Showing papers by "Stefan Karlsson published in 1985"

Transfer of genes into hematopoietic cells using recombinant DNA viruses.

Abstract: The ability of recombinant DNA viruses to transfer genes into hematopoietic cells has been explored. A recombinant simian virus 40 (SV40) in which the early region had been replaced with the chloramphenicol acetyltransferase (CAT) gene driven by the promoter from Rous sarcoma virus (RSV), was constructed. This virus transferred the CAT gene more efficiently into mouse and human bone marrow cells and into the K562, MEL, and WEHI hematopoietic tissue culture cell lines, than the classical calcium phosphate DNA transfer procedure, as shown by assay for CAT activity 48 hr after infection. Recombinant SV40 virions were also shown to be capable of stably transforming Chinese hamster ovary cells by use of an early region recombinant containing the methotrexate-resistant dihydrofolate reductase (DHFR) gene driven by the RSV promoter. The entire DHFR transcriptional unit could be detected in the genome of transformed cells that were also shown to be resistant to methotrexate. A recombinant adenovirus stock containing the neomycin-resistance gene driven by the SV40 early promoter was used to infect the K562 and MEL hematopoietic cell lines to resistance to the antibiotic G418. Transformation frequency was 10- to 100-fold higher than that obtained with calcium phosphate-precipitated DNA. Most or all of the recombinant adenovirus genome was integrated as 1-3 copies in the transformed cells. These studies show the feasibility of using DNA viruses for introduction of new genetic material into hematopoietic cells.

Cis-acting sequences that affect the expression of the human fetal gamma-globin genes.

Abstract: We have identified the sequences in the human gamma-globin gene promoter that are required for efficient and accurate transcription by using deletion mutants and in vitro site-directed mutagenesis. More than 131 bp (which include the 'CACA' sequence) upstream from the Cap site, are required for efficient transcription of the gamma-promoter. Furthermore, the 27 bp tandemly duplicated segment including the conserved 'CCAAT' box (represented only once in the beta promoter and required for its function) is not essential for the gamma-promoter, since scanning-linker mutants lacking part or all of the proximal 'CAT' box exhibited a 1.5 to 4.0 fold increase in the promoter function compared to the wild type. These data and the recently described point mutations in the regions of the A gamma and G gamma promoters associated with hereditary persistence of fetal hemoglobin, support the notion that the DNA sequences of the gamma-promoter region should be involved in the developmental regulation of globin genes. Molecular analysis of naturally occuring deletion-mutations in the beta-globin gene cluster leading to increased production of HbF in adult life, showed that irrespective of the nature of the deletions, they exhibit identical clinical and cellular phenotypes on interaction with the beta S gene. Erythroid progenitor cells from these individuals respond in vitro, to the same extent to the modulating effects of exogenous factors such as the 'switching activity' of fetal sheep serum with respect to their pattern of hemoglobin synthesis.