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.

SourceResearch connectionFrom yearThrough year

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

At least 451 records · Page 25Linked to original sources

PubMed1987

Genetic organization of the ovine MHC class II region.

To study the class II genes of the major histocompatibility region of the sheep genome, human HLA class II genes corresponding to the known subregions in man (DR, DQ, DP, DO, and DZ) were used for Southern hybridization analysis of sheep DNA and to probe a sheep genomic library. Hybridizing bands were noted for all probes except DP alpha. DQ alpha and beta and DR beta appear to be present as multicopy genes, while DR alpha-, DZ alpha-, and DO beta- like genes appear to be single copy. All bands detected with the DP beta probe were also detectable with other beta chain probes. From eight lambda-bacteriophage clones of a sheep genomic library nine distinct class II genes were identified. These genes were characterized by differential hybridization analysis and restriction mapping. Two genes were DR beta-like, three DQ alpha-like and four DQ beta-like. The extensive cross-hybridization observed with beta chain probes was not seen with alpha chain probes. The results of this study suggest that the major histocompatibility complex class II region of the sheep has a similar genetic organization to that of man, with the provisional exception of the DP subregion.

Animals↗

PubMedDate not supplied

[Construction and use of phasmid vectors of large capacity].

lambda vector phages--pUC19 phasmid hybrids were constructed. The hybrids, phasmids lambda pMYF131 and lambda pSL51, were used as vector molecules for making genomic libraries. Vector phasmids exist as plasmids in vivo. They contain all the genes and specific sites necessary for lytic development, but the DNA molecules are not long enough to be packaged in lambda capsid. Elongation of the molecule due to insertion of a foreign DNA fragment renders phasmid all features of non-defective phage. Output of recombinants is up to 3 X 10(6) per 1 microgram of phasmid vector DNA. The fraction of non-recombinants in libraries is less than 1-0.1%. The capacity of the vectors is 19.6 and 22 kb for lambda pMYE131 and lambda pSL51 accordingly. It is possible to clone DNA fragments with blunt ends and various sticky-end fragments obtained by digestion with 14 prototype restrictases, into nine unique restriction sites of vector lambda pMYF131. The created vectors allowed to construct more than 30 representative genomic libraries of eukaryotic and prokaryotic organisms. 13 individual genes were identified within the libraries.

Bacteriophage lambda↗

PubMed1992

The mouse alpha 1(XII) and human alpha 1(XII)-like collagen genes are localized on mouse chromosome 9 and human chromosome 6.

Type XII collagen is a member of the FACIT (fibril-associated collagens with interrupted triple helices) group of extracellular matrix proteins. Like the other members of this group, collagen types IX and XIV, type XII has alternating triple-helical and non-triple-helical domains. Because of its structure, its association with collagen fibrils, and its distribution in dense connective tissues, type XII is thought possibly to act as a cross-bridge between fibrils and resist shear forces caused by tension. A portion of the ffuse gene was isolated by screening a genomic library with a chicken alpha 1 (XII) cDNA probe, followed by subcloning and sequence analysis. Comparison of exon sequences with the sequence of a mouse cDNA clone allowed the mouse gene to be identified as the alpha 1 (XII) collagen gene. In the mouse, Col12a1 is located on chromosome 9, as determined by linkage analysis using DNA from interspecific backcrosses with Mus spretus. Screening of a human genomic library also allowed the isolation of a human alpha 1(XII)-like gene (CoL12A1). This gene was mapped to chromosome 6 by blot hybridization to DNA from human/hamster hybrid cell lines. This information should prove useful in determining the role of type XII collagen genes as candidate genes in inheritable connective tissue diseases.

Amino Acid Sequence↗

PubMed1992

The human 5-HT2 receptor is encoded by a multiple intron-exon gene.

