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At least 163 records · Page 9Linked to original sources
PubMed1986

In vivo repackaging of recombinant cosmid molecules for analyses of Salmonella typhimurium, Streptococcus mutans, and mycobacterial genomic libraries.

Strains of Escherichia coli K-12 were constructed that permitted the amplification of in vitro-packaged recombinant cosmid-transducing particles by in vivo repackaging of recombinant cosmid molecules. Thermal induction of these thermoinducible, excision-defective lysogens containing recombinant cosmid molecules yielded high titers of packaged recombinant cosmids and low levels of PFU. These strains were used to amplify packaged recombinant cosmid libraries of Mycobacterium leprae, Mycobacterium vaccae, Salmonella typhimurium, and Streptococcus mutans DNA. Contiguous and noncontiguous libraries were compared for the successful identification of cloned genes. Construction of noncontiguous libraries allowed the dissociation of desired genes from genes that were deleterious to the survival of a cosmid recombinant and permitted selection for unlinked traits that resulted in a selected phenotype. In vivo repackaging of recombinant cosmids permitted amplification of the original in vitro-packaged collection of transducing particles, storage of cosmid libraries as phage lysates, facilitation of complementation screening, expression analysis of repackaged recombinant cosmids after UV-irradiated cells were infected, in situ enzyme or immunological screening, and facilitation of recovery of recombinant cosmid molecules containing transposon inserts.

ATP-Dependent Proteases↗
PubMed1995

The CIC library: a large insert YAC library for genome mapping in Arabidopsis thaliana.

A new Arabidopsis thaliana (ecotype Columbia) genomic library has been constructed in Yeast Artificial Chromosomes: the CIC library (for CEPH, INRA and CNRS). Optimization of plant culture conditions and protoplast preparation allowed the recovery of large amounts of viable protoplasts. Mechanical shearing of DNA was minimized by isolation of DNA from protoplasts embedded in agarose. Cloning of large inserts was favored by including two successive size fractionation steps (after partial EcoRI digestion and after ligation with the vector arms), which selected DNA fragments larger than 350 kb. The library consists of 1152 clones with an average insert size of 420 kb. Clones carrying chloroplast DNA and various nuclear repeated sequences have been identified. Twenty-one per cent of the clones are found to contain chloroplast DNA. Therefore, the library represents around four nuclear genome equivalents. The clones containing 5S rDNA genes, 18S-25S rDNA sequences and the 180 bp paracentromeric repeated element account for 3.6%, 8.9% and 5.8%, respectively. Only one clone was found to carry the 160 bp paracentromeric repeated element. Given the smaller size of clones carrying Arabidopsis repeated DNA, the average size of remaining clones is around 480 kb. The library was screened by PCR amplification using pairs of primers corresponding to sequences dispersed in the genome. Seventy out of 76 pairs of primers identified from one to seven YAC clones. Thus at least 92% of the genome is represented in the CIC library. The survey of the library for clones containing unlinked DNA sequences indicates that the proportion of chimeric clones is lower than 10%.

Arabidopsis↗
PubMed1994

Exon amplification from complete libraries of genomic DNA using a novel phage vector with automatic plasmid excision facility: application to the mouse neurofibromatosis-1 locus.

The identification of transcription units in the vicinity of chromosomal lesions found in tumours is an essential step in the identification of new oncogenes. Here, we describe a lambda phage vector system for genomic exon-trapping (lambda GET), which dramatically simplifies the task of exon amplification from genomic DNA. The vector accommodates about 6.5 to 19 kb of DNA and allows inserts to be automatically subcloned as multi-copy plasmids containing splice donor and acceptor sites positioned flanking the inserted genomic DNA. RNA transcripts derived from such plasmids are processed in vivo and exons contained within the inserted genomic fragments become flanked by known sequences in the resulting mRNAs. RNA-based PCR can then be used for subsequent cloning and sequence analysis of trapped exons. We have exploited the large cloning capacity of lambda GET to construct highly redundant complete genomic libraries from Sau3AI partially digested vertebrae DNAs. Using this system, we have analysed a region of about 1 MB around the mouse neurofibromatosis-1 locus and have identified novel transcription units flanking the Nf-1 gene.

Amino Acid Sequence↗
PubMed1986

Factors which equalize the representation of genome segments in recombinant libraries.

