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At least 973 records · Page 54Linked to original sources
PubMed1993

Sorbitol dehydrogenase. Full-length cDNA sequencing reveals a mRNA coding for a protein containing an additional 42 amino acids at the N-terminal end.

A cDNA clone encoding rat sorbitol dehydrogenase (SDH) was isolated from a rat testis lambda ZAP II cDNA library. The full-length cDNA insert contained 2277 base pairs (bp), starting 182 bp upstream from an ATG codon where translation to the active enzyme SDH is presumed to be initiated. A second ATG codon, however, was found 126 bp upstream, aligned in the same reading frame as that of the active enzyme. Therefore, the coding sequence for SDH can be translated into an additional 42-amino-acid polypeptide linked to the N-terminal amino acid of the enzyme, generating a pre-sorbitol dehydrogenase. The sequence data indicate that the nucleotide environment around this ATG codon is more favorable towards it being the actual open reading frame (ORF) for a pre-SDH than the ATG codon preceding the nucleotide sequence for SDH. Since no known SDH starts with the additional 42 amino acids, it may be that post-translational removal of this polypeptide accompanies the release of the active enzyme. Next, the 3' untranslated region of the cDNA contained a non-coding 1021 bp downstream from the TAA stop codon. The latter sequence included three putative poly(A) signals: one at nucleotides 1362-1367, the second at nucleotides 1465-1470, and the third at nucleotides 2212-2217 [17 bp away from the poly(A) tail]. In addition to the above findings we also report a variance in one of the amino acids in the SDH cDNA sequence. This variance occurs at position 957-960, where threonine is coded for instead of aspartic acid; in the rat testis SDH cDNA, we find the sequence is ACG instead of GAC, as was reported for the rat liver SDH cDNA. Northern-blot hybridization analysis showed that SDH mRNA is a doublet, one band of 4 kb and the other of 2.3-2.4 kb, in both the rat liver and the rat lens, further confirming that the isolated SDH cDNA constituted a full-length cDNA.

Amino Acid Sequence↗
PubMed2005

Heat shock response of Archaeoglobus fulgidus.

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.

Amino Acid Sequence↗
PubMed2004

TorI, a response regulator inhibitor of phage origin in Escherichia coli.

The torI gene has been identified by using a genetic multicopy approach as a negative regulator of the torCAD operon that encodes the trimethylamine N-oxide reductase respiratory system in Escherichia coli. The negative effect was due to a previously unidentified small ORF (66 aa) of phage origin that we called torI for Tor inhibition. Overexpression of torI led to an 8-fold decrease of the torCAD operon transcription. This operon is positively regulated, in the presence of trimethylamine N-oxide, by a four-step phosphorelay involving the TorS sensor and the TorR response regulator. Epistatic experiments showed that TorI acts downstream of TorS and needs the presence of TorR. In vitro experiments showed that it is neither a TorR phosphatase nor a histidine kinase inhibitor and that it binds to the effector domain of TorR. Unexpectedly, TorI did not impede TorR DNA binding, and we propose that it may prevent RNA polymerase recruitment to the torC promoter. This study thus reveals a previously uncharacterized class of response regulator inhibitors.

Amino Acid Sequence↗
PubMed2006

Characterization of White bream virus reveals a novel genetic cluster of nidoviruses.

