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PubMed2002

Nucleotide sequence shows that Bean leafroll virus has a Luteovirus-like genome organization.

The complete nucleotide sequence of the Bean leafroll virus (BLRV) genomic RNA and the termini of its smallest subgenomic RNAs were determined to better understand its mechanisms of gene expression and replication and its phylogenetic position within the Luteoviridae: The number and placement of open reading frames (ORFs) within the BLRV genome was Luteovirus-like. The nucleotide and predicted amino acid sequences of BLRV were most similar to those of Soybean dwarf virus (SbDV). Phylogenetic analyses employing the neighbour-joining method and sister-scanning analysis indicated that the BLRV nonstructural proteins were closely related to those of Barley yellow dwarf virus-PAV (BYDV-PAV), a luteovirus: The region surrounding the frameshift at the junction between ORFs 1 and 2 also contained sequences very similar to those of BYDV-PAV and a Dianthovirus, Red clover necrotic mosaic virus. Similar analyses showed that the structural proteins were most similar to those of the Polerovirus genus. The 3'-noncoding regions downstream of ORF5 contained sequences similar to translational control elements identified in the BYDV-PAV genome. These data suggest that BLRV, like SbDV, is derived either through selection from a common ancestor with BYDV-PAV or that BLRV is the product of two recombination events between luteovirus-like and polerovirus-like ancestors where the 5' 2900 nt and 3' 700 nt of the BLRV genome are from a Luteovirus and the intervening sequences are derived from a Polerovirus:

3' Untranslated Regions↗
PubMed1995

Identification and characterization of the orf virus type I topoisomerase.

Vaccinia virus (VV) and Shope fibroma virus (SFV), representatives of the orthopox and leporipox genera, respectively, encode type I DNA topoisomerases. Here we report that the 957-nt F4R open reading frame of orf virus (OV), a representative of the parapox genus, is predicted to encode a 318-aa protein with extensive homology to these enzymes. The deduced amino acid sequence of F4R has 54.7 and 50.6% identity with the VV and SFV enzymes, respectively. One hundred forty amino acids are predicted to be conserved in all three proteins. The F4R protein was expressed in Escherichia coli under the control of an inducible T7 promoter, partially purified, and shown to be a bona fide type I topoisomerase. Like the VV enzyme, the OV enzyme relaxed negatively supercoiled DNA in the absence of divalent cations or ATP and formed a transient covalent intermediate with cleaved DNA that could be visualized by SDS-PAGE. Both the noncovalent and covalent protein/DNA complexes could be detected in an electrophoretic mobility shift assay. The initial PCR used to prepare expression constructs yielded a mutant allele of the OV topoisomerase with a G-A transition at nt 677 that was predicted to replace a highly conserved Tyr residue with a Cys. This allele directed the expression of an enzyme which retained noncovalent DNA binding activity but was severely impaired in DNA cleavage and relaxation. Incubation of pUC19 DNA with the wild-type OV or VV enzyme yielded an indistinguishable set of DNA cleavage fragments, although the relative abundance of the fragments differed for the two enzymes. Using a duplex oligonucleotide substrate containing the consensus site for the VV enzyme, we demonstrated that the OV enzyme also cleaved efficiently immediately downstream of the sequence CCCTT.

Amino Acid Sequence↗
PubMed2005

Identification and characterization of two polyhydroxyalkanoate biosynthesis loci in Pseudomonas sp. strain 3Y2.

