The protein kinase inhibitor 2-aminopurine (2AP) blocks the induction of the human beta-interferon gene by virus or poly(I)-poly(C) at the level of transcription. This inhibition is specific, since 2AP does not inhibit induction of either the hsp70 heat-shock gene by high temperature or the metallothionein gene by cadmium or dexamethasone. However, 2AP does block the induction of the c-fos and c-myc proto-oncogenes by serum growth factors or virus, suggesting that a protein kinase may be involved in the regulation of these genes, as well as of the beta-interferon gene.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
March 1988
Virus or poly(I).poly(C) induction of human beta-interferon gene expression requires a 40-base-pair DNA sequence designated the interferon gene regulatory element (IRE). Previous studies have shown that the IRE contains both positive and negative regulatory DNA sequences.
View Article and Find Full Text PDFProc Natl Acad Sci U S A
August 1987
The product of the Drosophila segmentation gene Krüppel was produced in cultured insect cells using the baculovirus expression system. When a cloned Krüppel cDNA sequence was inserted into the viral genome downstream from the promoter of the polyhedrin gene, a polypeptide with an apparent molecular weight of approximately equal to 72,000 was observed in the nuclei of infected cells. Antibodies were raised against this protein and used to detect Krüppel in Drosophila embryos.
View Article and Find Full Text PDFA G to T transversion at the fifth nucleotide of the first intervening sequence (IVS-1) of the beta-globin gene has been identified in cloned beta-thalassemia genes of two unrelated individuals, one of Mediterranean and the other of Anglo Saxon ancestry. In each patient the mutation was present in a different beta globin gene framework, defined by intragenic restriction site polymorphisms, thereby suggesting the occurrence of independent mutations. The study of the RNA products of one of these cloned genes, after transfer and transient expression in HeLa cells, showed partial inactivation of the normal donor splice site of IVS-1 and activation of two major and one minor cryptic splice sites.
View Article and Find Full Text PDFMolecular genetics approaches have been used to identify and characterize cis-acting DNA sequences required for eukaryotic gene regulation. These sequences are modular in nature, consisting of arrays of short (10- to 12-base pair) recognition elements that interact with specific transcription factors. Some transcription factors have been extensively purified and the corresponding genes have been cloned, but the mechanisms by which they promote transcription are not yet understood.
View Article and Find Full Text PDFA small set of distinctive short RNA molecules are found in the nuclei of all higher eukaryotic cells and yeast, in protein complexes known as 'small nuclear ribonucleoprotein particles', or snRNPs. Recent work has confirmed early suggestions that these particles form part of the machinery by which primary RNA transcripts are processed to their mature, functional form. In particular, snRNPs have been shown to be an integral part of the 'spliceosome', a multi-component complex involved in the removal of intron sequences from the coding regions of messenger RNA precursors.
View Article and Find Full Text PDFTo evaluate the bioavailability of a new theophylline preparation suitable for once-a-day (od) oral administration, we performed a nonrandomized crossover study in which the absorption of the OD and a standard twice-a-day (bid) preparation were compared. Eight stable asthmatic patients, after having achieved steady-state, received an average of 975 mg of OD preparation at 8 PM. The protocol was later repeated with the same subjects receiving 487.
View Article and Find Full Text PDFAnalysis of the in vitro splicing products of RNA precursors containing tandem duplications of the 5' or 3' splice sites of human beta-globin IVS 1 revealed that exon sequences play an important role in the relative use of the duplicated sites. These studies also show that the proximity of the 5' and 3' splice sites is an important determinant in the selection of splice-sites. Deletion, substitution, or even subtle changes of exon sequences can alter the pattern of splice-site selection, and in many cases the splice site adjacent to the altered exon is not used.
View Article and Find Full Text PDFInterspecific heterokaryons were formed by fusing adult mouse erythroleukemia (MEL) cells and human embryonic/fetal erythroid (K562) cells with each other, or with a variety of mouse and human nonerythroid cell types. Analysis of total cellular RNA isolated 24 hr after fusion revealed that normally inactive globin genes can be activated in these "transient" heterokaryons, in which the nuclei do not fuse. In general, the types of globin genes expressed in the donor erythroid cell are activated in the nucleus of the recipient cell.
