 85 kb. All exon/intron splice junctions follow the GT/AG rule. The results of primer extension analysis imply that the OGDH transcription start point is a thymine residue 55 bp upstream from the ATG start codon. The 5'-flanking region of OGDH lacked canonical TATA or CAAT boxes. Using DNAs from human x rodent somatic cell hybrids that segregate human chromosomes in conjunction with fluorescence in situ hybridization, human OGDH was assigned to chromosome arm 7p, an area that corresponds to the boundary between bands 7p13 and 7p14.

Amino Acid Sequence↗
PubMed2004

Cloning and sequence analysis of the glyceraldehyde-3-phosphate dehydrogenase gene from the zygomycetes fungus Rhizomucor miehei.

Rhizomucor miehei is important from a biotechnological aspect in consequence of its content of aspartic proteinase, which has high milk-clotting activity. A genomic library of R. miehei NRRL 5901 has been constructed in a phage (Lambda Fix II) vector. The glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated from this library by hybridization of the recombinant phage clones with a gpd-specific gene probe generated by polymerase chain reaction. The complete nucleotide sequence encodes a putative polypeptide chain of 336 amino acids interrupted by 5 introns. The predicted amino acid sequence of this gene shows a high degree of sequence similarity to the glyceraldehyde-3-phosphate dehydrogenase proteins from yeast and filamentous fungi. The promoter region, containing a consensus TATA box, and 246-bp downstream from the putative stop codon were also determined. The possibility of using the gpd promoter in the construction of new transformation vectors is discussed.

Amino Acid Sequence↗
PubMed1992

Basic methodology in the molecular characterization of genes.

PURPOSE: During the past two decades, molecular biology techniques have had an increasing impact upon hypertension research. This article will thus review the basic methodology in this field. CONTENTS: Protocols are described for the establishment of a genomic library and its use for the cloning of specific genes, as well as methods for the detection and sequencing of DNA. In addition, techniques to detect and quantify specific messenger RNA, such as Northern blotting, ribonuclease protection assay and in situ hybridization, and the reporter gene approach for the analysis of regulatory gene sequences, are included. The polymerase chain reaction which, as a newly established technique to detect and amplify DNA, has exerted a strong influence upon all areas of molecular biology is the subject of the concluding paragraph. CONCLUSIONS: Molecular biology techniques may be of substantial help in revealing the cause of hypertension and developing tools to prevent and treat this disorder.

Animals↗
PubMed1999

Cloning and characterization of rat leukotriene B(4) receptor.

Leukotriene B(4) (LTB(4)) is a potent chemoattractant for neutrophils and eosinophils. cDNAs for LTB(4) receptor (BLT) have been cloned from human, mouse, and guinea pig. Here we report the isolation of BLT from rat genomic library. Rat BLT consists of 351 amino acids with homologies of 80.2, 93.2, and 71.6%, to human, mouse, and guinea pig BLT, respectively. When expressed in human embryonic kidney (HEK)-293 cells, rat BLT showed a specific and high-affinity binding to LTB(4) with a Kd value of 0.68 nM (mean, n = 3). Northern blot analysis showed that BLT is exclusively expressed in polymorphonuclear leukocytes. Furthermore, the expression of BLT was high in proteosepeptone-activated peritoneal macrophages, while the resident macrophages did not show significant expression. The present results suggest important roles of LTB(4) in macrophage recruitment and activation.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and characterization of the promoter region of the mouse regulatory subunit RII beta of type II cAMP-dependent protein kinase.

The promoter and exon 1 of the regulatory subunit (RII beta) of type II cAMP-dependent protein kinase were isolated from a mouse genomic library. The 5'-flanking DNA lacked TATA and CAAT sites but contained GC rich regions typically found in constitutively expressed house keeping genes. Fusion gene constructs, containing RII beta 5'-flanking sequences and the bacterial CAT structural gene, were transfected into NB2a neuroblastoma cells and CHO cells. The NB2a cells expressed high levels of CAT activity. CHO cells expressed CAT activity at 5% of the level seen in the NB2a cells. Transfection of deletion constructs into both cell lines was used to define the core promoter and enhancer elements. The core promoter was situated between bp -291/-121. An enhancer element was located between bp -1426/-1018.

Amino Acid Sequence↗
PubMed1991

Isolation and structural characterization of the murine tryptophan hydroxylase gene.

The mouse tryptophan hydroxylase gene was isolated and its intron/exon boundaries and putative regulatory sequences identified. To isolate the gene a mouse mastocytoma cDNA clone encoding tryptophan hydroxylase was used to identify and isolate ten overlapping DNA fragments from a mouse genomic library. Restriction mapping and sequence analysis of the clones revealed that the gene contains 11 exons and covers a region of DNA of approximately 21 kb. The transcription initiation site was mapped and the major site of initiation yields an untranslated leader sequence of 124 nucleotides. A minor initiation site is located 9 nucleotides 3' of the major site. The 5' untranslated sequence is interrupted by the first intron. Analysis of the sequence upstream of the initiation site showed the presence of several putative promoter and regulatory sequences. Nine of the ten intron/exon boundaries of tryptophan hydroxylase are conserved with tyrosine hydroxylase and phenylalanine hydroxylase, further delineating the evolutionary relationship of these three genes.