A Pseudomonas strain, 3Y2, that produced polyhydroxyalkanoate (PHA) polymers consisting of 3-hydroxybutyric acid (3HB) and medium-chain-length 3-hydroxyalkanoate (mcl-HA) units, with up to 30% 3HB, was isolated. Two PHA biosynthesis loci (pha ( Ps-1) and pha ( Ps-2)) from 3Y2 were cloned by polymerase chain reaction amplification techniques. The pha ( Ps-2) locus was similar to the PHA biosynthesis loci of other PHA-producing Pseudomonas strains, with five tandem open reading frames (ORFs) located in the order ORF1( Ps-2)-phaC1 ( Ps-2)-phaZ ( Ps-2)-phaC2 ( Ps-2)-phaD ( Ps-2). The pha ( Ps-1) locus that contains phaC1 ( Ps-1)-phaZ ( Ps-1) appears to have arisen by a duplication event that placed it downstream of a gene (ORF1( Ps-1)), encoding a putative glucose-methanol-choline flavoprotein oxidoreductase. The PHA synthases 1 encoded by phaC1 ( Ps-1) and phaC1 ( Ps-2) were investigated by heterologous expression in Wautersia eutropha PHB(-)4. Both synthases displayed similar substrate specificities for incorporating 3HB and mcl-HA units into PHA. The ability of PhaC1( Ps-1) to confer PHA synthesis, however, appeared reduced compared to that of PhaC1( Ps-2), since cells harboring PhaC1( Ps-1) accumulated 2.5 to 4.6 times less PHA than cells expressing PhaC1( Ps-2). Primary sequence analysis revealed that PhaC1( Ps-1) had markedly diverged from the other PHA synthases with a relatively high substitution rate (14.9 vs 2% within PhaC1( Ps-2)). The mutations affected a highly conserved C-terminal region and the surroundings of the essential active site cysteine (Cys296) with a loss of hydrophobicity. This led us to predict that if phaC1 ( Ps-1) produces a protein product in the native strain, it is likely that PhaC1( Ps-1) may be destined for elimination by the accumulation of inactivating mutations, although its specialization to accommodate different substrates cannot be eliminated.

Acyltransferases↗
PubMed1996

Identification and analyses of glycoprotein B of human herpesvirus 7.

The gene for the human herpes virus 7 (HHV-7) glycoprotein B (gB) has been identified by sequencing a molecularly cloned HHV-7 DNA fragment. A 2.5-kb open reading frame (ORF) encoded a protein of 822 amino acids with characteristics of a transmembrane glycoprotein, and showed the strongest similarity (56.5%) with the human herpesvirus 6 (HHV-6) gB. The genes for the transport/capsid assembly protein (tp/cap) and the DNA polymerase (pol) existed upstream and downstream of the gB gene, respectively. This arrangement was the same as that of HHV-6. Antisera were generated by immunizing mice with a glutathione S-transferase-carboxy terminal gB fusion protein. Immunofluorescent tests demonstrated that the antisera reacted specifically with HHV-7 antigens in cytoplasm of infected cells. The antisera immunoprecipitated proteins with apparent molecular masses of 51, 63 and 112 kDa from HHV-7 infected cells by pulse-chase analysis. In the presence of tunicamycin, the protein with a molecular mass of 112 kDa was replaced by a protein with a molecular mass of 88 kDa, and this size was consistent with the predicted size of the primary translation product of the HHV-7 gB gene. These results suggested that the protein with a molecular mass of 112 kDa was a glycoprotein synthesized by addition of N-linked oligosaccharides to a non-glycosylated precursor of the protein with a molecular mass of 88 kDa and then cleaved into the proteins with molecular masses of 51 and 63 kDa in HHV-7 infected cells.

Amino Acid Sequence↗
PubMed2001

Characterization of the prfA virulence gene cluster insertion site in non-hemolytic Listeria spp.: probing the evolution of the Listeria virulence gene island.

The prfA virulence gene cluster is present between prs and ldh in the pathogenic L. monocytogenes and L. ivanovii, but absent from the non-pathogenic L. innocua and L. welshimeri. To probe the evolution of this virulence gene cluster, we sequenced the prs-ldh intergenic region in L. welshimeri and L. innocua. Two ORFs (ORFA and ORFB) were found in both species as well as in L. monocytogenes. Another ORF of unknown function (ORFZ) was found in L. monocytogenes and L. innocua, while two unique ORFs were present in L. welshimeri. ORFA and ORFB showed significant functional constraint, suggesting that further investigations in the functions of these genes, including possible roles in horizontal gene transfer or sequence deletion, are warranted. DNA sequences homologous to Tn1545 integration consensus sequences were found downstream of prs and ORFB, thus defining the likely junctions of the virulence gene island and indicating that the prs-ldh intergenic region may represent a Tn insertion hot spot. Our results are consistent with the hypothesis that a combination of horizontal gene transfer and deletion events mayhave been involved in the evolution of the prfA virulence gene cluster in Listeria.

