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At least 631 records · Page 35Linked to original sources

PubMed1998

Forced evolution reveals the importance of short open reading frame A and secondary structure in the cauliflower mosaic virus 35S RNA leader.

Cauliflower mosaic virus pregenomic 35S RNA begins with a long leader sequence containing an extensive secondary structure and up to nine short open reading frames (sORFs), 2 to 35 codons in length. To test whether any of these sORFs are required for virus viability, their start codons were mutated either individually or in various combinations. The resulting viral mutants were tested for infectivity on mechanically inoculated turnip plants. Viable mutants were passaged several times, and the stability of the introduced mutations was analyzed by PCR amplification and sequencing. Mutations at the 5'-proximal sORF A and in the center of the leader resulted in delayed symptom development and in the appearance of revertants. In the central leader region, the predicted secondary structure, rather than the sORF organization, was restored, while true reversions or second-site substitutions in response to mutations of sORF A restored this sORF. Involvement of sORF A and secondary structure of the leader in the virus replication cycle, and especially in translation of the 35S RNA via ribosome shunting, is discussed.

Base Sequence↗

PubMed1997

The aquaporin-Z water channel gene of Escherichia coli: structure, organization and phylogeny.

Aquaporin water channel proteins are found throughout the plant and animal kingdoms, but the first prokaryotic water channel gene, aqpZ, was only recently identified in wild type Escherichia coli (Calamita G et al (1995) J Biol Chem 270, 29063-29066). Here we define the organization of aqpZ in E coli, produce the AqpZ protein and compare the AqpZ phylogeny to that of some known bacterial homologs. Physical mapping and sequence analyses confirmed the location of aqpZ at minute 19.7 on the E coli chromosome where it is transcribed counterclockwise. The monocistronic nature of aqpZ was clearly indicated by the structural organization of its surrounding genes, ybjD and ybjE' and by the presence of a typical Rho-independent transcriptional terminator following the aqpZ stop codon. Computer sequence analysis indicated the -35/-10 region located 72 bases upstream of the aqpZ start codon as the most likely aqpZ promoter. A series of potential cis-regulatory elements were found in the 400 bp region preceding the aqpZ ORF. The AqpZ protein, produced under T7 phi 10 control, showed a size of about 20 kDa by SDS-PAGE. Striking similarities were found between the E coli aqpZ and a gene included in the genome of the cyanobacterium Synechocystis sp PCC6803, a species permanently living a fresh water. These results may represent a fundamental step to characterize the regulation and the physiological features of the AqpZ water channel in prokaryotes.

Aquaporins↗

PubMed1997

Improved translational efficiency of subtilisin YaB gene with different initiation codons in Bacillus subtilis and alkalophilic Bacillus YaB.

The ale gene specifying the subtilisin YaB produced by alkalophilic Bacillus YaB, has an unusual start codon UUG. Changing this codon to AUG and GUG increased expression of the ale gene in B. subtilis DB104 and in an ale deficient mutant strain YaB-DEC4. The relative translational efficiency order of the three initiation codons is AUG > GUG > UUG in B. subtilis DB104 and in YaB-DEC4. These data suggest that the preferred initiation codon is AUG for ale gene expression in Bacillus.

Bacillus subtilis↗

PubMed2004

Specific residues of the GDP/GTP exchange factor Bud5p are involved in establishment of the cell type-specific budding pattern in yeast.

Cells of the budding yeast undergo oriented cell division by choosing a specific site for growth depending on their cell type. Haploid a and alpha cells bud in an axial pattern whereas diploid a/alpha cells bud in a bipolar pattern. The Ras-like GTPase Rsr1p/Bud1p, its GDP-GTP exchange factor Bud5p, and its GTPase-activating protein Bud2p are essential for selecting the proper site for polarized growth in all cell types. Here we showed that specific residues at the N terminus and the C terminus of Bud5p were important for bipolar budding, while some residues were involved in both axial and bipolar budding. These bipolar-specific mutations of BUD5 disrupted proper localization of Bud5p in diploid a/alpha cells without affecting Bud5p localization in haploid alpha cells. In contrast, Bud5p expressed in the bud5 mutants defective in both budding patterns failed to localize in all cell types. Thus, these results identify specific residues of Bud5p that are likely to be involved in direct interaction with spatial landmarks, which recruit Bud5p to the proper bud site. Finally, we found a new start codon of BUD5, which extends the open reading frame to 210 bp upstream of the previously estimated start site, thus encoding a polypeptide of 608 amino acid residues. Bud5p with these additional N-terminal residues interacted with Bud8p, a potential bipolar landmark, suggesting that the N-terminal region is necessary for recognition of the spatial cues.

