Translational enhancement by an element downstream of the initiation codon in Escherichia coli.
(9/852)
The translation initiation of Escherichia coli mRNAs is known to be facilitated by a cis element upstream of the initiation codon, called the Shine-Dalgarno (SD) sequence. This sequence complementary to the 3' end of 16 S rRNA enhances the formation of the translation initiation complex of the 30 S ribosomal subunit with mRNAs. It has been debated that a cis element called the downstream box downstream of the initiation codon, in addition to the SD sequence, facilitates formation of the translation initiation complex; however, conclusive evidence remains elusive. Here, we show evidence that the downstream box plays a major role in the enhancement of translation initiation in concert with SD. (+info)
Design of 5' untranslated sequences in retroviral vectors developed for medical use.
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Utilizing genetic modules of simple retroviruses, we have developed a novel generation of gene transfer vectors with improved therapeutic potential. In the 5' untranslated "leader" sequences, all AUG codons which may aberrantly initiate translation and all viral coding sequences were removed. Thus, the probability of expressing unwanted peptides and the potential for homologous recombination with retroviral genes were largely reduced, and the cloning capacity was increased. The transgene was inserted to replace the viral gag sequences, and a new minimal splice acceptor was introduced, resulting in increased expression with all genes tested (those coding for human multidrug resistance 1 and enhanced green fluorescent protein, as well as the lacZ gene). These vectors may represent attractive tools for human gene therapy, because they increase the efficiency of transgene expression and may also increase safety in medical applications. (+info)
Expression of murine IL-12 is regulated by translational control of the p35 subunit.
(11/852)
IL-12 is a heterodimer of two subunits, p35 and p40, encoded by separate genes that are regulated independently. To investigate the mechanisms underlying the regulation of the p35 gene, we characterized murine p35 expression in the B cell lymphoma line A20 and in bone marrow-derived dendritic cells. Multiple transcription start sites were identified in both cell types, resulting in four p35 mRNA isoforms (types I-IV) that differ in the number and position of upstream ATGs in their 5' untranslated regions. In nonstimulated cells, the predominant forms of p35 message (types II and IV) contained an additional upstream ATG, whose presence was shown to inhibit the downstream translation of the p35 subunit. After LPS stimulation, however, transcription initiated from alternate positions, so that the proportion of transcripts not containing this upstream ATG (types I and III) was significantly increased in the population of p35 mRNA. These type I and type III transcripts readily supported translation of the p35 subunit and its incorporation into bioactive IL-12. Furthermore, p35 mRNA levels were substantially up-regulated after LPS stimulation in both cell types. Thus, our results show that p35 gene expression is highly regulated by both transcriptional and translational mechanisms. (+info)
An mtDNA mutation in the initiation codon of the cytochrome C oxidase subunit II gene results in lower levels of the protein and a mitochondrial encephalomyopathy.
(12/852)
A novel heteroplasmic 7587T-->C mutation in the mitochondrial genome which changes the initiation codon of the gene encoding cytochrome c oxidase subunit II (COX II), was found in a family with mitochondrial disease. This T-->C transition is predicted to change the initiating methionine to threonine. The mutation load was present at 67% in muscle from the index case and at 91% in muscle from the patient's clinically affected son. Muscle biopsy samples revealed isolated COX deficiency and mitochondrial proliferation. Single-muscle-fiber analysis revealed that the 7587C copy was at much higher load in COX-negative fibers than in COX-positive fibers. After microphotometric enzyme analysis, the mutation was shown to cause a decrease in COX activity when the mutant load was >55%-65%. In fibroblasts from one family member, which contained >95% mutated mtDNA, there was no detectable synthesis or any steady-state level of COX II. This new mutation constitutes a new mechanism by which mtDNA mutations can cause disease-defective initiation of translation. (+info)
Structural organization and splice variants of the POLE1 gene encoding the catalytic subunit of human DNA polymerase epsilon.
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The catalytic subunit of human DNA polymerase epsilon, an enzyme involved in nuclear DNA replication and repair, is encoded by the POLE1 gene. This gene is composed of 51 exons spanning at least 97 kb of genomic DNA. It was found to encode three alternative mRNA splice variants that differ in their 5'-terminal sequences and in the N-termini of the predicted proteins. A CpG island covers the promoter region for the major transcript in HeLa cells. This promoter is TATA-less and contains several putative binding sites for transcription factors typical of S-phase-up-regulated and serum-responsive promoters. Potential promoter regions were also identified for the two other alternative transcripts. Interestingly, no nuclear polyadenylation signal sequence was detected in the 3'-untranslated region, although a poly(A) tail was present. These results suggest a complicated regulatory machinery for the expression of the human POLE1 gene, including three alternative transcripts expressed from three promoters. (+info)
Genomic cloning and characterization of the rat glutathione S-transferase-A3-subunit gene.