Serotonin (5-hydroxytryptamine; 5-HT) mediates many central and peripheral nervous system functions by its interaction with specific neuronal receptors. Here we report the genomic structure of the human 5-HT2 receptor. The SacI-EcoRI restriction fragment of rat 5-HT2 receptor cDNA was used as a probe to identify and isolate two positive clones of 8.5 and 7.0 kb from an EcoRI restriction digest of a chromosome 13 specific EcoRI fragment lambda-phage human genomic library. Subcloning and sequencing of these fragments showed the 8.5 kb fragment (designated lambda SE-5) contained the first two exons of the 5-HT2 receptor gene. The 7.0 kb insert (lambda SE-2) contained an incomplete third exon. A HindIII-EcoRI fragment of this insert was used as a probe to isolate a 9.0 kb clone (lambda SH-2), which contained the entire third exon, from a chromosome 13 specific HindIII-fragment lambda-phage human genomic library. The isolation of these three clones (lambda SE-5, lambda SE-2 and lambda SH-2) shows that the human 5-HT2 receptor gene consists of three exons separated by two introns and spans over 20 kb. The deduced amino acid sequence of the human, mouse and rat 5-HT2 receptors are highly conserved and all three share a 90% sequence similarity.

Amino Acid Sequence↗

PubMed1985

Cloning determinants of pathogenesis from Pseudomonas syringae pathovar syringae.

Transposon mutagenesis and a cosmid genomic library of DNA from the bean pathogen Pseudomonas syringae pathovar syringae were used to identify and isolate sequences essential for pathogenesis. Strain PS9021, derived by Tn5 mutagenesis, was determined to be nonpathogenic on Phaseolus vulgaris cultivar Red Mexican and incapable of inducing a hypersensitive response in Nicotiana tabacum. This mutant also produced fluidal rather than firm colonies on selected agar media. A Tn5-containing EcoRI fragment from PS9021 was cloned and used to probe 1500 members of a genomic library constructed with DNA from the pathogenic parent strain and the wide host range cosmid pVK102. One member that hybridized to the probe contained a cosmid with a 30-kilobase-pair insert (pOSU3101) that complemented the mutant phenotypes when mobilized into PS9021. A restriction endonuclease cleavage site map of pOSU3101 was constructed and sequences essential for pathogenesis were determined by subcloning. Approximately 8.5 kilobase pairs of the insert were essential for restoration by complementation of pathogenesis and hypersensitive response and wild-type colony morphology in strain PS9021.

Journal Article↗

PubMed1998

Cloning of the groE operon of the marine bacterium Vibrio harveyi using a lambda vector.

groES and groEL genes encode two co-operating proteins GroES and GroEL, belonging to a class of chaperone proteins highly conserved during evolution. The GroE chaperones are indispensable for the growth of bacteriophage lambda in Escherichia coli cells. In order to clone the groEL and groES genes of the marine bacterium Vibrio harveyi, we constructed the V. harveyi genomic library in the lambdaEMBL1 vector, and selected clones which were able to complement mutations in both groE genes of E. coli for bacteriophage lambda growth. Using Southern hybridization, in one of these clones we identified a DNA fragment homologous to the E. coli groE region. Analysis of the nucleotide sequence of this fragment showed that the cloned region contained a sequence in 71.7% homologous to the 3' end of the groEL gene of E. coli. This confirmed that the lambda clone indeed carries the groE region of V. harveyi. The positive result of our strategy of cloning with the use of the genomic library in lambda vector suggests that the same method might be useful in the isolation of the groE homologues from other bacteria. The V. harveyi cloned groE genes did not suppress thermosensitivity of the E. coli groE mutants.

Bacteriophage lambda↗

PubMed2000

[Molecular cloning and nucleotide sequence of soybean glycinin gene Gy5(A3B4)].

The glycinin gene family encoding the glycinin subunits in soybean plants is composed of at least 5 gene members, i.e.: Gy1-Gy5. A genomic clone containing the Gy5 gene from a genomic library of cv. Williams was isolated by using Gy5 cDNA probe. The complete nucleotide sequence of this gene has been determined. It is 2819 bp long consisting of four exons and three introns. These exons and introns are as follows: exonl(292 bp), intronl(358 bp), exon2(263 bp), intron2(425 bp), exon3(645 bp), intron3(485 bp), exon4(351 bp). The gene encodes 517 amino asids. This is the first time to report the complete Gy5 gene sequence from a genomic library.