Alanine↗

PubMed1992

Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus.

The N-terminal amino acid sequence of purified acetaldehyde dehydrogenase II (AcDH-II) from ethanol-grown cells of Alcaligenes eutrophus was determined. By using oligonucleotides deduced from this sequence the structural gene for AcDH-II, which was referred to as acoD, was localized on a 7.2-kbp EcoRI restriction fragment (fragment D), which has been cloned recently (C. Fründ, H. Priefert, A. Steinbüchel, and H. G. Schlegel, J. Bacteriol. 171:6539-6548, 1989). A 2.8-kbp PstI subfragment of D, which harbored acoD, was sequenced. It revealed an open reading frame of 1,518 bp, encoding a protein with a relative molecular weight of 54,819. The insertions of Tn5::mob of two transposon-induced mutants of A. eutrophus, which were impaired in the catabolism of acetoin, were mapped 483 or 1,359 bp downstream from the translational start codon of acoD. The structural gene was preceded by a putative Shine-Dalgarno sequence. The transcriptional start site 57 bp upstream of acoD was identified and was preceded by a sequence which exhibited a striking homology to the enterobacterial sigma 54-dependent promoter consensus sequence. This was in accordance with the observation that the expression of acoD and of other acetoin-catabolic genes depended on the presence of an intact rpoN-like gene. Alignments of the amino acid sequence deduced from acoD with the primary structures of aldehyde dehydrogenases from other sources revealed high degrees of homology, amounting to 46.5% identical amino acids.

Acetoin↗

PubMed2004

Cloning of a gene encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase from Bordetella sp. 10d.

Bordetella sp. 10d produces a novel dioxygenase catalyzing the meta-cleavage of 4-amino-3-hydroxybenzoic acid, 4-amino-3-hydroxybenzoate 2,3-dioxygenase (4A3HBA23D). A gene encoding 4A3HBA23D was cloned and named ahdA. The deduced amino acid sequence of ahdA showed 29.2-24.2% identities to those of prokaryotic and eukaryotic 3-hydoxybenzoate 3,4-dioxygenases in reported meta-cleavage dioxygenases. However, no identities were observed in the amino-terminal sequences of the first 29 amino acid residues. An ORF was found downstream of ahdA. The deduced amino acid sequence of the ORF showed identities to those of LysR family regulators involved in protocatechuate metabolism and contained motifs conserved in the regulators. On the basis of these results, the ORF was named ahdR encoding a putative LysR family regulator. The transcription start point of ahdA was localized 414-bp upstream of the start codon of ahdA. Two DNA-binding motifs of LysR family regulators were found upstream of the transcription start point. These observations suggest that a LysR family regulator encoded by ahdR regulates the expression of ahdA.

Amino Acid Motifs↗

PubMed1987

The unusual translational initiation codon AUU limits the expression of the infC (initiation factor IF3) gene of Escherichia coli.

The expression of infC, the structural gene for translational initiation factor IF3, has been studied in different constructs under the control of the lambda PL and tac promoters. The amount of synthesized IF3 has been determined by a quantitative functional test and the levels of IF3-specific mRNA have been estimated. The synthesis of IF3 is strongly enhanced when the unusual AUU initiation codon is changed to AUG by site-directed mutagenesis. Removal of the sequence upstream from the start codon including most of the Shine-Dalgarno sequence, as well as part of a 10 bp region with potential complementarity to an internal region of the 16S rRNA, which is unique to the IF3 mRNA, reduced but did not completely abolish the high expression of infC obtained after introduction of the AUG initiation codon. The level of IF3 mRNA was found to be positively influenced by the presence of the rplT gene in the plasmid downstream from the infC gene. In vivo accumulation of a large excess of IF3, obtained when the infC gene was placed under the control of an incompletely repressed tac promoter, was not accompanied by any noticeable adverse phenotype.

Base Sequence↗

PubMed1993

Identification of proximal and distal regulatory elements of the rat connexin32 gene.

