Cloning of rat Brca2 and linkage mapping to chromosome 12.
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Fifty-eight new anonymous simple sequence repeats (SSR) were generated and mapped to various rat chromosomes. Among them two genes (rat homologs for human cadherin-14 and mouse fibroblast growth factor-related protein) were mapped on Chromosomes (Chrs) 2 and 11 respectively. The majority of markers were generated from a small insert genomic library specific to Chr 11, 13, 14, and 15. Twenty new markers were mapped to Chr 13, which is known to contain a blood pressure quantitative trait locus (QTL). Several approaches to obtain microsatellite markers are described. The protocols and newly generated markers should be useful for ongoing rat genome project.
Seventy-six novel microsatellite markers with various simple sequence repeat (SSR) motifs are reported in this paper. They were generated on the basis of non-radioactive library screening procedures from flow-sorted rat Chromosome (Chr) 5-specific DNA, and were mapped in three rat backcross populations. Fifty-four of these markers mapped to Chr 5, while the other 22 mapped to other chromosomes of the rat genome. The marker D3Uwm8 is a new microsatellite marker for the rat syndecan 4 (ryudocan) gene. A genotyping protocol based on agarose gel electrophoresis is also provided in this paper.
We describe a general method of determining the recombination fraction between a polymorphic locus and the centromere in any species where single oocytes can be obtained. After removal of the first polar body, each oocyte is analyzed by PCR. The frequency of oocytes heterozygous at the polymorphic locus is used to estimate the recombination fraction. We estimate a recombination fraction of 0.15 between the mouse major histocompatibility complex (H-2) and the centromere of chromosome 17.
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The yeast two-hybrid system was used to catalog all detectable interactions among the P2 nonstructural cleavage products of poliovirus type 1 (Mahoney). Evidence has been obtained for specific associations among 2A(pro), 2BC, 2C, and 2B. Specifically, 2A(pro) can interact with itself and 2BC and its cleavage products (2B and 2C) interact in all possible combinations, with the exception of 2C/2C. Detected interactions were confirmed in vitro by a glutathione S-transferase pulldown assay, which allowed us to detect 2C/2C association. transdominant-negative mutants of 2B (K. Johnson and P. J. Sarnow, J. Virol. 65:4341-4349, 1991) were examined and were found to retain interaction with wild-type 2B, perhaps reflecting a need for 2B multimerization in viral RNA replication. The multimerization of 2B was examined further by screening a mutagenized library for 2B variants that have lost the ability to bind wild-type 2B. The screen identified two nonconservative missense mutations within a central hydrophobic region, as well as truncations and frameshifts that implicate the C terminus in homointeraction. Introduction of the missense mutations into the genome of the virus conferred a quasi-infectious phenotype, an observation strongly suggesting that the 2B/2B interaction is required for replication of the viral genome.
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The brain fatty acid-binding protein (B-FABP) is involved in brain development and adult neurogenesis. We have determined the sequence of the gene encoding the B-FABP in zebrafish. The zebrafish B-FABP gene spans 2370 bp and contains four exons interrupted by three introns. The coding sequence of zebrafish B-FABP gene is identical to its cDNA sequence and the coding capacity of each exon is the same as that for the human and mouse B-FABP genes. A 1249 bp sequence 5' upstream of exon 1 of the zebrafish B-FABP gene was cloned and sequenced. Several brain development/growth-associated transcription factor binding elements, including POU-domain binding elements and the proposed lipogenic-associated transcription factor NF-Y elements, were found within the 5' region of the B-FABP gene. RT-PCR analysis using mRNA extracted from different tissues of adult zebrafish demonstrated that the zebrafish B-FABP mRNA was predominant in brain with lower levels in liver, testis and intestine, but not in ovary, skin, heart, kidney and muscle. Quantitative RT-PCR revealed a similar tissue-specific distribution for zebrafish B-FABP mRNA except that very low levels of B-FABP mRNA, normalized to beta-actin mRNA, were detected in the heart and muscle RNA, but not in liver RNA. Zebrafish B-FABP mRNA was detected by RT-PCR in embryos beyond 12 h postfertilization, suggesting a correlation of zebrafish B-FABP mRNA expression with early brain development. Radiation hybrid mapping assigned the zebrafish B-FABP gene to linkage group 17. Conserved syntenies of the zebrafish B-FABP gene and the human and mouse orthologous B-FABP genes were observed by comparative genomic analysis.
High-density genetic maps of individual human chromosomes will permit accurate localization of disease-associated genes and provide signposts that will be useful in the construction of physical maps. We have constructed a genetic map of chromosome 7 with 63 polymorphic DNA markers by using segregation data from 23 three-generation families and recently developed multilocus linkage-analysis techniques. The map spans 250 centimorgans in females and 170 centimorgans in males, with much of the difference being concentrated in a few intervals. The density and informativeness of the markers are such that there is a high probability of detecting linkage to any disease gene on this chromosome for which 20 phase-known meioses are available.
We have constructed a genetic map of the human chromosomal region 11q22-23 by multipoint linkage analysis of 13 DNA polymorphisms that we have condensed into eight loci. An analysis for linkage disequilibrium between tightly linked probe/enzyme systems allows us to make specific recommendations for future DNA typing at these loci. The resulting sex-averaged multipoint map spans approximately 80 cM and differs considerably from previously reported genetic maps of this region. Our mathematically derived "most likely order" of the markers is compatible with physical mapping data using somatic cell hybrids. The known localizations of at least 14 functional genes and several disease loci to 11q22-23, including ataxia telangiectasia, make the mapping of this region especially relevant to studies of disease pathogenesis.