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Biomedical subjects

R A Flavell

Publications and source records attributed to R A Flavell.

At least 361 records · Page 20Linked to original sources
PubMed1983

Structure of the murine immune response I-A beta locus: sequence of the I-A beta gene and an adjacent beta-chain second domain exon.

The murine major histocompatibility complex I region encodes two class II antigens, I-A and I-E. From a mouse spleen DNA cosmid library of the b haplotype, we isolated a clone containing the entire I-A beta gene and a separate exon encoding a beta-chain second domain (A beta 2). The A beta gene, encompassing more than 6 kb, is encoded by six exons corresponding to the different domains of the A beta polypeptide. The translated A beta amino acid sequence displays 73% homology to human DC beta chains; homologies to other subsets of human beta chains are lower, establishing that I-A corresponds structurally to DC. The A beta 2 exon is about 20 kb centromeric to the A beta gene. Its translated amino acid sequence includes all the conserved amino acids of other class II beta-chain second domains. It shows about 60% homology to each of three subsets of human beta chains available for comparison, and to the A beta chain. No A beta 2 first domain exon has been detected with A beta or DC beta probes.

Amino Acid Sequence↗
PubMed1983

DNA methylation and the regulation of globin gene expression.

We have studied the effect of DNA methylation on human gamma-globin gene expression, using a novel in vitro DNA methylation technique. With this method we have methylated specific segments of the gamma-globin gene and its 5'-flanking region, which were cloned for this purpose in the bacteriophage vector M13mp8. The vitro methylated DNA was introduced into mouse L-cells by cotransfer using the herpes simplex virus thymidine kinase gene as a selective marker. Transformed cell lines were isolated to examine the inheritance of the segmental methylation pattern and its effect on the expression of the gamma-globin gene. Cells transformed with DNA methylated throughout the M13 vector and gamma-globin DNA sequences do not express the gamma-globin gene. Methyl-C residues in either the M13 DNA sequences or the gamma-globin structural gene have, however, no inhibitory effect on globin transcription. In contrast, methylation in the 5' region of the gamma-globin gene (from nucleotides -760 to +100) prevents transcription. These data indicate that DNA methylation in the 5' region of a gene might play a direct role in the regulation of gene expression.

5-Methylcytosine↗
PubMed1982

The construction of cosmid libraries which can be used to transform eukaryotic cells.

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.

Animals↗
PubMed1982

Sequence requirements for the transcription of the rabbit beta-globin gene in vivo: the -80 region.

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.

Animals↗
PubMed1982

Expression of murine H-2Kb histocompatibility antigen in cells transformed with cloned H-2 genes.

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.

Animals↗
PubMed1982

Isolation and characterization of a human collagen alpha 1(I)-like gene from a cosmid library.

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.

Base Sequence↗
PubMed1982

The DNA sequence of the 5' flanking region of the human beta-globin gene: evolutionary conservation and polymorphic differences.

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.

Animals↗
PubMed1982

DNA sequences necessary for transcription of the rabbit beta-globin gene in vivo.

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.

Animals↗