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At least 73 records · Page 4Linked to original sources
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↗
PubMedDate not supplied

[Vectors--derivatives of phage lambda for construction and analysis of genome libraries].

Basic features of lambda phage derived, cosmid and plasmid vectors are described. Plasmid vectors combine the most useful features of phage and plasmid vectors. Plasmids can exist in vivo as a plasmid or as a phage. Plasmid vectors are similar to large capacity phage vectors, but can be maintained in vivo without a stuffer fragment. That is why plasmids are easier in preparation for cloning than phage vectors. The yield of recombinants is higher with plasmid vectors (up to 3.10(6)) and the background of non-recombinants in the library is lower. Analysis of recombinant plasmids is more simple and effective than analysis of recombinant phages and plasmids. Probably plasmid vectors will soon be widely used instead of phage or cosmid vectors for genomic libraries construction and analysis.

Bacteriophage lambda↗
PubMed1981

Multigene family of actin-related sequences isolated from a soybean genomic library.

We have investigated the actin-related sequences in soybean using heterologous actin DNA probes from Dictyostelium, Drosophila, and yeast. Southern blot analysis of restriction digests of soybean DNA indicates that actin is encoded in a small multigene family. In order to isolate individual members of this gene family, we have constructed a soybean genomic library in the lambda vehicle Charon 4A. A partial characterization of this library shows it to be nearly complete. We have isolated from this library a number of recombinant clones that hybridize to actin-coding sequences from all three heterologous probes. We have identified the fragments containing the actin-related sequences on the physical maps of two of these clones lambda SAc1 and lambda SAc3. These fragments were subcloned in the plasmid vehicle pBR322. Using electron microscope heteroduplex mapping we show that the subclones, pSAc1 and pSAc3, share homology with the entire actin-coding sequence (1.1 kb) of Drosophila and Dictyostelium. Furthermore, pSAc1 and pSAc3 have additional homology of approximately 0.22 kb at the 5' ends of their coding sequences. No homology is detected in the 3' flanking regions of these clones. The actin sequence in pSAc1 contains an interruption of approximately 0.30 kb located 0.39 kb from the 5' end of the actin polypeptide coding region.

Actins↗
PubMed1992

The construction of Streptomyces cyaneus genomic libraries in Escherichia coli is dependent upon the use of Mcr-deficient strains.

Streptomyces cyaneus genomic DNA ligated into either lambda phage or plasmid vectors was very inefficiently cloned into standard Escherichia coli host strains. However, the same material could be efficiently cloned using Mcr-deficient E. coli strains. These results suggest that the S. cyaneus genome contains 5-methylcytosine residues, some of which occur within the recognition sequences of the E. coli Mcr restriction system.

Cloning, Molecular↗
PubMed1987

Screening of a bovine genomic library for myosin heavy-chain genes.

Restriction enzyme digests of bovine genomic DNA were hybridized against a .37-kilobase (kb) quail embryonic myosin heavy-chain (MHC) copy deoxyribonucleic acid (cDNA) probe; containing both translated and nontranslated regions surrounding the 3' end of the gene. These experiments revealed seven to eight different bands of hybridization, indicative of multiple genes of MHC in the bovine genome. Additionally, a bovine genomic recombinant DNA library was screened with the .37-kb probe. Of the 10(6) phage screened, 11 clones containing portions of the MHC genome were identified, and four were selected for further analyses. Characterization of these four clones was carried out by constructing partial restriction enzyme maps of the inserts using six restriction enzymes singly or in combination. Orientation of the inserts with respect to the arms of the vector and with respect to direction of transcription was determined by hybridizing the DNA fragments against either the .37-kb Pst 1 fragment of pcC128 or to the .23-kb Pst I fragment of pcC128. The .23-kb fragment is located upstream from the .37-kg fragment and contains only coding sequence. Therefore, the differential hybridization pattern of these two probes provided a means for determining the probable direction of transcription. These data provide evidence for a myosin multigene family in cattle, as well as illustrating that the organization of these genes around the 3' end is unique for each of the genes analyzed.

