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At least 919 records · Page 51Linked to original sources
PubMed2003

Efficient large-scale development of microsatellites for marker and mapping applications in Brassica crop species.

A set of 398 simple sequence repeat markers (SSRs) have been developed and characterised for use with genetic studies of Brassica species. Small-insert (250-900 bp) genomic libraries from Brassica rapa, B. nigra, B. oleracea and B. napus, highly enriched for dinucleotide and trinucleotide SSR motifs, were constructed. Screening the clones with a mixture of oligonucleotide repeat probes revealed positive hybridisation to between 75% and 90% of the clones. Of these, 1230 were sequenced. Primer pairs were designed for 398 SSR clones, and of these, 270 (67.8%) amplified a PCR product of the expected size in their focal and/or closely related species. A further screen of 138 primers pairs that produced a PCR product in B. napus germplasm found that 86 (62.3%) revealed length polymorphisms within at least one line of a test array representing the four Brassica species. The results of this screen were used to identify 56 SSRs and were combined with 41 SSRs that had previously shown polymorphism between the parents of a B. napus mapping population. These 97 SSR markers were mapped relative to a framework of RFLP markers and detected 136 loci over all 19 linkage groups of the oilseed rape genome.

Brassica↗
PubMed1994

Use of non-radioactively labeled DNA probes for the detection of a baculovirus from Penaeus monodon by in situ hybridization on fixed tissue.

Clones isolated from a genomic library of the baculovirus PmSNPV (= MBV) were used to prepare DNA probes for detection of PmSNPV in shrimp tissue. Davidson's AFA fixed shrimp tissues were probed using DNA that had been labeled with the hapten, digoxigenin, by an in situ hybridization assay. The DNA probes readily distinguished PmSNPV-infected from uninfected shrimp tissue. The reactions were localized to the hepatopancreas and midgut, the sites where this virus is detected by standard histological methods. The probes did not react to shrimp infected with Baculovirus penaei (BP = PvSNPV).

Animals↗
PubMed1996

Cloning and heterologous expression of the entire set of structural genes for nikkomycin synthesis from Streptomyces tendae Tü901 in Streptomyces lividans.

A genomic library from Streptomyces tendae raised in shuttle cosmid vector pKC505 was screened with a previously isolated 8-kb DNA fragment containing the orfP1 gene, which is involved in nikkomycin biosynthesis. The entire set of structural genes for nikkomycin synthesis was heterologously expressed in S. lividans TK23 by introducing recombinant cosmids p24/32 and p9/43-2, carrying inserts of about 31 and 27 kb, respectively, overlapping by 15 kb. S. lividans transformants synthesized nikkomycins X, Z, I, and J, which were identified by high-pressure liquid chromatography analyses of culture filtrates.

Aminoglycosides↗
PubMed1996

Isolation and characterization of an auxin-inducible glutathione S-transferase gene of Arabidopsis thaliana.

Genes homologous to the auxin-inducible Nt103 glutathione S-transferase (GST) gene of tobacco, were isolated from a genomic library of Arabidopsis thaliana. We isolated a lambda clone containing an auxin-inducible gene, At103-1a, and part of a constitutively expressed gene, At103-1b. The coding regions of the Arabidopsis genes were highly homologous to each other and to the coding region of the tobacco gene but distinct from the GST genes that have been isolated from arabidopsis thusfar. Overexpression of a cDNA clone in Escherichia coli revealed that the AT103-1A protein had GST activity.

Amino Acid Sequence↗
PubMed1992

Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.

Allosteric Regulation↗
PubMed1995

Molecular cloning and structural characterization of the human histidase gene (HAL).

