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Biomedical subjects

R G Filby

Publications and source records attributed to R G Filby.

6 recordsLinked to original sources
PubMed1990

Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15.

We report the isolation of two distinct subfamilies of alpha satellite DNA (pTRA-20 and -25) from human chromosome 15. In situ hybridization experiments indicated that both subfamilies are highly specific for this chromosome. Southern analysis of a somatic hybrid cell line carrying human chromosome 15 revealed a likely higher-order genomic band of 2.5 kb for pTRA-20. Similar analysis for pTRA-25 showed multiple higher-order bands of 3.5, 4.5, and 5 kb at moderately high hybridization stringency, but a predominance of the 4.5-kb species at very high stringency. Direct comparison with human genomic DNA confirmed the authenticity of these higher-order structures and demonstrated polymorphic variations using both probes. The origin of the different alphoid subfamilies on chromosome 15 is discussed. These sequences should be useful for the construction of centromere-based genetic linkage maps for human chromosome 15 and, in conjunction with the other alphoid sequences already reported for chromosomes 13, 14, 21, and 22, should allow a concerted analysis of the evolution and the possible etiological role of these DNAs in aberrations commonly seen in these chromosomes.

Blotting, Southern↗
PubMed1988

Homologous alpha satellite sequences on human acrocentric chromosomes with selectivity for chromosomes 13, 14 and 21: implications for recombination between nonhomologues and Robertsonian translocations.

We report a new subfamily of alpha satellite DNA (pTRA-2) which is found on all the human acrocentric chromosomes. The alphoid nature of the cloned DNA was established by partial sequencing. Southern analysis of restriction enzyme-digested DNA fragments from mouse/human hybrid cells containing only human chromosome 21 showed that the predominant higher-order repeating unit for pTRA-2 is a 3.9 kb structure. Analysis of a "consensus" in situ hybridisation profile derived from 13 normal individuals revealed the localisation of 73% of all centromeric autoradiographic grains over the five acrocentric chromosomes, with the following distribution: 20.4%, 21.5%, 17.1%, 7.3% and 6.5% on chromosomes 13, 14, 21, 15 and 22 respectively. An average of 1.4% of grains was found on the centromere of each of the remaining 19 nonacrocentric chromosomes. These results indicate the presence of a common subfamily of alpha satellite DNA on the five acrocentric chromosomes and suggest an evolutionary process consistent with recombination exchange of sequences between the nonhomologues. The results further suggests that such exchanges are more selective for chromosomes 13, 14 and 21 than for chromosomes 15 and 22. The possible role of centromeric alpha satellite DNA in the aetiology of 13q14q and 14q21q Robertsonian translocations involving the common and nonrandom association of chromosomes 13 and 14, and 14 and 21 is discussed.

Base Sequence↗
PubMed1987

Genomic organization of human centromeric alpha satellite DNA: characterization of a chromosome 17 alpha satellite sequence.

We characterized a recombinant clone E7 containing a 1.6-kb Eco RI insert of human alpha satellite DNA (alpha DNA) which hybridized in situ predominantly to the centromere of chromosome 17. Three thousand copies of this sequence were detected on chromosome 17, although a lesser number of copies were also found on the centromeres of chromosomes 11, X, and the other human chromosomes, except Y. In the human genome, sequences homologous to E7 were organized principally as five major polymorphic (Pst I) forms of tandem alpha DNA repeats with molecular weights between 2.0 and 2.7 kb. We We studied the higher-order organization of these major forms using a series of 12 cosmid clones. Close linkage of the different polymorphic forms was demonstrated, with no two cosmids showing an identical linkage pattern. Six of the cosmid clones carried a considerable amount (20-25%) of nonhomologous (non-alpha) DNA, indicating that the repeat arrays are relatively frequently interrupted by other genomic DNA. In none of the cosmid inserts were the repeat arrays bound on both sides by non-alpha DNA, suggesting that short arrays are not common. However, some of the intervening non-alpha DNA sequences were relatively short, and vary in size from 6 to 24 kb. Our results suggest an irregular and complex pattern of organization of alpha DNA in the human genome.

Chromosome Mapping↗
PubMed1986

Vectors for expression and amplification of cDNA in mammalian cells: expression of rat phenylalanine hydroxylase.

We have constructed two recombinant plasmid vectors for direct expression and amplification of cDNA in mammalian cells. Each vector carries two dominant selectable markers (the bacterial neo gene and the mouse DHFR gene), a promoter sequence (viral LTR in pAV009/A+, and sheep metallothionein promoter in pMT010/A+), a polyadenylation signal sequence, and a Bam HI site to allow insertion of cDNA. We have used these vectors to prepare recombinant clones for the expression of rat phenylalanine hydroxylase (PH) in LTK- cells. Selection of transformants with neomycin followed by selection of the transformants in methotrexate led to a 30- to 60-fold amplification of the DHFR marker and co-amplification of the PH cDNA, with a corresponding increase in the level of PH mRNA and enzyme polypeptide. The expressed enzyme has a subunit molecular weight of 50,000 which corresponds to the W- allele of rat liver PH. PH activity was detected in the transfected cells by enzymatic measurement of the conversion of [14C]phenylalanine to [14C]tyrosine, and by growth of these cells in a tyrosine-free culture medium. Expression of rat PH in cell culture should facilitate the analysis of the biochemical properties of this enzyme.

Animals↗