Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A modified procedure for preparing cosmid libraries from genomic DNA is described. Genomic DNA was partially digested with a restriction endonuclease, and DNA fragments of appropriate size fractionated by agarose gel electrophoresis. A cosmid library was prepared, prescreened, and used to isolate gene inserts with previously published procedures. In one series of experiments, a modified cosmid vector containing stuffer fragments was used to prepare cosmid libraries containing partial SphI digests of 25 to 35 kb. From 10(5) to 10(7) clones were obtained per microgram of size-fractionated genomic DNA. From 10 to 100 hybridization-positive clones of a single copy gene (COL2A1) were obtained from plates that were positive in the prescreening step. Restriction mapping of over 20 clones and nucleotide sequencing of over 20,000 bp in each of two clones indicated that the inserts were faithful copies of the gene. In another experiment, a standard cosmid vector was used to prepare a cosmid library containing partial BamHI fragments of 30 to 45 kb. Genomic libraries can be prepared with 5 to 20 micrograms of genomic DNA and a large number of clones containing 25 to 45 kb fragments of a single copy gene can be isolated in about three weeks.
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PMID:Efficient procedure for preparing cosmid libraries from microgram quantities of genomic DNA fragments size fractionated by gel electrophoresis. 208 10

Recombinant DNA probes specific for the human pro alpha 1(II) and pro alpha 1(III) collagen chains have been used for the chromosomal localization of the two genes. Restriction endonuclease analysis of DNA from human-rodent hybrid cell lines in conjunction with in situ hybridization of human metaphasic chromosomes have shown that the gene coding for the pro alpha 1 chain of type II collagen (COL2A1) is located on chromosome 12 in the segment 12q131----12q132. Likewise, the gene coding for the pro alpha 1 chain of type III collagen (COL3A1) was assigned to the segment 2q31----2q323 of chromosome 2.
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PMID:Further evidence for the dispersion of the human fibrillar collagen genes. 300 2

Stickler syndrome is one of the milder phenotypes resulting from mutations in the gene that encodes type-II collagen, COL2A1. All COL2A1 mutations known to cause Stickler syndrome result in the formation of a premature termination codon within the type-II collagen gene. COL2A1 has 10 in-frame CGA codons, which can mutate to TGA STOP codons via a methylation-deamination mechanism. We have analyzed these sites in genomic DNA from a panel of 40 Stickler syndrome patients to test the hypothesis that mutations that cause Stickler syndrome preferentially occur at these bases. Polymerase chain reaction (PCR) amplification of genomic DNA containing each of the in-frame CGA codons was done by one of two methods: either using primers that amplify DNA that includes the CGA codon, or using allele-specific primers that either amplify normal sequence containing a CGA codon or amplify a mutant sequence containing a TGA codon. Analysis of PCR products by restriction endonuclease digestion or sequencing demonstrated the presence of a normal or mutated codon. TGA mutations were identified in eight patients, at five of the 10 in-frame CGA codons. The identification of these mutations in eight of 40 patients demonstrates that these sites are common sites for mutations in individuals with Stickler syndrome and, we propose, should be analyzed as a first step in the search for mutations that result in this disorder.
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PMID:Rapid determination of COL2A1 mutations in individuals with Stickler syndrome: analysis of potential premature termination codons. 1098 70