Histidase (EC 4.3.1.3) is a cytosolic enzyme that catalyzes the nonoxidative deamination of histidine to urocanic acid. Histidinemia, resulting from reduced histidase activity as reported in Cambridge stock his/his mice and in humans, is the most frequent inborn metabolic error in Japan. The histidase chromosomal gene (HAL) was isolated from a lambda EMBL-3 human genomic library using the human histidase cDNA as a probe. Restriction mapping and Southern blot analysis of the isolated clones reveal a single-copy gene spanning approximately 25 kb and consisting of 21 exons. Exon 1 encodes only 5' untranslated sequence of liver histidase mRNA, with protein coding beginning in exon 2. A rarely observed 5' GC, similar to that reported in the human P-450 (SCC) gene, is present in intron 20. All other splicing junctions adhere to the canonical GT/AG rule. A TATA box sequence is located 25 bp upstream of the liver histidase transcription initiation site determined by S1 nuclease protection analysis. Several liver- and epidermis-specific transcription factor binding sites, including C/EBP, NFIL6, HNF5, AP2/KER1, MNF, and others, are also identified in the 5' flanking region. Consistent with the hepatic and epidermal expression of histidase, this finding suggests that histidase transcription may be regulated by these factors. We further identify a polymorphism (A to G transition) in the histidase coding region of exon 16. The human histidase genomic structure presented here should facilitate the molecular investigation of symptomatic and asymptomatic forms of histidinemia.

Alternative Splicing↗
PubMed2004

A new member of the bacterial ribonuclease inhibitor family from Saccharopolyspora erythraea.

We have identified Sti, the gene of a ribonuclease inhibitor from Saccharopolyspora erythraea, by using a T7 phage display system. A specific phage has been isolated from a genome library by a biopanning procedure, using RNase Sa3, a ribonuclease from Streptomyces aureofaciens, as bait. Sti, a protein of 121 amino acid residues, with molecular mass 13059 Da, is a homolog of barstar and other microbial ribonuclease inhibitors. To overexpress its gene in Escherichia coli, we optimized the secondary structure of its mRNA by introducing a series of silent mutations. Soluble protein was isolated and purified to homogeneity. Inhibition constants of complex of Sti and RNase Sa3 or barnase were determined at pH 7 as 5 x 10(-12) or 7 x 10(-7), respectively.

Amino Acid Sequence↗
PubMed1999

Purification, characterization, and gene analysis of a chitosanase (ChoA) from Matsuebacter chitosanotabidus 3001.

The extracellular chitosanase (34,000 M(r)) produced by a novel gram-negative bacterium Matsuebacter chitosanotabidus 3001 was purified. The optimal pH of this chitosanase was 4.0, and the optimal temperature was between 30 and 40 degrees C. The purified chitosanase was most active on 90% deacetylated colloidal chitosan and glycol chitosan, both of which were hydrolyzed in an endosplitting manner, but this did not hydrolyze chitin, cellulose, or their derivatives. Among potential inhibitors, the purified chitosanase was only inhibited by Ag(+). Internal amino acid sequences of the purified chitosanase were obtained. A PCR fragment corresponding to one of these amino acid sequences was then used to screen a genomic library for the entire choA gene encoding chitosanase. Sequencing of the choA gene revealed an open reading frame encoding a 391-amino-acid protein. The N-terminal amino acid sequence had an excretion signal, but the sequence did not show any significant homology to other proteins, including known chitosanases. The 80-amino-acid excretion signal of ChoA fused to green fluorescent protein was functional in Escherichia coli. Taken together, these results suggest that we have identified a novel, previously unreported chitosanase.

Amino Acid Sequence↗
PubMed1991

A family of repeated DNA sequences in Toxoplasma gondii: cloning, sequence analysis, and use in strain characterization.

A Toxoplasma gondii genomic library was constructed in lambda EMBL3. Repeated fragments were detected by hybridization with radiolabeled total DNA from the parasite and one recombinant was chosen due to its strong hybridization signal. By using electrophoretic and hybridization analysis, four cross-hybridizating restriction fragments were selected and sequenced. The determined nucleotide sequence of these fragments (TGR1A, TGR1E, TGR2, and TGR4) has shown a complex system of conserved and degenerated repeats in which TGR1E corresponds to the most conserved element. This last sequence was used to investigate restriction fragment length polymorphisms among several T. gondii strains by Southern blotting.

Animals↗
PubMed1992

Isolation and characterization of the Schizosaccharomyces pombe rad3 gene, involved in the DNA damage and DNA synthesis checkpoints.

