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PubMed1992

The E.coli fis promoter is subject to stringent control and autoregulation.

The DNA binding protein FIS is involved in processes like site specific DNA inversion, lambda excision and stimulation of stable RNA synthesis in Escherichia coli. The amount of FIS protein is subject to dramatic changes during growth. We demonstrate that fis is part of an operon with one ORF of unknown function preceding the fis gene. Regulation of fis synthesis occurs at the transcriptional level. Within 15 min after nutritional upshift a large burst of fis mRNA is produced which levels off when cells begin to grow. By mutational analysis using promoter-lacZ fusions we demonstrate that the fis promoter is autoregulated by FIS. Growth phase regulation of the fis promoter depends on the presence of a GC motif downstream of the -10 region. We show that the fis promoter is subject to stringent control and discuss this unusual feature with respect to the known and putative functions FIS serves in E. coli.

Amino Acid Sequence↗
PubMed1998

Mushroom bacilliform virus RNA: the initiation of translation at the 5' end of the genome and identification of the VPg.

Mushroom bacilliform virus (MBV) is often found in cultivated mushrooms (Agaricus bisporus) with La France disease. MBV has a 4-kb ssRNA genome of positive-sense encoding four major open reading frames (ORFs). The arrangement of ORFs at the 5' end of the genome and the deduced amino-acid sequences of two of the putative gene products (protease and RNA-dependent RNA polymerase) show remarkable similarity to some plant viruses, particularly subgroup II luteoviruses. We show that this similarity extends to the translation strategy at the 5' end of the genome, the presence of a genome-linked protein (VPg), and the location of the VPg downstream of the protease motifs in the polypeptide encoded by ORF2.

Agaricus↗
PubMed1994

Mapping of Marek's disease virus genome: identification of junction sequences between unique and inverted repeat regions.

Polymerase chain reaction (PCR) to amplify MDV DNA and subsequent sequencing identified the junction of TRL/UL, UL/IRL, IRS/US, and US/TRS. The TRL/UL junction is located 192 bp downstream of the last EcoRI site in the TRL region, while the UL/IRL junction is located 192 bp upstream of the first EcoRI restriction enzyme site in the IRL region. The IRS/US junction is located 950 bp downstream of the second EcoRI site in the IRS region, while the US/TRS junction is located 950 bp upstream of the first EcoRI restriction enzyme site in the TRS region. BamHI restriction enzyme mapping of one of the PCR products identified two novel DNA subfragments, BamHI-U2 and -P4, upstream of the US/TRS junction of the MDV genome. Sequencing of the BamHI-D fragment revealed a novel open reading frame (ORF) encoding a 155 amino acid protein. The TRL/UL junction is located in this ORF. The N-terminal 65 amino acids of this protein is homologous to the N-terminal region of the previously reported pp38, which is located in the UL/IRL region. Computer-assisted analysis indicated that both are transmembrane proteins and that they share an antigenic domain.

Amino Acid Sequence↗
PubMed1989

Structure and expression in Escherichia coli K-12 of the L-asparaginase I-encoding ansA gene and its flanking regions.

Escherichia coli contains two L-asparaginase isozymes: a secreted high-affinity enzyme, L-asparaginase II (AnsII), and a low-affinity cytoplasmic enzyme, L-asparaginase I (AnsI), which is encoded by the ansA gene. The nucleotide sequence of ansA and flanking regions, comprising 2156 bp, has been determined. The ansA gene product has been identified and has a calculated Mr of 35,388; gel filtration of cell extracts indicates that the active form of the enzyme is a dimer. The deduced amino acid sequence of AnsI shows discernible similarity to AnsII in a region immediately adjacent to the proposed active-site peptide of asparaginase II as previously determined by substrate analogue binding experiments. A second open reading frame (ORF1), encoding a protein of Mr 23,336, is found 10 bp downstream from ansA; the ribosome-binding site of ORF1 overlaps the stop codon of ansA. Deletions within the 5' region of ansA abolish expression of ansA and also reduce expression of ORF1. Together, these observations suggest that ansA and ORF1 constitute an operon. A palindromic sequence exists in the 3' region of ORF1 which may function as a bidirectional transcription terminator both for the ansA-ORF1 operon and a second, convergent, ORF.