Animals↗
PubMed1992

Isolation of chromosome 21-specific yeast artificial chromosomes from a total human genome library.

A new approach for the isolation of chromosome-specific subsets from a human genomic yeast artificial chromosome (YAC) library is described. It is based on the hybridization with an Alu polymerase chain reaction (PCR) probe. We screened a 1.5 genome equivalent YAC library of megabase insert size with Alu PCR products amplified from hybrid cell lines containing human chromosome 21, and identified a subset of 63 clones representative of this chromosome. The majority of clones were assigned to chromosome 21 by the presence of specific STSs and in situ hybridization. Twenty-nine of 36 STSs that we tested were detected in the subset, and a contig spanning 20 centimorgans in the genetic map and containing 8 STSs in 4 YACs was identified. The proposed approach can greatly speed efforts to construct physical maps of the human genome.

Base Sequence↗
PubMedDate not supplied

A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors.

The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5' phosphorylated blunt end, and an overlapping or staggered 5' hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.

Animals↗
PubMed1992

Dominant genetics using a yeast genomic library under the control of a strong inducible promoter.

In Saccharomyces cerevisiae, numerous genes have been identified by selection from high-copy-number libraries based on "multicopy suppression" or other phenotypic consequences of overexpression. Although fruitful, this approach suffers from two major drawbacks. First, high copy number alone may not permit high-level expression of tightly regulated genes. Conversely, other genes expressed in proportion to dosage cannot be identified if their products are toxic at elevated levels. This work reports construction of a genomic DNA expression library for S. cerevisiae that circumvents both limitations by fusing randomly sheared genomic DNA to the strong, inducible yeast GAL1 promoter, which can be regulated by carbon source. The library obtained contains 5 x 10(7) independent recombinants, representing a breakpoint at every base in the yeast genome. This library was used to examine aberrant gene expression in S. cerevisiae. A screen for dominant activators of yeast mating response identified eight genes that activate the pathway in the absence of exogenous mating pheromone, including one previously unidentified gene. One activator was a truncated STE11 gene lacking approximately 1000 base pairs of amino-terminal coding sequence. In two different clones, the same GAL1 promoter-proximal ATG is in-frame with the coding sequence of STE11, suggesting that internal initiation of translation there results in production of a biologically active, truncated STE11 protein. Thus this library allows isolation based on dominant phenotypes of genes that might have been difficult or impossible to isolate from high-copy-number libraries.

Base Sequence↗
PubMed1994

Are there two DNA methyltransferase gene families in plant cells? A new potential methyltransferase gene isolated from an Arabidopsis thaliana genomic library.

Using the 1kb 3' terminal DNA fragment of the mouse methyltransferase cDNA as a probe and low stringent hybridisation conditions, a new potential methyltransferase (MTase) gene family was isolated from an Arabidopsis thaliana genomic DNA library. One clone (MTase-11), which gave the strongest signal at the Northern blot, was entirely sequenced (11483 bp) and further characterised. Under consideration of the likely open reading frames and our preliminary cDNA experiments we propose that the clone 11 gene encodes for an approximately 90 kD protein. As deduced form the DNA sequence this protein contains all conserved sequence motifs specific for the 5m cytosine MTases. MTase-11 gene expression was demonstrable in callus and during germination but not in one month old plants or in leaves.

Amino Acid Sequence↗
PubMed1992

[The construction of rich genomic library of bovine Y chromosome].

According to the fundamentals of DNA renatured kinetics, we made a study in which genomic DNA from male cattle was completely digested with Sau 3A, while female DNA was sheared to an average size of 500 bp fragments. The Y derived DNA sequence rich library of bovine has been contracted with pUC19 plasmid as vector and JM109 as host bacteria by reassociation of male and female DNA. Male and female DNA probes, were used. The result of colony hybridization showed that out of 1000 recombinants analysed, 6 were found to be stronger signal with male probe. Analysis of Pvu' I digested DNA fragment revealed that most of the inserts were in the 90-300 bp.

Animals↗