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PubMed1999

Analysis of transcripts expressed from the UL47 gene of human cytomegalovirus.

The UL47 gene (b 60390-b 60338) located in the unique long region of the human cytomegalovirus (HCMV) AD169 strain genome was analyzed by RNA mapping. Northern blot analysis showed that the UL47 gene was expressed at late times after infection (72 h postinfection). The 9.7-kb transcript was expressed in the infected cells but not in phosphonoformate-treated cells at 72 hpi, indicating that the UL47 gene was only expressed at late times after infection. To map the 5'-end and 3'-end of UL47 transcripts, primer extension and RNase protection analysis were performed. Primer extension analysis revealed that the transcription initiation site of UL47 was located in 27 bp downstream (b 60323) of the TATA box motif. The sizes of UL47 ORF (approximately 2.9-kb) and UL48 ORF (approximately 6.7-kb) deduced from computer sequence analysis suggest that the expressed 9.7-kb transcript of UL47 uses the 3'-end polyadenylation signal of UL48. The result of RNase protection determined that the 3'-end of UL47 RNA utilized the 3'-end polyadenylation signal of UL48, which is located in HCMV genome b 70082.

Base Sequence↗
PubMed1999

The S gene of canine coronavirus, strain UCD-1, is more closely related to the S gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus.

To gain insight into the genetic relationships among six canine coronavirus (CCV) strains, the variable region of the spike (S) protein gene was sequenced. The CCV strains were: two ATCC reference strains, the Insavc-1 vaccine strain, the National Veterinary Services Laboratories (Ames, IA) challenge strain, and two California field isolates (UCD-1 and UCD-2) from the 1970s. All six strains, downstream of the nucleocapsid (N) protein gene, had sufficient size for an ORF 7b, and thus, none were transmissible gastroenteritis virus (TGEV)-like since TGEV lacks ORF 7b. By sequence analysis of the variable domain at the 5' end of the S gene, five of the six CCV strains had a high degree of identity with feline infectious peritonitis virus (FIPV). However, one CCV field isolate (UCD-1) was different and had a high degree of identity with the 5' end of the TGEV S gene. This suggests that RNA recombination occurred at this site between antigenically related coronaviruses. The low passage field isolates, UCD-1 and UCD-2, varied in their initial infectivity for swine testicular cells suggesting that sequence differences in the variable domain of the S gene may account for biological variation among CCVs.

Amino Acid Sequence↗
PubMed2000

Lessons from sequenced genomes. Overlapping genes in Methanococcus jannaschii?

This paper describes our finding on overlapping genes in Methanococcus jannaschii genome. We found that one of the open reading frames (ORFs) within the M. jannaschii genome contains the nucleotide sequence of tRNA(Ser), which raises a serious question of the correctness of the initiation codon assignment for that ORF. We suggest that there are two other possible AUG initiation codons downstream from the TTG triplet, which was initially considered as a translation start site. Only one of the AUG triplets is preceded by the Shine-Dalgarno sequence that seems to be required for binding the ribosome and initiation of translation.

Amino Acid Sequence↗
PubMed1995

pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin.

Nucleotide sequencing of a region downstream from the Pseudomonas aeruginosa 1244 pilin structural gene, pilA, revealed an ORF potentially able to code for a protein of M(r) 50,862. This ORF, called pilO, was flanked by a tRNAthr gene, which was followed by a transcriptional termination sequence. The tRNAthr gene and the termination sequence were nearly identical to sequences found immediately adjacent to the pilA gene of several P. aeruginosa strains. A 2200 base mRNA strand, which contained both the pilO and pilA transcripts, was produced from this region, while a 650 base transcript containing only pilA was present in a 100-fold excess over the longer transcript. Hyperexpression of the pilA gene in a PilO- strain resulted in normal pilus-specific phage sensitivity and twitching motility. The pilin produced by this strain had a lower apparent M(r) and a more neutral pl compared to that produced by a strain containing a functional pilO gene. This pilin failed to react with a sugar-specific reagent which recognized pilin produced by the strain containing a functional pilO gene.

Amino Acid Sequence↗
PubMed2000

Identification of a gene within a pathogenicity island of enterotoxigenic Escherichia coli H10407 required for maximal secretion of the heat-labile enterotoxin.

Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island.

Adhesins, Bacterial↗
PubMed2006

Abortive phage resistance mechanism AbiZ speeds the lysis clock to cause premature lysis of phage-infected Lactococcus lactis.