Amino Acid Sequence↗
PubMed2001

The Streptomyces venezuelae pikAV gene contains a transcription unit essential for expression of enzymes involved in glycosylation of narbonolide and 10-deoxymethynolide.

In Streptomyces venezuelae, four polyketide synthase (PKS) polypeptides encoded by pikAI-pikAIV are used to generate 10 and 12-membered macrocyclic structures, narbonolide and 10-deoxymethynolide. Sequence analysis suggests these genes are translationally coupled with downstream genes, pikAV (encoding a type II thioesterase), desVIII-desVI (encoding enzymes responsible for production of the final glycosylated products pikromycin, narbomycin, methymycin and neomethymycin) and desR (a resistance gene). Type II thioesterases have been suggested to have an editing function in polyketide biosynthesis and deletion of the corresponding genes often leads to decreased levels of polyketide production. Surprisingly an in-frame deletion of 687 bp of the 843 bp pikAV ORF led to a strain SC1022 that produced normal yields of polyketide products, but only in the aglycone form. Plasmid-based expression of the desVIII-VI and desR in the SC1022 strain completely restored production of glycosylated products, despite the absence of a functional pikAV gene product. Under these conditions the PikAV TEII therefore does not play an important role in polyketide biosynthesis, and its function remains an enigma. These observations also demonstrate that the region of pikAV DNA deleted in strain SC1022 contains a transcription unit essential for expression of the des genes. A sequence alignment of PikAV with members of the highly conserved type II thioesterases revealed a short divergent region at the carboxy terminus, suggesting a region of pikAV that might contain such a transcription unit. DNA containing this region of pikAV was shown to be able to increase plasmid-based expression of both crotonyl CoA reductase gene (ccr) and the erythromycin resistance gene (ermE) in S. venezuelae.

Acyl-CoA Dehydrogenases↗
PubMed2001

Molecular characterizations of the equine herpesvirus 1 ETIF promoter region and translation initiation site.

The equine herpesvirus 1 (EHV-1) homolog of the herpes simplex virus type 1 (HSV-1) tegument phosphoprotein, alphaTIF (Vmw65; VP16), was identified previously as the product of open reading frame 12 (ORF12), was shown to trans-activate immediate-early (IE) gene promoters, and was described as a 60-kDa virion component designated ETIF. However, the ETIF promoter region and transcription initiation site were not identified. The poly(A) signal of the gene 11 (UL49 homolog) lies just upstream of the first ETIF translation initiation codon, indicating that the first ATG may not be used for initiating ETIF translation. Another in-frame translation initiation codon (ATG2) is located 88 bp downstream of the first ETIF initiation codon (ATG1). Western blot analysis showed that the expressed ETIF protein migrated in SDS-PAGE with an apparent molecular mass of approximately 56 kDa, the same molecular weight identified in SDS-PAGE analysis of the KyD EHV-1 virion preparations. The ETIF expression vector pCETIF, which contains ATG2, trans-activated the IE promoter more efficiently than the pC12 containing both ATG1 and ATG2. S1 nuclease analyses mapped the 5' initiation site of the 1.4-kb transcript approximately 17 to 21 nt downstream of the ATG1. The nucleotide sequence upstream of the ATG1 did not have any promoter activity, while the nucleotide sequence upstream of the ATG2 had promoter activity. In transient transfection assays, the pETIFM2 vector, which was mutated in the ATG2, did not trans-activate the IE promoter; however, the pETIFM1 vector, which was mutated in the ATG1, trans-activated the IE promoter. These results demonstrated that the ATG2 of the ETIF ORF is the ETIF translation initiation codon. ETIF trans-activated only the IE promoter, not early (EICP0, EICP22, EICP27, and thymidine kinase) or late (IR5) promoters, confirming that EICP0, EICP22, and EICP27 are early genes.

Amino Acid Sequence↗
PubMed2004

Divergent structures of carbazole degradative car operons isolated from gram-negative bacteria.

