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PubMed2002

Characterization of a gene encoding tRNA nucleotidyltransferase from Candida glabrata.

A gene encoding ATP (CTP):tRNA nucleotidyltransferase (EC2.7.7.25) was isolated from Candida (Torulopsis) glabrata by complementation in Saccharomyces cerevisiae. The predicted amino acid sequence of the protein revealed a large region with high sequence similarity to members of the Class II group of the nucleotidyltransferase superfamily and an N-terminal region characteristic of a mitochondrial targeting sequence. The essential role of the carboxylates within the conserved DXD and RRD motifs was confirmed by mutagenesis. C. glabrata strains bearing truncated CCA1 genes that lacked sequences encoding the putative mitochondrial targeting peptide were unable to grow on non-fermentable carbon sources but were able to grow on a fermentable carbon source. These results suggest that, as in S. cerevisiae, the C. glabrata CCA-adding enzyme is a sorting isozyme that functions in multiple cellular compartments. Mapping of the 5'-ends of primary transcripts of CCA1 revealed multiple transcription start sites located both upstream of and between two in-frame start codons. When the cells were cultured on a non-fermentable carbon source the longer transcripts appeared more abundant, suggesting that the choice of transcription start sites was influenced by carbon source. The shorter transcripts, which lacked sequences encoding the mitochondrial targeting information, were more predominant in cells grown on glucose. These observations suggest that expression of CCA-adding isozymes in C. glabrata may be regulated. The DNA sequence has been assigned GenBank Accession No. AF098803.

Amino Acid Sequence↗

PubMed1986

The trfB region of broad host range plasmid RK2: the nucleotide sequence reveals incC and key regulatory gene trfB/korA/korD as overlapping genes.

We report the nucleotide sequence of the trfB region of broad host range plasmid RK2. This region encodes the following loci: trfB, identical to korA and korD, which encodes a key transcriptional repressor of certain RK2 operons; incC, which appears to be involved in plasmid maintenance, possibley through post-transcriptional regulation of trfA product levels; the start of korB, which encodes a second transcriptional repressor of operons involved in stable inheritance of RK2. These loci are expressed as part of the trfB operon. In combination with deletion analysis, transcriptional and translation fusions and 'maxicell' analysis of polypeptides, the DNA sequence allows a number of conclusions to be drawn. First, the korB ORF start codon overlaps the incC ORF stop codon, suggesting the possibility of translational coupling between these two genes. Second, the trfB ORF lies entirely within the first third of the incC ORF using a different phase. Third, the incC ORF appears to contain a second transcriptional start whose function appears to be coupled to translation of the trfB ORF. Analysis of codon usage in the region of overlap between incC and trfB suggests that the incC gene may have evolved before the trfB gene. Determination of the DNA sequence of a mutant in which the product of trfB is rendered defective for transcriptional repression reveals an amino acid alteration within a region of this polypeptide which exhibits homology to the alpha helix-turn-alpha helix motif characteristic of many DNA binding proteins, and which is probably responsible for recognition of the trfB operator by this protein.

Amino Acid Sequence↗

PubMed1991

Initiation of translation at AUC, AUA and AUU codons in Escherichia coli.

A truncated form of the HBL murein hydrolase, encoded by the temperate bacteriophage HB-3, was cloned in a pUC-derivative and translated in Escherichia coli using AUC as start codon, as confirmed by biochemical, immunological, and N-terminal analyses. Using site-directed mutagenesis, we have changed this AUC codon into AUA, AUU and AUG codons. The relative translation efficiencies for these triplets were about 5% for AUC and AUU and 7.5% for AUA compared to that of AUG codon. In the same gene arrangement E. coli beta-galactosidase was also translated at moderate efficiency using AUC as initiator.

Amino Acid Sequence↗

PubMed2000

Escherichia coli DipZ: anatomy of a transmembrane protein disulphide reductase in which three pairs of cysteine residues, one in each of three domains, contribute differentially to function.

DipZ is a bacterial cytoplasmic membrane protein that transfers reducing power from the cytoplasm to the periplasm so as to facilitate the formation of correct disulphide bonds and c-type cytochromes in the latter compartment. Topological analysis using gene fusions between the Escherichia coli dipZ and either E. coli phoA or lacZ shows that DipZ has a highly hydrophobic central domain comprising eight transmembrane alpha-helices plus periplasmic globular N-terminal and C-terminal domains. The previously assigned translational start codon for the E. coli DipZ was shown to be incorrect and the protein to be larger than previously thought. The experimentally determined translational start position indicates that an additional alpha-helix at the N-terminus acts as a cleavable signal peptide so that the N-terminus of the mature protein is located in the periplasm. The newly assigned 5' end of the dipZ gene was shown to be preceded by a functional ribosome-binding site. The hydrophobic central domain and both of the periplasmic globular domains each have a pair of highly conserved cysteine residues, and it was shown by site directed mutagenesis that all six conserved cysteine residues contribute to DipZ function.