Genomic segments which contain inverted repetitions longer than 300 bp are frequently lost from recombinant libraries grown on rec+ hosts. We have found that 9% of phage lambda clones that contain 15-20-kb insertions of human or Drosophila DNA are inhibited on rec+ hosts and as a result will become under-represented in amplified genomic libraries. We have therefore examined several factors of both host and vector origin which affect the fidelity of representation of genomic sequences in recombinant DNA libraries constructed in bacteriophage lambda vectors. This loss may be diminished if the vector carries either a chi element or a functional gam gene. The most successful approach, however, involves using a host with mutations in recB, recC, and sbcB, or in recD. We have shown that recombinant clones which require such mutant hosts for growth are somewhat more likely to contain DNA derived from loci in the genome which are polymorphic than are clones recovered on conventional hosts.

Animals↗
PubMed1998

The genomic and sequence analysis of rat histone H2B genes.

In addition to previously characterized testis-specific and somatic H2B histone genes, two somatic histone H2B genes, hereafter called sH2B-2 and sH2B-3, were isolated from a rat genomic library, genomic organization was determined, and their promoter was sequenced. Like many other H2B genes, sH2B-2 gene was closely linked to H2A gene whereas H3 gene was located upstream of sH2B-3 gene. Deletion and mutation analysis of 5' sequence fused to CAT reporter gene revealed that the interaction between CCAAT and octamer binding factors is important for S-phase-specific activation of sH2B-3 gene.

3T3 Cells↗
PubMed2000

Onchocerca volvulus superoxide dismutase genes: identification of functional promoters for pre-mRNA transcripts which undergo trans-splicing.

The genes encoding three forms of superoxide dismutase, the cytosolic and extracellular CuZn superoxide dismutases and the mitochondrial Mn superoxide dismutase, were isolated from an Onchocerca volvulus lambda fix II genomic library. Genomic Southern blot analyses indicate single-copy genes in the O. volvulus genome. The O. volvulus cytosolic and extracellular CuZnSOD genes (Ov-sod-1 and Ov-sod-2) are separated by 0.8 kb of sequence and are convergently transcribed. Since the transcripts from all three sod genes are trans-spliced, the transcription start point of each gene was determined in a heterologous system that lacks trans-splicing machinery by in vitro transcription using Drosophila embryo nuclear extracts, followed by primer extension experiments. The ability of the 5' flanking region of the genes encoding the three Ov-SODs to promote transcription was further examined in transient transfections of Chinese hamster ovary cells. In firefly luciferase reporter assays, the Ov-sod-1 and -2 and the MnSOD (Ov-sod-3) gene promoters showed minimal, strong, and moderate levels of activity in these cells, respectively. Both Ov-sod-2 and -3 gene promoter regions showed an initial increase in activity in response to 5' deletions. The results from the in vitro transcription experiments and the luciferase reporter assays were consistent and suggest the presence of Inr-like elements in the promoter regions of the Ov-sod genes.

Animals↗
PubMed1996

[Construction of Chinese genomic cosmid library].

A Chinese genomic library has been constructed using SuperCos1 cosmid vector. 6.09 x 10(5) clones were obtained with an everage insert size of 40 kb ranging from 32 to 45 kb, which cover approximately 8.12 fold human genomic DNA. DNA pools prepared from the total library were screened with 6 known markers distributed on different chromosomes, which all were tested positive.

Asian People↗
PubMed1995

Human androgen-induced growth factor in prostate and breast cancer cells: its molecular cloning and growth properties.

Androgen-induced growth factor (AIGF) has hormone-regulated properties in the mouse Shionogi carcinoma cell line. To investigate whether or not it is involved in growth of human hormone-responsive cancers, we isolated the human AIGF gene from a placental genomic library. Genomic analyses suggested that the AIGF gene was about 6.5 kilobases in length containing five exons. The deduced amino acid sequence of human AIGF was completely identical with that of the mouse. RT-PCR analyses showed that prostate and breast cancer cell lines, LNCaP, PC-3, and MCF-7, slightly expressed the AIGF gene. Recombinant AIGF enhanced the growth of the human prostate cancer LNCaP cells, and it also markedly stimulated the growth of fibroblasts. These in vitro findings suggest that AIGF might be a possible autocrine or paracrine factor in hormone-responsive cancers.

3T3 Cells↗