Animals↗
PubMed1994

Characterization of the gene encoding the human transcriptional elongation factor TFIIS.

The transcriptional elongation factor TFIIS causes stimulation of RNA polymerase II elongation and readthrough of some of the elongation blocks. We present cloning and sequence characterization of the human TFIIS gene and a pseudogene. The intron-less organization of both of these genes indicates that previously identified cDNAs which suggested the presence of an intron were the products of cloning artifacts. The gene is organized in an uninterrupted ORF which codes for 301 amino acids, whereas the pseudogene lacks an ORF able to code for a full-length protein. The potential promoter for the gene has two putative GC-box-type consensus sequences, two CCAAT-box consensus sequences, and is bounded by a human Alu sequence. Two potential transcriptional termination signal sequences downstream from the consensus polyadenylation signal are proposed.

Base Sequence↗
PubMed1998

Genetic organization and DNA sequence of early region 4 of bovine adenovirus type 3.

We have identified and sequenced 3614 nucleotides located at the extreme right-end of the bovine adenovirus type 3 (BAV3) genome from map units 89.5-100. Analysis of the sequence revealed an inverted terminal repeat (ITR) of 195 bp, and identified five open reading frames (ORFs) designated ORF1, ORF2, ORF3, ORF4 and ORF5. When compared with known E4 ORFs of other adenoviruses, ORFs 1, 2 and 4, which code for proteins of 143, 69 and 143 amino acids respectively, were found to be unique to BAV3. ORFs 3 and 5, which code for proteins of 268 and 219 amino acids respectively, showed partial homology to the E4 34 kDa protein of human adenovirus 2. Nucleotide sequence analysis also identified two potential TATA boxes upstream of ORF1 and a potential polyadenylation signal downstream of ORF5 suggesting that E4 transcripts may be 3' co-terminal.

Adenoviridae↗
PubMed1998

An 8 kb nucleotide sequence at the 3' flanking region of the sspC gene (184 degrees) on the Bacillus subtilis 168 chromosome containing an intein and an intron.

As part of the Bacillus subtilis genome sequencing project, we determined the complete nucleotide sequence of an 8000-bp fragment downstream of the sspC gene (184 degrees) of the B. subtilis 168 chromosome. The sequence analysis shows that the sspC gene is located inside of the SP beta region, which differs from the current genetic map of B. subtilis 168. This region contains 12 putative ORFs (yojQ through yojZ and sspC). A homology search for the deduced products of the ORFs shows significant similarities to enzymes involved in deoxyribonucleotide metabolism: ribonucleotide reductase (Nrd) E, NrdF, thioredoxin and dUTPase. Interestingly, this DNA fragment includes two split genes, yojP containing conserved motifs of an intein and yojQ and yojS with an 808-bp intervening sequence for a putative intron structure. In addition, the yojR gene includes a putative new DNA replication terminator.

Amino Acid Sequence↗
PubMed1999

PhaF, a polyhydroxyalkanoate-granule-associated protein of Pseudomonas oleovorans GPo1 involved in the regulatory expression system for pha genes.

The phaC1 gene codes for the medium-chain-length polyhydroxyalkanoate (mcl PHA) synthase of Pseudomonas oleovorans GPo1, which produces mcl PHA when grown in an excess of carbon source and under nitrogen limitation. In this work, we have demonstrated, by constructing a recombinant P. oleovorans strain carrying a phaC1::lacZ reporter system, that the phaC1 gene is expressed efficiently in the presence of octanoic acid while its expression is repressed when glucose or citrate is used as the carbon source. Moreover, a P. oleovorans GPo1 mutant (strain GPG-Tc6) expressing higher levels of the reporter gene than the wild-type strain in the presence of glucose or citrate has been generated by mini-Tn5 insertional mutagenesis. Characterization of this mutant allowed us to conclude that phaF, a gene located downstream of the pha gene cluster, was knocked out in this strain. P. oleovorans GPG-Tc6 regained the ability to control phaC1 gene expression when complemented with the phaF wild-type gene. Sequencing data revealed the presence of three complete open reading frames (ORFs) in this region: ORF1 and phaI and phaF genes. The amino acid sequences of the phaI gene product and the N-terminal half of the PhaF protein showed a significant degree of similarity. Furthermore, the primary structure of the PhaF C terminus identifies this protein as a member of the histone H1-like group of proteins. Northern blot analysis showed two transcription units containing phaF, i.e., phaF and phaIF transcripts. Expression of the phaIF operon is more efficient in the presence of octanoic acid and is enhanced by the lack of the PhaF protein. In addition, it has also been demonstrated that both PhaF and PhaI proteins are bound to PHA granules produced by P. oleovorans. A model for the role of PhaF in regulating PHA synthesis is presented.

