RESUMO
Human DNA restriction fragments containing high numbers of Alu repeat sequences can be preferentially detected in the presence of other human DNA restriction fragments in DNA from human: rodent somatic cell hybrids when the DNA is fragmented with enzymes that cleave mammalian DNA infrequently. This ability to lower the observed human DNA complexity allowed us to develop an approach to order rapidly somatic hybrid cell lines retaining overlapping human genomic domains. The ordering process also generates a relative physical map of the human fragments detected with Alu probe DNA. This process can generate physical mapping information for human genomic domains as large as an entire chromosome (100,000 kb). The strategy is demonstrated by ordering Alu-detected NotI fragments in a panel of mouse: human hybrid cells that span the entire long arm of human chromosome 17.
Assuntos
Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 17 , Variação Genética , Sequências Repetitivas de Ácido Nucleico , Humanos , Células Híbridas , Cariotipagem , Hibridização de Ácido NucleicoRESUMO
Genomic mapping studies frequently employ retrovirus-mediated transfer of dominant selectable markers to specific target chromosomes. DNA probes containing sequences adjacent to inserted proviruses are valuable mapping tools in such studies. We have implemented a strategy for amplification of chromosomal sequences flanking the 5' LTR of MoMuLV-based vectors. Probes derived from these amplification products successfully differentiated murine versus human proviral localization in retrovirus-infected mouse-human chromosome 17q hybrid cells.
Assuntos
DNA/isolamento & purificação , Vírus da Leucemia Murina/genética , Reação em Cadeia da Polimerase/métodos , Provírus/genética , Animais , Sequência de Bases , Southern Blotting , Linhagem Celular , Cromossomos Humanos Par 17 , Clonagem Molecular , Sondas de DNA , DNA Viral/isolamento & purificação , Ligação Genética , Vetores Genéticos , Humanos , Camundongos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
The enzyme glyceraldehyde-3-phosphate dehydrogenase from Drosophila melanogaster has been purified, and these preparations contain two subunits forms which have molecular weights of 37,000 and 35,500, respectively. Each subunit is found in crude extracts, and two activity bands are seen in nondenaturing acrylamide gels. Translation of Drosophila poly(A)-containing RNA results in two products which are precipitable with anti-glyceraldehyde-3-phosphate dehydrogenase serum. Two recombinant DNA clones have been isolated from a genomic library of Drosophila DNA. Each of these clones has the ability to hybrid select mRNAs which translate into both subunit forms. These clones have been genetically characterized by in situ hybridization and restriction mapping. One clone hybridizes to region 13F and the other to region 43E of the Drosophila cytogenetic map. Therefore, it appears that the Drosophila melanogaster genome contains two unlinked genes for glyceraldehyde-3-phosphate dehydrogenase; one of them encodes a protein of 37,000 daltons, the other a protein of 35,500 daltons.