Alkaline Phosphatase↗

PubMed2000

Transcriptional regulation of the pas gene of enterohemorrhagic Escherichia coli.

The Pas protein plays a key role in the pathogenesis of enterohemorrhagic Escherichia coli (EHEC), being required for the secretion of the Esp proteins. Here, the transcriptional regulation of the pas gene was analyzed through the construction of a pas::lacZ translational fusion. When bacteria were grown in Luria Bertani medium or tissue culture medium supplemented with HEPES, a bimodal activation curve was observed. The early phase of induction was not significantly modified by the incubation temperature (either 25 or 37 degrees C), whereas the second phase, which overlaps with the late exponential growth phase, was enhanced at 37 degrees C. The early phase was also stimulated by growth on tissue culture medium and by the addition of Ca(2+), Mn(2+)or Mg(2+) to the M9-glucose minimal medium. Primer extension analysis showed the presence of two major starts of transcription, which were located 58 and 60 bp upstream of the ATG-start codon of the Pas protein, respectively. Although these sites are very close to each other, the transcripts produced during the early induction phase mainly start on the -60 position, whereas the -58 start was activated during the second induction phase.

Amino Acid Sequence↗

PubMed1990

Multiple copies of a bile acid-inducible gene in Eubacterium sp. strain VPI 12708.

Eubacterium sp. strain VPI 12708 is an anaerobic intestinal bacterium which possesses inducible bile acid 7-dehydroxylation activity. Several new polypeptides are produced in this strain following induction with cholic acid. Genes coding for two copies of a bile acid-inducible 27,000-dalton polypeptide (baiA1 and baiA2) have been previously cloned and sequenced. We now report on a gene coding for a third copy of this 27,000-dalton polypeptide (baiA3). The baiA3 gene has been cloned in lambda DASH on an 11.2-kilobase DNA fragment from a partial Sau3A digest of the Eubacterium DNA. DNA sequence analysis of the baiA3 gene revealed 100% homology with the baiA1 gene within the coding region of the 27,000-dalton polypeptides. The baiA2 gene shares 81% sequence identity with the other two genes at the nucleotide level. The flanking nucleotide sequences associated with the baiA1 and baiA3 genes are identical for 930 bases in the 5' direction from the initiation codon and for at least 325 bases in the 3' direction from the stop codon, including the putative promoter regions for the genes. An additional open reading frame (occupying from 621 to 648 bases, depending on the correct start codon) was found in the identical 5' regions associated with the baiA1 and baiA3 clones. The 5' sequence 930 bases upstream from the baiA1 and baiA3 genes was totally divergent. The baiA2 gene, which is part of a large bile acid-inducible operon, showed no homology with the other two genes either in the 5' or 3' direction from the polypeptide coding region, except for a 15-base-pair presumed ribosome-binding site in the 5' region. These studies strongly suggest that a gene duplication (baiA1 and baiA3) has occurred and is stably maintained in this bacterium.

Amino Acid Sequence↗

PubMed1993

Cloning and characterization of the guinea pig neutrophil cationic peptide-1 and -2 genes.

Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP-1 and GNCP-2 in the granules. Using cDNA probes, four different GNCP gene clones were isolated from a guinea pig genomic library. Two clones encoded GNCP-1 and other two clones encoded GNCP-2. The nucleotide sequence of GNCP-1 and GNCP-2 gene clones were highly homologous (> 97%) in the region sequenced. The GNCP-1 and GNCP-2 genes spanned 3 kb, and consisted of three exons and two introns. Exon 1 encoded the 5' untranslated region, exon 2 encoded the prepro-peptide region, and exon 3 encoded the mature peptide region and the 3' untranslated region. The transcription start site was located to a thymidine residue 93 bp upstream of the start codon (ATG) of GNCP-1 or GNCP-2 mRNA, and TATA-like sequence was located 24 to 30 bp upstream of the transcription start site. Glucocorticoid regulating element-like sequence was found in the 5' flanking region of the GNCP-1 or GNCP-2 gene, and the GNCP gene transcription rate of bone marrow cells was upregulated by dexamethasone treatment. Together these observations indicate that GNCP-1 and GNCP-2 are encoded by the very homologous but different genes, and that GNCP gene expression is regulated by glucocorticoid.