Acyltransferases↗
PubMed2003

Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence.

Plasmid SCP2* is a 31 kb, circular, low-copy-number plasmid originally identified in Streptomyces coelicolor A3(2) as a fertility factor. The plasmid was completely sequenced. The analysis of the 31 317 bp sequence revealed 34 ORFs encoding putative proteins from 31 to 710 aa long, most of them lacking similarity to known proteins. Three functional regions had been identified previously: the replication region, the transfer and spreading region, and the stability region. Three genes were identified in the stability region which contribute to the stability of SCP2 as shown by plasmid stability testing. The first gene, mrpA, encodes a new member of the lambda integrase family of site-specific recombinases. The two genes downstream of mrpA were called parA and parB. The gene product, ParA, shows similarity to a family of ATPases involved in plasmid partition. An increase of plasmid stability could be seen only when both genes were present. By deletion analysis, the replication region could be narrowed down to a 1.6 kb region, consisting of a 650 bp non-coding region and two genes, repI and repII, encoding proteins of 161 and 131 aa. Only RepI exhibits similarities to DNA binding elements and contains a putative helix-turn-helix motif. The traA gene that is essential for DNA transfer and pock formation was identified previously. Upstream of traA, 10 ORFs were found in the same orientation as traA which might be involved in conjugation and DNA spreading, together with one gene in the opposite orientation with similarities to transcriptional regulators of DNA transfer. Two transposable elements were found on SCP2*. IS1648 belongs to the IS3 family of insertion sequences. The second element, Tn5417, shows the highest similarity to the Tn4811 element located in the terminal inverted repeats of the Streptomyces lividans chromosome.

Bacterial Proteins↗
PubMed1995

Cloning and genomic characterization of LST1: a new gene in the human TNF region.

The leucocyte specific transcript - 1 (LST1) represents the human homolog of the mouse B144 transcript, encoded within the tumor necrosis factor (TNF) region of the human major histocompatibility complex class III interval. The gene is localized about 4 kilobases upstream of the lymphotoxin beta gene. It spans a polymorphic genomic region encompassing the microsatellites TNFd and TNFe in intron 3 and a polymorphic Pvu II restriction site 260 base pairs downstream of the polyadenylation signal. Isolation of a full-length cDNA clone revealed that LST1 codes for IFN-gamma-inducible 800 nt transcripts, which are present in lymphoid tissues, T cells, macrophages, and histiocyte cell lines. The cDNA contains three long open reading frames (ORF) with the most likely ORF encoding a transmembrane protein. Its close linkage to the TNF genes and pattern of expression point toward a possible role for LST1 in the immune response.

Amino Acid Sequence↗
PubMed2004

ORFeome projects: gateway between genomics and omics.

The availability of entire genome sequences is expected to revolutionize the way in which biology and medicine are conducted for years to come. However, achieving this promise still requires significant effort in the areas of gene annotation, cloning and expression of thousands of known and heretofore unknown protein-encoding genes. Traditional technologies of manipulating genes are too cumbersome and inefficient when one is dealing with more than a few genes at a time. Entire libraries composed of all protein-encoding open reading frames (ORFs) cloned in highly flexible vectors will be needed to take full advantage of the information found in any genome sequence. The creation of such ORFeome resources using novel technologies for cloning and expressing entire proteomes constitutes an effective gateway from whole genome sequencing efforts to downstream 'omics' applications.