Base Sequence↗

PubMed1990

Characterization of the gene for human plasminogen, a key proenzyme in the fibrinolytic system.

The organization and structure of the gene coding for plasminogen has been determined by a combination of in vitro amplification of leukocyte DNA from normal individuals and isolation of unique clones from three different human genomic libraries. These clones were characterized by restriction mapping, Southern blotting, and DNA sequencing. The gene for human plasminogen spanned about 52.5 kilobases of DNA and consisted of 19 exons separated by 18 introns. DNA sequence analysis revealed that the five kringle structures in plasminogen were coded by two exons. The nucleotides in the introns at the intron-exon boundaries were GT-AG analogous to those found in other eukaryotic genes. Three polyadenylation sites for plasminogen mRNA were also identified. When the amino acid sequences deduced from the genomic DNA and cDNAs of plasminogen were compared with that of the plasma protein determined by amino acid sequence analysis, an apparent amino acid polymorphism was observed in several positions of the polypeptide chain. Nucleotide sequence analysis of the amplified genomic DNAs and genomic clones also revealed that the plasminogen gene was very closely related to several other proteins, including apolipoprotein(a). This protein may have evolved via duplication and exon shuffling of the plasminogen gene. The presence of another plasminogen-related gene(s) in the human genomic library was also observed.

Amino Acid Sequence↗

PubMedDate not supplied

Population structure of tropical abalone (Haliotis asinina) in coastal waters of Thailand determined using microsatellite markers.

Three partial genomic libraries were constructed from genomic DNA of the tropical abalone (Haliotis asinina) that was digested with AluI, vortexed/sonicated, and digested with mixed enzyme (AluI, HincII, and RsaI). The libraries yielded 0.02%, 0.42%, and 1.46% positive microsatellite-containing clones, respectively. Eleven clones each of perfect, imperfect, and compound microsatellites were isolated. Ten primer pairs (CUHas1-CUHas10) were analyzed to evaluate their polymorphic level. The numbers of alleles per locus, observed heterozygosity (H0), and expected heterozygosity (He) ranged from 3 to 26 alleles, and varied between 0.27 and 0.85 and between 0.24 and 0.93, respectively. Three microsatellite loci (CUHas2, CUHas3, and CUHas8) were further used for examination of genetic diversity and differentiation of natural H. asinina in coastal waters of Thailand. Genetic variabilities in terms of the effective number of alleles (n(e)), H0, and He were higher in 2 samples from the Gulf of Thailand (n(e)=9.37, 7.66; H0=0.62, 0.78; and He=0.87, 0.86) than those of one sample (n(e)=6.04; H0=0.58; and He=0.62) derived from the Andaman Sea. Assessment of genetic heterogeneity, including allele frequency comparison and pairwise F(ST) analysis, indicated interpopulational differentiation, between natural H. asinina from the Gulf of Thailand and that from the Andaman Sea (P<0.0001).

Animals↗

PubMed1992

Cloning and expression of midecamycin 4"-acylase gene in spiramycin producing strains.

