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V C Sheffield

Publications and source records attributed to V C Sheffield.

150 records · Page 9Linked to original sources
PubMed1989

Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes.

Denaturing gradient gel electrophoresis (DGGE) can be used to distinguish two DNA molecules that differ by as little as a single-base substitution. This method detects approximately 50% of all possible single-base changes in DNA fragments ranging from 50 to approximately 1000 base pairs. To increase the number of single-base changes that can be distinguished by DGGE, we used the polymerase chain reaction to attach a 40-base-pair G + C-rich sequence, designated a GC-clamp, to one end of amplified DNA fragments that encompass regions of the mouse and human beta-globin genes. We show that this GC-clamp allows the detection of mutations, including the hemoglobin sickle (HbS) and hemoglobin C (HbC) mutations within the human beta-globin gene, that were previously indistinguishable by DGGE. In addition to providing an easy way to attach a GC-clamp to genomic DNA fragments, the polymerase chain reaction technique greatly increases the sensitivity of DGGE. With this approach, DNA fragments derived from less than 5 ng of human genomic DNA can be detected by ethidium bromide staining of the gel, obviating the need for radioactive probes. These improvements extend the applicability of DGGE for the detection of polymorphisms and mutations in genomic and cloned DNA.

Animals↗
PubMed1985

Copy number of the chicken type II procollagen gene.

The copy number of the type II procollagen gene has been determined by quantitative hybridization of cloned DNA to Southern blots of total cellular DNA from chicken. Two different DNA probes have been used. One contains the 289 base pair exon coding for the junction between the triple helical domain and the carboxyl-terminal propeptide and the other contains an 162 base pair exon coding for amino acids 712-765 of the triple helical domain. Both fragments hybridize to single bands present at one copy per haploid genome in genomic DNA from chicken liver or sternal cartilage after these DNAs are cleaved with either HinfI of PstI. When the hybridization stringency and washing conditions were modified sufficiently to stabilize hybridization of heteroduplexes with up to 31% mismatch, no additional hybridizing fragments were detected. These results show that there is not a second copy of this gene or a pseudogene which is more closely related to the type II procollagen gene than the alpha 1 type I procollagen gene. These studies suggest that the cartilage 3 alpha collagen chain, which appears to be very similar to the alpha 1 (II) collagen chain, is encoded by the type II procollagen gene but is differently processed than the major type II gene product, and that the alpha 1 (II) Minor chain identified in bovine type II collagen preparations most likely represents an allelic form of type II collagen rather than a product of a different gene.

Animals↗
PubMed1982

Construction and partial characterization of two recombinant cDNA clones for procollagen from chicken cartilage.

Type II procollagen mRNA has been partially purified from embryonic chick sternal cartilage by guanidine hydrochloride extraction, sucrose gradient sedimentation and Sepharose 4B chromatography. Double stranded cDNA was synthesized using AMV reverse transcriptase and E. coli DNA polymerase I, tailed using terminal transferase and inserted into the Pst I site of pBR322. Two putative type II procollagen cDNA clones have been characterized. Both plasmids hybridize to 2 sternal RNA species, a major species of 5.3 kb and a minor species of 7 kb. These RNAs are present in total RNA from sterna and differentiated limb bud cultures but are not detected in RNA from stage 24 limb bud which has not yet differentiated to cartilage or in RNA from calvaria. The time of appearance of these RNAs during the differentiation of limb mesenchyme in culture parallels the appearance of translatable type II procollagen mRNA.

Animals↗
PubMed1980

Impaired energy metabolism as an initial step in the mechanism for 6-aminonicotinamide-induced limb malformation.

The analogue and antagonist of nicotinamide, 6-aminonicotinamide (6-AN), impairs cartilage formation and results in shortening of the limbs when administered to chick embryos. Studies have shown that 6-AN forms an abnormal NAD analogue which inhibits the activity of NAD-dependent enzymes associated with production of ATP. To determine if an effect on ATP synthesis might be associated with the mechanism of teratogenesis in the chick embryo, ATP levels of cartilage from day-8 chick embryos treated in vitro were assayed in relation to biosynthesis of protein, DNA and chondroitin sulfate. Incorporation of 35SO4= was inhibited by 6 h or treatment with 10 microgram/ml of 6-AN, whereas incorporation of [3h]thymidine and [3H]amino acid was not inhibited until 12 h. Incorporation of [3H]glucosamine was increased at all treatment times. A decrease in the level of ATP preceded any detectable inhibition of precursor incorporation. These results are consistent with the hypothesis that 6-AN inhibits chondroitin sulfate synthesis through a reduction in the level of ATP in chondrocytes.

6-Aminonicotinamide↗
PubMedDate not supplied

Focal microsatellite mutations in relatives with prostatic adenocarcinoma.

Instability of short tandem repeat sequences, microsatellite instability (MI), has been reported to play an important role in the tumorigenesis of various adenocarcinomas, including prostatic adenocarcinoma. Although prostate cancer is not widely recognized as a heriditary cancer, familial clustering is well known. To investigate the frequency of microsatellite instability in familial prostatic adenocarcinomas we analyzed archival tumor tissue from seven paired first degree relatives with prostatic adenocarcinoma. Twelve dinucleotide, nine trinucleotide, six tetranucleotide repeats and the CAG repeat of the androgen receptor gene were screened for MI. Solitary mutations were observed in four separate cases (28.6%) and widespread somatic alterations were not identified. No statistical correlation to pathological characteristics was determined. Our data indicate that microsatellite instability is an uncommon phenomenon in prostatic adenocarcinoma within first degree relatives. Those changes present appear to manifest as focal mutations in contrast to the more global changes seen in MI.

Adenocarcinoma↗