Animals↗
PubMed2000

Identification of a two-component signal transduction system involved in fimbriation of Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontopathogen, is an oral anaerobic gram-negative bacterium with numerous fimbriae on the cell surface. Fimbriae have been considered to be an important virulence factor in this organism. We analyzed the genomic DNA of transposon-induced, fimbria-deficient mutants derived from ATCC 33277 and found that seven independent mutants had transposon insertions within the same restriction fragment. Cloning and sequencing of the disrupted region from one of the mutants revealed two adjacent open reading frames (ORFs) which seemed to encode a two-component signal transduction system. We also found that six of the mutants had insertions in a gene, fimS, a homologue of the genes encoding sensor kinase, and that the insertion in the remaining one disrupted the gene immediately downstream, fimR, a homologue of the response regulator genes in other bacteria. These findings suggest that this two-component regulatory system is involved in fimbriation of P. gingivalis.

Amino Acid Sequence↗
PubMed2006

Yeast autonomous linear plasmid pGKL2: ORF9 is an actively transcribed essential gene with multiple transcription start points.

A pair of linear plasmids, pGKL1 (8.9 kb) and pGKL2 (13.4 kb), resides in the cytoplasm of Kluyveromyces lactis killer strains. The smaller element, actually conferring the killer phenotype, strictly depends on the larger autonomous pGKL2. Here, we have examined the previously uncharacterized pGKL2 open reading frame (ORF)9 (1.34 kb). Northern analysis of a killer plasmid carrying Saccharomyces cerevisiae strain applying an ORF9-specific probe revealed a single transcript closely matching the size of the ORF9 coding region. Multiple transcriptional start points, determined by primer extension analysis, are located 16 nt downstream of a conserved sequence element regarded as the cytoplasmic promoter. In vivo disruption of pGKL2/ORF9 using the cytoplasmically expressible marker-gene LEU2* resulted in the establishment of a three-plasmid system composed of the native cytoplasmic elements pGKL1/2 and a hybrid of the latter, which only remained stable under selective conditions. The native pGKL2, however, did not segregate during prolonged subcultivations, proving an essential function of ORF9 for plasmid maintenance.

Base Sequence↗
PubMed1999

Characterization of a novel methyl-accepting chemotaxis gene, dmcB, from the oral spirochete Treponema denticola.

Immediately downstream from the previously isolated Treponema denticola ATCC 35405 prtB gene coding for a chymotrypsinlike protease activity, an open reading frame, ORF3, was identified which shared significant homology with the highly conserved domains (HCDs) of bacterial methyl-accepting chemotaxis proteins (MCPs). Nucleotide sequencing of this ORF revealed that the gene would code for a protein with a size of approximately 41 kDa. In addition, this sequence contained a domain which was virtually identical to the HCD of a recently characterized MCP, DmcA, of strain 35405. Therefore, this ORF was named dmcB. Northern blot analysis suggested that dmcB was part of an operon structure containing prtB. Insertional inactivation of dmcB utilizing an ermF-ermAM cassette resulted in a mutant with decreased chemoattraction toward nutrient supplements. In addition, the mutant displayed an altered pattern of methylated proteins under conditions of chemotaxis. Inactivation of the dmcB gene also attenuated the methylation of the DmcA protein. These results suggest that the dmcB gene codes for an MCP in T. denticola which may interact with other MCPs in these organisms.

Amino Acid Sequence↗
PubMed1992

Receptor properties of two varicella-zoster virus glycoproteins, gpI and gpIV, homologous to herpes simplex virus gE and gI.

The varicella-zoster virus (VZV) genome contains 70 reading frames (ORF), 5 of which encode the glycoproteins gpI, gpII, gpIII, gpIV, and gpV. ORF 67 and 68 lie adjacent to each other in the unique short region of the VZV genome and code for gpIV and gpI, respectively. These two genes, which are contained within the HindIII C fragment of the VZV genome, were subcloned in the correct orientation downstream from the promoter regions of the eukaryotic expression vectors pCMV5 and pBJ. After transfection, 5 to 20% of the Cos cells bound antibody specific for the given glycoprotein. In this study, it was shown that only the cells transfected with the gpI construct bound to the Fc fragment of human immunoglobulin G. Neither the transfected gpIV gene product nor the vector only bound to the Fc fragment. Thus, VZV gpI is confirmed to be the VZV-encoded Fc-binding glycoprotein. Like the wild-type form of gpI expressed in VZV-infected cells, gpI precipitated from transfected cells contained both N-linked and O-linked glycans and was heavily sialated. In addition, the transfected gpI gene product was phosphorylated both in cell culture and in protein kinase assays by mammalian casein kinases I and II. Extensive computer-assisted analyses of the VZV gpI sequence, as well as those of alphaherpesviral homolog glycoproteins, disclosed properties similar to those of other cell surface receptors; these included (i) exocytoplasmic regions rich in cysteine residues, (ii) membrane-proximal regions with potential O-linked glycosylation sites, and (iii) cytoplasmic domains with consensus phosphorylation sites.