The order Nidovirales comprises viruses from the families Coronaviridae (genera Coronavirus and Torovirus), Roniviridae (genus Okavirus), and Arteriviridae (genus Arterivirus). In this study, we characterized White bream virus (WBV), a bacilliform plus-strand RNA virus isolated from fish. Analysis of the nucleotide sequence, organization, and expression of the 26.6-kb genome provided conclusive evidence for a phylogenetic relationship between WBV and nidoviruses. The polycistronic genome of WBV contains five open reading frames (ORFs), called ORF1a, -1b, -2, -3, and -4. In WBV-infected cells, three subgenomic RNAs expressing the structural proteins S, M, and N were identified. The subgenomic RNAs were revealed to share a 42-nucleotide, 5' leader sequence that is identical to the 5'-terminal genome sequence. The data suggest that a conserved nonanucleotide sequence, CA(G/A)CACUAC, located downstream of the leader and upstream of the structural protein genes acts as the core transcription-regulating sequence element in WBV. Like other nidoviruses with large genomes (>26 kb), WBV encodes in its ORF1b an extensive set of enzymes, including putative polymerase, helicase, ribose methyltransferase, exoribonuclease, and endoribonuclease activities. ORF1a encodes several membrane domains, a putative ADP-ribose 1"-phosphatase, and a chymotrypsin-like serine protease whose activity was established in this study. Comparative sequence analysis revealed that WBV represents a separate cluster of nidoviruses that significantly diverged from toroviruses and, even more, from coronaviruses, roniviruses, and arteriviruses. The study adds to the amazing diversity of nidoviruses and appeals for a more extensive characterization of nonmammalian nidoviruses to better understand the evolution of these largest known RNA viruses.

Coronaviridae↗
PubMed1997

Cloning and characterization of a copy of Tirant transposable element in Drosophila melanogaster.

A Tirant element, inserted at the 5' end of the mitochondrial glutamine synthetase (mt-gs) gene in a mutant allele giving rise to a recessive female sterility phenotype, was cloned and utilized to characterize this novel retrotransposable element of the Drosophila melanogaster genome. The 5.3 kb element present in the fs(2) PM11-19 mt-gs allele possesses a 417 bp long terminal repeat (LTR) at both ends. There is a serine tRNA binding site downstream of the 5' LTR sequence and a polypurine tract upstream of the 3' LTR end. The insertion leads to the duplication of a host-site CGCG sequence. In situ hybridization to salivary glands chromosomes showed evidence of the mobile nature of the element. The DNA sequencing of the cloned 5.3 kb element revealed that Tirant possesses an open reading frame (ORF) that shows similarity with the envelope protein encoded by the gypsy and 297 retrotransposons. In addition, the cloned element appears to be a subgenomic fragment of a not yet identified complete element, because only the integrase domain of the reverse transcriptase gene is found.

Amino Acid Sequence↗
PubMed1992

Sequence and in vitro expression of the M2 gene of turkey rhinotracheitis pneumovirus.

Negative-stranded virion RNA and oligonucleotide primers complementary to fusion (F) protein gene sequences were used to generate cDNA clones, revealing that the gene 5'-proximal to the F protein corresponded to the M2 (22K) gene, as in respiratory syncytial (RS) virus. The transcription start signal, GGGACAAGU, was identical to that of the F and matrix (M) proteins of turkey rhinotracheitis virus (TRTV). There were two sequences with the potential to function as transcription termination/poly(A) signals, located at nucleotides 751 to 762 and 777 to 787; 15 clones derived from mRNA indicated that the first of these sequences formed the major signal. Part of the next downstream (5') gene was sequenced; unlike mammalian pneumoviruses the TRTV M2 gene did not overlap the beginning of the 5'-proximal gene. Northern blotting indicated that infected Vero cells contained less M2 mRNA than F mRNA and that about half of the M2 mRNA was present as a F-M2 dicistronic mRNA. The M2 gene contained two overlapping open reading frames (ORFs 1 and 2), as with RS virus. ORF 1 comprised 558 nucleotides with the coding potential for a 186 amino acid polypeptide, M(r) 20959, eight or nine residues shorter than for human RS virus strains. The overall amino acid identity was 40%, the N-terminal one-third of the proteins sharing 62% of residues, the remainder 29%. A hydropathy plot of the TRTV M2 protein had close similarity to that of the M2 or RS virus. The protein was predicted to have a basic character with no N-terminal signal sequence or other major highly hydrophobic sequences. In vitro translation of a transcript comprising both ORFs 1 and 2 produced a single product of apparent M(r) 23000, corresponding to the M2 product of ORF 1. Site-directed mutagenesis confirmed that this product was derived from ORF 1 and that frameshifting was not involved. The second ORF was expressed only from a transcript which lacked the AUG codons of ORF 1 and, although occupying a similar position to that in the RS virus M2 gene, had virtually no amino acid identity in its 73 residue length and was approximately 25% shorter than the corresponding RS virus ORF 2. The hydropathy plot of the potential products of the second ORFs of TRTV and RS virus showed little resemblance. Taken together these results suggest that ORF 2 is unlikely to be expressed in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Characterization of an insertion sequence (IS53) located within IS51 on the iaa-containing plasmid of Pseudomonas syringae pv. savastanoi.