Animals↗

PubMed1996

Structure and organization of the gene encoding human selenoprotein.

We have isolated a genomic clone encoding human selenoprotein P including the putative promoter region. The gene spans 12 kb and consists of five exons with a start codon in the second exon. A typical TATA sequence, the recognition motifs for a GATA-binding factor and the liver-specific factors, HNF-1 and HNF-3, were detected upstream from the transcription start point.

Base Sequence↗

PubMed1991

The mouse transition protein 1 gene contains a B1 repetitive element and is located on chromosome 1.

The gene for mouse transition protein 1 (mTP1) was isolated, sequenced, and chromosomally mapped. The nucleotide sequence of 1895 bp of a 6.4-kb mTP1 genomic subclone was determined to include 788 bp of 5' flanking region, 564 bp of coding region including a 396-bp intron and a TAA stop codon, and 543 bp of 3' flanking region. The mTP1 gene contains a B1 repeat sequence within the only intron of the gene. The transcriptional start site of the mTP1 mRNA was determined to be located 31 bases upstream of the ATG translational start codon. Southern blot analysis demonstrated the presence of sequences homologous to the mTP1 cDNA in the genomes of the rat, hamster, bull, boar, dog, horse, ram, human, and two marsupials (the American opossum and Monodelphis), suggesting that the mTP1 gene sequence is widely conserved. The TP1 gene has been mapped by analysis of restriction fragment length variants (RFLV) in an interspecific backcross to a position 0.7 +/- 0.4 cM telomeric of Mylf and 1.2 +/- 0.5 cM centromeric of Vil on mouse chromosome 1.

Amino Acid Sequence↗

PubMed1995

Genomic structure and expression of the ADH7 gene encoding human class IV alcohol dehydrogenase, the form most efficient for retinol metabolism in vitro.

Human alcohol dehydrogenase (ADH) consists of a family of five evolutionarily related classes of enzymes that collectively function in the metabolism of a wide variety of alcohols including ethanol and retinol. Class IV ADH has been found to be the most active as a retinol dehydrogenase, thus it may participate in retinoic acid synthesis. The gene encoding class IV ADH (ADH7) has now been cloned and subjected to molecular examination. Southern blot analysis indicated that class IV ADH is encoded by a single unique gene and has no related pseudogenes. The class IV ADH gene is divided into nine exons, consistent with the highly conserved intron/exon structure of other mammalian ADH genes. The predicted amino acid sequence of the exon coding regions indicates that a protein of 373 amino acids, excluding the amino-terminal methionine, would be translated, sharing greater sequence identity with class I ADH (69%) than with classes II, III or V (59-61%). Expression of class IV ADH mRNA was detected in human stomach but not liver. This correlates with previous protein studies, which have indicated that class IV ADH is the major stomach ADH but unlike other ADHs is absent from liver. Primer extension studies using human stomach RNA were performed to identify the transcription initiation site lying 100 base pairs upstream of the ATG translation start codon. Nucleotide sequence analysis of the promoter region indicated the absence of a TATA box sequence often located about 25 base pairs upstream of the start site as well as the absence of GC boxes, which are quite often seen in promoters lacking a TATA box. The class IV ADH promoter thus differs from the other ADH promoters, which contain either a TATA box (classes I and II) or GC-boxes (class III), suggesting a fundamentally different form of transcriptional regulation.

Alcohol Dehydrogenase↗

PubMed1995

The gene and pseudogenes of rat S-adenosyl-L-homocysteine hydrolase.

Two rat liver genomic DNA libraries constructed in lambda DASH and lambda Charon 4A were screened for sequences with similarity to S-adenosyl-L-homocysteine (AdoHcy) hydrolase cDNA. Of 36 clones purified, two contained the AdoHcy hydrolase gene sequence and 34 contained pseudogene sequences. The AdoHcy hydrolase gene, which has been sequenced in its entirety, spans approximately 15 kb and consists of 10 exons. Primer extension and S1 experiments show that transcription is initiated from two major initiation sites located at positions -63 and -62 from the starting codon and from several minor sites. The promoter region is located in a CpG island, sequence TATTTAAA is present 23 bases upstream from the transcription start site, and an inverted CCAAT box is located 285 bp upstream from the transcription start site. Other potential transcription-factor binding sites including SP1, AP-2, GRE and Oct-1 sites were identified in the 5'-flanking region. Several different processed pseudogenes were found and analyzed.

