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Transcriptional and translational expression kinetics of the UL25 homologue of bovine herpesvirus 1.1.

We investigated whether the bovine herpesvirus 1.1 (BHV1) ORF, a homologue of the herpes simplex virus 1 (HSV1) UL25 gene, represented a functional gene. The BHV1 UL25 ORF, which is located at positions 60602<--62398 of the viral genome, generated a 4.5 kb transcript accumulating at low abundance as soon as 3 hours p.i. after which the levels increased up to 12h p.i. and remained constant up to 24 hours p.i. In addition, UL25 transcription initiated at 303 bases upstream from the translation initiation codon, corresponding to 26 and 354 b downstream from putative TATA and CAAT boxes, respectively, thus providing evidence that these elements function as the UL25 promoter. Western blotting of BHV1-infected cell lysates, using a BHV1-UL25 specific antiserum generated against a T7-Tag/UL25 fusion recombinant protein expressed in E. coli, detected a 63 kDa protein of the expected size (63,083 Da) whose expression profile followed that of its transcript. The synthesis of the 63 kDa protein was abrogated by a DNA synthesis inhibitor, unambiguously demonstrating that the viral specific protein expressed from the BHV1 UL25 ORF belongs to the gamma2 class.

Animals↗

Programmed +1 frameshifting stimulated by complementarity between a downstream mRNA sequence and an error-correcting region of rRNA.

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site. This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA. A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold. Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting. Its function depends on strict spacing from the site of frameshifting. Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons. Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.

Amino Acid Sequence↗

Putative lmbI and lmbH genes form a single lmbIH ORF in Streptomyces lincolnensis type strain ATCC 25466.

The lincomycin-production gene cluster of the industrial overproduction strain Streptomyces lincolnensis 78-11 has been sequenced (Peschke et al. 1995) and twenty-seven putative open reading frames with biosynthetic or regulatory functions (lmb genes) identified. Two distinct hypothetical genes, lmbI and lmbH, were found downstream of the lmbJ gene, coding for LmbJ protein, which is believed to participate in the last lincomycin biosynthetic step, i.e. conversion of N-demethyllincomycin (NDL) to lincomycin. In the present study, we demonstrate the presence of a single larger open reading frame, called lmbIH, in the lincomycin low-production type strain Streptomyces lincolnensis ATCC 25466, instead of two smaller lmbI and lmbH genes. The product, LmbIH, is a protein of an unknown function and is homologous with the T1dD protein family. Escherichia coli T1dD protein was previously shown to be involved in the control of DNA gyrase by LetD protein. Moreover, our experiments indicate co-regulation of lmbJ and lmbIH expression. This translation coupling probably reflects an eight nucleotide overlap between the lmbJ and lmbIH genes, as well as the lack of a Shine-Dalgarno sequence upstream of the lmbIH gene.

Amino Acid Sequence↗

Sequence and transcriptional analysis of a near-terminal region of the orf virus genome.

A 3605 bp region located approximately 6.6 kb from the left end of the orf virus genome (strain NZ2) was sequenced. The sequence revealed two open reading frames, which we have designated G2L and B1L. The predicted amino acid sequences of G2L and B1L were found to be homologous to the vaccinia virus (VAC) F11L and F12L gene products, respectively, and were found to be arranged on the genome in the same orientation and relative position as their VAC counterparts. Transcriptional analysis of both G2L and B1L showed they were transcribed toward the genome terminus during the early phase of infection. S1 nuclease and primer-extension analyses showed that the transcriptional start sites of both genes were located a short distance downstream from A+T-rich sequences, similar to vac virus early promoters.

Amino Acid Sequence↗

Rhizobium meliloti lacking mosA synthesizes the rhizopine scyllo-inosamine in place of 3-O-methyl-scyllo-inosamine.