The conjugative plasmid pTR2030 has been used extensively to confer phage resistance in commercial Lactococcus starter cultures. The plasmid harbors a 16-kb region, flanked by insertion sequence (IS) elements, that encodes the restriction/modification system LlaI and carries an abortive infection gene, abiA. The AbiA system inhibits both prolate and small isometric phages by interfering with the early stages of phage DNA replication. However, abiA alone does not account for the full abortive activity reported for pTR2030. In this study, a 7.5-kb region positioned within the IS elements and downstream of abiA was sequenced to reveal seven additional open reading frames (ORFs). A single ORF, designated abiZ, was found to be responsible for a significant reduction in plaque size and an efficiency of plaquing (EOP) of 10(-6), without affecting phage adsorption. AbiZ causes phage phi31-infected Lactococcus lactis NCK203 to lyse 15 min early, reducing the burst size of phi31 100-fold. Thirteen of 14 phages of the P335 group were sensitive to AbiZ, through reduction in either plaque size, EOP, or both. The predicted AbiZ protein contains two predicted transmembrane helices but shows no significant DNA homologies. When the phage phi31 lysin and holin genes were cloned into the nisin-inducible shuttle vector pMSP3545, nisin induction of holin and lysin caused partial lysis of NCK203. In the presence of AbiZ, lysis occurred 30 min earlier. In holin-induced cells, membrane permeability as measured using propidium iodide was greater in the presence of AbiZ. These results suggest that AbiZ may interact cooperatively with holin to cause premature lysis.

Bacterial Proteins↗
PubMed1996

Effects of mutations in the gene-start and gene-end sequence motifs on transcription of monocistronic and dicistronic minigenomes of respiratory syncytial virus.

Preceding and following each gene of respiratory syncytial virus (RSV) are two conserved sequences, the gene-start (GS) and gene-end (GE) motifs, respectively, which are thought to be transcription signals. The functions and boundaries of these signals and the process of sequential transcription were analyzed with cDNA-encoded RNA analogs (minigenomes) of nonsegmented negative-sense RSV genomic RNA. Two minigenomes were used. The monocistronic RSV-CAT minigenome consists of the chloramphenicol acetyltransferase (CAT) translational open reading frame (ORF) bordered by the GS and GE motifs and flanked by the 3' leader and 5' trailer extragenic regions of genomic RNA. The dicistronic RSV-CAT-LUC minigenome is a derivative of RSV-CAT into which the ORF for luciferase (LUC), bordered by GS and GE motifs, was inserted downstream of the CAT gene with an intergenic region positioned between the two genes. Each minigenome was synthesized in vitro and transfected into RSV-infected cells, where it was replicated and transcribed to yield the predicted polyadenylated subgenomic mRNA(s). The only RSV sequences required for efficient transcription and RNA replication were the 44-nucleotide 3' leader region, the last 40 nucleotides of the 5' trailer region, and the 9- to 10-nucleotide GS and 12- to 13-nucleotide GE motifs. The GS and GE motifs functioned as self-contained, transportable transcription signals which could be attached to foreign sequences to direct their transcription into subgenomic mRNAs. Removal of the GS motif greatly reduced transcription of its gene, and the requirement for this element was particularly strict for the gene in the downstream position. Ablation of the promoter-proximal GS signal was not associated with increased antigenome synthesis. Consistent with its proposed role in termination and polyadenylation, removal of the CAT GE signal in RSV-CAT resulted in the synthesis of a nonpolyadenylated CAT mRNA, and in RSV-CAT-LUC the same mutation resulted in readthrough transcription to yield a dicistronic CAT-LUC mRNA. The latter result showed that a downstream GS signal is not recognized for reinitiation by the polymerase if it is already engaged in mRNA synthesis; instead, it is recognized only if the polymerase first terminates transcription at an upstream termination signal. This result also showed that ongoing transcription did not open the downstream LUC gene for internal polymerase entry. Removal of both the GS and GE signals of the upstream CAT gene in RSV-CAT-LUC silenced expression of both genes, confirming that independent polymerase entry at an internal gene is insignificant. Remarkably, whereas both genes were silent when the CAT GS and GE signals were both absent, restoration of the CAT GE signal alone restored a significant level (approximately 10 to 12% of the wild-type level) of synthesis of both subgenomic mRNAs. This analysis identified a component of sequential transcription that was independent of the promoter-proximal GS signal and appeared to involve readthrough from the leader region.

Animals↗
PubMed1998

Cloning, sequencing and characterisation of a downstream region of ksdDI operon of Arthrobacter simplex.