A 2568-base pair (bp) insertion sequence (IS53) was cloned from the iaa-containing plasmid pIAA2 of Pseudomonas syringae subsp. savastanoi. IS53 is located downstream from the iaa operon and is inserted into IS51, a previously described element on pIAA2. IS53 causes an 8-bp target duplication and has nearly perfect terminal inverted repeats of 27 bp. Several degenerate copies of the inverted repeats are located internal, and in direct orientation to the terminal inverted repeats. A putative transposase gene conforms to the transposase ORF/opposite strand overlapping ORF motif described in many gram-negative IS elements. The region 5' to the presumptive transposase gene contains sequences that are homologous to the consensus E. coli heat-shock (sigma 32) promoter.

Amino Acid Sequence↗
PubMed1992

Nucleotide sequence of the 3' terminal region of belladonna mottle virus-Iowa (renamed Physalis mottle virus) RNA and an analysis of the relationships of tymoviral coat proteins.

The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.

Amino Acid Sequence↗
PubMed2004

Identification and phenotypic characterization of the cell-division protein CdpA.

Analysis of the automated computer annotation of the early draft phase genome of Lactobacillus acidophilus NCFM revealed the previously discovered S-layer gene slpA and an additional partial ORF with weak similarities to S-layer proteins. The entire gene was sequenced to reveal a 1799-bp gene coding for 599 amino acids with a calculated molecular mass of 64.8 kDa. No transcription or translation signals could be determined in close proximity to the 5'-region. However, a strong putative terminator with a free energy of -16.84 kcal/mol was identified directly downstream of the gene. A PSI-Blast analysis showed similarities to members of S-layer proteins, cell-wall associated proteinases and hexosyl-transferases. Calculation of an unrooted phylogenetic tree with other examples of S-layer proteins and proteinases placed the deduced protein separately from both groups. A derivative of L. acidophilus NCFM was constructed by targeted integration into the gene. SDS-PAGE analysis of non-covalently linked proteins of the cell wall of the mutant, compared to the wild type, revealed the loss of a cell-surface protein. Phenotypic analyses of the mutant revealed significant changes in cell morphology, altered responses to various environmental stresses, and lowered cell adhesion. Based on the in silico and functional analyses, we ascertained that this protein plays a role in cell-wall processing during the growth and cell-cell separation and designated the gene as cell-division protein, cdpA.

Bacterial Adhesion↗
PubMed2000

Characterization of the DNA replication module of bacteriophage A2 and use of its origin of replication as a defense against infection during milk fermentation by Lactobacillus casei.

Adjacent to the lysis/lysogeny cassette of the A2 phage genome lies a stretch of over 8 kb, which contains a series of genes probably involved in DNA replication. Fifteen open reading frames (orfs) were identified, 13 of which are encoded on the main coding strand and only two on the complementary strand. Database searches and comparative analyses allowed the identification of an open reading frame (orf455) that shows similarity with DNA helicases and contains a variant zinc-finger motif known from the phage T7 helicase/primase. Orf770 showed similarity to putative plasmid and phage DNA primases. Downstream of orf770 is a noncoding 258-bp region rich in direct and inverted repeats, which specifically binds to proteins whose synthesis is induced during phage infection. When present in a plasmid, this region can direct a partial bacteriophage resistance phenotype due to interference with phage DNA replication, both under laboratory conditions and during milk fermentation. It is deduced that this stretch contains the origin of replication of phage A2.

Amino Acid Sequence↗
PubMed1997

Screening and identification of yeast sequences that cause growth inhibition when overexpressed.

To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter. In all, 240,000 yeast transformants were screened for lethality on galactose medium. From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained. Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII. Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition. The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule. Overexpression of the BNI1 deltaN and BOI1 deltaN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation. Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively. The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA.