The Rhizobium meliloti Rm220-3 mos locus producing the rhizopine scyllo-inosamine (SI) in nodules is shown to consist of three ORFs (ORF1, mosB and mosC) arranged in an operon structure. This differs from the R. meliloti L5-30 mos locus, which produces 3-O-methyl-scyllo-inosamine (3-O-MSI), by the complete absence of mosA. The deletion covers a region of 1235 nt and includes the entire mosA gene as well as the sequence both upstream and downstream. As a result, Rm220-3 mos ORF1 shares a 5' region common with L5-30 ORF1 but includes an additional 10 bp insertion and a section in the L5-30 mosA and mosB intercistronic region. Antibodies against L5-30 Mos proteins detected MosB and MosC proteins in Rm220-3-induced nodules but no translation product for either ORF1 or mosA was detected. A construct prepared from the L5-30 mos locus which has a truncated mosA gene produces SI rather than 3-O-MSI, confirming this gene is involved in a methylation step in the production of 3-O-MSI. Further, changes made to this ORF result in reduced levels of the rhizopine.

Amino Acid Sequence↗

Complete nucleotide sequence of Sesbania mosaic virus: a new virus species of the genus Sobemovirus.

The complete nucleotide sequence of the Sesbania mosaic virus (SeMV) genomic RNA was determined by sequencing overlapping cDNA clones. The SeMV genome is 4149 nucleotides in length and encodes four potential overlapping open reading frames (ORFs). Comparison of the nucleotide sequence and the deduced amino acid sequence of the four ORFs of SeMV with that of other sobemoviruses revealed that SeMV was closest to southern bean mosaic virus Arkansas isolate (SBMV-Ark, 73% identity). The 5' non-coding regions of SeMV, SBMV and southern cowpea mosaic virus (SCPMV) are nearly identical. However ORF1 of SeMV which encodes for a putative movement protein of M(r) 18370 has only 34% identity with SBMV-Ark. ORF 2 encodes a polyprotein containing the serine protease, genome linked viral protein (VPg) and RNA dependent RNA polymerase domains and shows 78% identity with SBMV-Ark. The N-terminal amino acid sequence of VPg was found to be TLPPELSIIEIP, which mapped to the region 326-337 of ORF2 product and the cleavage site between the protease domain and VPg was identified to be E325-T326. The cleavage site between VPg and RNA dependent RNA polymerase was predicted to be E445-T446 based on the amino acid sequence analysis of the polyprotein from different sobemoviruses. ORF3 is nested within ORF2 in a--1 reading frame. The potential ribosomal frame shift signal and the downstream stem-loop structure found in other sobemoviruses are also conserved in SeMV RNA sequence, indicating that ORF3 might be expressed via--1 frame shifting mechanism. ORF4 encodes the coat protein of SeMV, which shows 76 and 66% identity with SBMV-Ark and SCPMV, respectively. Thus the comparison of the non-coding regions and the ORFs of SeMV with other sobemoviruses clearly revealed that it is not a strain of SBMV. Phylogenetic analysis of six different sobemoviruses, including SeMV, suggests that recombination event is not frequent in this group and that SeMV is a distinct member of the genus sobemovirus. The analysis also shows sobemoviruses infecting monocotyledons and dicotyledons fall into two distinct clusters.

Base Sequence↗

Sequences, organization and analysis of the hupZMNOQRTV genes from the Azotobacter chroococcum hydrogenase gene cluster.

Hydrogen-uptake (Hup) activity in Azotobacter chroococcum depends upon a cluster of genes spread over 13,687 bp of the chromosome. Six accessory genes of the cluster, hupABYCDE, begin 4.8 kb downstream of the structural genes, hupSL, and are required for the formation of a functional [NiFe] hydrogenase. The sequencing of the intervening 4.8 kb of hup-specific DNA has now been completed. This revealed eight additional closely linked ORFs, which we designated hupZ, hupM, hupN, hupO, hupQ, hupR, hupT and hupV. These genes potentially encode polypeptides with predicted masses of 27.7, 22.3, 11.4, 16.2, 31.3, 8.1, 16.2 and 36.7 kDa, respectively. All eight genes are transcribed from the same strand as hupSL and hupABYCDE. A chroococcum, therefore, has a total of 16 contiguous genes affecting hydrogenase activity beginning with hupS and ending with hupE. The amino acid sequence deduced from hupZ has the characteristics of a b-type cytochrome. Insertion mutagenesis of hupZ resulted in a mutant incapable of supporting O2-dependent H2 oxidation. The deduced amino acid sequence of hupR shares high homology with bacterial rubredoxins. HupZ and HupR may both be involved in transferring electrons from hydrogenase to the electron transport chain. A mutation in hupV knocked out hydrogenase activity entirely; this gene may be involved in processing the large subunit of hydrogenase. It is now clear that the genes controlling [NiFe] hydrogenase activity in many bacteria including Azotobacter chroococcum, Alcaligenes eutrophus, Rhizobium leguminosarum, Rhodobacter capsulatus and Escherichia coli are highly conserved, organized in much the same manner, and likely derived from a common ancestor.

