http://repub.eur.nl/res/aut/7883/ List of Publications en http://repub.eur.nl/static-eur/img/logo.png http://repub.eur.nl http://repub.eur.nl/res/pub/2352/ 1994-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/2350/ 1982-01-01T00:00:00Z http://repub.eur.nl/res/pub/2362/ 1982-01-01T00:00:00Z 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. http://repub.eur.nl/res/pub/2342/ 1981-04-01T00:00:00Z A human gene library was constructed using an improved cloning technique for cosmid vectors. Human placental DNA was partially digested with restriction endonuclease MboI; size-fractionated and ligated to BamHI-cut and phosphatase-treated cosmid vector pJB8. After packaging in lambda phage particles, the recombinant DNA was transduced into Escherichia coli 1400 or HB101 followed by selection on ampicillin for recombinant E. coli. 150 000 recombinant-DNA-containing colonies were screened for the presence of the human beta-globin related genes. Five recombinants were isolated containing the human beta-globin locus and encompassing approx. 70 kb of human DNA. 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. http://repub.eur.nl/res/pub/2354/ 1981-01-01T00:00:00Z The isolation of a human genomic cosmid hybrid containing the interferon beta gene has recently been reported (Gross et al., 1981). This hybrid was mapped using single and double digests and cross-hybridisation with the sub-cloned EcoRI and BgIII fragments. Purified fragments and subclones were used as hybridisation probes against chromosomal "Southern" blots to show that at least half of the region has been cloned without alteration. This cannot at present be confirmed for the rest of the region due to the presence of highly repetitive DNA on these cloned fragments. Sequencing data on the 5'-end of the fibroblast IFN-beta gene shows a high density of direct and inverted repeats. The IFN-beta mRNA coding region contains no intron, although the possibility of other transcription starts is not ruled out. The cloned region shows no similarities to known genomic clones containing IFN-alpha genes.