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At least 145 records · Page 8Linked to original sources
PubMed2000

Rapid cloning of metK encoding methionine adenosyltransferase from Corynebacterium glutamicum by screening a genomic library on a high density colony-array.

The genes SAM1 and SAM2 encoding the two different methionine adenosyltransferases (EC 2.5.1.6) in Saccharomyces cerevisiae were used as templates to generate specific DNA-probes. This heterologous mixture of DNA-probes was hybridized under low stringency hybridization conditions to a Corynebacterium glutamicum colony-array representing the complete genome. Subsequently, one genomic fragment was isolated which contained the C. glutamicum methionine adenosyltransferase gene metK (1.224 kb). When overproduced in Escherichia coli, MetK (44.2 kDa) of C. glutamicum had methionine adenosyltransferase activity. In addition, overexpression of metK in C. glutamicum led to an increased intracellular S-adenosylmethionine concentration. The metK transcript was detected by reverse transcription PCR in C. glutamicum cells in the exponential growth phase but not in the stationary phase.

Blotting, Southern↗
PubMed1997

Construction and characterization of a Plasmodium vivax genomic library in yeast artificial chromosomes.

Here we describe the construction of a representative YAC library for the human malarial parasite Plasmodium vivax. As P. vivax cannot be maintained continuously under laboratory conditions, the P. vivax DNA necessary for the library construction was isolated from a single human patient presenting himself with vivax malaria to a local hospital in the Brazilian Amazon. Thus, this YAC library is the first of its kind to be generated from patient-derived material. The YAC library consists of 560 clones with an average insert size of 180 kb. Of 9 published P. vivax genes, 8 were found to be present in the library. In addition, 12 P. vivax telomeric YAC clones were identified.

Animals↗
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↗
PubMed1990

A phosphate group at the cos ends of phage lambda DNA is not a prerequisite for in vitro packaging: an alternative method for constructing genomic libraries using a new phasmid vector, lambda pGY97.

It is shown here that the phosphate groups at the cos ends of phage lambda DNA are not a prerequisite for in vitro packaging. Molecules with phosphatase-treated cos ends are packaged in vitro as efficiently as native lambda DNA. This observation can be used for an alternative strategy to improve the efficiency of gene library construction, since cos-cos ligation decreases in vitro encapsidation and infectivity. Dephosphorylated cos ends and a new phasmid vector lambda pGY97 have been used to construct a representative gene bank of alfalfa in a Mcr- (5-methylcytosine restriction deficient) Escherichia coli host strain. These recombinant clones can be propagated as phages or more conveniently as plasmids in recA- E. coli, to prevent possible homologous recombination events between repetitive sequences of the insert that would otherwise interfere with clone stability. The 5-19-kb inserts can be easily recloned as plasmids from the recombinant phasmids with simple EcoRI digestion and re-ligation. This observation also implies that the construction of gene libraries in cosmid vectors can be made more efficient if cos-cos ligates were cleaved by lambda terminase just before in vitro packaging.

Bacteriophage lambda↗
PubMed1994

The P1 vector system for the preparation and screening of genomic libraries.

In retrospect, it is remarkable how swiftly the P1 cloning system has progressed in only a few years from a novel cloning system to one now widely used for the production of recombinant libraries and the building of physical maps. As the libraries become larger, better characterized and more widely distributed, we certainly will see a blossoming of research articles and techniques based on the use of P1 recombinant clones. Specifically, we can look forward to scanning P1 clones for expressed sequences (N. Sternberg, personal communication), routine retrofitting of P1 clones with a combination of transposon and P1 transduction techniques (3), the random or loxP-directed (68,69) insertion of P1 clones into host genomes and the subsequent production of transgenic animals (63), a further use of P1 clones in the building of contigs and physical maps, an a higher in vitro cloning efficiency due to the purification of the P1 pacase proteins used during in vitro packaging (70). In summary, P1 bacteriophage cloning is favorably impacting research today and will continue to fill an important niche as a genomic cloning system.

Animals↗
PubMed1994

[Construction of the genomic library for cyanobacteria Synechocystis sp. PCC 6803 using a cosmid].

The gene library of the phototrophic cyanobacterium Synechocystis PCC6803 was constructed in the cosmid vector Lorist6. About -3 x 10(3) recombinant clones were obtained, of which 760 were selected for further work. Each clone was kept in an individual well of a microtiter plate. The clone grids obtained were hybridized with two DNA probes, which contained the ndhE and dfr genes of Synechocystis PCC6803. Two groups of probe-overlapping cosmids (11 and 8 clones respectively), containing insertions averaging of 39 kb, were isolated. The capacity of the gene library is nine equivalents of the Synechocystis PCC6803 genome and its representativity exceeds 99%.

Cloning, Molecular↗
PubMed1993

Construction of a bovine genomic library of large yeast artificial chromosome clones.

We have constructed a yeast artificial chromosome (YAC) library derived from bull fibroblasts in primary culture. The library consists of 21,500 clones arranged on 224 gridded plates (96 or 8 x 12 positions) and in 2 x 224 96-well microplates maintained as permanent frozen stocks. An average insert size of 750 kb was estimated from the analysis of 200 randomly selected YACs, giving a sixfold coverage of the bovine genome for the total library. A screening strategy based on the polymerase chain reaction that allows the identification of individual clones has been devised. Screening of the library with six locus-specific markers of interest led to the isolation of 34 positive clones. The proportion of chimeric YACs was estimated from the analysis of a subset of the positive clones: 3 of 9 YACs were found to contain noncontiguous DNA.

Animals↗
PubMed1995

Construction of a human genomic library of clones containing poly(dG-dA).poly(dT-dC) tracts by Mg(2+)-dependent triplex affinity capture. DNA polymorphism associated with the tracts.