Amino Acid Sequence↗

Chromosomal sequences from Klebsiella pneumoniae flank the SHV-5 extended-spectrum beta-lactamase gene in pACM1.

The nucleotide sequence was determined for Anon 13, a 1250-bp SmaI fragment located approximately 2.8 kb downstream from bla(SHV-5) in pACM1. Anon 13 is 99% identical to a segment of the unpublished sequence of the Klebsiella pneumoniae chromosome. Genes of the K. pneumoniae sequence are undefined, but conceptual amino acid translations of two ORFs in Anon 13 are homologous to L-fuculose-1-phosphate aldolase (FucA) and a conserved hypothetical protein present in the chromosomes of several species of bacteria. In addition, restriction mapping indicates that the region of homology between the K. pneumoniae chromosome and pACM1 is as least 7.9 kb and includes both Anon 13 and bla(SHV). These observations demonstrate the chromosomal origin of the bla(SHV-5) on pACM1.

Chromosomes, Bacterial↗

Characterization of the ecdysteroid UDP-glucosyltransferase gene of a single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria.

A putative ecdysteroid UDP-glucosyltransferase (egt) gene was identified in the single nucleocapsid nucleopolyhedrovirus of Buzura suppressaria (BusuNPV). This is the first egt gene to be characterized in a SNPV, suggesting that egt genes are prevalent in nucleopolyhedroviruses and possibly in all baculoviruses. The open reading frame (ORF) of the gene is 1539 nucleotides (nt) long, encoding a putative protein (EGT) of 513 amino acids (aa) with a M of 58922. The 5' noncoding region contains three possible TATA boxes. A polyadenylation signal, AATAAA, was found downstream of the translation stop codon. A putative signal peptide of 16 residues was present at the N-terminus of the EGT. The BusuNPV egt gene has a high degree of nucleotide and amino acid sequence homology to multiple nucleocapsid (M) NPV egt genes, the highest being to the Spodoptera exigua MNPV egt. A phylogenetic tree of eleven known EGTs was constructed using maximum parsimony analysis.

Amino Acid Sequence↗

Genomic localization and nucleotide sequence of a lef-8 gene of the Spodoptera littoralis nucleopolyhedrovirus.

A gene similar to lef-8 of the Autographa californica (Ac) nucleopolyhedrovirus (MNPV) was identified in the Spodoptera littoralis (Spli) MNPV. The SpliMNPV lef-8-like gene was localized on the genomic map between 26.9 and 29 map units and is flanked by a chitinase gene and p47 gene. This gene arrangement differs from that of similar genes in the AcMNPV genome, where the lef-8 gene is located about 62 kbp from the chitinase gene and about 7 kbp from the p47 gene. Sequence analyses of the lef-8 gene revealed an ORF of 2730 nucleotides, predicted to encode a protein with Mr 104876. The putative protein is 60.9% identical to the AcMNPV LEF-8 and contains an identical sequence of a conserved motif of DNA-dependent RNA polymerases. Sequences downstream of the lef-8 gene contain two sequence repeats which resemble a repeated motif of the SpliMNPV enhancer element and other repetitive sequences from the viral genome.

Amino Acid Sequence↗

Isolation and characterization of a new chromosomal virulence gene of Agrobacterium tumefaciens.

