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At least 127 records · Page 7Linked to original sources
PubMed2002

Adrenergic receptor genes: cDNA and genomic library construction.

Adrenergic receptors mediate the central and peripheral actions of norepinephrine and epinephrine and are pharmacologically divided into three major types, alpha-1, alpha-2, and beta. These types are further subdivided into alpha-1A, alpha-1B, and alpha-1D; alpha-2A, alpha-2B, and alpha-2C; and beta-1, beta-2, and beta-3, respectively. Adrenergic receptor sequence information is presented in three tables with respect to species, subtype identification, GenBank accession number, source of the nucleic acid sequence, the presence of a 5' flanking region upstream of the transcription start site, and the nucleotides defined as introns, coding regions, or 3' and/or 5' untranslated but transcribed (UTR) regions. Sequences have been assigned to adrenergic subtype categories based on sequence comparison using either FASTA or denogram of Pileup from the GCG sequence analysis program rather than as described in the author definition line. Sequence information found in these tables can be important for probe development for screening libraries for isolating adrenergic receptor genes from species other than the most common species. Where commercial libraries for specific tissue or species needs are not available, we have described construction of genomic cosmid libraries or PCR-based synthesis of a cDNA library using a microgram of RNA.

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↗
PubMed1999

Zebrafish YAC, BAC, and PAC genomic libraries.

Numerous positional cloning projects directed at isolating genes responsible for the myriads of observed developmental defects in the zebrafish are anticipated in the very near future. In this chapter, we have reviewed the YAC, BAC, and PAC large-insert genomic resources available to the zebrafish community. We have discussed how these resources are screened and used in a positional cloning scheme and have pointed out frequently formidable logistical considerations in the approach. Despite being extremely tedious, positional cloning projects in the zebrafish will be comparatively easier to accomplish than in human and mouse, because of unique biological advantages of the zebrafish system. Moreover, the ease and speed at which genes are identified and cloned should rapidly increase as more mapping reagents and information become available, thereby paving the way for meaningful biological studies.

Animals↗
PubMed1997

[Construction of mycobacterium tuberculosis genomic library].

OBJECTIVE: To provide effective tools for identification and characterization of M. tuberculosis genes/antigenes and to evaluate their roles in diagnosis, vaccination, drug resistance, and pathogenesis. METHODS: M. tuberculosis genomic DNA obtained from H 37 Ra strain was partially digested with DNase I. The DNA fragments ranging from 4-8 kb were isolated from agarose gel and ligated to EcoR I adaptor, and the products were linked to lamda gt11 arms and packaged using an in vitro packaging extract. The different diluted bacteriophages were used to transfect bacteria Y1090. RESULTS: The efficiency and titer of the library were 85% and 3 x 10(5) pfu/ml, respectively. The library contained 1.3 x 10(5) individual recombinant phage whose foreign DNA inserted fragment size was 3.5 kb on average. CONCLUSION: The genomic DNA library constructed here can provide sufficient clone to cover H37Ra gene.

Genome, Bacterial↗
PubMedDate not supplied

An indexed genomic library for Paramecium complementation cloning.

Recent pioneering work opened the way for cloning genes in Paramecium by functional complementation of mutants. We present here the construction and pilot utilization of a new indexed library of Paramecium macronuclear DNA. The library is made of 61,440 clones containing inserts mostly between 6 and 12 kilobases. It has already allowed the complementation cloning of four new genes, and this library has proven to be very useful for rapid hybridization cloning.

Animals↗
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↗