We have cloned the Schizosaccharomyces pombe rad3 gene which is involved in G2 arrest following DNA damage, and in the dependence of mitosis on the completion of DNA replication. The gene was cloned by complementation of the sensitivity to UV light and gamma rays of the rad3-136 mutant with an Sz. pombe genomic library. Sublocalization of the complementing activity and sequencing of the clone identified an intronless 3210-bp open reading frame capable of encoding a 1070-amino acid protein with an M(r) of 121974. The rad3 gene is a new gene with no homologs in existing sequence databases. The gene is poorly expressed, with a codon bias index of -0.01. A disruption mutant affecting the coding region was only slightly more sensitive to UV light than the original rad3-136 mutant. The rad3 gene was mapped to NotI fragment C on chromosome II.

Base Sequence↗
PubMed1999

Molecular cloning, sequence analysis, and characterization of a penicillin-resistant DD-carboxypeptidase of Myxococcus xanthus.

We have cloned a gene, pdcA, from the genomic library of Myxococcus xanthus with an oligonucleotide probe representing conserved regions of penicillin-resistant DD-carboxypeptidases. The amino- and carboxy-terminal halves of the predicted pdcA gene product showed significant sequence similarity to N-acetylmuramoyl-L-alanine amidase and penicillin-resistant DD-carboxypeptidase, respectively. The pdcA gene was expressed in Escherichia coli, and the characteristics of the gene product were similar to those of DD-carboxypeptidase (VanY) of vancomycin-resistant enterococci. No apparent changes in cell growth, sporulation, or germination were observed in pdcA deletion mutants.

Amino Acid Sequence↗
PubMed1998

Molecular cloning of bovine thyrotropin-releasing hormone receptor gene.

The genomic DNA encoding bovine Thyrotropin-Releasing Hormone Receptor (TRHR) was isolated from a bovine (Holstein) genomic library. Using PCR fragments of bovine candidate TRHR Transmembrane domain (TMD-III-IV) and C-terminus domain of mouse TRHR cDNA as probes, 9 x 10(5) plaques were screened to obtain several clones each containing the N-terminus or C-terminus domain. The bovine TRHR gene encoded 398 amino acids and has a long intron. The identity of the deduced amino acid sequence of bovine TRHR exceeded 88% that of mouse, rat or human. RT-PCR analysis indicated TRHR mRNA to be expressed in the pituitary and brain.

Amino Acid Sequence↗
PubMed1991

Intracellular survival and expression of virulence determinants of Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease is able to live and multiply within macrophages as well as within protozoan organisms. Legionella strains inhibit phagosome-lysosome fusion and phagosome acidification. By using two different cell culture systems, one derived from human macrophages and the other from human embryo lung fibroblastic cells, it is demonstrated that Legionella strains lose their virulence following cultivation in the laboratory. In order to study the mechanisms involved in intracellular survival of Legionella a genomic library of strain Legionella pneumophila Philadelphia I was established in Escherichia coli K-12. By cosmid cloning technique we were able to clone five putative virulence factors, two of which exhibit hemolytic activities and three of which represent membrane-associated proteins of 19, 26 and 60 kilodalton. One of the hemolytic proteins, termed legiolysin, represents a new toxin which specifically lyses human erythrocytes. The other hemolysin exhibits proteolytic properties in addition and is cytolytic for Vero and CHO cells. Further studies will be necessary to determine the exact role of the cloned proteins in the pathogenesis of Legionella.

Bacterial Toxins↗
PubMed1990

Molecular cloning and characterization of rat liver catechol-O-methyltransferase.

The coding sequence of rat liver catechol-O-methyl-transferase (COMT; EC 2.1.1.6) was determined from rat cDNA and genomic libraries were screened with DNA probes and specific antiserum. The open reading frame consisted of 663 nucleotides coding for a 221-amino acid (aa) polypeptide with a deduced Mr of 24,747. No obvious hydrophobic signal sequence, membrane-spanning domains, or potential N-glycosylation sites were found in this sequence. The identity of the clone and the accuracy of the sequence was verified by direct aa sequencing of the tryptic peptides derived from the purified rat liver enzyme. Primer extension analysis showed that the transcription start point of the rat liver COMT mRNA was 450 bp upstream from the translation start codon. A putative polyadenylation signal (ATTAAA) was found in the 3'-noncoding region. The predicted size of the COMT transcript was 1.8-2.0 kb, which could be confirmed from Northern hybridization analyses of the isolated rat liver mRNA. One polypeptide of 25 kDa, could be immunoprecipitated with anti-COMT antibody from in vitro translation of rat liver mRNA. Employing the DNA blot analysis only one COMT-encoding gene was found in the rat genome.

