SEARCH · PubMed Health

Results for “Genomic Library”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 55 records · Page 3Linked to original sources

PubMed1986

Rapid isolation and characterization of hybridization selected recombinants from lambda genomic libraries.

This paper describes an efficient protocol for the screening of lambda genomic libraries, plaque and DNA purification, and probe characterization by a combination of new and recently described techniques. The protocol has allowed large numbers of human subchromosome-specific probes to be rapidly generated from an EMBL3 library of human-mouse somatic cell hybrid DNA. The protocol affords considerable savings in time and effort over previous procedures.

Animals↗

PubMed1982

Characterization of histone genes isolated from Xenopus laevis and Xenopus tropicalis genomic libraries.

Using a cDNA clone for the histone H3 we have isolated, from two genomic libraries of Xenopus laevis and Xenopus tropicalis, clones containing four different histone gene clusters. The structural organization of X. laevis histone genes has been determined by restriction mapping, Southern blot hybridization and translation of the mRNAs which hybridize to the various restriction fragments. The arrangement of the histone genes in X. tropicalis has been determined by Southern analysis using X. laevis genomic fragments, containing individual genes, as probes. Histone genes are clustered in the genome of X. laevis and X. tropicalis and, compared to invertebrates, show a higher organization heterogeneity as demonstrated by structural analysis of the four genomic clones. In fact, the order of the genes within individual clusters is not conserved.

Amino Acid Sequence↗

PubMed1992

A mouse genomic library in the bacteriophage P1 cloning system: organization and characterization.

Using the bacteriophage P1 cloning system, we have constructed a two to three times coverage, high-molecular-weight (HMW) genomic library from mouse C127 fibroblast cells. The library consists of about 127,500 clones with an average insert size of about 70 kb that are organized into 300 primary pools containing approximately 425 clones per pool. For screening purposes the primary pools are combined into secondary pools (approximately 4250 clones each) and tertiary pools (approximately 21,250 clones each). Screening is performed by the polymerase chain reaction (PCR) with DNA isolated from the secondary and tertiary pools. We have screened the library for 13 different mouse sequences and have detected 11. Clones generated from two of the eleven positive screens were isolated from the library (those containing the c-fos and G alpha i2 genes) and were further characterized. Direct double-stranded sequencing of DNA from P1 clones with primers bordering the insert provided sequence information from each end of the cloned DNA.

Animals↗

PubMed1994

Construction of an ordered genomic library of Mycoplasma genitalium.

As a first step towards sequencing the chromosome of the suspected human pathogen Mycoplasma genitalium, we attempted to clone its entire genome in a set of ordered cosmids. Cosmid libraries were established by partial digestion of M. genitalium genomic DNA with Sau3AI or EcoRI. A chromosome-walking strategy was used to identify 20 overlapping cosmid clones which contained over 99% of the genome. The final 5.1 kb could not be cloned in cosmids, and was eventually obtained from a genomic library established in a lambda vector. Correspondence of cloned and genomic EcoRI fragments indicated no detectable major deletions or rearrangements in the library. The library was oriented on established XhoI and SmaI physical maps of the chromosome with restriction sites present at the expected locations in the library. The genome contained 74 EcoRI fragments which added up to a total genome size of 578 kb. These were arranged in a partial EcoRI physical map, and those containing the MgPa major attachment protein-encoding operon and its repeat sequences were identified. The existence of this ordered genomic library, which accurately and completely encompasses the entire M. genitalium genome, should serve as a valuable tool for many future studies of this organism and facilitate our long-term goal of sequencing its genome.

Bacterial Proteins↗

PubMed1992

Versatile shuttle vectors and genomic libraries for use with Schizosaccharomyces pombe.

We have constructed a variety of pUC-based vectors designed for maintenance in Schizosaccharomyces pombe. These can be used for both gene bank construction and subcloning. Plasmids pUR18 and pUR19 are modifications of pUC vectors containing the Sc. pombe ars1 and ura4 sequences and retaining the lacZ XGal blue-white selection system for screening for DNA inserts. These vectors have been used to construct representative Sc. pombe and Saccharomyces cerevisiae genomic libraries. To assist in the creation of gene deletions, we have constructed another two plasmids. Combined with the technique of partially filling-in 5' overhangs created with restriction enzymes, these plasmids simplify the replacement of all or part of an open reading frame by a functional ura4 gene. Furthermore, such constructs can be excised with SfiI as a linear fragment for use in Sc. pombe transformations. When integrated into the Sc. pombe genome, the site of integration can be easily mapped by pulsed-field gel electrophoresis using the presence of a novel NotI site.

