Eukaryotic gene expression can be regulated through selective translation of specific mRNA species. Nevertheless, the limited number of known examples hampers the identification of common mechanisms that regulate translation of specific groups of genes in mammalian cells. We developed a method to identify translationally regulated genes. This method was used to examine the regulation of protein synthesis in HL-60 cells undergoing monocytic differentiation. A partial screening of cellular mRNAs identified five mRNAs whose translation was specifically inhibited and five others that were activated as was indicated by their mobilization onto polysomes. The specifically inhibited mRNAs encoded ribosomal proteins, identified as members of the 5-terminal oligopyrimidine tract mRNA family. Most of the activated transcripts represented uncharacterized genes. The most actively mobilized transcript (termed TA-40) was an untranslated 1.3-kilobase polyadenylated RNA with unusual structural features, including two Alu-like elements. Following differentiation, a significant change in the cytoplasmic distribution of Alu-containing mRNAs was observed, namely, the enhancement of Alu-containing mRNAs in the polysomes. Our findings support the notion that protein synthesis is regulated during differentiation of HL-60 cells by both global and gene-specific mechanisms and that Alu-like sequences within cytoplasmic mRNAs are involved in such specific regulation.
The pathway for the utilization of L-arginine, agmatine, L-ornithine, and putrescine as the sole nitrogen source by Escherichia coli K-12 has been elucidated. Mutants impaired in the utilization of one or more of the above compounds were isolated, and their growth on the different compounds as a sole source of nitrogen and the activities of enzymes of the putative pathway were examined. Our results show that L-arginine is first decarboxylated to agmatine, which is hydrolyzed to urea and putrescine. L-Ornithine is decarboxylated to putrescine. Putrescine is transaminated to y-aminobutyraldehyde, which is oxidized to y-aminobutyric acid.-y-Aminobutyric acid is degraded to succinate.
Cationic polysaccharides were synthesized by conjugation of various oligoamines to oxidized polysaccharides by reductive amination and tested for antiprion activity. Polycations of dextran, pullulan and arabinogalactan grafted with oligoamines of 2 to 4 amino groups were investigated for their ability to eliminate PrP(Sc), the protease-resistant isoform of the prion protein, from chronically infected neuroblastoma cells, ScN2a-M. The proteinase K (PK)-resistant PrP elimination depends on both the concentration of the reagent and the duration of exposure. The most potent compound was found to be dextran-spermine that caused depletion of PrP(Sc) to undetectable levels at concentration of 31 ng/mL after 4 days of exposure. Activity analysis revealed that grafted oligoamine indentity of the polycation plays a significant role in elimination of PK-resistant PrP from chronically infected N2a-M cells, regardless of the polysaccharide used. Dextran-spermine conjugates were modified with oleic acid and with methoxypoly(ethylene glycol) (MPEG) at various degrees of substitution for further studies and their antiprion activity was examined. Substitution of dextran-spermine with MPEG or oleic acid slightly decreases its activity as a function of MPEG/oleic acid content. These findings confirm previous reports that polycations are effective in eliminating PrP(Sc) in vitro.
The gabCTDP gene cluster, which specifies and regulates synthesis of the y-aminobutyrate (GABA) transport carrier, of glutamate-succinic semialdehyde transaminase, and of succinic semialdehyde dehydrogenase, responsible for the uptake and metabolism of Y-aninobutyric acid in Escherichia coli K-12, was doned in vivo, using the mini-Mu replicon bacteriophage Mu dI5086 as the vector. A subclone containing a 7.8-kilobase (kb) EcoRI-HindJI fragment complemented ail of our Gab-mutants. By restriction mapping, this DNA fragment was located at kb 2800.5 to 2808.5 on the physical map of the E. coli K-12 chromosome. A subclone containing a 1.8-kb EcoRI-Sall fragment complemented the gab-repressed strain CS1O1A (wild-type gabC) but did not complement any gab structural gene mutants. The gab genes are divergently transcribed from promoters located in the vicinib of the unique BamHI site. Transcription in both directions is under dual control of catabolite repression and nitogen regulation. Using a procaryotic DNA-directed translation system, we observed three insert-coded polypeptide bands of 53 to 55, 45 to 48, and 40 to 43 kilodaltons (kDa). In vivo studies with subcloned fragments of the gab DNA identified the 53-to 55-and 45-to 48-kDa bands as products of the BamHI-Sall fragment and the 40-to 43-kDa band as the product of the EcoRI-Sall fragment. An additional 26-to 28-kDa band was identified as the product of the BamHI-HindM fragment. Furthermore, the BamHI-Sall fragment was shown to specify synthesis of the two GABA enzymes, whereas synthesis of the GABA carrier was specified by the BamHI-HindM fragment. No catalytic function in addition to its regulatory role could be attributed to the EcoRI-SaII gene product.
The regulation of the synthesis of the enzymes involved in the utilization of L-arginine, L-ornithine, agmatine, and putrescine as a sole nitrogen source in Escherichia coli K-12 was examined. The synthesis of agmatine ueohydrolase, putrescine aminotransferase, and pyrroline dehydrogenase is dually controlled by catabolite repression and nitrogen availability. Catabolite repression of agmatine ureohydrolase, but not that of putrescine aminotransferase or pyrroline dehydroge!nase, is relieved by the addition of cAMP. Agmatine ureohydrolase synthesis im addition is subject to induction by L-arginine and agmatine. Arginine decarboxylase and ornithine decarboxylase synthesis is not sensitive to catabolite repression or to stimulation by nitrogen limitation or subject to substrate induction.
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