Amino Acid Sequence↗
PubMed1994

Cloning and expression of putative cytotonic enterotoxin-encoding genes from Aeromonas hydrophila.

A genomic library from a diarrheal isolate, SSU, of Aeromonas hydrophila was constructed in a cosmid vector, pHC79, and in bacteriophage lambda EMBL3. Cell lysates from various Escherichia coli clones containing the recombinant cosmid were examined for their ability to elongate Chinese hamster ovary (CHO) cells, which is a typical enterotoxic response. Based on restriction analysis, a 4.0-kb SalI DNA fragment from one of the clones that exhibited enterotoxic activity was subcloned into a bacteriophage T7 RNA polymerase/promoter hyperexpression system. The cell lysate from this E. coli [pSL24] clone caused CHO cells to elongate and revealed the presence of a major 35-kDa polypeptide by [35S]methionine labeling and sodium dodecyl sulfate (SDS)-polyacrylamide-gel electrophoresis (PAGE). The toxin was biologically heat labile, losing all activity within 20 min at 56 degrees C. In addition, another enterotoxin-producing clone, E. coli[pSBS32], was isolated from cosmid and lambda bacteriophage libraries. We localized this heat-stable (56 degrees C/20 min) enterotoxin to a 4.8-kb SalI-BamHI fragment. Both enterotoxins caused elevation of cyclic adenosine monophosphate (cAMP) in CHO cells. The DNA fragments encoding these enterotoxins did not hybridize with each other. However, a 4.8-kb SalI-BamHI DNA fragment encoding a heat-stable enterotoxin hybridized to a 3.5-kb BamHI DNA fragment of a plasmid, pHPC100, that contained a cytotonic enterotoxin-encoding gene isolated from A. trota. Our data suggest Aeromonas species produce different structural types of cytotonic enterotoxins that are functionally similar.

Aeromonas hydrophila↗
PubMed1990

Location of the cis-acting auxin-responsive region in the promoter of the par gene from tobacco mesophyll protoplasts.

We have isolated a genomic clone of an auxin-regulated par gene, which is expressed during the transition from G0 phase to S phase in the early stage of tobacco mesophyll protoplasts cultured in vitro, from a tobacco genomic library using the par cDNA as a probe. When a chimeric gene, in which a reporter gene for bacterial beta-glucuronidase (GUS) was placed downstream of the 5' flanking sequences of the par gene, was introduced into tobacco mesophyll protoplasts by electroporation, the chimeric gene elicited auxin-regulated expression of GUS activity. Because deletion of a 111-base-pair (bp) direct repeat in the 5' flanking sequences of the par gene abolished the auxin-induced GUS activity, it is deduced that in the 111-bp direct repeat of the par gene promoter is localized an auxin-responsive region, which regulates auxin-mediated activation of transcription.

Amino Acid Sequence↗
PubMed1999

The sae locus of Staphylococcus aureus encodes a two-component regulatory system.

Sae is a regulatory locus that activates the production of several exoproteins in Staphylococcus aureus. A 3.4-kb fragment of a S. aureus genomic library, screened with a probe adjacent to the transposon insertion of a sae::Tn551 mutant, was cloned into a bifunctional vector. This fragment was shown to carry the sae locus by restoration of exoprotein production in sae mutants. The sae locus was mapped to the SmaI-D fragment of the staphylococcal chromosome by pulse-field electrophoresis. Sequence analysis of the cloned fragment revealed the presence of two genes, designated saeR and saeS, encoding a response regulator and a histidine protein kinase, respectively, with high homology to other bacterial two-component regulatory systems.

Amino Acid Sequence↗
PubMed1998

Cloning of the Lactococcus lactis adhE gene, encoding a multifunctional alcohol dehydrogenase, by complementation of a fermentative mutant of Escherichia coli.

The Lactococcus lactis adhE gene, which encodes a multifunctional alcohol dehydrogenase, has been cloned and characterized. A DNA fragment encoding the putative alcohol dehydrogenase domain of the AdhE protein was cloned by screening an L. lactis genomic library in a fermentative mutant of Escherichia coli and selecting for the ability to grow anaerobically. Further analysis of the clone obtained allowed the cloning of the entire adhE gene sequence. Analysis of adhE expression in L. lactis during anaerobiosis showed induction at the transcriptional level, especially in medium containing glucose. Constructed mutant strains produced reduced amounts of ethanol under anaerobic conditions. With the L. lactis gene as a probe, adhE homologs were found in other industrially relevant lactic acid bacteria.

Alcohol Dehydrogenase↗
PubMed2002

Expression of the rice vdac isoform2: histochemical localization and expression level.

