http://repub.eur.nl/res/aut/8276/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/2388/ 1985-01-01T00:00:00Z The class I major histocompatibility complex (MHC) antigens are highly polymorphic cell-surface proteins whose expression is essential for the cellular immune response against virus-infected, abnormal and foreign cells. Transformation of primary rat cell cultures by the oncogenic adenovirus 12 (Ad12) results in suppression of the transplantation antigens, thus enabling the transformed cells to escape the immune response and efficiently form tumours in vivo. In contrast, transformation of the same cells with the non-oncogenic adenovirus 5 (Ad5) does not suppress the transplantation antigens and, consequently, they elicit an effective (MHC-restricted) immune response. Here, however, we show that infection of mouse embryo cells with both viruses initially increases the level of transcripts from the H-2Kb transplantation antigen gene. Both the adenovirus E1a (12S RNA) and E1b genes are required for activation of the H-2K gene and measurement of the relative rate of transcription indicates that the increase in the level of H-2K messenger RNA following infection is at least in part due to a gene-specific transcriptional activation. The newly transcribed H-2Kb mRNA is then properly transported to the cytoplasm. http://repub.eur.nl/res/pub/2374/ 1984-01-01T00:00:00Z The transplantation antigens H-2K, H-2D and H-2L are developmentally regulated, highly polymorphic cell surface proteins encoded by the major histocompatibility gene complex (MHC). First detectable on the early embryo, they are subsequently expressed on most somatic cells of the adult mouse in association with the protein beta2-microglobulin (beta 2 M; ref. 5). Cultured F embryonal carcinoma (EC) cells can be induced to differentiate along alternative pathways to form either parietal or visceral9 extra-embryonic endoderm, each concomitant with a change in morphology and pattern of gene expression. Previous reports have demonstrated an increased level of transplantation antigens in differentiated F9 EC cells, but the cell types expressing them were not defined. Here we show that the level of MHC H-2Kb and beta 2 M transcripts is increased during both pathways of this differentiation. Expression of a foreign MHC H-2Kbm1 gene was found to be regulated in a similar manner when the gene was introduced into EC cells. In contrast, an introduced rabbit beta-globin gene was not regulated but expressed constitutively. http://repub.eur.nl/res/pub/2375/ 1984-01-01T00:00:00Z We introduced into MEL cells rabbit beta-globin gene deletion mutants and two sets of hybrid genes constructed from the inducible human beta-globin gene and noninducible human gamma-globin gene or the murine H-2Kbm1 class I MHC gene. S1 nuclease analysis of gene transcripts before and after MEL differentiation showed that induction of the rabbit beta-globin gene did not require more than 58 bp of DNA 5' to the transcription initiation site. Hybrid genes were constructed with human beta-globin DNA sequences from either 5' or 3' of the translation initiation site linked to the complementary parts of the gamma or H2Kbm1 genes. Both types of constructs were inducible during MEL differentiation. The relative rates of transcription of the 5' gamma-3' beta and 5'H2-3' beta hybrid genes show that induction of the hybrid gene transcripts results at least in part from transcriptional activation of the genes. We suggest that DNA sequences that regulate beta-globin gene transcription during MEL differentiation are located both 5' and 3' to the translation initiation site. http://repub.eur.nl/res/pub/2376/ 1984-01-01T00:00:00Z We have introduced into murine erythroleukaemia (MEL) cells a series of human globin gene cosmids and two sets of hybrid genes constructed from the human beta-globin gene and the human gamma-globin or murine H-2Kbm1 genes. S1-nuclease analysis of the mRNA products from these genes before and after MEL cell differentiation showed that the human beta-globin gene, but not the human epsilon- or gamma-globin or H-2Kbm1 genes, is induced specifically. Hybrid genes containing human beta-globin DNA sequences from either 5' or 3' side of the translation initiation site were both inducible. Measurement of the relative rate of transcription showed this induction to be the result of transcriptional activation. We therefore suggest that DNA sequences which regulate beta-globin gene expression during MEL differentiation are located both 5' and 3' to the translation initiation site. http://repub.eur.nl/res/pub/2366/ 1983-01-01T00:00:00Z MspI essentially fails to cut the sequence GGCmCGG at enzyme concentrations which give total digestion of CCGG, CmCGG and GGCCGG sites. This result explains why certain sites in mammalian DNA are resistant to both MspI and HpaII and shows that this results from an idiosynchracy of MspI rather than a novel form of DNA methylation at this site in mammalian cells. http://repub.eur.nl/res/pub/2367/ 1983-01-01T00:00:00Z Chemically induced differentiation of cultured murine erythroleukaemia (MEL) cells results in a several hundred-fold increase in transcription of the adult mouse globin genes and thus serves as a model for gene activation during erythropoiesis. One approach to study gene regulation in this system has been to analyse the expression of foreign globin genes introduced into MEL cells. By introducing cosmid DNA containing the human adult(beta), fetal(gamma) and embryonic(epsilon)-globin genes, we have shown here that expression of the beta, but not the gamma or epsilon genes, is regulated during MEL differentiation. Regulated expression of the human beta-globin gene was observed when it was introduced either as part of the intact globin gene cluster or as an individual gene with 1.5 kilobases (kb) of 5' flanking DNA. Transcription from a herpes simplex virus (HSV) promoter adjacent to the thymidine kinase (tk) gene is also inducible in MEL cells. http://repub.eur.nl/res/pub/2347/ 1981-01-01T00:00:00Z We have cloned the single beta-globin gene from an Italian patient who is a double heterozygote for beta o/delta beta o thalassaemia. RNA isolated from nucleated red cells from this patient can be translated in vitro to give detectable levels of A gamma- G gamma and alpha-globin, but no beta-globin. S1-mapping transcription studies show that beta-globin mRNA is present at about 1-3% of the level of alpha- and gamma-globin mRNA. In addition, the expression of this gene has been studied by reversed genetics. SV40-plasmid-beta o-globin gene recombinants have been transfected into Hela cells and analysed for beta-globin mRNA. In contrast to the results obtained with mRNA isolated directly from the blood of this patient, in the transfected Hela cells the same level of beta-globin mRNA is seen for the beta o thalassaemic globin gene and for a normal beta-globin gene. To elucidate the nature of the lesion, the entire DNA sequence of the beta-globin gene of this patient has been determined. The sequence shows that this gene contains a termination codon at position 39 (CAG - greater than UAG). Otherwise, there is a remarkable conservation of the entire DNA sequence.