Showing papers by "Richard A. Flavell published in 1982"
TL;DR: Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method and newly introduced genes are expressed accurately in L cells.
Abstract: Cosmid vectors have been developed which carry selective markers for growth in bacteria (beta lactamase gene) and animal cells (the Herpes Simplex virus thymidine kinase gene, the transposon Tn-5 aminoglycosyl 3' phosphotransferase gene and the E. coli guanine phosphoribosyltransferase gene). The design of the cosmids allows the exchange of the eukaryotic markers in recombinant cosmids. Human and mouse cosmid libraries containing DNA inserts of about 40kb have been generated by an improved method. Several clones from the human beta-globin locus were isolated. These cosmids transform mouse L cells at high efficiency in both circular and linear form. The newly introduced genes are expressed accurately in L cells.
266 citations
TL;DR: Comparison of the level of β-globin transcripts in a variety of deletion mutants shows that for efficient transcription, both the ATA or Goldberg–Hogness box, and a region between 100 and 58 base pairs in front of the site at which transcription is initiated, are required.
Abstract: The DNA sequences required for the expression of the rabbit-beta-globin gene in vivo have been examined. A variety of mutant rabbit beta-globin gene templates were linked to a simian virus 40-plasmid recombinant and introduced into HeLa cells; in these conditions the rabbit beta-globin gene is expressed from its own promoter. Comparison of the level of beta-globin transcripts in a variety of deletion mutants shows that for efficient transcription, both the ATA or Goldberg-Hogness box, and a region between 100 and 58 base pairs in front of the site at which transcription is initiated, are required. Deletion of either of these regions results in a decrease in the level of beta-globin transcripts by an order of magnitude; deletion of the ATA box causes an additional loss in the specificity of the site of initiation of RNA synthesis. The DNA sequences downstream from the ATA box, including the natural beta-globin mRNA cap site, are dispensable for transcription in vivo.
230 citations
TL;DR: Results show that expression of the H–2Kb gene product on the L-cell surface is sufficient to make it a target for specific T-cell killing.
Abstract: Expression of marine H–2Kb histocompatibility antigen in cells transformed with cloned H–2 genes
84 citations
TL;DR: Analysis of DNA sequences in the -80 region of the rabbit beta-globin gene shows that the conserved GGCCAATCT sequence is required for efficient transcription in vivo and further identifies another sequence in the region from about -81 to -96 which is also required for transcription in vitro.
Abstract: We have performed a detailed analysis of DNA sequences in the -80 region of the rabbit beta-globin gene that are required for transcription. A variety of rabbit beta-globin gene templates deleted at various sites in the -80 region were linked to an SV40-plasmid recombinant and introduced into HeLa cells; under these conditions the rabbit beta-globin gene is expressed from its own promotor. The results show that the conserved GGCCAATCT sequence is required for efficient transcription in vivo and further identify another sequence in the region from about -81 to -96 which is also required for transcription in vivo.
73 citations
TL;DR: The DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene is determined and it is not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.
Abstract: We have determined the DNA sequence of a 1464 bp segment immediately flanking the 5' side of the human beta-globin gene. The sequence shows little similarity to the corresponding regions of the epsilon- or gamma-globin genes. There is about 75% homology, however, between the 5' extragenic regions of the beta-globin genes of man, goat and rabbit respectively. The mouse beta minor globin gene, but not the mouse beta major globin gene, also shares this extensive homology. A short segment of simple sequence DNA is found from about 1418 to 1388 bp upstream from the human beta-globin gene which consists of repeats of the sequence (TTTTA). Similar DNA sequences are also found at several sites in the large intron of the beta-globin gene. We have compared the DNA sequence of the 5' extragenic region of the normal beta-globin gene with the same segment of the beta-globin gene of a patient with beta thalassaemia. Of the two nucleotide differences observed, one generates a polymorphic HinfI site present 990 bp upstream from the beta-globin gene in the thalassaemic beta-globin and absent in the normal gene. A second beta thalassemic beta-globin gene which has the same molecular defect as the above mentioned case, however, lacks this HinfI site. It is therefore not yet clear whether this HinfI site will have any value in prenatal diagnosis of beta thalassaemia.
52 citations
TL;DR: Results show that expression of the H-2Kb gene product on the L-cell surface is sufficient to make it a target for specific T-cell killing.
Abstract: Cosmids containing H-2 histocompatibility antigen genes of the H-2b haplotype have been isolated. One of these genes expresses a 45,000 molecular weight protein, indistinguishable from H-2Kb when introduced into mouse L cells. These H-2Kb transformed L cells can be killed by allospecific anti-H-2Kb cytotoxic T cells. Moreover, when infected with influenza virus, they can be killed by an H-2Kb-restricted, influenza virus-specific cytotoxic T cell line. These results show that expression of the H-2Kb gene product on the L-cell surface is sufficient to make it a target for specific T-cell killing.
43 citations
TL;DR: This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.
Abstract: A procedure has been developed to allow the recovery of an integrated plasmid genome from a transformed cell, together with large areas of the flanking DNA sequences. DNA from Saccharomyces cerevisiae BAS2, in which the pBR322--ura 3 plasmid (Y1p5) is integrated at the yeast histone H2A and H2B locus, was used to generate a cosmid library, using a new cosmid vector (pTL5) that is ampicillin sensitive and tetracycline resistant. Colonies were selected for ampicillin resistance, which was conferred by the incorporation of the integrated pBR322 beta-lactamase gene into the recombinant cosmid. Restriction enzyme and blot hybridization analyses show that the rescued clones contain the yeast histone genes in addition to the Y1p5 sequences; a total of approximately 50 kilobase pairs of DNA sequences flanking the plasmid was recovered as a series of overlapping cosmids. This approach should allow the recovery of most genes that can be linked to a marker pBR322 sequence and for which a specific phenotype can be selected in a recipient eukaryotic cell.
40 citations
TL;DR: A human collagen alpha 1(I)-like gene from a cosmid library is isolated and shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations.
Abstract: We have isolated a human collagen alpha 1(I)-like gene from a cosmid library. The clone which contains 37kb of human DNA has been shown to contain this gene by DNA sequencing, hybrid arrest and hybrid selection assays and Northern blot hybridizations. The collagen gene sequence extends through most of the cloned DNA and must, therefore, be at least 35kb in length.
38 citations
TL;DR: The 37 Kb cosmid H col. 1, which contains the human collagen α1(I) gene, was used as a probe for detecting Restriction Fragment Length Polymorphisms (RFLP's) and a highly polymorphic Hind III site in the gene was found on chromosome No. 7, using competitive hybridization techniques.
Abstract: The 37 Kb cosmid H col. 1, which contains the human collagen α1(I) gene, was used as a probe for detecting Restriction Fragment Length Polymorphisms (RFLP's). A highly polymorphic Hind III site in the gene was found on chromosome No. 7, using competitive hybridization techniques.
6 citations
4 citations
01 Jan 1982
TL;DR: It is shown that an H-2K b gene when introduced into mouse L cells renders these cells a target for both allospecific and H- 2 restricted, virus-specific cytotoxic T cell killing.
Abstract: The use of DNA-mediated gene transfer for the study of the rabbit s-globin promotor, the molecular basis of s + thalassaemia and the specific induction of human s-globin expression in mouse erythroid cells is described. In addition, we describe the isolation of the class I mouse MHC genes from the H-2 b haplotype. We further show that an H-2K b gene when introduced into mouse L cells renders these cells a target for both allospecific and H-2 restricted, virus-specific cytotoxic T cell killing.