Base Sequence↗

PubMed1994

Coleopterancidal delta-endotoxin and constructing its genomic library.

The composition of crystal proteins and plasmid patterns in five new coleopterancidal strains of Bacillus thuringiensis were investigated. Larvae of Plagiodera versicolora were chosen as a model insect in bioassay. Strain YM-03 had the highest toxicity. Plasmid patterns were detected by rapid agarose gel electrophoresis. It was demonstrated that plasmid patterns of five Bt strains were quite different. The partial amino acid sequences of the N-terminal of crystal protein from YM-03 were analyzed by amino acid analyzer. A genomic library of delta-endotoxin of YM-03 was constructed. The EcoRI fragments (20-30 kb) from the total DNA were ligated with the vector DNA of pLAFRI. Thirteen clones which contained both of pLAFRI and foreign DNA fragments were obtained from 17 recombined plasmids. The frequency of recombinant clones was 76%. Three positive clones, LE392 (pBYM2), LE392 (pBYM3) and LE392 (pBYM4) were detected from 1200 resistant clones by using a synthesized 18bp probe from a coleopterancidal delta-endotoxin gene. It was shown that LE392 (pBYM3) and LE392 (pBYM4) had the same EcoRI digestion patterns and that they all contained coleoptera-specific delta-endotoxin gene but showed a different level of expression.

Amino Acid Sequence↗

PubMed1986

Cloning of open reading frames and promoters from the Saccharomyces cerevisiae genome: construction of genomic libraries of random small fragments.

We have developed a novel efficient method, carrier-facilitated insertion, to insert small (150-600 bp) DNA fragments into plasmid vectors. This method employs a carrier segment of vector DNA to circumvent the difficulties in ligating two fragments together to generate a recombinant circle efficiently. We have used carrier-facilitated insertion to construct three genomic libraries of random (DNase I-generated) fragments from the Saccharomyces cerevisiae genome. One of these was an expression library, and the other two were promoter-cloning libraries. 87-90% of the Escherichia coli colonies in each library contained recombinant plasmids, and less than 3% of the recombinants contained more than one insert. Detection of open reading frames among the inserts in the expression library was accomplished by testing for beta-galactosidase activity. This methodology, unencumbered by the intrinsic disproportionality of cDNA libraries, can be used to identify and clone DNA that codes for a specific antigenic determinant. When used in combination with a method to detect and isolate random constitutive, repressible and inducible yeast promoters, these libraries should permit a comprehensive analysis of the yeast genome and its expression.

Cloning, Molecular↗

PubMed1987

Filter transfer of genomic libraries in a state accessible to DNA-binding proteins.

I have developed a method for transferring plaque DNA of lambda genomic libraries onto 3MM filters in a state accessible to DNA-binding proteins. DNA bound to 3MM is available to proteins as large as Escherichia coli RNA polymerase and maintains template activity similar to that in free solution. Lambda Plaques can be lifted onto 3MM filter disks, deproteinized, and used for transcription assays in vitro. The RNA synthesized is complementary to phage rather than to E. coli DNA and plaques can be identified by autoradiography. Furthermore, the filters can subsequently be probed with radioactive nucleic acids under standard hybridization conditions. Finally, colorimetric assays can be employed with lactate dehydrogenase (LDH) A in which plaques are identified by the localized reduction of nitroblue tetrazolium.

Animals↗

PubMed1994

A versatile shuttle cosmid vector for the efficient construction of genomic libraries and for the cloning of fungal genes.

A shuttle cosmid vector, pANsCos1, has been constructed for Escherichia coli and filamentous fungi. This vector contains two cos sequences separated by a single XbaI restriction site. pANsCos1 allows the efficient construction of representative genomic libraries from as little as 15-20 micrograms of genomic DNA. Due to the presence of a functional hygromycin B phosphotransferase gene (hph) transformation of fungal protoplasts with pAN-sCos1, or derivatives of it, results in the formation of hygromycin B-resistant transformants. The T7 and T3 RNA polymerase promoter sequences flanking the cloning site, in combination with two adjacent NotI sites facilitate genomic walking and the rapid construction of restriction maps of cloned inserts.