Amino Acid Sequence↗
PubMed1994

Novel exon 1 of the aromatase gene specific for aromatase transcripts in human brain.

Aromatase in the brain is supposed to participate in sexual differentiation of the brain. The presence of a brain-specific exon 1 and promoter in the human aromatase gene was examined in the 5' regions of aromatase cDNAs isolated from the amygdala and hypothalamus areas. The DNA sequences of all the isolated clones were different in the 5' regions encoded by exon 1 from those encoded by any alternative exons 1 of human aromatase gene. This unique sequence was also found in the clones isolated from a human genomic library and indicated to be that of a new exon 1. It involved putative TATA and CAAT boxes, Ad4 sequence, and the androgen responsive element in the promoter region. Reverse transcriptase-polymerase chain reaction analysis of mRNAs in various tissues revealed that expression of this unique species of aromatase mRNA is specific for the brain, in particular in the fetal brain. These show that the aromatase gene in the human brain is expressed by means of the brain-specific exon 1 and promoter, and may be responsible for sexual differentiation of the brain in the fetal period.

Amygdala↗
PubMed1996

Nucleotide sequence of 5'-upstream region and expression of a silkworm gene encoding a new member of the attacin family.

A genomic clone encoding a new member of attacin, an insect antibacterial protein, was isolated from a genomic library of the silkworm, Bombyx mori, and the nucleotide sequence of the 5'-upstream region was determined. The region contained Bm 1, a highly repetitive element of B. mori and a lipopolysaccharide (LPS) response element (RE)(NF-kappaB binding site), CAAT box and TATA box. Northern blot analysis showed that the attacin gene expression was rapidly induced by bacterial cell wall components such as LPS from Escherichia coli and peptidoglycan (PG) from Micrococcus luteus, suggesting that attacin plays an important role in an early phase of the self-defense system upon bacterial infection.

Animals↗
PubMed1997

Genomic structure and chromosomal localization of human uroguanylin.

Uroguanylin, a member of the guanylin peptide family, is a novel peptide regulator in intestinal salt and water transport. We isolated the gene for uroguanylin from a human genomic library and determined its structure. This gene consists of three exons and two introns within an overall length of 2.5 kb. The 5' flanking region has TATA and CAAT boxes. The gene also has multiple binding sites for promoter-specific transcription factor, activator protein-1, and activator protein-2, and a cAMP-regulated enhancer element. Fluorescence in situ hybridization showed the uroguanylin gene at human chromosome 1p33-p34. RNA blot analysis showed that human uroguanylin mRNA is expressed in the gastric fundus and pylorus as well as in the intestine.

Amino Acid Sequence↗
PubMed2006

Involvement of LuxR, a quorum sensing regulator in Vibrio harveyi, in the promotion of metabolic genes: argA, purM, lysE and rluA.

Quorum sensing, involving signal transduction via the two-component response regulator LuxO to its downstream target LuxR, controls luminescence in the marine bacterium Vibrio harveyi. LuxR is a DNA binding protein that acts as both activator of the lux operon and repressor of its own gene. In order to determine if any other genes are affected by quorum sensing in V. harveyi, an assay for luxR-dependent promotion was devised using a genomic library maintained in a novel luxAB (luciferase) reporter. Screening in Escherichia coli DH-21 (lacI(sq)) entailed the addition of a second plasmid containing luxR under plac control. Four out of 5000 colonies showed luminescence stimulation upon IPTG induction of luxR. The four luxR-dependent promoters were upstream of argA, purM, lysE, and rluA, genes involved in arginine and purine biosyntheses, amino acid efflux, and pseudouridine synthesis, respectively. Based on analysis of luxR-dependent promoters, particularly that of argA, we describe a LuxR binding site, and implicate the coordination of LuxR with ArgR.

Amino-Acid N-Acetyltransferase↗