The voltage-dependent anion-selective channel (VDAC) is a mitochondrial outer membrane ion channel. The putative promoter of the rice vdac isoform2 (osvdac2) was isolated by screening a rice genomic library. Computer-based analyses predicted a TATA box, a putative transcription start and several transcription factor-binding sites including pollen specific elements. The promoter region was fused to the gus reporter gene and introduced into rice by Agrobacterium-mediated transformation. Histochemical and cell-type localizations indicated an overall expression of this promoter with a strong expression in actively growing lateral roots and in the pollen grains. Quantitative experiments showed that the osvdac2 promoter has a strong specific activity in both root and shoot. Thus, the osvdac2 promoter could be a good alternative to viral promoters (e.g. CaMV 35S) to overexpress genes in transgenic Poaceae.

Base Sequence↗
PubMed1990

Molecular cloning and characterization of human pyruvate dehydrogenase beta subunit gene.

A genomic clone encompassing the entire gene for the human pyruvate dehydrogenase beta subunit (PDH beta) has been isolated by screening a leukocyte genomic library with a nick-translated human foreskin fibroblast PDH beta cDNA probe. The 18-kilobase clone was characterized by restriction enzyme analysis, extensive DNA sequencing, and primer-extension analysis. The PDH beta structural gene is composed of 10 exons and 9 introns. All intron-exon splice junctions follow the GT/AG rule. The Alu family was found in introns 2 and 8. The 5' flanking region of the PDH beta gene contains a "CAAT" consensus promoter sequence but no "TATA" sequence. Primer-extension analysis indicated that the PDH beta gene transcription start site is an adenine residue located 132 bases upstream from the initiation codon in exon 1.

Amino Acid Sequence↗
PubMed2001

[Cloning and expression of D-arabitol dehydrogenase gene from Acetobacter suboxydans in Escherichia coli].

The partial genomic library of Acetobacter suboxydans was constructed using Yeast-E. coli shuttle plasmid YEp352 as vector. Two positive transformants, designated as DH5 alpha(pAD91) and DH5 alpha(pAD98), were obtained by screening the growth of transformants on the agar plate in which D-arabitol was used as the sole carbon source. The results of Southern blot and restriction endonuclease analysis showed that the two recombinants are identical. The insert is about 2.3 kb. Arabitol dehydrogenase activity assay indicated that the transformants could produce D-xylulose-forming D-arabitol dehydrogenase. Hence, the gene encoding D-arabitol dehydrogenase exists in the cloned DNA fragment.

Acetobacter↗
PubMed1994

Isolation and structure of the Drosophila corazonin gene.

A recombinant DNA clone containing the corazonin gene was isolated from a genomic library of Drosophila melanogaster. Its nucleotide sequence predicts a preprocorazonin consisting of an 19 amino acid putative signal peptide, the 11 amino acid corazonin sequence, a Gly used for amidation, a Lys-Arg proteolytic processing site, and a 39 amino acid corazonin-precursor-related peptide (CPRP). CPRP has an internal Arg-Arg sequence and thus could possibly be further processed into a tripeptide and a 34-mer. Neither CPRP or its possible products are structurally related to any known neuropeptide, and their physiological function is unknown. The structure of the predicted preprocorazonin is remarkably similar to the preprohormone of adipokinetic hormone, which suggests that corazonin and adipokinetic hormone have a common evolutionary origin.

Amino Acid Sequence↗
PubMedDate not supplied

Highly instable (GATA)n-containing sequences of the mouse during the cloning process.

Mouse DNA fingerprints were obtained by HaeIII digestion of genomic DNA and in-gel hybridization with the (GATA)4 oligonucleotide probe. In order to obtain locus-specific probes that hybridize with only one fragment of the (GATA)4 DNA fingerprint, a genomic library of size-selected inserts was constructed using a system of direct subcloning from the phage clones. During the cloning procedure, the phage as well as the plasmid insert DNAs changed primarily within their repetitive DNA but also within adjacent nonrepetitive sequences, as was demonstrated for several clones by in-gel hybridization with the (GATA)4 probe as well as by sequence analysis. Isolated subclones varied within their (GATA)n repeats, resulting in different insert lengths. Several "metastable" as well as stable (GATA)4-positive subclones could be isolated. Also, vector sequences were affected by alterations during the cloning process. These phenomena are discussed within the context of possible mechanisms for cloning artifacts.

Animals↗
PubMedDate not supplied

Cloning and characterization of an ATPase gene from Pneumocystis carinii which closely resembles fungal H+ ATPases.

A gene encoding a P-type cation translocating ATPase was cloned from a genomic library of rat-derived Pneumocystis carinii. The nucleotide sequence of the gene contains a 2781 base-pair open reading frame that is predicted to encode a 101,401 dalton protein composed of 927 amino acids. The P. carinii ATPase protein (pcal) is 69-75% identical when compared with eight proton pumps from six fungal species. The Pneumocystis ATPase is less than 34% identical to ATPase proteins from protozoans, vertebrates or the Ca++ ATPases of yeast. The P. carinii ATPase contains 115 of 121 residues previously identified as characteristic of H+ ATPases. Alignment of the Pneumocystis and fungal proton pumps reveals five homologous domains specific for fungal H+ ATPases.

Adenosine Triphosphatases↗