An 8-kb region downstream of the ketosteroid dehydrogenase (ksdD)-ketosteroid isomerase (ksdI) genes of Arthrobacter simplex was cloned. The nucleotide sequence of the first 3-kb segment downstream of ksdD-ksdI operon was determined. Three open reading frames (ORFs) preceded by Shine-Dalgarno (SD) sequences have been found. Homology search revealed that the putative product encoded by ORF3 has high level of similarity with the 3-oxosteroid dehydrogenases from A. simplex and P. testosteroni (90% identity in their putative active sites). The role of ORF3 product as a FAD-containing dehydrogenase in steroid degradation pathway is discussed.

Amino Acid Sequence↗
PubMed1987

Analysis of the regulatory region of the protease III (ptr) gene of Escherichia coli K-12.

The ptr gene of Escherichia coli encodes protease III (Mr 110,000) and a 50-kDa polypeptide, both of which are found in the periplasmic space. The gene is physically located between the recC and recB loci on the E. coli chromosome. The nucleotide sequence of a 1167-bp EcoRV-ClaI fragment of chromosomal DNA containing the promoter region and 885 bp of the ptr coding sequence has been determined. S1 nuclease mapping analysis showed that the major 5' end of the ptr mRNA was localized 127 bp upstream from the ATG start codon. The open reading frame (ORF), preceded by a Shine-Dalgarno sequence, extends to the end of the sequenced DNA. Downstream from the -35 and -10 regions is a sequence that strongly fits the consensus sequence of known nitrogen-regulated promoters. A signal peptide of 23 amino acids residues is present at the N terminus of the derived amino acid sequence. The cleavage site as well as the ORF were confirmed by sequencing the N terminus of mature protease III.

Amino Acid Sequence↗
PubMed1994

Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; physiological relevance of an autoactivated nifU2-rpoN superoperon.

The alternative sigma factor, RpoN (sigma 54) is responsible for recruiting core RNA polymerase to the promoters of genes required for diverse physiological functions in a variety of eubacterial species. The RpoN protein in Rhodobacter capsulatus is a putative sigma factor specific for nitrogen fixation (nif) genes. Insertional mutagenesis was used to define regions important for the function of the R. capsulatus RpoN protein. Insertions of four amino acids in the predicted helixturn-helix or in the highly conserved C-terminal eight amino acid residues (previously termed the RpoN box), and an in-frame deletion of the glutamine-rich N-terminus completely inactivated the R. capsulatus RpoN protein. Two separate insertions in the second hydrophobic heptad repeat, a putative leucine zipper, resulted in a partially functional RpoN protein. Eight other linkers in the rpoN open reading frame (ORF) resulted in a completely or partially functional RpoN protein. The rpoN gene in R. capsulatus is downstream from the nifHDKU2 genes, in a nifU2-rpoN operon. Results of genetic experiments on the nifU2-rpoN locus show that the rpoN gene is organized in a nifU2-rpoN superoperon. A primary promoter directly upstream of the rpoN ORF is responsible for the initial expression of rpoN. Deletion analysis and insertional mutagenesis were used to define the primary promoter to 50 bp, between 37 and 87 nucleotides upstream of the predicted rpoN translational start site. This primary promoter is expressed constitutively with respect to nitrogen, and it is necessary and sufficient for growth under nitrogen-limiting conditions typically used in the laboratory. A secondary promoter upstream of nifU2 is autoactivated by RpoN and NifA to increase the expression of rpoN, which ultimately results in higher expression of RpoN-dependent genes. Moreover, rpoN expression from this secondary promoter is physiologically beneficial under certain stressful conditions, such as nitrogen-limiting environments that contain high salt (> 50 mM NaCl) or low iron (< 400 nM FeSO4).

Alleles↗
PubMed1993

Adelaide river rhabdovirus expresses consecutive glycoprotein genes as polycistronic mRNAs: new evidence of gene duplication as an evolutionary process.

