Topic
genomic DNA
About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.
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TL;DR: This method has been successfully applied to the isolation and purification of DNA from eight different adult insects and can be cleaved with restriction endonucleases, ligated efficiently using standard cloning vectors, and hybridized to synthetic oligonucleotides.
188 citations
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TL;DR: It is demonstrated that genes required for pyoluteorin production were expressed in situ by the biological control bacterium Pseudomonas fluorescens Pf-5.
Abstract: A 21-kb region required for the biosynthesis of the polyketide antibiotic pyoluteorin by the biological control agent Pseudomonas fluorescens Pf-5 was identified and cloned. Seven previously isolated mutants deficient in pyoluteorin production (Plt(sup-)) had Tn5 insertions spanning the 21-kb region. Sequences flanking Tn5 inserts were cloned from genomic DNA of three Plt(sup-) mutants and used as probes to identify wild-type alleles of the plt loci from a genomic library of Pf-5. Five cosmids containing overlapping regions of genomic DNA hybridized to one or more of the probes. One cosmid, pJEL1938, contained the entire 21-kb region and, when introduced into a Plt(sup-) mutant, partially restored pyoluteorin production. To study the expression of the genes required for pyoluteorin biosynthesis, the transposon Tn3-nice, which contains a promoterless ice nucleation gene (inaZ) and a type I neomycin phosphotransferase gene, was introduced into the genomic plt region of Pf-5. Carbon sources that influenced pyoluteorin production by Pf-5 had parallel effects on ice nucleation activity of Pf-5 containing a genomic plt::Tn3-nice fusion, indicating that inaZ was transcribed from a promoter of the plt region. Cells of Pf-5 containing a genomic plt::Tn3-nice fusion expressed ice nucleation activity on cotton and cucumber seeds planted in field soil. The expression of plt genes by Pf-5 in the cucumber spermosphere was delayed in comparison with expression in the cotton spermosphere. This study demonstrates that genes required for pyoluteorin production were expressed in situ by the biological control bacterium.
187 citations
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TL;DR: A library of genomic DNA segments has been constructed from the DNA of a somatic cell hybrid carrying a portion of human chromosome 11 on a Chinese hamster ovary cell background, and using a nucleic acid hybridization technique that distinguishes human and Chinese hamsters interspersed, repetitive DNA, this approach promises implications for human genetics generally, for the human genetic diseases, and possibly for understanding of gene regulation in normal and abnormal differentiation.
Abstract: Recombinant DNA techniques have been combined with somatic cell genetic methods to identify, isolate, and amplify fragments of human DNA localized at specific regions of human chromosome 11 selected as a model system. A library of genomic DNA segments has been constructed, in λ Charon 4A bacteriophage, from the DNA of a somatic cell hybrid carrying a portion of human chromosome 11 on a Chinese hamster ovary cell background. Using a nucleic acid hybridization technique that distinguishes human and Chinese hamster interspersed, repetitive DNA, we have been able to distinguish recombinant phages carrying DNA segments of human origin from recombinant phages carrying DNA segments of Chinese hamster origin. We have isolated 50 human DNA segments thus far and have characterized 5 in detail. For each DNA segment characterized, a subsegment that carries no repetitive human DNA sequences has been identified. These segments have been used as hybridization probes in experiments that localize the DNA fragment on the chromosome. In each case an unequivocal chromosomal localization has been obtained with reference to a panel of hybrid cell clones each of which carries a deletion of a portion of the short arm of chromosome 11. At least one DNA segment has been identified which maps to each of the four regions on the short arm defined by the panel of hybrid cell clones used. The approaches described here appear to be general. They can be extended to produce a fine structure map of human chromosome 11 and other human chromosomes. This approach promises implications for human genetics generally, for the human genetic diseases, and possibly for understanding of gene regulation in normal and abnormal differentiation.
187 citations
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TL;DR: It is suggested that the GLI-related genes encode a family of DNA-binding proteins with related target sequence specificities, and sequence similarity aside from the zinc finger region suggests that other aspects of function are shared among the members of this gene family.
Abstract: The GLI oncogene, discovered by virtue of its amplification in human tumors, encodes a sequence-specific DNA-binding protein containing five zinc fingers. We have now characterized one member of a family of GLI-related zinc finger genes. A previously identified fragment of GLI3 genomic DNA was used to localize GLI3 to chromosome 7p13 and to isolate cDNA clones. Sequence analysis of these clones and identification of the GLI3 protein by using polyclonal antisera demonstrated that GLI3 encodes a protein of 1,596 amino acids and an apparent molecular mass of 190 kilodaltons. Amino acid sequence comparison with GLI demonstrated seven regions of similarity (53 to 88% identity), with the zinc fingers representing the most similar region. Furthermore, when produced in vitro, the GLI3 protein bound specifically to genomic DNA fragments containing GLI-binding sites. Amino acid sequence comparison with the product of another member of the GLI family, the Drosophila segment polarity gene cubitus interruptus Dominant, revealed additional similarity that was not shared with GLI. These studies suggest that the GLI-related genes encode a family of DNA-binding proteins with related target sequence specificities. In addition, sequence similarity aside from the zinc finger region suggests that other aspects of function are shared among the members of this gene family.
187 citations
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TL;DR: Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and PerissodactylA, but that at least one gene is represented outside the hooved species.
Abstract: The pregnancy-associated glycoproteins (PAGs) are structurally related to the pepsins, thought to be restricted to the hooved (ungulate) mammals and characterized by being expressed specifically in the outer epithelial cell layer (chorion/trophectoderm) of the placenta. At least some PAGs are catalytically inactive as proteinases, although each appears to possess a cleft capable of binding peptides. By cloning expressed genes from ovine and bovine placental cDNA libraries, by Southern genomic blotting, by screening genomic libraries, and by using PCR to amplify portions of PAG genes from genomic DNA, we estimate that cattle, sheep, and most probably all ruminant Artiodactyla possess many, possibly 100 or more, PAG genes, many of which are placentally expressed. The PAGs are highly diverse in sequence, with regions of hypervariability confined largely to surface-exposed loops. Nonsynonymous (replacement) mutations in the regions of the genes coding for these hypervariable loop segments have accumulated at a higher rate than synonymous (silent) mutations. Construction of distance phylograms, based on comparisons of PAG and related aspartic proteinase amino acid sequences, suggests that much diversification of the PAG genes occurred after the divergence of the Artiodactyla and Perissodactyla, but that at least one gene is represented outside the hooved species. The results also suggest that positive selection of duplicated genes has acted to provide considerable functional diversity among the PAGs, whose presence at the interface between the placenta and endometrium and in the maternal circulation indicates involvement in fetal–maternal interactions.
187 citations