We have investigated the genetic basis of the transcriptional regulation of the rat connexin32 gene which encodes the major gap junction protein in rat liver. Primer extension analysis of RNA isolated from adult rat liver identified multiple initiation sites clustered between -110 bp and -50 bp upstream from the translation start codon. An approx. 760 bp genomic DNA fragment upstream of the first exon which included the mRNA start sites was cloned 5' to the luciferase reporter cassette in p19LUC to yield pCx32-800/-33-LUC. Transfection of pCx32-800/-33-LUC resulted in a 200-fold increase in luciferase activity above p19LUC in the human hepatoma cell line HuH-7. Using a series of vectors containing 5' deletions of the 760 bp fragment, a basal promoter was localized between -179 bp and -134 bp. Three DNA:protein complexes were identified with the basal promoter fragment by DNA mobility shift assay using nuclear extracts from HuH-7 cells. Two of the DNA-binding complexes appeared to be related to the transcription factor Sp1. In addition, three DNase hypersensitive (HS) sites were identified within the genomic locus of connexin32 in adult rat liver. Two of the DNase HS regions behaved as silencer elements with both the native promoter and a heterologous promoter in HuH-7 cells. These data demonstrate that (1) the active promoter responsible for rat connexin32 mRNA transcription is located upstream of the first exon, (2) a basal promoter region was localized to a 50 bp region which formed multiple DNA:protein complexes, and (3) multiple proximal and distant regulatory elements are involved in the expression of connexin32.

Animals↗

PubMed1986

Transcription and processing signals in the 3-phosphoglycerate kinase (PGK) gene from Aspergillus nidulans.

The 3-phosphoglycerate kinase gene from Aspergillus nidulans contains two 57-bp introns and codes for a 421-amino acid (aa) protein with considerable homology to the Saccharomyces cerevisiae (68%) and mammalian (64%) proteins. Almost total conservation is found in Aspergillus of residues thought to be important to the structure and function of the yeast enzyme, and the introns fall between coding sequences for postulated structures in the N-domain. The strong codon preference found is more similar to that in other filamentous fungi than in yeast. The transcription start point (+1) has been mapped 32 bp upstream from the start codon, and the promoter region contains potential homologies for CAAT (-80 bp) and TATA (-30 bp) sequences, and certain other features common to other highly expressed genes in ascomycetes. There are three major termini 23, 83 and 115 bp beyond the stop codon and two of these are preceded by the polyadenylation consensus sequence and contain potential secondary structure.

Amino Acid Sequence↗

PubMed2001

Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter.

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.

Base Sequence↗

PubMed2001

Gene structure, chromosomal localization and analysis of 3-ketosteroid reductase activity of the human 3(alpha-->beta)-hydroxysteroid epimerase.

Following our previous characterization of the first human 3(alpha-->beta)hydroxysteroid epimerase (hHSE), we determined the genomic structure and chromosomal localization of the hHSE gene using fluorescent in situ hybridization (FISH) in this study. The gene spans 23 kb and contains five exons and four introns. FISH mapping assigned this gene to chromosome band 12q13. Primer extension analysis allowed the identification of a single transcription start site at 179 bp upstream from the ATG start codon. The 5'-flanking sequence lacks a typical TATA box in the proximal region of the transcription start site. However, analysis of the 2 kb promoter region revealed the presence of multiple potential transcription factor binding sites. Furthermore, we studied the 3-ketosteroid reductase activity demonstrated by hHSE in intact cells stably expressing the enzyme. It has been known that, in vitro, 3beta-hydroxysteroid dehydrogenase (3beta-HSD) shows both oxidative and reductive activity. Our results showed that hHSE catalyzes the reduction of 3-ketosteroids to form 3beta-hydroxysteroids while 3beta-HSD cannot catalyze this reaction in intact cells. However, hHSE showed 3-keto reductase activity in both microsomal fractions and intact cells. Since intact cells constitute a system which closely reflects in vivo intracellular conditions, we propose that hHSE might contribute to the cellular 3-ketosteroid reductase activity in the peripheral tissues.

3-Hydroxysteroid Dehydrogenases↗

PubMed1997

Sequencing and analysis of the Thermus thermophilus ribosomal protein gene cluster equivalent to the spectinomycin operon.

