Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Genomic Library”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 847 records · Page 47Linked to original sources
PubMed1995

Structure of histone H2B and H4 genes of the sea cucumber Holothuria tubulosa.

A genomic library of the sea cucumber Holothuria tubulosa was screened with human and murine histone gene hybridization probes. A recombinant phage carrying an H4 gene was isolated and sequenced. Hybridization analysis of the entire 20 kb phage insert with probes for H1, H2A, H2B and H3 histones was negative except for H2B. This solitary arrangement of the two neighbouring histone H4 and H2B genes is in contrast to the organization of 'cleavage stage' histone genes, which are arranged in tandem quintets of genes encoding the 5 histone classes. Gene organization and sequence features indicate that the two Holothuria genes are the equivalents of a known pair of late H2B and H4 genes which have been described in the genome of sea urchins. This result shows that the simultaneous occurrence of tandem repeats of histone gene quintets and smaller groups of structurally distinct histone gene subtypes is not unique for sea urchins, but also applies to other echinodermata, such as the sea cucumber Holothuria tubulosa.

Amino Acid Sequence↗
PubMed2001

[Identification and cloning of partial mbh2 gene cluster of hyperthermophile Aquifex pyrophilus].

A 0.8 kb fragment of mbhS2 gene of Aquifex pyrophilus was obtained by PCR with designed primers basing mbhS2 gene of A. aeolicus. It showed 85% homology with the corresponding region of A. aeolicus. Using it as probe, a 5.0 kb Nco I fragment was fished out from the partial genomic library of A. pyrophilus. Then this fragment was cloned, subcloned and sequenced. The result revealed that the fragment contains the full length gene for the mbhS2, the gene orf1 and the first 366 bp of orf2. Comparison with mbhS2 and orf963 of A. aeolicus shows 81% and 60% homologies in amino acid sequence, respectively.

Amino Acid Sequence↗
PubMed1992

Isolation, sequencing and expression in E. coli of the urocanase gene from white clover (Trifolium repens).

The urocanase gene was detected in a clone obtained from a genomic library of white clover. The entire gene has been sequenced and expressed in the pT7-7/E. coli BL 21 (DE 3) system. The deduced sequence of the plant urocanase is 72% homologous with that of the well-characterized urocanase from Pseudomonas putida. The purification procedure, as well as kinetic and electrophoretic behaviour, of the new enzyme are described.

Amino Acid Sequence↗
PubMed1994

Sequence of the 5'-flanking region of the gene encoding human muscle glycogen synthase.

The 5'-flanking region of the gene encoding human muscle glycogen synthase was isolated from a human placental genomic library and sequenced. The sequence is TATA-less and G+C-rich, and putative transcription-controlling sequences were identified. Furthermore, a simple (dC-dA)n sequence repeat was identified about 4 kb upstream from the start codon. This sequence was highly polymorphic and five alleles were typed in the Japanese population using the polymerase chain reaction.

Animals↗
PubMed1992

Cloning, sequencing, and expression of the Escherichia coli peptide methionine sulfoxide reductase gene.

The gene encoding peptide methionine sulfoxide reductase was cloned from an Escherichia coli genomic library using an oligonucleotide probe based on the amino-terminal sequence of the protein. The nucleotide sequence revealed that the gene codes for a polypeptide of 212 amino acid residues with a calculated molecular weight of 23,314. The protein has been overexpressed in E. coli and is present as a soluble active species.

Amino Acid Sequence↗
PubMed1998

Construction and extensive characterization of a goat bacterial artificial chromosome library with threefold genome coverage.

A goat Bacterial Artificial Chromosome (BAC) library of 61,440 independent clones was constructed and characterized. The average size of the inserts was estimated at 153 kilobases by analyzing almost 500 clones using Not1 digestion followed by FIGE (Field Inverted Gel Electrophoresis) analysis. The library represents about three genome equivalents, which yields a theoretical probability of 0.95 of isolating a particular DNA sequence. After individual growth, the clones were arrayed in 40 superpools, which were organized in three dimension pools. A rapid technique for pool DNA preparation by microwave treatment was set up. This technique was compatible with PCR analysis. Primer pairs from 166 sequences (microsatellites, coding sequences from goat, and conserved Expressed Sequence Tags (ESTs) from humans) enabled the library to be successfully searched in 165 cases, with an average of 3.52 positive superpools. Only one sequence could not be found. The degree of chimerism was evaluated by FISH analysis with DNA from over 110 clones and was estimated at 4%. This BAC library will constitute an invaluable tool for positional cloning in ruminants, as well as for more general comparative mapping studies in mammals.