Cloning, Molecular↗

PubMed1984

Use of a recombination-deficient phage lambda system to construct wheat genomic libraries.

The poor cloning efficiency of wheat (Triticum aestivum cv. Yamhill) DNA in conventional cloning vectors has previously prevented the preparation of complete genomic libraries. We show here that while wheat DNA does not clone efficiently using the vector Ch4A, it can be cloned efficiently using Ch32. Ch32 clones are red- gam+ and therefore can be propagated on recombination-deficient hosts. These results suggest that instability of wheat sequences in conventional lambda vector systems has frustrated previous attempts to prepare libraries.

Bacteriophage lambda↗

PubMed1984

Isolation of a series of HLA class I clones from a human chromosome 6 genomic library.

Human metaphase chromosomes were fractionated by a fluorescent-activated cell sorter (FACS II) and the chromosome 6 fraction was sorted. A genomic library was constructed in lambda gtWES cloning arms using a partial EcoRI digest of the chromosomal DNA. We estimate that at least 60% of our library represents chromosome 6 material, and as 1.2 x 10(5) recombinants were obtained, this indicates that the majority of clonable chromosome 6 sequences are represented. The library was screened with a mouse H-2 class I clone and 18 HLA class I recombinants were isolated from 4 x 10(4) plaques.

Cell Fractionation↗

PubMed1989

Cloning of apoprotein AI gene and observation on DNA polymorphism of apoprotein AI/CIII gene-loci in genomic library from a liver of Chinese.

For the research on the structure and function of the HDL apoproteins, we have successfully constructed a complete genomic library, in self-prepared EMBL3 lambda vector and the packaging extracts of lysogenic strains BHB2688, BHB2690, from a Chinese fetal liver, which will be widely used in the research on the structure and function of apoproteins. In this study, a DNA sequence polymorphism, revealed by digestion of human DNA with the restriction endonuclease Sst1 and hybridization with an apoprotein AI cDNA probe, has been shown to be located at apoAI/CIII gene-loci. Three gene related fragments (5.7 Kb, 4.0 Kb and 3.0 Kb) were detected. The 5.7 Kb fragment is common and the polymorphism is demonstrated by the presence of either a 4.0 Kb fragment (S1 allele) or the 3.0 Kb fragment (S2 allele). Individuals were genotyped S1S1 or S1S2. Our result showed that the frequency of genotype S1S2 was higher in hypertriglyceridemic subjects than that in normolipidemic subjects.

Apolipoprotein A-I↗

PubMed1990

Optimizing restriction fragment fingerprinting methods for ordering large genomic libraries.

We present a statistical analysis of the problem of ordering large genomic cloned libraries through overlap detection based on restriction fingerprinting. Such ordering projects involve a large investment of effort involving many repetitious experiments. Our primary purpose here is to provide methods of maximizing the efficiency of such efforts. To this end, we adopt a statistical approach that uses the likelihood ratio as a statistic to detect overlap. The main advantages of this approach are that (1) it allows the relatively straightforward incorporation of the observed statistical properties of the data; (2) it permits the efficiency of a particular experimental method for detecting overlap to be quantitatively defined so that alternative experimental designs may be compared and optimized; and (3) it yields a direct estimate of the probability that any two library members overlap. This estimate is a critical tool for the accurate, automatic assembly of overlapping sets of fragments into islands called "contigs." These contigs must subsequently be connected by other methods to provide an ordered set of overlapping fragments covering the entire genome.

Genomic Library↗

PubMed1985

Cloning of genes encoding pectolytic enzymes from a genomic library of the phytopathogenic bacterium, Erwinia chrysanthemi.