A recombinant plasmid p66B containing the midecamycin 4"-acylase gene was obtained by cloning this gene into plasmid vetor pIJ680 from the primary clone pCN6C5, presumably harboring the midecamycin biosynthetic gene. The expression of the midecamycin 4"-acylase gene (p66B) in spiramycin producing strains resulted mainly in the production of 4"-isovalerylspiramycin. Another positive clone pCN10F5 was discovered from the genomic library of S. mycarofacians 1748 by probing with p66B DNA BamHI-BamHI 2.3kb fragment. A BamHI-BamHI 8.0kb homologous region on pCN10F5 was determined by Southern hybridization and was subcloned into plasmids pWHM3 and pIJ680. Recombinant plasmids pWF5 and p6F5 with molecular size about 15.2kb and 13.3kb, respectively, were obtained. Transformation of spiramycin producing strains with these plasmids resulted in the production of two major components. Based on their physicochemical properties and spectral evidences, component I was identified as 4"-propionylspiramycin III, and component II as 4"-propionylspiramycin II. Southern hybridization confirmed that the BamHI-BamHI 8.0kb fragment was cloned in the spiramycin producing strain. Only pCN10F5 clone was identified from the genomic library of S. mycarofaciens 1748 when the 4"-isovaleryltransferase gene of carbomycin producing strain S. thermotolerans was used as a probe in colony hybridization. It suggests that there is a difference between the 4"-acyltransferase genes in the pCN6C5 and pCN10F5 clones.

Acyltransferases↗

PubMed1995

A mutant approach and molecular strategy to study fungal cell walls.

The current study describes recombinant plasmids which complement the hypersusceptibility to killing bleomycin of blm1-1 mutant cells of Saccharomyces cerevisiae, and a strategy developed and used to recover active clones from a stable yeast genomic library. The resistance of a spontaneous revertant isolated from the original blm1-1 mutant strain and of mutant cells transformed with each of several recombinant plasmids which complemented the recessive blm1-1 mutation was comparable to the resistance of the parental (non-mutant) strain from which the original blm1-1 mutant was derived. The strategy for cloning S. cerevisiae DNA was based on complementation and in situ hybridization. This strategy employed 32P-labelled 6.6-kb BamHI and 3.8-kb BamHI-ClaI probes from a cloned DNA fragment to recover clones which either fully or partially complemented the hypersensitivity of mutant cells to killing by bleomycin. This method considerably reduced the time and effort required to recover biologically active clones from a genomic library.

Bleomycin↗

PubMed1999

Isolation and mapping of rFUS6, a rice orthologue of Arabidopsis thaliana FUS6.

COP9 complex is one of the most important components that act in repressing photomorphogenesis in Arabidopsis thaliana. FUS6 has been identified as one of eight subunits of the COP9 complex in Arabidopsis. Using Arabidopsis Fus6 cDNA as a probe, we screened a rice root cDNA library and a rice genomic library. A 1730-bp cDNA was obtained, which has an open reading frame corresponding to 441-amino-acid. This 441 amino acids putative protein has 67% identity with Arabidopsis COP11/FUS6 (AtFUS6) and 40% identity with human GPS1, an AtFUS6 orthologue. So we designated this novel gene as rFUS6. The 6.2-kb genomic sequence of rFUS6 was also obtained. Sequence comparison showed that the rFUS6 gene had six exons and five introns. Sequence inspection of the 5'-flanking region revealed the presence of some potential light-regulated cis-elements such as a G-box, GT-1 binding sites, and a TGACG motif. Southern hybridization with rice total DNA showed that rFUS6 was perhaps a single copy gene. The rFUS6 locus was mapped by hybridization with a rice BAC library membrane and the results showed that rFUS6 had a locus at 16.3 cM of chromosome 1.

Amino Acid Sequence↗

PubMed2000

Toward structural and functional genomics of Agrobacterium tumefaciens: linkage map of the left region of linear chromosome.

Genome of A. tumefaciens contains a linear and a circular chromosome. As an initial step of elucidating the structural and functional genomics of this bacterium, linkage map of the left region of its linear chromosome was constructed. Total genomic libraries of A. tumefaciens MAFF301001 were constructed in BAC vectors namely, pFOS1 and pBeloBAC11. Upon construction of sub-libraries, minimum overlapping clones needed to cover the left region was determined. So far, four contigs have been assembled with a total of 19 overlapping clones. Detailed EcoRI physical map of contig III was constructed and it covers a 110 kb region of the Pme5 fragment of the linear chromosome. Seven end regions of the linking clones were partially sequenced but no gene existence was determined due to low homology.

Agrobacterium tumefaciens↗

PubMed2005

A microsatellite-based, gene-rich linkage map for the AA genome of Arachis (Fabaceae).

