RESUMO
Shotgun collections of Charon 3A bacteriophages containing Eco RI fragments of human and mouse DNA were constructed with the use of in vitro packaging. Plaques were screened by hybridization, and globin-specific clones were isolated from both human (Charon 3AHs51.1) and mouse (Charon 3AMm30.5). The fragments cloned were detected in unfractionated genomic DNA by the Southern method of hybridization.
Assuntos
Genes , Globinas/genética , Animais , Colífagos/genética , Enzimas de Restrição do DNA , DNA Recombinante , Hemoglobina Fetal/genética , Humanos , Métodos , Camundongos , Hibridização de Ácido Nucleico , Poli A , Poli TRESUMO
The Charon lambda bacteriophages have been developed as vectors for cloning. Their construction incorporates mutations that make them simple to use and also greatly increases their safety for the biological containment of cloned recombinant DNA. Three of the Charon vector phages, 3A, 4A, and 16A, have been certified for use as EK2 vector-host systems, when propagated in bulk in a special bacterial host, DP50SupF. We present here some of the data on which the safety of these systems was evaluated. DNA fragments ranging in size from 0 to 2.2 X 10(4) base pairs can be cloned in these EK2 Charon phages.
Assuntos
Colífagos/metabolismo , DNA Recombinante/metabolismo , DNA Viral/metabolismo , Projetos de Pesquisa/normas , Mapeamento Cromossômico , Enzimas de Restrição do DNA/metabolismo , DNA Bacteriano/metabolismo , Galactosidases/metabolismo , Genes , Lisogenia , Peso Molecular , Mutação , Terminologia como Assunto , Transcrição Gênica , Replicação ViralRESUMO
Restriction fragments of Epstein-Barr virus (EBV; B95-8) DNA were cloned in the Tc gene of pBR322 (HindIII-F, -G, -I, -J, -K, -L, and -M) and in Charon3A (EcoRI-GI and -G2). Altogether these cloned fragments covered 39% of the entire viral genome. The cloned EcoRI-G2 fragment of EBV (B95-8) DNA was shown to contain, in addition to HindIII-J, two more HindIII-fragments : HindIII-M, which had not been located on the linkage map of the viral genome (Given and Kieff, 1978) and HindIII-N, which had been unrecognized up to now. The utility of this cloning method is discussed in regard to the detailed mapping of a viral genome and large-scale production of the viral DNA.
Assuntos
DNA Viral/genética , Herpesvirus Humano 4/genética , Bacteriófago lambda/genética , Clonagem Molecular/métodos , Enzimas de Restrição do DNA/metabolismo , Escherichia coli/genética , PlasmídeosRESUMO
We have developed an autoradiographic/electrophoretic assay capable of distinguishing mouse and human beta2-microglobulin (beta2m) in spent culture media. The method is applicable to mouse and human lines and to hybrid cell lines made from them. With this technique, mouse/human hybrid cell lines were tested for the presence of human beta2m. Isozyme and karyotype analyses were also carried out with the hybrids. The combined results of these studies show that the structural gene for human beta2m is on the long arm of chromosome 15.