5-Methylcytosine↗

PubMed1986

Influence of modification next to the anticodon in tRNA on codon context sensitivity of translational suppression and accuracy.

Effects on translation in vivo by modification deficiencies for 2-methylthio-N6-isopentenyladenosine (ms2i6A) (Escherichia coli) or 2-methylthio-N6-(4-hydroxyisopentenyl)adenosine (ms2io6A) (Salmonella typhimurium) in tRNA were studied in mutant strains. These hypermodified nucleosides are present on the 3' side of the anticodon (position 37) in tRNA reading codons starting with uridine. In E. coli, translational error caused by tRNA was strongly reduced in the case of third-position misreading of a tryptophan codon (UGG) in a particular codon context but was not affected in the case of first-position misreading of an arginine codon (CGU) in another codon context. Misreading of UGA nonsense codons at two different positions was codon context dependent. The efficiencies of some tRNA nonsense suppressors were decreased in a tRNA-dependent manner. Suppressor tRNA which lacks ms2i6A-ms2io6A becomes more sensitive to codon context. Our results therefore indicate that, besides improving translational efficiency, ms2i6A37 and ms2io6A37 modifications in tRNA are also involved in decreasing the intrinsic codon reading context sensitivity of tRNA. Possible consequences for regulation of gene expression are discussed.

Anticodon↗

PubMed1994

Molecular characterization of the extracellular poly(3-hydroxyoctanoic acid) [P(3HO)] depolymerase gene of Pseudomonas fluorescens GK13 and of its gene product.

phaZPfi, the gene encoding the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, was cloned, sequenced, and characterized. It comprises 837 bp and is transcribed as a monocistronic message of about 950 bp from a putative sigma 70-like promoter 32 bp upstream of the ATG start codon. The deduced protein of 278 amino acids reveals a typical leader peptide at its N terminus. When expressed in Escherichia coli, the mature depolymerase started with Ala-23, whereas the mature enzyme purified from P. fluorescens GK13 started with both Leu-34 and Arg-35 determining proteins of 26,687 and 26,573 Da, respectively. The depolymerase is a strongly hydrophobic protein and includes the lipase consensus sequence Gly-X-Ser-X-Gly, which is known for serine hydrolases. Replacement of the central residue, Ser-172, in the corresponding sequence (Gly-Ile-Ser-Ser-Gly) of PhaZPfl with alanine resulted in complete loss of enzyme activity, indicating that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.

Amino Acid Sequence↗

PubMed1995

Characterization and regulation of two testicular inhibin/activin beta B-subunit messenger ribonucleic acids that are transcribed from alternate initiation sites.

We and others have shown that the inhibin/activin beta B-subunit gene is expressed differently in the gonads. Two species of 4.8- and 3.7-kilobase (kb) beta B-subunit messenger RNA (mRNA) with equal concentrations were identified in the testis, whereas 1 predominant 4.8-kb and a minor 3.7-kb mRNA were observed in the ovary. In this study, we analyzed the structures of these 2 mRNAs in rat testis and showed that both 4.8- and 3.7-kb beta B-subunit mRNAs were terminated at the region proximal to 2.2 kb down-stream from the translation stop codon. However, only 4.8-kb mRNA could be detected when RNA probes prepared from the 5'-region 1 kb up-stream from the translation start site were used for Northern blot analysis. Our observations suggested that the 2 heterogeneously sized beta B-subunit mRNAs are transcribed from different initiation sites. Transcription of the 4.8-kb mRNA was initiated at 3 adjacent nucleotides, GGA, 1.1 kb up-stream from the translation start codon ATG, whereas multiple transcription initiation sites spreading over 150 nucleotides upstream from the ATG codon were previously identified for 3.7-kb mRNA. Neither of the 2 transcripts contained TATA and CAAT boxes in their promoters. The 5'-flanking DNAs required for transcription of the 4.8- and 3.7-kb mRNA were examined by their ability to induce transient expression of the chloramphenicol acetyltransferase (CAT) gene in MA-10 Leydig tumor cells. A marked increase in CAT activity was detected when the 5'-flanking DNA for the 4.8- or 3.7-kb transcript was progressively shortened from its 5'-end. Maximal CAT activity was observed when -409 and -139 basepair beta B-subunit DNA up-stream from the 4.8- and 3.7-kb transcription initiation site, respectively, were fused to the CAT gene, suggesting the presence of a negative regulatory element(s) at the up-stream regions of these promoters. Although putative AP-2 sites were identified, treatment of the transfected cells with cAMP and/or phorbol 12-myristate 13-acetate did not apparently change CAT activity driven by either the 4.8- or 3.7-kb promoter. Our results concluded that 1) the two inhibin/activin beta B-subunit mRNAs were transcribed from different initiation sites; 2) both promoters may be controlled by up-stream negative regulatory elements; and 3) neither of these promoters is responsive to cAMP and/or phorbol esters under the conditions employed.