A mutant (strain B119) of Agrobacterium tumefaciens with a chromosomal mutation was isolated by transposon (Tn5) mutagenesis. The mutant exhibited growth rates on L agar and minimal medium (AB) plates similar to those of the parent strain (strain A208 harboring a nopaline-type Ti plasmid). The mutant was avirulent on all host plants tested: Daucus carota, Cucumis sativus, and Kalanchoe diagremontiana. The mutant was not impaired in attachment ability to carrot cells. The mutant had one insertion of Tn5 in its chromosome. The avirulent phenotype of B119 was shown to be due to the Tn5 insertion in the chromosome by the marker exchange technique. A wild-type target chromosomal segment (3.0 kb) which included the site of mutation was cloned and sequenced. Two open reading frames, ORF-1 (468 bp) and ORF-2 (995 bp), were identified in the 3.0-kb DNA segment. Tn5 was inserted in the middle of ORF-2 (acvB gene). Introduction of the acvB gene into the mutant B119 strain complemented the avirulent phenotype of the strain. Homology search found no genes homologous to acvB, although it had some similarity to the open reading frame downstream of the virA gene on the Ti plasmid. Thus, the acvB gene identified in this study seems to be a new chromosomal virulence gene of A. tumefaciens.

Agrobacterium tumefaciens↗

Molecular cloning and characterization of the structural gene coding for the developmentally regulated lysosomal enzyme, alpha-mannosidase, in Dictyostelium discoideum.

The gene coding for the Dictyostelium discoideum lysosomal enzyme, alpha-mannosidase, has been cloned and sequenced. To accomplish this, the mature 60- and 58-kDa subunits of the enzyme were purified and subjected to liquid-phase N-terminal amino acid sequencing. Sequence information was obtained for both of the mature subunits, and a 48-mer oligonucleotide was synthesized based on the determined amino acid sequence of the 58-kDa subunit. Using this oligonucleotide as a probe, an 8-kilobase HindIII fragment of genomic DNA was isolated and subjected to Sanger dideoxy DNA sequencing. The first 4400 nucleotides contained the complete alpha-mannosidase gene and 1100 nucleotides of 5'-flanking DNA. Primer extension analysis indicated that transcription begins at multiple sites -48 to -64 nucleotides upstream of the first nucleotide of the predicted translation initiation codon. A single open reading frame (ORF) of 3015 nucleotides was found that was interrupted by a single intron and that contained the amino acid sequences of the N termini of the two mature alpha-mannosidase subunits; a polyadenylation signal was also found just downstream of the termination codon. A potential cleavable signal sequence was identified in the first 22 amino acids of the predicted precursor protein, and two propeptide regions (Pro I and II) were identified that were immediately upstream of the N termini of the 60- and 58-kDa mature subunits, respectively. These propeptide regions are not present in the mature protein and are therefore predicted to be proteolytically removed as the membrane associated 140-kDa precursor is transported to lysosomes and processed to the soluble 60- and 58-kDa mature forms of the enzyme. In fact, potential proteolytic cleavage sites were identified flanking the Pro I and Pro II regions. Pro I, which immediately follows the signal sequence, consists of 18 amino acids, most of which are highly charged and hydrophilic residues, while Pro II, found in the central portion of the precursor, is very hydrophobic. While no obvious transmembrane regions were identified, several short hydrophobic amino acid stretches were found to be localized in and around the Pro II region, and these may be responsible for attachment of precursors to membranes.

Amino Acid Sequence↗

Genetic organization of the Francisella plasmid pFNL10.