To assess the organization of the Thermus thermophilus ribosomal protein genes, a fragment of DNA containing the complete S10 region and ten ribosomal protein genes of the spc region was cloned, using an oligonucleotide coding for the N-terminal amino acid (aa) sequence of T. thermophilus S8 protein as hybridization probe. The nucleotide sequence of a 4290 bp region between the rps17 and rpl15 genes was determined. Comparative analysis of this gene cluster showed that the gene arrangement (S17, L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15) is identical to that of eubacteria. However, T. thermophilus ribosomal protein genes corresponding to the Escherichia coli S10 and spc operons are not resolved into two clusters: the stop codon of the rps17 gene (the last gene of the S10 operon in E. coli) and the start codon of the rpl14 gene (the first gene of the spc operon in E. coli) overlap. Most genes, except the rps14-rps8 intergenic spacer (69 bp), are separated by very short (only 3-7 bp) spacer regions or partially overlapped. The deduced aa sequences of T. thermophilus proteins share about 51-100% identities with the sequences of homologous proteins from thermophile Thermus aquaticus and Thermotoga maritima and 27-70% identities with the sequences of their mesophile counterparts.

Cloning, Molecular↗

PubMed1999

Expression and heat-responsive regulation of a TFIIB homologue from the archaeon Haloferax volcanii.

Multiple divergent genes encoding the eukaryal-like TFIIB (TFB) transcription initiation factor have been identified in the archaeon Haloferax volcanii. Expression of one of these TFB-encoding genes, referred to here as tfb2, was induced specifically in response to heat shock at the transcription level. A time course for tfb2 induction demonstrated that mRNA levels increased as much as eightfold after 15 min at 60 degrees C. A transcription fusion of the tfb2 promoter region with a stable RNA reporter gene confirmed the heat responsiveness of the tfb2 core promoter, and immunoblot analysis using antibodies generated against a recombinant His-tagged TFB2 showed that the protein levels of one TFB increased slightly in response to elevated temperatures. An archaeal consensus TATA element (5'-TTTATA-3') was located 110 bp upstream of the translation start site and appeared to be used for both basal and heat shock-induced expression. The long DNA leader region (79 bp) preceding the predicted AUG translation start codon for TFB2 contained a T-rich sequence element located 22 bp downstream of the transcription start site. Using an in vivo transcription termination assay, we demonstrated that this T-rich element can function as a sequence-dependent transcription terminator, which may serve to downregulate expression of the tfb2 gene under both non-heat shock and heat shock conditions.

Amino Acid Sequence↗

PubMed1998

Neonatal fulminant hepatitis B: structural and functional analysis of complete hepatitis B virus genomes from mother and infant.

Transmission of hepatitis B virus (HBV) from anti-hepatitis B e (anti-HBe)-positive carrier mothers to their infants may result in neonatal fulminant hepatitis B (FHB). We investigated whether HBV variants with a particular DNA sequence and functional phenotype, responsible for FHB, are selected during transmission. Full-length HBV genomes from a mother-infant pair were completely sequenced and transfected into human hepatoma cells. The dominant neonatal and maternal HBV populations were nearly identical (homology 99.8%) and showed a precore stop codon mutation, T-1762 and A-1764 substitutions in the core promoter region, and pre-S2 start codon mutations. Cells transfected with variants from mother and child, compared with wild-type virus, synthesized and released a similar number or fewer HBV DNA-containing particles. In conclusion, no particular HBV strain emerged during neonatal FHB. In this case, a de novo infection with variants showing a defect in HBe antigen and pre-S2 protein synthesis but not a high replication competence probably contributed to the fulminant disease course.

Adult↗

PubMed1992

Sequencing and analysis of the Bacillus subtilis lytRABC divergon: a regulatory unit encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and its modifier.