A 3914 nucleotide region of the Adelaide River virus (ARV) genome, located immediately downstream of the M2 gene, has been cloned and sequenced. The region contains two long open reading frames (ORFs). The first encodes a protein comprising 660 amino acids which shares extensive sequence homology with the virion G protein of bovine ephemeral fever virus (BEFV) and less but significant homology with other rhabdovirus glycoproteins. The size and structural characteristics of the product indicate that it represents the 90-kDa ARV virion G protein. The second ORF encodes a polypeptide of 609 residues with nine potential glycosylation sites which is most closely related to the BEFV non-structural glycoprotein (GNS). In infected mammalian cells, the ARV G and GNS genes are transcribed primarily as a polycistronic mRNA which appears to extend from the consensus sequence (AACAG) at the start of the G gene to the next recognized polyadenylation signal (CATG[A]7) located 697 nucleotides downstream of the GNS protein termination codon. Less abundant mRNAs which appeared to initiate at consensus sequences immediately preceding and following the GNS ORF and terminate at the same polyadenylation signal were also detected. Polyadenylation-like sequences at the end of each ORF do not appear to be recognized as transcription stop signals. Multiple sequence alignments and phylogenetic analyses indicated that the ARV G and GNS glycoproteins, like those of BEFV, are structurally related and appear to have evolved at different rates from a common ancestral gene. A copy-choice mechanism, involving upstream relocation of the polymerase during replication, is proposed to account for the evolution of the tandem glycoprotein genes.

Amino Acid Sequence↗
PubMed1994

Expression of the rubella virus nonstructural protein ORF and demonstration of proteolytic processing.

To analyze the proteins produced from the rubella virus (RUB) nonstructural protein open reading frame (NSP-ORF), a DNA containing the RUB NSP-ORF was introduced into the expression vector pTM3 in which the sequences to be expressed are downstream from a T7 RNA polymerase promoter. In cells infected with a vaccinia virus recombinant which expresses T7 RNA polymerase and transfected with this plasmid, three RUB-specific products with electrophoretic mobilities of 200, 150, and 97 kDa were clearly visible. By computer alignment, the presence of a cysteine protease was predicted within the NSP-ORF (A. E. Gorbalenya et al., FEBS Lett. 288, 201-205, 1991). When the Cys proposed as the catalytic residue of this protease (Cys1151) was mutated to a Gly, only the 200-kDa product was produced, demonstrating that the Cys is important in the activity of the protease responsible for the processing of the RUB NSPs and that the 150- and 97-kDa species are processing products. Transfections with deletion mutants revealed that the 150-kDa processing product is derived from the amino-terminal two-thirds of the ORF and that both the protease and the cleavage site on the COOH-terminal side of the 150-kDa product are between amino acids 1005 and 1507 of the ORF.

Animals↗
PubMed1998

Cloning and sequence analysis of the Pichia pastoris TRP1, IPP1 and HIS3 genes.

The Pichia pastoris TRP1 and HIS3 genes were cloned by complementation of the Saccharomyces cerevisiae trip1 and his3 mutants, respectively, and their nucleotide sequence was determined. The P. pastoris TRP1 gene includes an open reading frame (ORF) of 714 nucleotides corresponding to a polypeptide of 237 amino acids whose sequence shares about 40% identity with that of TRP1 encoding proteins in other yeast species. DNA sequencing showed that an ORF of 858 nucleotides, encoding a protein of 285 amino acids with high homology to inorganic pyrophosphatases (IPP1), is located downstream of the P. pastoris TRP1 gene. Both genes converge in this chromosomal region, showing a genetic organization analogous to that found in the Kluyveromyces lactis genome. The P. pastoris HIS3 gene possesses an ORF of 675 nucleotides, encoding a polypeptide of 224 amino acids which shows 74.1% identity to the homologous S. cerevisiae protein. The hexameric consensus GCN4 binding sequence (TGACTC), characteristic of many amino acid biosynthetic genes, is present in the promoter region.

Aldose-Ketose Isomerases↗
PubMed2002

Transcriptional analysis and preliminary characterization of ORF Bm42 from Bombyx mori nucleopolyhedrovirus.

Clusters of closely spaced genes are frequently present in baculovirus genomes. We report here the detailed transcriptional mapping of one such gene cluster from Bombyx mori nucleopolyhedrovirus (BmNPV) comprising five ORFs, viz., Bm42, Lef-10, VP1054, Bm44, and Bm45, which are closely spaced and transcribed in the same orientation. This region is homologous to ORFs 53, 53a, 54, 55, and 56, respectively, on the AcMNPV genome. Multiple, overlapping sets of polycistronic transcripts from this region, that initiate independently but terminate at a common 3' end, were detected following BmNPV infection. Expression of these genes followed a temporal pattern where the shorter transcripts initiating downstream appeared early, and with the progress of infection the longer transcripts initiated from upstream. The first ORF in this cluster, Bm42, is conserved in almost all baculoviruses, but has not been characterized so far. The protein was localized in the cytosol, predominantly near the nucleus-cytoplasm boundary in BmNPV-infected BmN cells. Bm42 was present in the budded virions and was not associated with occluded virus particles or the polyhedral matrix.