Amino Acid Sequence↗
PubMed2002

Cloning and characterization of linR, involved in regulation of the downstream pathway for gamma-hexachlorocyclohexane degradation in Sphingomonas paucimobilis UT26.

In Sphingomonas paucimobilis UT26, LinD and LinE activities, which are responsible for the degradation of gamma-hexachlorocyclohexane, are inducibly expressed in the presence of their substrates, 2,5-dichlorohydroquinone (2,5-DCHQ) and chlorohydroquinone (CHQ). The nucleotide sequence of the 1-kb upstream region of the linE gene was determined, and an open reading frame (ORF) was found in divergent orientation from linE. Because the putative protein product of the ORF showed similarity to the LysR-type transcriptional regulator (LTTR) family, we named it linR. The fragment containing the putative LTTR recognition sequence (a palindromic TN(11)A sequence), which exists immediately upstream of linE, was ligated with the reporter gene lacZ and was inserted into the plasmid expressing LinR under the control of the lac promoter. When the resultant plasmid was introduced into Escherichia coli, the LacZ activity rose in the presence of 2,5-DCHQ and CHQ. RNA slot blot analysis for the total RNAs of UT26 and UT102, which has an insertional mutation in linR, revealed that the expression of the linD and linE genes was induced in the presence of 2,5-DCHQ, CHQ, and hydroquinone in UT26 but not in UT102. These results indicated that the linR gene is directly involved in the inducible expression of the linD and linE genes.

Amino Acid Sequence↗
PubMed2000

Further studies on the GS element. A novel mycobacterial insertion sequence (IS1612), inserted into an acetylase gene (mpa) in Mycobacterium avium subsp. silvaticum but not in Mycobacterium avium subsp. paratuberculosis.

We have recently described the GS element, found in Mycobacterium avium subsp. paratuberculosis (MAP), Mycobacterium avium subsp. silvaticum (MAS) and some isolates of Mycobacterium avium subsp. avium serotype 2 (MAAs2), which contains a set of genes of low GC% content, putatively associated with the biosynthesis, modification and transference of fucose to cell wall glycopeptidolipids. Here we describe a further gene of low GC% content (mpa), within the GS element in MAP. mpa is a putative acetyltransferase with homology to genes directly responsible for host specificity and virulence in Salmonella typhimurium and Shigella flexneri. Unlike other GS genes, strong homologues of mpa have not been found in related species, including Mycobacterium tuberculosis (MTB). In MAP, mpa encodes an ORF of 445aa, however, in MAS and MAAs2 mpa contains a single inserted copy of a novel insertion sequence. This element (IS1612) has two sets of inverted repeats at each terminus and encodes two ORFs with good homologies to transposase and helper proteins of IS21 (E. coli) and IS1415 (R. erythropolis). Sequence comparisons between mpa in MAP and MAS indicate the target site for IS1612 is duplicated on insertion to give a direct repeat at each end of the element. Immediately, downstream of the mpa gene in both MAP and MAS are a group of three genes with good homology to the daunorubicin resistance cluster. This cluster has a high GC% content which suggests a 'border' for the GS element. A short motif present at the beginning of this cluster matches with an inverted repeat of this motif at the beginning of the first gene in the GS element. This encapsulates the whole of this group of low GC% genes in MAP and further suggests its cassette-like nature. Homologues of the GS element in MTB show a marked similarity of organisation, suggesting a parallel role for these genes in both pathogens.

Acetylesterase↗