The regulatory unit of Bacillus subtilis strain 168 encompassing the structural genes of the N-acetylmuramoyl-L-alanine amidase and of its modifier has been sequenced, and found to be a divergon consisting of divergently transcribed operons lytABC and lytR. Proteins LytA, LytB and LytC are endowed with export signal peptides. Mature LytA is a 9.4 kDa, highly acidic polypeptide whose deduced amino acid sequence points to a lipoprotein. LytB and LytC, the modifier and the amidase, are highly basic. After cleavage of the signal sequence their molecular masses are 74.1 and 49.9 kDa, respectively. These two proteins share considerable homology in their N-terminal moieties and have three GSNRY consensus motifs, characteristic of nearly all amidases. The C-terminal moiety of LytB exhibits homology to the product of spoIID. LytR is a 35 kDa protein which acts as an attenuator of the expression of both lytABC and lytR operons. Transcription of the lytABC operon proceeds from two promoters: PD, identified as P28-7 (Gilman et al., 1984), and an upstream PA. The former only is subject to LytR attenuation. Translational initiation of lytB and lytC is directed by UUG start codons, suggesting that lytA, B and C undergo coupled translation. Transcription of lytR is initiated at two start sites, one of which corresponds to a highly intense PA promoter whereas the other does not seem to share much homology with any of the known promoter consensus sequences. Both promoters are attenuated by LytR. It is confirmed that the synthesis of the amidase is controlled at least in part by SigD, i.e. that it belongs to the fla regulon and that its activity, or part of it, is co-regulated with flagellar motility. The role of the mutations conferring the Sin, Fla and Ifm phenotypes in the expression of the lytABC operon is discussed.

Amino Acid Sequence↗

PubMed1990

Drosophila choline acetyltransferase uses a non-AUG initiation codon and full length RNA is inefficiently translated.

RNA from a partial cDNA clone containing the entire protein coding sequence of Drosophila melanogaster acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6; choline acetyltransferase) can be translated into active enzyme. This is unusual since this partial cDNA clone contains no appropriate ATG (AUG) initiation codon. In this study we use in vitro deletion and point mutants to identify GTG as the starting codon for protein translation. We also report the sequence of a full length Drosophila choline acetyltransferase cDNA and demonstrate that RNA produced by this clone is translated into active choline acetyltransferase but at a significantly reduced efficiency when compared to the partial cDNA clone. These results indicate that translational control may be an important regulatory step in production of Drosophila choline acetyltransferase.

Amino Acid Sequence↗

PubMed2000

Cloning and characterization of the 5'-flanking region for the mouse phospholipase C-delta1 gene.

To date, little is known about the molecular mechanisms controlling the regulation of phospholipase C-delta1 (PLC-delta1) gene expression. To understand the mechanisms responsible for the regulation of PLC-delta1 gene expression, the 5'-flanking region of the mouse PLC-delta1 gene was isolated from a mouse genomic DNA library. Primer extension analysis revealed that there is a single transcriptional start site located at 127 bases upstream from the translation start codon in the mouse PLC-delta1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA or CAAT boxes. Transient expression of a luciferase reporter gene under the control of serially deleted 5'-flanking sequences revealed that the 160-base-pair region from -622 to -462 upstream of the transcriptional start site includes a positive cis-acting element(s) for the efficient expression of the PLC-delta1 gene. Gel retardation analysis suggests that multiple transcription factors bind to separate sites on the promoter region. Based on these results, our study suggests that the minimal essential region located at -622 to +70 is fully sufficient to confer high-level transcriptional activity and contains high-affinity binding elements for multiple transcription factors.

Animals↗

PubMed1991

Different promoter regions control level and tissue specificity of expression of Agrobacterium rhizogenes rolB gene in plants.

Expression of the rolB gene of A. rhizogenes T-DNA triggers root differentiation in transformed plant cells. In order to study the regulation of this morphogenetic gene, the GUS reporter gene was placed under the control of several deleted fragments of the rolB 5' non-coding region: carrot disc transformations and the analysis of transgenic tobacco plants containing these constructions identified the presence of distinct regulatory domains in the rolB promoter. Two regions (located from positions -623 to -471 and from -471 to -341, from the translation start codon) control the level but not the tissue specificity of rolB expression: progressive deletions of the rolB promoter starting from position -1185 to -341, although at different levels, maintained the same pattern of GUS expression-maximal in root meristems and less pronounced in the vascular tissue of aerial organs. Further deletion of 35 bp, from -341 to -306, drastically affected tissue specificity: GUS activity was still clearly detectable in the vascular tissue of the aerial organs while expression in the root meristem was totally suppressed. Analysis of transgenic embryos and seedlings confirmed that distinct promoter domains are responsible for meristematic (root) and differentiated (vascular) expression of rolB. Finally, we present data concerning the effects of plant hormones on the expression of rolB-GUS constructions.

Cell Differentiation↗