Microsatellite DNA is a useful tool for detecting DNA polymorphisms among species or individuals, especially those among closely related individuals. We constructed a library of clones that contained poly(dG-dA).poly(dT-dC) tracts from human genomic DNA by Mg(2+)-dependent triplex DNA formation. Examination of triplex DNA formation in the presence of various metal ions Mg2+, Mn2+, or Zn2+ revealed that the procedure worked best in the presence of Mg2+. Affinity enrichment was performed with AluI-digested chromosomal DNA mixed with biotinylated (dG-dA)17 in the presence of Mg2+. A library constructed after three cycles of affinity enrichment showed that over 80% of the clones contained at least one poly(dG-dA).poly(dT-dC) tract. Most of them contained a perfect (dG-dA)n repeat 30-84 base pairs in length, while some contained variants such as (dC-dT)10-(dC)-(dC-dT)9. Using the clones from the library as a probe, we detected DNA polymorphisms associated with the repeat length of the tracts in the Japanese population. We also detected a microsatellite instability among the tracts in a cancer tissue sample.

Base Sequence↗
PubMed2000

[Construction of genomic library from L. lactis AL2 and isolation of entire nisin biosynthesis gene cluster].

A total DNA library of Lactococcus lactis AL2 with high yield of nisin was successfully constructed using lambda EMBL3 as vector. 3900 plaques were obtained, which is much more than the required number of recombinants that represents an entire L. lactis genome according to Clarke and Carbon formula. Southern hybridization, PCR amplification and DNA sequencing revealed that the entire nisin biosynthesis gene cluster was isolated from the constructed library.

Base Sequence↗
PubMed1987

The physical map of the whole E. coli chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library.

Thirty-four hundred lambda phage clones containing segments of the E. coli chromosome were isolated and used to construct a 4700 kb long integrated restriction map for eight six-base-recognizing enzymes by a rapid mass-analysis method. Our strategy was to measure the sizes of partial restriction enzyme digests by hybridization with a vector probe in a manner analogous to nucleotide sequencing. The data were sorted into groups by a computer program and the boundary clones were further correlated with each other using a mass hybridization method. These clones can be exploited for the isolation of any desired E. coli genes if their map positions are known. Also, the strategy is applicable to analyses of the genomes of other organisms.

Chromosome Mapping↗
PubMed1987

Predefined gene transfer for expression of a glycosphingolipid antigen by transfection with a cosmid genomic library prepared from a cell line in which the specific glycosphingolipid is highly expressed.

The deliberate transfer of globotriaosylceramide (Gb3) expression in mouse lymphoma L5178 cells was achieved by transfection with a cosmid DNA library prepared from human Burkitt lymphoma Ramos cells in which Gb3 was highly expressed. The recipient mouse lymphoma cells did not contain Gb3 but did contain its direct precursor, lactosylceramide. The transfected cells expressed Gb3, detected both chemically and immunologically, and contained human DNA detected by an Alu sequence probe. This model demonstrates a general method for studying glycosyltransferase genes and other factors necessary for the expression of glycosphingolipid antigens.

Animals↗
PubMed1990

Cloning and sequencing the gene encoding Escherichia coli ribonuclease I: exact physical mapping using the genome library.

The amino acid (aa) sequence of the N terminus of Escherichia coli RNase I was determined. A mixed oligodeoxynucleotide coding for that sequence was used to probe the 476 lambda clones of Kohara et al. [Cell 50 (1987) 495-508]. DNA from these clones carry almost the entire E. coli chromosome in overlapping segments. Two overlapping clones hybridized to the probe sequence. From one of them DNA containing the rna gene was subcloned and sequenced. The inferred protein contains 245 aa residues and has an Mr of 27,156, which agrees with earlier estimates from sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. RNase I is close to twice the size of pancreatic RNase A, but both enzymes contain eight Cys and four His; those aa are important for structure and function of RNase A. Proximal to the rna gene is a sequence that would code for a 23-aa peptide which conforms to consensus rules for signal peptides, and thus should transport this periplasmic enzyme. Sites for eight restriction enzymes had been mapped on each lambda clone. By relating to the map for that specific region, it was possible to position the rna gene exactly at 659 kb from the thr locus (time zero on a time scale of 100 min). This physical mapping gave a more precise (exact) map position based on distance than was possible using genetic mapping based on a time scale derived from conjugation, and should be applicable for mapping many other E. coli genes.

Amino Acid Sequence↗
PubMed1997

Shearing DNA for genomic library construction.

Methods and reagents is a unique monthly column that highlights current discussion in the newsgroup bionet.molibio.methds-reagnts, available on the internet. This month's column discusses the pros and cons of various techniques used to shear DNA for shotgun cloning. For details on how to partake in the newsgroup, see the accompanying box.

Chromatography, High Pressure Liquid↗
PubMed1981

Cloning of a representative genomic library of the human X chromosome after sorting by flow cytometry.

A library of 50,000 recombinants representative of the human X chromosome has been constructed. Human X chromosomes were physically separated using a fluorescence-activated cell sorter. The DNA was purified from the chromosomes, digested to completion with the restriction enzyme EcoRI and cloned into the phage lambda gtWES.lambda B. The X-derived nature of the recombinants was confirmed by hybridization to rodent/human cell line DNA containing only the human X chromosome. Such libraries will be particularly useful for the investigation of genetic diseases such as Duchenne muscular dystrophy, where the basic defect has not been elucidated, and of neoplasia, where several specific chromosomal anomalies, particularly for the leukaemias, have been identified.

Bacteriophage lambda↗
PubMed1982

Construction and characterization of genomic libraries from specific human chromosomes.

Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.

Cell Line↗