Cultivated peanut (Arachis hypogaea) is an important crop, widely grown in tropical and subtropical regions of the world. It is highly susceptible to several biotic and abiotic stresses to which wild species are resistant. As a first step towards the introgression of these resistance genes into cultivated peanut, a linkage map based on microsatellite markers was constructed, using an F(2) population obtained from a cross between two diploid wild species with AA genome (A. duranensis and A. stenosperma). A total of 271 new microsatellite markers were developed in the present study from SSR-enriched genomic libraries, expressed sequence tags (ESTs), and by "data-mining" sequences available in GenBank. Of these, 66 were polymorphic for cultivated peanut. The 271 new markers plus another 162 published for peanut were screened against both progenitors and 204 of these (47.1%) were polymorphic, with 170 codominant and 34 dominant markers. The 80 codominant markers segregating 1:2:1 (P<0.05) were initially used to establish the linkage groups. Distorted and dominant markers were subsequently included in the map. The resulting linkage map consists of 11 linkage groups covering 1,230.89 cM of total map distance, with an average distance of 7.24 cM between markers. This is the first microsatellite-based map published for Arachis, and the first map based on sequences that are all currently publicly available. Because most markers used were derived from ESTs and genomic libraries made using methylation-sensitive restriction enzymes, about one-third of the mapped markers are genic. Linkage group ordering is being validated in other mapping populations, with the aim of constructing a transferable reference map for Arachis.

Arachis↗

PubMed1992

Primary sequences of two P-glycoprotein genes of Entamoeba histolytica.

Two P-glycoprotein genes (EhPgp1 and EhPgp2) from the protozoan parasite Entamoeba histolytica were sequenced from a genomic library made with the DNA of an emetine-resistant ameba mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene. The open reading frames for EhPgp1 and EhPgp2 were 1302 and 1310 amino acids long, respectively, and showed a 67% positional identity with each other and 41% and 40% positional identities, respectively, with human mdr1 gene. Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins. Hydropathy plots of the ameba P-glycoproteins were nearly superimposable on that of the human mdr 1, showing 2 homologous halves, each containing an ATP-binding site and 6 hydrophobic transmembrane domains that form the putative channel. A phylogenetic tree showed that the Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins. Also identified in the E. histolytica genomic library were 2 P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site. In conclusion, the 2 E. histolytica P-glycoproteins encoded by the EhPgp1 and EhPgp2 genes are similar in structure to the mammalian P-glycoproteins and so may be involved in energy-dependent drug efflux by this human parasite.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2003

Characterization of the 5'-flanking region of the rat AJ18 gene.

Krüppel-associated box (KRAB) domains are present in one-third of all C(2)H(2) zinc finger containing proteins, making the KRAB/C(2)H(2) proteins one of the largest known families of putative transcription repressors. AJ18 has been identified as a novel KRAB/C(2)H(2) gene that is involved in the differentiation of osteogenic cells. To study the regulation of expression of the AJ18 gene, the 5'-flanking region of the AJ18 gene was obtained by screening a rat genomic library. This region was sequenced, and the transcription start site mapped by primer extension. The AJ18 gene consists of at least four exons, the first exon coding for an unusually long 2.3 kb 5'-UTR region. A putative internal ribosome entry site, immediately upstream of the translation initiation site, is indicated from the complementarity of a 12 nucleotide sequence with a region in the rat 18S rRNA. Chimeric constructs encompassing the region surrounding the transcription start site (-77-+171), as well as constructs with additional 1.9 kb upstream from this region revealed strong transcriptional activity when ligated to a luciferase reporter gene and tested in transient transfection assays. This activity was lost on deletion of the 5'-flanking region to -77. In addition, transcriptional activity was progressively lost with the inclusion of downstream sequences extending into the 5'-UTR. Several known response elements for proteins such as Runx2, NFkappaB, Smads, Sp1, and Ets1 are retained within the conserved sequences of rat and mouse AJ18, which was retrieved from mouse genomic libraries. Interestingly, the transcriptional activity was approximately 100-fold higher in the osteocarcinoma cell line ROS 2.8/17 compared to the fibroblast-like C3H10T1/2. Notably, this is the first gene promoter from the large KRAB/C(2)H(2) zinc finger family of proteins to be identified and characterized.