Animals↗
PubMed1997

Purification, characterization and cloning of a novel variant of the superantigen Yersinia pseudotuberculosis-derived mitogen.

The novel superantigen designated as Yersinia pseudotuberculosis-derived mitogen typeB (YPMb) was purified from the cell lysate of Y. pseudotuberculosis O:6 strain (R-104). Proliferative response of human peripheral blood mononuclear cells to the purified YPMb was detectable at a concentration of as low as 1 pg/ml, that is comparable to the previously documented YPM (YPMa). The Vbeta repertoire specificity (3, 9, 13.1 and 13.2) of YPMb was also the same as that of YPMa. A gene (ypmB) encoding YPMb was cloned and its nucleotide sequence was determined. The open reading frame (ORF 453 bp) of the ypmB encodes a protein with 150 amino acid residues, though the precursor protein of YPMa consists of 151 amino acid residues. There is a nucleotide sequence homology of 88.9% between ypmB and ypmA. The low homology in the downstream of the structural gene between ypmB and ypmA and the difference in the GC content in the ORF of ypmB and ypmA from that of the base usage of Y. pseudotuberculosis suggest that the ypm gene originates from another organism. The alignment of the amino acid sequences of mature proteins of YPMb and YPMa revealed that there is 83% homology (108 amino acid residues are identical). Between YPMa and YPMb, the central region is less homologous than the N- and C-terminal regions. Based on the functional similarity of two superantigen molecules, the less homology suggested that the central region is less important for the function of both YPM molecules.

Amino Acid Sequence↗
PubMed1994

Computer aided identification of a potential 5'-3' exonuclease gene encoded by Escherichia coli.

The predicted amino acid sequence of a previously overlooked open reading frame (ORF) from E. coli has been subjected to sequence analysis. The ORF, sequenced independently by two research groups, appears to encode a protein of 251 amino acids residues (25 kDa). The protein sequence shares a high degree of similarity with the amino acid termini of E. coli DNA polymerase I and other bacterial DNA polymerases (and phage-encoded enzymes) known to possess 5'-3' exonuclease activity. The newly identified E. coli gene, designated exo, is positioned downstream of signals characteristic of an efficient translation initiation sequence. Codon bias analysis indicates that the ORF has good protein coding potential. These observations indicate that the gene is likely to be expressed. The gene is 60% identical with the 5'-region of the E. coli DNA PolI gene (polA) over a 260 base-pair region. These two genes may have arisen by duplication of an ancestral gene or polA may have arisen by a recombination event involving exo. A comparison of the predicted secondary structures of 5'-3' exonucleases revealed the presence of several conserved regions of secondary structure, including a potential helix-turn-helix motif.

Amino Acid Sequence↗
PubMed1990

A major transcript of human papillomavirus type 16 in transformed NIH 3T3 cells contains polycistronic mRNA encoding E7, E5, and E1--E4 fusion gene.

We have cloned cDNA of the major 1.8 kb mRNA from HPV 16-transformed NIH 3T3 cells (PM3T3). The entire nucleotide sequences of this cDNA were determined and compared with prototype HPV 16 genomic DNA sequences. The 5'-end of the cDNA was flanked by approximately 300 bp of cellular sequences, and the 3'-end of the cDNA sequences contained poly A residues following at nt 4230. HPV 16 sequences began at nt 124, downstream of a major viral p97 promoter, within the E6 open reading frame (ORF). The first splice donor site was at nt 226 and the splice acceptor site was at nt 409, suggesting that the E6 gene is inert. Second splice donor and acceptor sites were located at nt 880 and at nt 3357, respectively. This mRNA was thus shown to consist of three exons, resulting in polycistronic mRNA containing three potentially functional virus early genes--E7, E1--E4, and E5--actively transcribed in the transformant.

Amino Acid Sequence↗
PubMed1991

Cloning and sequence of an infectious bovine rhinotracheitis virus (BHV-1) gene homologous to glycoprotein H of herpes simplex virus.

A homologue to the glycoprotein H (gH) gene of herpes simplex virus (HSV) has been identified in the genome of infectious bovine rhinotracheitis virus (IBR, BHV-1). The gene is located immediately downstream from the thymidine kinase gene, and codes for an open reading frame (orf) of 842 amino acids. The orf has the characteristics of a membrane glycoprotein, including an N-terminal hydrophobic region resembling a signal sequence, a C-terminal region which is probably a transmembrane domain, and six potential sites for N-linked glycosylation. This orf shows significant homology to the gH sequences of both HSV and pseudorabies virus (PRV). We conclude that this gene encodes BHV-1 gH.

Base Sequence↗