Animals↗
PubMed1995

Sequence data and chromosomal localization of human type I and type II hair keratin genes.

A cDNA library constructed with poly(A)+ RNA from human scalp was screened with selected fragments of both murine type I and type II hair keratin cDNAs. Two keratin clones, one type I, phKI-2, and one type II, phKII-1, were isolated and sequenced. In Northern blots, cDNA probes containing the 3'-noncoding sequences of the clones specifically hybridized to scalp mRNA species. Based on sequence homology comparisons with the four known murine type I hair keratins mHa1-4, the phKI-2 encoded keratin could be identified as human hair keratin hHa2. Similarly, sequence comparison with the four type II sheep wool keratins K2.9-12 revealed an orthologous relationship between the largest member of the type II wool keratin subfamily, K2.9 (i.e., sHb1) and the phKII-1 encoded human hair keratin (hHb1). The specific 3'-noncoding sequences of hHa2 and hHb1 were also used to isolate genomic fragments for both keratins from human genomic libraries which were than used for fluorescence in situ hybridization to human metaphase chromosomes. The hHa2 gene could be mapped to the long arm of chromosome 17, whereas the hHb1 gene was found on the long arm of chromosome 12. DAPI banding of the chromosomes allowed sublocalization of the hHa2 gene to 17q12-q21 and the hHb1 gene to 12q13, i.e., gene loci that have also been previously determined for human type I and type II epithelial keratins.

Amino Acid Sequence↗
PubMed1995

Cloning of genes required for hypersensitivity and pathogenicity in Pseudomonas syringae pv. aptata.

A genomic library of Pseudomonas syringae pv. aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv. syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P. syringae pv. aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis of EcoRI-digested genomic DNA of P. syringae pv. aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P. syringae pv. aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11. These results indicate that P. syringae pv. aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P. syringae pv. syringae.

Blotting, Southern↗
PubMed1993

Vault ribonucleoprotein particles from rat and bullfrog contain a related small RNA that is transcribed by RNA polymerase III.

Vaults are large cytoplasmic ribonucleoprotein particles with a sedimentation value of about 150 S. These particles contain a unique small RNA (vault RNA (vRNA)). We have determined the sequence of the RNA associated with vaults purified from both rat and bullfrog. The rat vRNA is 141 bases in length, whereas the bullfrog vRNA is present as two highly related species of 89 and 94 bases. Despite the differences in length the predicted secondary structures of the three vRNAs are clearly related. All of the vRNAs contain sequences related to the internal promoter elements necessary for transcription by RNA polymerase III. The gene for the rat vRNA was isolated and sequenced from a rat genomic library, and its transcription by RNA polymerase III was verified using an in vitro transcription assay. The rat vRNA gene was efficiently transcribed in vitro, producing a single transcript of about 140 bases. Unlike most RNA polymerase III genes, the rat vRNA is present as a single copy gene and has a distinct tissue-specific expression pattern.

Animals↗
PubMed2006

Targeted gene mutation in Phytophthora spp.

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosisinducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

Algal Proteins↗
PubMed2003

Novel chitosanase from Streptomyces griseus HUT 6037 with transglycosylation activity.

Streptomyces griseus HUT 6037 inducibly produced two chitosanases when grown on chitosan. To elucidate the mechanism of degradation of chitinous compound by this strain, chitosanases I and II of S. griseus HUT 6037 were purified and characterized. The purified enzymes had a molecular mass of 34 kDa. Their optimum pH was 5.7, and their optimum temperature was 60 degrees C. They hydrolyzed not only partially deacetylated chitosan, but also carboxymethylcellulose. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanases were obtained for identification of the anomeric form of the reaction products. Both chitosanases produced the beta-form specifically, indicating that they were retaining enzymes. These enzymes catalyzed a glycosyltransfer reaction in the hydrolysis of chitooligosaccharides. The N-terminal and internal amino acid sequences of chitosanase II were identified. A PCR fragment corresponding to these amino acid sequences was used to screen a genomic library for the entire gene encoding chitosanase II. Sequencing of the choII gene showed an open reading frame encoding a protein with 359 amino acid residues. The deduced primary structure was similar to endoglucanase E-5 of Thermomonospora fusca, which enzyme belongs to family 5 of the glycosyl hydrolases. This is the first report of a family 5 chitosanase with transglycosylation activity.

