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F G Barr

Publications and source records attributed to F G Barr.

64 records · Page 4Linked to original sources
PubMed1991

Molecular and cytogenetic analysis of chromosomal arms 2q and 13q in alveolar rhabdomyosarcoma.

We present cytogenetic and molecular genetic analyses of two cases of alveolar rhabdomyosarcoma. The characteristic translocation between chromosomes 2 and 13, t(2;13)(q35;q14), has been identified in both cases. Using cell lines derived from these tumor specimens, we have performed Southern blot analysis to investigate the possibility of rearrangement of 14 candidate genes mapping to the relevant regions of 2q and 13q. These candidate genes can be divided into 5 groups: signal transduction proteins (RB1, inhibin alpha, FLT1, and HOX4B), muscle-specific products [myosin light chain, desmin, and nicotinic cholinergic receptor subunits gamma and delta (CHRNG and CHRND)], extracellular matrix proteins (collagen type VI alpha 3 chain, elastin, and fibronectin), transformation-associated products (intestinal alkaline phosphatase and L-plastin), and other genes (esterase D). Conventional gel electrophoresis followed by Southern blot analysis indicated no evidence of rearrangement within or near these genes except for a rearrangement in the CHRNG-CHRND locus, which occurred only in a subpopulation of the late recurrence tumor cells of one patient. In addition, we employed pulsed-field gel electrophoresis-Southern blot analysis to demonstrate the absence of detectable rearrangements within a larger region around each of these genes.

Abdominal Neoplasms↗
PubMed1991

Localization of the rhabdomyosarcoma t(2;13) breakpoint on a physical map of chromosome 13.

Previous investigations of the pediatric soft tissue tumor alveolar rhabdomyosarcoma have identified a characteristic translocation t(2;13)(q35;q14). We have employed a physical mapping strategy to localize the site of this translocation breakpoint on chromosome 13. Using a panel of somatic cell hybrid and lymphoblast cell lines with deletions and unbalanced translocations involving chromosome 13, we have mapped numerous probes from the 13q12-q14 region and demonstrate that this region is divisible into five physical intervals. These probes were then mapped with respect to the t(2;13) rhabdomyosarcoma breakpoint by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2. Our findings demonstrate that the t(2;13) breakpoint is localized within a map interval delimited by the proximal deletion breakpoints in lymphoblast lines GM01484 and GM07312. Furthermore, the breakpoint is most closely flanked by loci D13S29 and TUBBP2 within this map interval. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the alveolar rhabdomyosarcoma translocation. In addition, this physical map will permit rapid determination of the proximity of new cloned sequences to the translocation breakpoint.

Animals↗
PubMed1990

Application of a subtraction hybridization technique involving photoactivatable biotin and organic extraction to solution hybridization analysis of genomic DNA.

We have adapted a subtraction hybridization technique involving photoactivatable biotin, streptavidin binding, and organic extraction for solution hybridization analysis of mammalian genomic DNA. By combining maximal hybridization conditions of high salt, dextran sulfate, and formamide with successive hybridization steps and sequence enrichment by agarose gel electrophoresis, up to 97% of tracer DNA can be reproducibly driven to hybridize with photobiotinylated driver DNA. We demonstrate that the fractionation of hybridized from unhybridized sequences by this technique differs from hydroxyapatite chromatography with respect to the handling of nondenatured tracer, foldback sequences, and tracer-tracer hybrids. Strategies are presented to control the contribution of these species to the final subtracted product thereby making this technology a useful adjunct to solution hybridization approaches such as deletion cloning.

Azides↗
PubMed1990

Analysis of the rearrangements associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have previously shown that chemical carcinogen treatment of RJK92 hamster cells activates the quiescent thymidine kinase (TK) gene and that 20% of the TK+ variants have a rearrangement in the region 5' to the TK gene (Barr et al. (1986) Mol. Cell. Biol. 6, 3023-3033). After cloning the wild type 5' region to obtain detailed mapping data and hybridization probes, we localized the rearrangement breakpoints by Southern blot analysis to a 1.5 kb region 6 kb 5' to the origin of transcription. This analysis also demonstrated that the rearrangements consist at least partly of a deletion of wild type sequences 5' to this breakpoint region. The region near the transcription origin in the rearranged TK genes has a DNase I-sensitive chromatin conformation and a DNase I hypersensitive site as well as the previously described domain of demethylation (Ibid.). Though this domain of demethylation extends into the breakpoint region, the rearranged region is not associated with DNase I sensitivity nor hypersensitive sites. The rearrangement also does not detectably alter the growth-related regulation of TK activity in these cells.

Animals↗
PubMed1986

Genomic hypomethylation and far-5' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.

Animals↗
PubMed1985

Distribution of 5-methyldeoxycytidine in products of staphylococcal nuclease digestion of nuclei and purified DNA.

We have compared the distribution of 5-methyldeoxycytidine (m5dC) between staphylococcal nuclease (SN) sensitive and resistant regions of human diploid fibroblast chromatin to the corresponding distribution in purified DNA. After SN digestion of fibroblast nuclei or purified DNA, nuclease-resistant products were separated from sensitive products by perchloric acid or ethanol precipitation; the radioactively labeled nucleosides were then fractionated by high-performance liquid chromatography and quantitated. Our results indicate that m5dC is preferentially associated with SN-resistant regions of both chromatin and purified DNA. The magnitudes of these preferences in fibroblast chromatin and DNA are similar; we find that the enrichment of m5dC content in SN-resistant fractions of nuclei and DNA relative to the corresponding sensitive fractions is approximately 2-3-fold. Therefore, highly methylated regions of DNA have an intrinsic resistance to digestion by SN that is of sufficient magnitude to explain the high degree of nuclease resistance of chromatin containing highly methylated DNA.

Cell Nucleus↗