Activins↗

PubMed1999

Genomic structure and chromosome location of RPL27A/Rpl27a, the genes encoding human and mouse ribosomal protein L27A.

The intron-containing genes encoding human and mouse ribosomal protein (r-protein) L27A were cloned and sequenced. The human r-protein L27A gene (RPL27A) shared an identical exon/intron structure with the mouse r-protein 27A gene (Rpl27a). The translational start codon ATG was separated from the main reading frame by the first intron sequence in both genes. An approximately 200-bp sequence upstream of the translational start site of both genes displayed remarkable similarity, and contained the putative promoters lacking canonical TATA, but harbored Sp1 binding sites and a short stretch of pyrimidine cluster, similar to other r-protein genes. Transcriptional regulatory elements, Box-A and GABP, found in the promoters of some other r-protein genes were also conserved in both genes. These structural features were included in the typical CpG island identified in the 5'-end sequences, suggesting that RPL27A/Rpl27a cloned here are authentic and transcriptionally active. Fluorescence in situ hybridization (FISH) analysis localized the mouse intron-containing Rpl27a to chromosome 7E2-F1 syntenic to human chromosome 11p15, where human RPL27A was located.

Animals↗

PubMed1994

The rat amyloid precursor protein promoter contains two DNA regulatory elements which influence high level gene expression.

We have investigated the transcriptional activity and regulatory elements of the rat amyloid precursor protein promoter. A DNA fragment containing 375 base pairs upstream of the start codon drives transcription in rat PC12 cells at a level greater than five-fold that of the SV40 promoter. This fragment contains a predominant transcription start point and several additional start points which are similar to those found in the human promoter. The strong promoter activity appears to be dependent upon two small DNA regulatory elements. Deletion of one element at positions -260 through -248 reduces activity by 85%. This is the first report of a positive regulatory element at this location. Deletion of a second element at positions -223 through -192 reduces activity by 30%. Gel mobility shift assays with nuclear extracts from whole rat brain suggest that nuclear proteins interact directly with the second element but not with the first one.

Amyloid beta-Protein Precursor↗

PubMed1992

Hepatitis A virus polyprotein synthesis initiates from two alternative AUG codons.

The genomic RNA of hepatitis A virus has two potential translation initiation sites for synthesis of a 251-kDa polyprotein. It is not known which of these AUG codons, located at positions 735-737 and 741-743, is used in vitro or in vivo. Site-directed mutagenesis was carried out to eliminate each start codon independently. Transcripts from the unmodified and modified cDNA clones were used either to program an in vitro translation system or for transfection of BS-C-1 cells. In vitro and in vivo translation data revealed preferential usage of the downstream AUG located at position 741 to 743, although either site could be utilized in the absence of the other. Both modified RNAs were able to induce productive infections in BS-C-1 cells. Deletion of almost all of the 5'-untranslated region (5'UTR) of the RNA, however, stimulated selection of AUG 735-737 in vitro resulting in equal utilization of both sites, suggesting a strong influence of the 5'UTR for directing the ribosome to a specific internal initiation site.

Animals↗

PubMed1985

"ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli.

A plasmid cloning vector system has been constructed that allows for the production of large quantities of foreign proteins or fragments thereof, in an unfused state. These vectors provide strong regulated trp-lac fusion promoters and the lacZ ribosome-binding site (RBS) followed by an ATG translation initiation codon at an appropriate distance from the RBS. The ATG codon is located within a unique NcoI restriction site (CCATGG). Digestion with NcoI exposes the ATG for fusion. Gene fragments lacking a prokaryotic RBS and/or ATG start codons can be inserted in several ways. Expression experiments using a truncated cI gene of bacteriophage lambda or a large portion of the coding region of the Herpes simplex virus type 1 glycoprotein D gene have been performed. The results of these studies show that the vectors are useful for the high-level expression of prokaryotic and eukaryotic genes in Escherichia coli.

Base Sequence↗