Amino Acid Sequence↗
PubMed1991

Characterization of legiolysin (lly), responsible for haemolytic activity, colour production and fluorescence of Legionella pneumophila.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires' disease in humans was constructed in Escherichia coli K12 and the recombinant clones were tested for haemolysis and other phenotypic properties. Seven clones were identified which were able to confer haemolysis of human, sheep, and canine erythrocytes but which were unable to mediate proteolytic activities or cytotoxic effects on CHO- or Vero cells. Clones that exhibited this haemolytic property were also able to produce a brown colour and a yellow-green fluorescence activity detected on M9 plates containing tyrosine. The genetic determinant encoding these properties, termed legiolysin (lly) was mapped by Tn1000 mutagenesis and by subcloning experiments. Southern hybridization with an lly-specific gene probe showed that this determinant is part of the genome of L. pneumophila but is not identical to a protease gene of L. pneumophila which also mediates haemolysis. Minicell analysis of lly-specific plasmids exhibited a protein of 39 kDa. Polyclonal antibodies generated against a LacZ-Lly hybrid protein also recognized a 39 kDa protein produced either by the recombinant legiolysin-positive E. coli K12 clones or by L. pneumophila wild-type strains.

Animals↗
PubMed2004

A genetic screen for the identification of thiamin metabolic genes.

A genetic screen was developed for the identification of genes related to thiamin biosynthesis and degradation. Genes conferring resistance to bacimethrin or 4-amino-2-trifluoromethyl-5-hydroxymethylpyrimidine were selected from Escherichia coli and Bacillus subtilis genomic libraries. Hits from the selection included the known thiamin biosynthetic genes thiC, thiE, and dxs as well as five genes of previously unknown function (E. coli yjjX, yajO, ymfB, and cof and B. subtilis yveN). The gene products YmfB and Cof catalyze the hydrolysis of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate to 4-amino-2-methyl-5-hydroxymethylpyrimidine phosphate. YmfB also converts thiamin pyrophosphate into thiamin phosphate.

Bacillus subtilis↗
PubMed1998

Cloning and characterization of the rat MAdCAM-1 cDNA and gene.

A cDNA clone for rat MAdCAM-1 homologue was isolated from mesenteric lymph nodes by RT-PCR using primers prepared from the exon sequences of the gene isolated from a genomic library of the WKAH rat, using mouse cDNA as a probe. A 1279 bp cDNA fragment contained an open reading frame (ORF) for a protein of 394 amino acids. Homology of nucleotide sequences between the mouse and rat MAdCAM-1 ORF was 85.1% with an amino acid identity of 80.5%. The rat MAdCAM-1 had two immunoglobulin-like domains, a mucinlike domain and the third immunoglobulin-like domain with a homology to alpha 3 domain of the rat MHC class I molecule. Northern blot analysis demonstrated transcripts in Peyer's patches and mesenteric lymph nodes but not in spleen. Organization of the gene in the rat was similar to that in the mouse, consisting of 5 exons located at about a 4 kbp genomic region.

Amino Acid Sequence↗
PubMedDate not supplied

Human spermidine synthase gene: structure and chromosomal localization.