View Article and Find Full Text PDFTuberculosis has not been well documented as a complication of the acquired immunodeficiency syndrome (AIDS). We studied 48 cases of mycobacterial diseases among a group of 136 adult patients with AIDS over a 43-month period. Twenty-nine of them had severe and unusual manifestations of disease due to Mycobacterium tuberculosis, predominantly extrapulmonary and disseminated.
View Article and Find Full Text PDFAlcohol dehydrogenase (ADH) is expressed in a complex temporal and spatial pattern from tandem promoters (proximal and distal) in Drosophila melanogaster, and from two closely linked genes (Adh-1 and Adh-2) in D. mulleri. The expression patterns of Adh-1 and the proximal promoter, and Adh-2 and the distal promoter are similar, but not identical.
View Article and Find Full Text PDFWe have used a DNAase I genomic footprinting procedure to detect interactions between cellular factors and the regulatory sequences of the human beta-interferon gene. Prior to induction with poly(I)-poly(C), factors that bind to DNA are detected in one region located between -94 and -167 from the mRNA cap site, and in another region located between -68 and -38. After induction these factors dissociate and another factor binds to a region located between -77 and -64.
View Article and Find Full Text PDFThe human beta-interferon gene is regulated by an inducible enhancer element. Analysis of the effect of deletions within this element on beta-interferon transcription indicates that this enhancer is under negative control. Deletion of sequences from the 3' end of the enhancer leads to a dramatic increase in the basal level of beta-interferon mRNA and a decrease in the induction ratio.
View Article and Find Full Text PDFA novel procedure for saturation mutagenesis of cloned DNA was used to obtain more than 100 single base substitutions within the promoter of the mouse beta-major globin gene. The effects of these promoter substitutions on transcription were determined by transfecting the cloned mutant genes into HeLa cells on plasmids containing an SV40 transcription enhancer, and measuring the levels of correctly initiated beta-globin transcripts after 2 days. Mutations in three regions of the promoter resulted in a significant decrease in the level of transcription: (i) the CACCC box, located between -87 and -95, (ii) the CCAAT box, located between -72 and -77, and (iii) the TATA box, located between -26 and -30 relative to the start site of transcription.
View Article and Find Full Text PDFMol Cell Biol
March 1986
beta-Interferon (beta-IFN) gene expression can be induced by poly(I)-poly(C) or virus, but there is considerable variation in the extent of induction between different cell lines. We characterized two poorly inducible human cell lines, HeLa and 143 thymidine kinase negative (143 tk-), to define cellular factors involved in the activation of the beta-IFN gene. We show that the deficiency in beta-IFN induction in these cells can be complemented by fusion to highly inducible mouse cells.
View Article and Find Full Text PDFCold Spring Harb Symp Quant Biol
June 1987
Studies on the effects of thalassemic mutations on gene function in vivo have clinical as well as scientific implications. Usually these studies have been performed on nucleated red blood cell (RBC) precursors normally present in bone marrow. Many patients with beta-thalassemia are splenectomized and may have high levels of nucleated RBC, orthochromatic normoblasts, in their peripheral blood (1-5% of total RBC).
View Article and Find Full Text PDFSingle base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis.
View Article and Find Full Text PDFNucleic Acids Res
October 1985
The D. melanogaster Adh gene is transcribed from two different promoters; a proximal (larval) promoter is active during late embryonic and larval stages, and a distal (adult) promoter is active primarily in third instar larvae and in adult flies (1). Genetic analyses suggest that several species of the mulleri subgroup (distant relatives of D.
View Article and Find Full Text PDFWe have identified six distinct factors necessary for pre-mRNA splicing in vitro by selective inactivation and complementation studies, and by fractionation procedures. Splicing factor 1 (SF1) is sensitive to micrococcal nuclease, and appears to consist of at least U1 and U2 snRNPs, since splicing is inhibited when the 5' termini of U1 and U2 snRNAs are removed by site-directed cleavage with RNAase H. SF2 is a micrococcal nuclease-resistant factor present in the nuclear extract but absent from an S100 extract.
View Article and Find Full Text PDFA new procedure for generating and isolating random single-base substitutions in cloned DNA fragments is presented. The mutations are generated by treatment of single-stranded DNA with various chemicals, followed by the synthesis of the complementary strand with reverse transcriptase. Misincorporation frequently occurs when the enzyme encounters a damaged base in the mutagenized template DNA.
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