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D Melloul

Publications and source records attributed to D Melloul.

25 records · Page 2Linked to original sources
PubMed1986

Muscle-specific activation of a methylated chimeric actin gene.

To understand how DNA methylation affects tissue-specific activation of genes, we have transfected in vitro methylated alpha-actin (skeletal) constructs into fibroblasts, which do not produce endogenous alpha-actin, and into a myogenic line, which is inducible for alpha-actin expression. Although methylation significantly inhibits the expression of these constructs in fibroblasts, it does not in myoblasts. The methylation pattern of the introduced methylated genes reveals specific demethylations in the transfected molecules in myoblasts but not in fibroblasts, and it precisely mimics the methylation pattern found in myoblasts in vivo.

Actins↗
PubMed1984

Developmentally regulated expression of chimeric genes containing muscle actin DNA sequences in transfected myogenic cells.

A recombinant plasmid containing 2/3 of the rat skeletal muscle actin structural gene plus 730 bp of its 5' flanking region, spliced to the 3' end of the human epsilon-globin gene, was introduced into cells of the rat myogenic line L8. Myogenic clones carrying the actin/globin chimeric gene were isolated. In many of these clones, the expression of the gene greatly increased during differentiation (up to greater than 50-fold) and, in some clones, the amount of the chimeric gene transcripts in the differentiated cultures exceeded that of the native muscle actin gene transcripts. Furthermore, the temporal relation between differentiation of the cultures and the accumulation of the transcripts from the transferred genes was very similar to that of the native skeletal muscle actin gene, suggesting a similar mechanism of regulation. Endonuclease S1 analysis indicated a correct initiation and termination of the mRNA but suggested that a fraction of the chimeric actin/globin transcripts was not properly processed. To test whether the increased expression of the transferred gene which occurred during differentiation was determined by DNA sequences in the 5' region of the muscle actin gene, a plasmid (p alpha-CAT) containing 730 bp of the 5' flanking region of the rat skeletal muscle actin gene (plus the exon of the 5' untranslated region, and 25 bp of the first intron), spliced to the bacterial structural gene coding for chloramphenicol acetyl transferase (CAT), was constructed and introduced into L8 cells. In the majority of the isolated clones containing this plasmid, CAT activity increased many-fold during differentiation.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1978

Interaction between membrane functions and protein synthesis in reticulocytes. Isolation of RNase M, a membrane component inhibiting protein synthesis through specific endonucleolytic activity.

An inhibitor of protein synthesis has been isolated from reticulocyte membranes by solubilization with Triton X-100; it has been purified using heat treatment, filtration on Amicon filters, DEAE-cellulose ion-exchange chromatography and Sephadex G-75 gel chromatography. A final purification of 120-fold was achieved. The purified inhibitor was found to be 95% homogenous when run on a dodecylsulfate/polyacrylamide gel system. Three independent methods were used to estimate the molecular weight of the purified inhibitor: Sephadex G-75 gel chromatography, dodecylsulfate/polyacrylamide gel electrophoresis and sucrose gradient all confirmed that the purified inhibitor was a small molecule with a sedimentation coefficient of 0.7 S and a molecular weight ranging between 5000 and 8000. The purified inhibitor was shown to possess a specific endonucleolytic activity, degrading the 28-S species of ribosomal RNA to species sedimenting between 10 and 14 S. Due to its membrane localisation the name RNase M is proposed. The purified inhibitor's endonucleolytic activity was characterized with regard to its kinetics, concentration dependence, pH optimum and its requirements for divalent cations. Kinetics showed that RNase M retained its specificity after 60 min of incubation with the RNA substrate. Specificity was also demonstrated by incubating the polysomal RNA with high concentrations of purified enzyme. The pH optimum was found to be between pH 6 and pH 7, and the enzyme did not require divalent cations for its activity. Pancreatic RNase B used at a similar protein synthesis inhibitory concentration as the RNase M caused a complete breakdown of ribosomal RNA to oligonucleotides and mononucleotides. The possible biological significance of the purified inhibitor in regulating protein synthesis in the maturing reticulocyte is discussed.

Animals↗
PubMed1978

Interaction between membrane functions and protein synthesis in reticulocytes: specific cleavage of 28-S ribosomal RNA by a membrane constituent.

A factor isolated from rabbit reticulocyte white ghosts by Triton X-100 treatment blocks protein synthesis at the elongation-termination stage. Factor-treated ribosomes were found to have an identical buoyant density to that of control ribosomes. When incubated with either reticulocyte ribosomes or ribosomal RNA, the factor products specific cuts in the 28-S ribosomal RNA compenent without damaging the 18-S RNA. Incubations of pancreatic or T1 RNase, with ribosomal RNA, at similar protein-synthesis inhibitory concentrations effected a complete breakdown to oligo and mononucleotides. When challenged with isolated 28-S or 18-S reticulocyte ribosomal RNA, the highly purified factor only attacked the 28-S RNA species. There was no accumulation of nucleotides or oligonucleotides and we concluded that the membrane factor causes inhibition of protein synthesis by having a specific endonucleolytic cleavage activity.

Animals↗