Amino Acid Sequence↗
PubMed1990

Transcription of the human cytomegalovirus glycoprotein gene family in the short unique component of the viral genome.

The HXLF (HindIII X left reading frame) genes are a group of six open reading frames (ORFs) within the short unique component of the human cytomegalovirus genome. Two or more ORFs of the HXLF gene family code for components of the virion envelope glycoprotein complex designated gcII. Transcription of the various HXLF genes in infected cells was analyzed using specific DNA, RNA, and synthetic oligonucleotide probes. Transcription from each of the six HXLF genes was analyzed at immediate early, early, and late times after infection. Even though an enhancer element in six copies is downstream of HXLF6, immediate early cytoplasmic RNAs from the HXLF genes was not detectable. In contrast, early and late cytoplasmic RNAs were detectable for all six HXLF genes. The HXLF1 and 2 genes and the HXLF3 and 4 genes are transcribed as bicistronic mRNAs of 1.6 and 1.7 kb, respectively. The HXLF5 and 6 genes are transcribed as monocistronic mRNAs. In the HXLF4, 5, and 6 transcription units cytoplasmic viral RNAs were detected from both strands. Bidirectional transcription upstream of the enhancer element occurs at only early and late times after infection. The significance of bicistronic mRNA to the formation of the glycoprotein complex gcII requires further investigation.

Blotting, Northern↗
PubMed1996

Molecular cloning and sequence analysis of the cDNA encoding rat liver cysteine sulfinate decarboxylase (CSD).

The taurine biosynthesis enzyme, cysteine sulfinate decarboxylase (CSD), was purified to homogeneity from rat liver. Three CSD peptides generated by tryptic cleavage were isolated and partially sequenced. Two of them showed a marked homology with glutamate decarboxylase and their respective position on the CSD amino acid sequence was postulated accordingly. Using appropriate degenerated primers derived from these two peptides, a PCR amplified DNA fragment was generated from liver poly(A)+ mRNA, cloned and used as a probe to screen a rat liver cDNA library. Three cDNAs, length around 1800 bp, were isolated which all contained an open reading frame (ORF) encoding a 493 amino acid protein with a calculated molecular mass of 55.2 kDa close to the experimental values for CSD. The encoded protein contained the sequence of the three peptides isolated from homogenous liver CSD. Our data confirm and significantly extend those recently published (Kaisaki et al. (1995) Biochim. Biophys. Acta 1262, 79-82). Indeed, an additional base pair found 1371 bp downstream from the initiation codon led to a shift in the open reading frame which extended the carboxy-terminal end by 15 amino acid residues and altogether modified 36 amino acids. The validity of this correction is supported by the finding that the corrected reading frame encoded a peptide issued from CSD tryptic cleavage that was not encoded anywhere in the CSD sequence previously reported.

Amino Acid Sequence↗
PubMed1993

Two putative insertion sequences flank a truncated glycogen branching enzyme gene in the thermophile Bacillus stearothermophilus CU21.

We have isolated a region from the Bacillus stearothermophilus CU21 chromosome hybridizing strongly to a fragment of the B. caldolyticus glycogen operon. Sequence analysis of this region revealed the presence of a truncated glgB gene encoding the N-terminus of branching enzyme. A region highly similar to an internal fragment of B. caldolyticus glgC encoding ADP-glucose pyrophosphorylase was located approximately 1kb downstream from the incomplete glgB gene. The two truncated genes appeared to flank a sequence with characteristics of bacterial Insertion Sequences, which was designated RSBst-alpha. The presence of RSBst-alpha at this position indicates that integration of (an) IS-like element(s) may have been involved in deletion formation in the putative glycogen operon. Upstream of glgB an additional incomplete ORF was found with significant similarity to putative transposases from bacterial Insertion Sequences. This region was designated RSBst-beta. Both RSBst-alpha and RSBst-beta appeared to be present in multiple copies in the B. stearothermophilus CU21 chromosome.