5' Flanking Region↗

PubMed1996

Mechanism of an evolutionary change in muscle cell differentiation in ascidians with different modes of development.

We have investigated the mechanism of an evolutionary change in ascidian muscle cell differentiation. The ascidians Molgula oculata and Molgula occulta are closely related species with different modes of development. M. oculata embryos develop into conventional tadpole larvae with a tail containing striated muscle cells, whereas M. occulta embryos develop into tailless larvae with undifferentiated vestigial muscle cells. The muscle actin gene MocuMA1 was isolated from an M. oculata genomic library. MocuMA1 is a single-copy, larval-type muscle actin gene which appears to lack introns. However, the 5' upstream region of MocuMA1 is sufficient to drive expression of a lacZ fusion construct in the larval muscle cells, implying that it is a functional gene. MocuMA1 mRNA first appears in the prospective muscle cells of M. oculata embryos during gastrulation, and transcripts continue to be present throughout embryogenesis. Muscle actin mRNA was not detected during M. occulta embryogenesis, although the same probe was capable of detecting muscle actin mRNA in more distantly related ascidian species with tail muscle cells. Interspecific hybrids produced by fertilizing M. occulta eggs with M. oculata sperm recover the ability to express muscle actin mRNA in the vestigial muscle cells, suggesting that trans-acting factors responsible for muscle actin gene expression are conserved in M. occulta. The presence of these trans-acting factors was confirmed by showing that the MocuMA1/lacZ fusion construct is expressed in the vestigial muscle cells of M. occulta larvae. The orthologous larval muscle actin genes MoccMA1a and MoccMA1b were isolated from a M. occulta genomic library. The coding regions of these genes contain deletions, insertions, and codon substitutions that would make their products nonfunctional. Although the 5' upstream regions of the M. occulta muscle actin genes also contain numerous changes, expression of MoccMA1a/lacZ and MoccMA1b/lacZ fusion constructs showed that they both retain specific promoter activity, although it is reduced in MoccMAlb. The results suggest that the regression of muscle cell differentiation is mediated by changes in the structure of muscle actin genes rather than in the trans-acting regulatory factors required for their expression.

Actins↗

PubMed1987

Structure and organization of the microsomal xenobiotic epoxide hydrolase gene.

The gene for the microsomal xenobiotic rat liver epoxide hydrolase has been isolated and characterized. Clones were obtained from a Wistar Furth Charon 35 genomic library by hybridization with a full-length epoxide hydrolase cDNA. The gene for the xenobiotic epoxide hydrolase is approximately 16 kilobases in length and consists of 9 exons ranging in size from 109 to 420 base pairs and 8 intervening sequences, the largest of which is 3.2 kilobases. S1-nuclease mapping, primer extension studies, and sequence analysis were used to determine the 5' cap site and the size of the first exon (170 base pairs). Regulatory sequences analogous to TATA, CCAAT, and core enhancer sequences were noted in the 5'-flanking region of the gene. The cDNA and gene for epoxide hydrolase displayed nucleotide sequence identity although they were isolated from different rat strains. Also, Southern blot analysis of restricted liver DNA from inbred Fischer 344 and Wistar Furth rat strains, and outbred Sprague-Dawley rats indicated a high degree of structural similarity for the epoxide hydrolase gene within these three strains. Only a single functional epoxide hydrolase gene was identified and no evidence of hybridization to the genes for the microsomal cholesterol epoxide hydrolase or the cytosolic epoxide hydrolase was observed. However, a pseudogene for the microsomal xenobiotic epoxide hydrolase was isolated and characterized from the genomic library.

Amino Acid Sequence↗