(14/852)
The rat glutathione S-transferase-A3-subunit (GSTA3) gene is a member of the class Alpha GSTs, which we have previously reported to be overexpressed in anti-cancer-drug-resistant cells. In this study, we report the isolation and characterization of the entire rat GSTA3 (rGST Yc1) subunit gene. The rat GSTA3 subunit gene is approximately 15 kb in length and consists of seven exons interrupted by introns of different lengths. Exon 1, with a length of 219 bp, contains only the 5'-untranslated region of the gene. Each exon-intron splicing junction exhibited the consensus sequence for a mammalian splice site. The transcription start site and exon 1 of rat GSTA3 were characterized by a combination of primer extension and rapid amplification of the cDNA ends. Position +1 was identified 219 bp upstream of the first exon-intron splicing junction. The proximal promoter region of the rat GSTA3 subunit gene does not contain typical TATA or CAAT boxes. A computer-based search for potential transcription-factor binding sites revealed the existence of a number of motifs such as anti-oxidant-responsive element, ras-response element, activator protein-1, nuclear factor-kappaB, cAMP-response-element-binding protein, Barbie box and E box. The functional activity of the regulatory region of the rat GSTA3 subunit gene was shown by its ability to drive the expression of a chloramphenicol acetyltransferase reporter gene in rat mammary carcinoma cells, and its activity was greater in melphalan-resistant cells known to have transcriptional activation of this gene by previous studies. The structure of the gene, with a large intron upstream of the translation-initiation site, may explain why the isolation of this promoter has been so elusive. This information will provide the opportunity to examine the involvement of the rat GSTA3 subunit gene in drug resistance and carcinogenesis. (+info)
Efficient transcription of the human angiotensin II type 2 receptor gene requires intronic sequence elements.
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To investigate mechanisms of human angiotensin II type 2 receptor (hAT2) gene regulation we functionally characterized the promoter and downstream regions of the gene. 5'-Terminal deletion mutants from -1417/+100 to -46/+100 elicited significant but low functional activity in luciferase reporter gene assays with PC12W cells. Inclusion into the promoter constructs of intron 1 and the transcribed region of the hAT2 gene up to the translation start enhanced luciferase activity 6.7+/-1.6-fold and 11.6+/-1.7-fold (means+/-S.E.M.) respectively, whereas fusion of the promoter to the spliced 5' untranslated region of hAT2 cDNA did not, which indicated an enhancement caused by intronic sequence elements. Reverse transcriptase-mediated PCR confirmed that the chimaeric hAT2-luciferase mRNA was regularly spliced in PC12W cells. A Northern blot analysis of transfected cells showed levels of luciferase mRNA expression consistent with the respective enzyme activities. Mapping of intron 1 revealed that a 12 bp sequence in the centre of the intron was required for the increase in promoter activity, whereas the 5' adjacent intronic region mediated a decrease in luciferase activity. Mutation of the 12 bp region led to altered protein binding and markedly decreased luciferase activity. Cloned into a promoterless luciferase vector, a 123 bp intron 1 fragment was able to direct reporter gene expression to the same activity as occurred in conjunction with the 5' flanking region. These results indicate that sequence elements in intron 1 are necessary for efficient transcription of hAT2. In reporter gene assays, intron 1 might by itself function as a promoter and initiate transcription from an alternative start point. (+info)
Cloning and expression of rat pancreatic beta-cell malonyl-CoA decarboxylase.
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To gain insight into the function and regulation of malonyl-CoA decarboxylase (MCD) we have cloned rat MCD cDNA from a differentiated insulin-secreting pancreatic beta-cell-line cDNA library. The full-length cDNA sequence shows 69% identity with the cDNA cloned previously from the goose uropygial gland, and predicts a 492 amino acid protein of 54.7 kDa. The open reading frame contains an N-terminal mitochondrial targeting sequence and the C-terminal part of the enzyme ends with a peroxisomal (Ser-Lys-Leu) targeting motif. Since the sequence does not reveal hydrophobic domains, MCD is most likely expressed in the mitochondrial matrix and inside the peroxisomes. A second methionine residue, located 3' of the mitochondrial presequence, might be the first amino acid of a putative cytosolic MCD, since the nucleotide sequence around it fits fairly well with a consensus Kozak site for translation initiation. However, primer extension detects the presence of only one transcript initiating upstream of the first ATG, indicating that the major, if not exclusive, transcript expressed in the pancreatic beta-cell encodes MCD with its mitochondrial presequence. The sequence also shows multiple possible sites of phosphorylation by casein kinase II and protein kinase C. mRNA tissue-distribution analysis indicates a transcript of 2.2 kb, and that the MCD gene is expressed over a wide range of rat tissues. The distribution of the enzyme shows a broad range of activities from very low in the brain to elevated in the liver and heart. The results provide the foundations for further studies of the role of MCD in lipid metabolism and metabolic signalling in various tissues. (+info)