1,4-alpha-Glucan Branching Enzyme↗
PubMed2001

Nucleotide sequence and transcriptional analysis of a putative basic DNA-binding protein of Helicoverpa armigera nucleopolyhedrovirus.

A putative basic DNA-binding protein (BDBP) gene was identified in the fragment EcoRI-K of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HearNPV) genome. The ORF is 330 nucleotides long encoding a basic protein of 109 amino acids with a molecular mass of 11.6 kDa. It is the first BDBP identified in single nucleocapsid NPVs and a homologue of Autographa californica MNPV (AcMNPV) P6.9. A consensus late transcription motif, ATAAG, was found at 57 nt upstream of the translational start codon and a polyadenylation signal was observed at 172 nt downstream of the stop codon. A major transcript of 620 nt was first observed in HearNPV-infected Hz2e5 cells 16 h post infection. Primer extension analysis revealed that this transcript initiated from the first residue of the consensus ATAAG late transcription start motif. Comparison with other baculoviral BDBPs showed that they all contained two conserved cAMP- and cGMP-dependent protein kinase phosphorylation motifs, R-R-R-S. The HearNPV P6.9 homologue is the longest BDBP found so far in baculoviruses.

Amino Acid Sequence↗
PubMed1989

The Bradyrhizobium japonicum fixBCX operon: identification of fixX and of a 5' mRNA region affecting the level of the fixBCX transcript.

The Bradyrhizobium japonicum fixX gene was identified and shown to be essential for symbiotic and free-living, microaerobic nitrogen fixation. The fixX gene encodes a ferredoxin-like protein which may be involved in a redox process (electron transport?) essential for nitrogenase activity. This gene was localized downstream of fixC and its expression was dependent on the fixB promoter, providing evidence for the existence of a fixBCX operon. Mutagenesis and sequence analysis of the unusually long, 709bp leader region between the fixB promoter and the fixB structural gene did not reveal the presence of a nif or fix gene that was absolutely essential for nitrogen fixation. However, a short open reading frame (ORF) within this region encoding a polypeptide of 35 amino acids (ORF35) was shown to be efficiently translated. Chromosomal deletion of a 400bp DNA fragment covering ORF35 resulted in a three-fold reduction of the fixBCX mRNA level, which in turn also reduced the nitrogen fixation activity of this mutant. This suggests a possible post-transcriptional control mechanism for the expression of the fixBCX operon involving the stabilization of fixBCX mRNA by ribosomes actively translating ORF35.

Amino Acid Sequence↗
PubMed2004

Comparative analysis of conserved genetic markers and adjacent DNA regions identified in beer-spoilage lactic acid bacteria.

AIMS: To conduct an inter-species comparative study on the nucleotide sequences of the conserved DNA regions surrounding ORF5, a genetic marker for differentiating beer-spoilage lactic acid bacteria. METHODS AND RESULTS: The conserved DNA regions surrounding ORF5 were examined by PCR analysis, using three beer-spoilage strains, Lactobacillus brevis ABBC45C, L. paracollinoides LA2T and Pediococcus damnosus ABBC478. As a result, the DNA regions containing ORF1-7, originally found in ABBC45C, appeared to be conserved among the three strains, while the downstream region was not found in L. paracollinoides LA2T and P. damnosus ABBC478. The sequencing analysis of the conserved DNA regions of LA2T and ABBC478 revealed ca 99% nucleotide sequence identities with that of ABBC45C. CONCLUSIONS: The nucleotide sequences of the ca 8.2 kb DNA regions containing ORF1-7 were virtually identical among the three strains belonging to different species. The internal organizations of the ORFs were found to be remarkably similar. SIGNIFICANCE AND IMPACT OF THE STUDY: The level of nucleotide sequence identities suggests the DNA regions surrounding ORF5 were horizontally acquired by these beer-spoilage strains belonging to the three different species of lactic acid bacteria.

Beer↗
PubMed1997

Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region.

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site (cosN). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left (cosL) and the right (cosR) of the cosN site were observed. Analysis of the 3.4 kb EcoRI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames (orfs). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orfs were translated into protein. From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.

Amino Acid Sequence↗