We report here the molecular characterization of pFNL10, a 3990-bp cryptic plasmid of Francisella novicida-like F6168. The plasmid was maintained in F. novicida Utah 112 and F. tularensis LVS strains. We sequenced the entire plasmid and found six open reading frames (ORFs)-ORF1, ORF2, ORF3, ORF4, ORF5, and ORFm. ORF3, ORF4, ORF5, and ORFm are located on the same strand, and we designated it the plus strand. ORF1 and ORF2 are on the complementary strand. The ORFs appear to be arranged in two operons, one comprising ORF5 and ORF4 and the other ORF1 and ORF2. There exist two distinct promoters similar to the Escherichia coli sigma(70) promoter, one 5' to ORF1-ORF2 operon and the other 5' to ORF5-ORF4 operon. We found that in both promoters the transcriptional start is an adenosine. ORF3 is positioned in tandem with ORF5-ORF4, but has its own transcriptional start, a thymidine. However, sequence analysis revealed no recognizable promoter in physical proximity to ORF3. Sequence analysis revealed transcriptional terminators immediately downstream of the two operons. Experimental results showed that the ORF1-ORF2 terminator is authentic. But we could not definitively confirm the ORF5-ORF4 terminator. Two sets of direct repeats, one 31 and the other 13 bp, characteristic of ori are positioned between the two promoters. ORF1 encodes a protein that bears homology to the replication initiation protein RepA of various bacteria, and disruption of this ORF indeed blocked pFNL10 replication. In contrast, ORF2 disruption caused formation of plasmid multimers, suggesting aberrant replication. Our analysis also suggests that pFNL10 replicates by the theta mode. The ORF5-ORF4 operon resembles the phd-doc operon of Escherichia coli bacteriophage P1, but the significance of this similarity is unclear.

Amino Acid Sequence↗

Scanning independent ribosomal initiation of the Sendai virus Y proteins in vitro and in vivo.

The Sendai virus P/C mRNA contains five ribosomal initiation sites between positions 81 and 201 from the 5' end. One of these sites initiates in the P open reading frame (ORF) (ATG/104), whereas four initiate in the C ORF (ACG/81 and ATGs/114, 183, 201), to give a nested set of C proteins (C', C, Y1, Y2). Introduction of new ATGs or physically breaking the mRNA upstream of these natural sites was used in vitro to prevent ribosomal scanning downstream. The results suggest that a minority of the ribosomes which initiate C (ATG/114) and all of those which initiate Y1 and Y2 (ATGs/183 and 201) do so by a scanning independent mechanism. When the leaky ACG/81 site is changed to a non-leaky ATG site in in vivo experiments, ribosomal initiation at Y is again not diminished, whereas that at C as well as at P becomes undetectable. Ribosomal initiation at Y appears to be scanning independent in vitro and in vivo. That at C is partly independent in vitro, but completely dependent in vivo. These results are discussed in terms of a model of internal initiation at Y.

Codon↗

Genomic organization of the Neurospora crassa gsn gene: possible involvement of the STRE and HSE elements in the modulation of transcription during heat shock.

Glycogen synthase, an enzyme involved in glycogen biosynthesis, is regulated by phosphorylation and by the allosteric ligand glucose-6-phosphate (G6P). In addition, enzyme levels can be regulated by changes in gene expression. We recently cloned a cDNA for glycogen synthase ( gsn) from Neurospora crassa, and showed that gsn transcription decreased when cells were exposed to heat shock (shifted from 30 degrees C to 45 degrees C). In order to understand the mechanisms that control gsn expression, we isolated the gene, including its 5' and 3' flanking regions, from the genome of N. crassa. An ORF of approximately 2.4 kb was identified, which is interrupted by four small introns (II-V). Intron I (482 bp) is located in the 5'UTR region. Three putative Transcription Initiation Sites (TISs) were mapped, one of which lies downstream of a canonical TATA-box sequence (5'-TGTATAAA-3'). Analysis of the 5'-flanking region revealed the presence of putative transcription factor-binding sites, including Heat Shock Elements (HSEs) and STress Responsive Elements (STREs). The possible involvement of these motifs in the negative regulation of gsn transcription was investigated using Electrophoretic Mobility Shift Assays (EMSA) with nuclear extracts of N. crassa mycelium obtained before and after heat shock, and DNA fragments encompassing HSE and STRE elements from the 5'-flanking region. While elements within the promoter region are involved in transcription under heat shock, elements in the 5'UTR intron may participate in transcription during vegetative growth. The results thus suggest that N. crassa possesses trans -acting elements that interact with the 5'-flanking region to regulate gsn transcription during heat shock and vegetative growth.