Erwinia chrysanthemi are phytopathogenic enterobacteria causing soft-rot disease due to pectolytic enzymes degrading plant cell walls. We constructed a genomic library from Sau3A-digested E. chrysanthemi B374 DNA cloned in the BamHI site of the broad-host-range cosmid pMMB33 grown in Escherichia coli. Out of 1500 kanamycin-resistant (KmR) transductants of E. coli, nine pectolytic-enzyme-positive clones were identified. One of these contained the pEW325 cosmid with a 35-kb insert of Erwinia DNA. Cell extracts of E. coli harboring the cosmid pEW325 were fractionated on a polyacrylamide electrofocusing gel; bands with pectolytic activity were found to co-focus with pectolytic enzymes of E. chrysanthemi B374 strain. Cosmid pEW325 encodes three pectolytic enzymes PL10, PL20 and PL130 with isoelectric points of about 9.3, 9.2 and 4.6, respectively. These enzymes are lyases that cleave polygalacturonate by transelimination, and give rise to unsaturated products. A 15-kb HindIII fragment coding for polygalacturonate lyases was subcloned in pBR322, and a physical map of the resulting plasmid pPL01 was constructed. Starting from the pPL01, various endonuclease-generated fragments were subcloned into pBR322. Genes encoding pectate lyases were localized within an 8-kb fragment (pPL04) and then in a 2.7-kb fragment (pPL03). Polygalacturonate lyases are expressed at various levels; they accumulated in the periplasmic space of E. coli host, whereas E. chrysanthemi secreted these enzymes into the culture medium.

Chromosome Mapping↗

PubMed1984

Nucleotide sequence and expression in E. coli of a human interferon-alpha gene selected from a genomic library using synthetic oligonucleotides.

The complete nucleotide sequence of a human interferon-alpha gene is reported. The gene, designated IFN-alpha M1, was isolated from a human genomic library in phage lambda Charon 4A using synthetic oligonucleotides as hybridization probes. Based on a comparison of nucleotide sequence data obtained from this recombinant phage with published interferon-alpha gene sequences, a region of DNA capable of coding for a pre-interferon of 189 amino acids was identified. An AluI fragment containing the coding region for the mature interferon was inserted into the HincII site of the phage M13mp11, resulting in a fusion of portions of the IFN-alpha M1 and the beta-galactosidase genes. Antiviral activity was detected in extracts from E. coli infected with the recombinant M13 phage carrying the fused gene. The antiviral activity was completely neutralized by antibodies to human interferon-alpha.

Amino Acid Sequence↗

PubMed1983

Isolation and characterization of snRNA and scRNA gene candidates from a human genomic library.

A simple procedure for the isolation and preparative gel electrophoresis of snRNA and scRNA is described. These small RNA species were used to select DNA sequences from a human genomic library which are able to protect hybridized snRNA or scRNA against T1-ribonuclease attack. The snRNA clones obtained contain only sequences for one snRNA species and only one copy of the respective gene. In contrast, more than one 7S RNA gene is present within the scRNA clones.

Cloning, Molecular↗

PubMed1986

Construction of a genomic library from germinating seedlings of mung bean (Vigna radiata). Evidence for the presence of a class of repetitive sequences and selection of beta-tubulin specific recombinant phage.

We have constructed a genomic library of Vigna radiata from its total DNA in the phage charon 4A. Recombinant phages were used to hybridize with a family of mung bean repetitive DNA. At least 30% of recombinant phages showed positive hybridization with a specific type of sequences. These phages were also used to screen for beta-tubulin-specific recombinants and a recombinant phage with beta-tubulin gene was identified.

Autoradiography↗

PubMed1983

Retrieval of human DNA from rodent-human genomic libraries by a recombination process.

Human Alu repeat ("BLUR") sequences have been cloned into the mini-plasmid vector piVX. The resulting piBLUR clones have been used to rescue selectively, by recombination, bacteriophage carrying human DNA sequences from genomic libraries constructed using DNA from rodent-human somatic cell hybrids. piBLUR clones are able to retrieve human clones from such libraries because at least one Alu family repeat is present on most 15 to 20 kb fragments of human DNA and because of the relative species-specificity of the sequences comprising the Alu family. The rapid, selective plaque purification achieved results in the construction of a collection of recombinant phage carrying diverse human DNA inserts from a specific subset of the human karyotype. Subfragments of two recombinants rescued from a mouse-human somatic cell hybrid containing human chromosomes X, 10, 13, and 22 were mapped to human chromosomes X and 13, respectively, demonstrating the utility of this protocol for the isolation of human chromosome-specific DNA sequences from appropriate somatic cell hybrids.

Animals↗