The human spermidine synthase (EC 2.5.1.16) gene was isolated from a genomic library constructed with DNA obtained from a human immunoglobulin G (IgG) myeloma cell line. Subsequent sequence analyses revealed that the gene comprised of 5,818 nucleotides from the cap site to the last A of the putative polyadenylation signal with 8 exons and 7 intervening sequences. The 5'-flanking region of the gene was extremely GC rich, lacking any TATA box but containing CCAAT consensus sequences. No perfect consensus sequence for the cAMP-responsive element for the AP-1 binding site was found, yet the gene contained seven AP-2 binding site consensus sequences. The putative polyadenylation signal was an unusual AATACA instead of AATAAA. Polymerase chain reaction analysis with DNA obtained from human x hamster somatic cell hybrids indicated that human spermidine synthase genomic sequences segregate with human chromosome 1. Transfection of the genomic clone into Chinese hamster ovary cells displaying a low endogenous spermidine synthase activity revealed that the gene was transiently expressed and hence in all likelihood represents a functional gene.

Animals↗
PubMed1996

Identification and mapping of a putative bombesin receptor gene on human chromosome 17q21.3+.

A mouse bombesin receptor cDNA was used as a probe to screen a human P1 genomic library. Clone HBR1 was isolated and used to localize a putative human bombesin receptor gene (HBRKS) on human chromosome 17q21.3 by fluorescent in situ hybridization (FISH). HBRKS was identified and mapped by polymerase chain reaction (PCR) amplification from a Yeast artificial chromosome (YAC) contig spanning 17q21-q23. In addition, a few candidate genes were found by exon-trapping from HBR1.

Animals↗
PubMed1992

Mouse dopamine beta-hydroxylase: primary structure deduced from the cDNA sequence and exon/intron organization of the gene.

Genomic clones for mouse dopamine beta-hydroxylase (DBH) were isolated from two genomic libraries derived from DBA/2J and 129/SV mouse strains, by plaque hybridization with the human DBH cDNA probe. Subsequently, cDNA encoding mouse DBH was amplified with reverse transcription-polymerase chain reaction (RT-PCR) method using primers corresponding to 5'- and 3'-portions of the mouse DBH mRNA, subcloned into a plasmid vector, and subjected to nucleotide sequence analysis. The clone encoded a protein of 621 amino acids with a calculated molecular mass of 70,186 daltons. The predicted amino acid sequence of mouse DBH showed 87%, 80% and 79% identities with the rat, bovine and human enzymes, respectively. Several potential amino acid sequences that are involved in the posttranslational modification and catalytic function of DBH were identified in mouse DBH protein. Nucleotide sequence analysis of the overlapping genomic clones showed that the mouse DBH gene was composed of 12 exons about 17 kb in length. Typical TATA and CCAAT boxes were observed in the 5'-upstream region of the gene. Northern blot analysis of adrenal gland RNA detected a single size species of the mouse DBH mRNA.

Adrenal Glands↗
PubMed1993

Gene cluster involved in melanin biosynthesis of the filamentous fungus Alternaria alternata.

The filamentous fungus Alternaria alternata produces melanin, a black pigment, from acetate via 1,8-dihydroxynaphthalene. To isolate a fungal gene required for melanin biosynthesis, we transformed an A. alternata Brm1- (light brown) mutant with the DNA of a wild-type strain genomic library constructed by use of a cosmid carrying the hygromycin B phosphotransferase gene. When hygromycin B-resistant transformants were screened for melanin production, 1 of 1,363 transformants appeared to regain melanin production, as judged by black pigmentation of the cultured mycelia. The cosmid, named pMBR1, was recovered by packaging nuclear DNA of the melanin-producing transformant into lambda phage. The gene on pMBR1 that enables the Brm1- mutant to produce melanin was designated BRM1. In addition to the BRM1 gene, pMBR1 was found to carry two more genes involved in melanin biosynthesis. These two genes, designated ALM and BRM2, transformed A. alternata Alm- (albino) and Brm2- (brown) mutants, respectively, to the wild-type phenotype. The three genes are located within a ca. 30-kb genomic region in the order ALM-BRM1-BRM2. Analysis of the gene transcripts indicated approximate sizes of 7.2, 4.0, and 0.9 kb for ALM, BRM1, and BRM2, respectively. The BRM1 and BRM2 transcripts are generated from the same strand, but the ALM transcript is generated from the opposite strand. The three mRNA species accumulate in cultured mycelia of the wild-type strain synchronously with mycelial melanization. The essential roles of the three genes in melanin biosynthesis were confirmed by transformation-mediated gene disruption experiments.

Alternaria↗