Base Sequence↗

Cloning and expression of two autolysin genes, cwIU and cwIV, which are tandemly arranged on the chromosome of Bacillus polymyxa var. colistinus.

The cwlV gene, which encodes Bacillus polymyxa var. colistinus autolysin was cloned and sequenced. cwlV comprises a 1497-bp ORF and encodes a polypeptide of 499 amino acid (aa) residues (Mr of 53,707 Da). The N-terminal sequence of the mature 23-kDa CwlV protein is NSXGKKVVVIDAGXGAKD(X, undetermined aa); this processed form corresponds to the C-terminal portion (183 aa, Mr of 20,050 Da) of the cwlV ORF. Sequencing of the flanking region revealed that another putative autolysin gene, cwlU, is located upstream of cwlV. cwlU encodes a polypeptide of 524 aa and its deduced sequence is 34.9% identical to the full-length sequence of CwlV. Downstream of cwlV, the genes for a deduced lipoprotein (OrfW), an endonuclease III homolog (Nth), a non-homologous OrfX, a glutathione peroxidase homolog (Gpx), and the N-terminal region of OrfZ containing a ATP/GTP-binding site motif were found. Northern blotting and primer-extension analyses revealed that cwlU is transcribed as a single cistron, but cwlV is transcribed with orfW. The unprocessed forms of CwlV and CwlU (VdeltaS and UdeltaS, respectively) and their predicted mature forms (Vcat and Ucat, respectively) were expressed in, and purified from, Escherichia coli. Enzyme analysis indicated that VdeltaS and Vcat exhibit low and high cell wall hydrolase activities toward B. polymyxa cell wall, respectively, but UdeltaS and Ucat exhibit almost no and low cell wall hydrolase activities, respectively.

Amino Acid Sequence↗

Genetic and molecular biological characterization of a vaccinia virus gene which renders the virus dependent on isatin-beta-thiosemicarbazone (IBT).

We have sequenced and analyzed the transcription of a gene capable of rendering vaccinia virus (VV) dependent upon isatin-beta-thiosemicarbazone (IBT) for growth. Marker rescue analysis of an IBT-dependent mutant of VV, IBTd-1, and a temperature-sensitive mutant of VV, ts56, both of which require IBT to grow at 40 degrees, showed that both lesions mapped to gene G2R. VV mutants with G2R deletions were constructed and shown to also be dependent upon IBT for growth. The nucleotide sequence changes responsible for IBTd-1, ts56, and the gene G2R deletion mutants were determined, and taken together show that IBT dependence results from inactivation of the orf G2R gene product. Gene G2R, which has the capacity to encode a 26-kDa protein, is transcribed solely early during infection. The 1.3-kb mRNA contains a 5' untranslated region of almost 600 nucleotides, and terminates about 20 nucleotides downstream from an early transcription termination signal. Transcription analyses of three flanking genes, as well as the map positions of the VV mutants ts11 and ts60 are also presented.

Adenine Nucleotides↗

Sequence analysis and characterization of the hmw gene cluster of Mycoplasma pneumoniae.

Mycoplasma pneumoniae (Mp) cytadherence requires the proper anchoring of cytadhesin proteins in the mycoplasmal membrane at an attachment organelle through their interaction with a cytoskeleton-like network of accessory proteins that includes HMW1 and HMW3. Approximately 8.25 kb of Mp DNA was sequenced, beginning at the 3' end of the hmw3 gene and continuing through hmw1. Comparison of the resulting deduced amino acid (aa) sequence with N terminus and internal peptide aa sequences from purified HMW1 permitted definitive identification of hmw1. HMW1 was characterized with respect to structure, hydrophobicity, possible phosphoacceptor sites and expression of the Mp recombinant protein in Escherichia coli. In addition, HMW1 membrane topography was examined for antibody accessibility on the mycoplasmal surface. hmw3 and hmw1 flank four open reading frames (ORFs) spanning approximately 4.3 kb and in the same orientation as the hmw genes. The sequences of their deduced products were evaluated for likely structural features and comparison with protein data banks. Finally, the Mp rpsD analog was identified immediately downstream from hmw1.

Amino Acid Sequence↗