Southern hybridization analysis of the genomes from the newly-isolated 10 carbazole (CAR)-utilizing bacteria revealed that 8 of the isolates carried gene clusters homologous to the CAR-catabolic car operon of Pseudomonas resinovorans strain CA10. Sequencing analysis showed that two car operons and the neighboring regions of Pseudomonas sp. strain K23 are nearly identical to that of strain CA10. In contrast to strains CA10 and K23, carEF genes did not exist downstream of the car gene cluster of Janthinobacterium sp. strain J3. In the car gene clusters, strains CA10, K23 and J3 have Rieske-type ferredoxin as a component of carbazole dioxygenase, although Sphingomonas sp. strain KA1 possesses a putidaredoxin-type ferredoxin. We confirmed that this putidaredoxin-type ferredoxin CarAc can function as an electron mediator to CarAa of strain KA1. In the upstream regions of the carJ3 and carKA1 gene clusters, ORFs whose deduced amino acid sequences showed homology to GntR-family transcriptional regulators were identified.

Amino Acid Sequence↗
PubMed2005

Analysis of full length ADAMTS6 transcript reveals alternative splicing and a role for the 5' untranslated region in translational control.

The ADAMTS (A Disintegrin and metalloproteinase with thrombospondin-1 type repeats) family of enzymes have been implicated in turnover of extracellular matrix. We previously showed that levels of ADAMTS6 mRNA in ARPE-19 cells were markedly increased following treatment with tumour necrosis factor alpha (TNFalpha). This study shows that the ADAMTS6 transcript contains unusually large untranslated regions (UTRs) at both the 5' and 3'end. The 5'UTR contains 11 AUG codons upstream of the predicted ADAMTS6 start codon and potently inhibits translation of a downstream reporter gene. However some translation can be restored by truncating the 5'UTR from the 5'end. The 5'UTR was tested for internal ribosome entry site activity using a bicistronic luciferase reporter plasmid, but none was detected. Using the 5' and 3'UTR sequences to screen the GenBank database we identified a full length ADAMTS6 cDNA of 7262 bp. This transcript is alternatively spliced at the 3'end of the open reading frame (ORF), resulting in an extended ORF containing 3 additional tsp-1 type repeats. Quantitative RT-PCR showed that the long and short form of the ADAMTS6 ORF are co-expressed in ARPE-19 cells, but the relative levels of the two forms is modulated by TNFalpha. The region of the transcript encoding the catalytic domain also contains several notable differences compared to the previously published ADAMTS6 cDNA sequence, including a redefinition of the predicted active site motif.

5' Untranslated Regions↗
PubMed2002

Identification, characterization, and expression of a second, bicistronic, operon involved in the production of lactocin S in Lactobacillus sakei L45.

Through the analysis of spontaneous insertion mutants of Lactobacillus sakei L45, a second operon involved in lactocin S production was identified and characterized. The new, bicistronic unit, termed lasXY, is situated immediately upstream of the previously characterized nine-open reading frame (ORF) lactocin S operon (lasA-W) and is transcribed in the opposite direction. The proximal of the two newly identified genes, lasX, specifies a 285-residue protein that is similar to a group of proteins with reported gene regulation functions in gram-positive bacteria. It was demonstrated that the lasX mutants have a strongly reduced level of lasA and lasA-W mRNA, thus indicating the likely cause of the Bac(-) phenotype of these mutants. The second ORF in the operon, lasY, specifies a 300-residue ABC transporter homolog, the function of which is currently obscure. Transcription initiation mapping of the lasXY operon demonstrates that the two lactocin S promoters overlap such that both transcripts initiate within the -35 region of the oppositely oriented promoter. This organization of promoters is unique among this group of regulons and may constitute a modulatory site in the proposed LasX-dependent expression of lasA and downstream genes.

Amino Acid Sequence↗
PubMed2006

Cloning and organisation analysis of a hepcidin-like gene and cDNA from Japan sea bass, Lateolabrax japonicus.

A hepcidin gene was amplified from the liver of Lateolabrax japonicus challenged with a mixed bacterial suspension. Using RT-PCR and RACE, a full length cDNA sequence of the hepcidin like antimicrobial peptide was determined. The complete hepcidin cDNA Hepc2 is 581 bases and contains an ORF of 258 bases with a coding capacity of 86 amino acids. The deduced amino acid sequence, which shares eight cysteines at the identical conserved positions, and gene organisation are conserved between Japan sea bass and other fish species. The predicted molecular weight of the peptide is 9.4 kDa. The 3'-untranslated region is composed of 225 bp with a polyadenylation signal AATAAA sequence appearing at 189 nt and the poly(A) tail at 212 nt downstream of stop codon TGA. The predicted signal peptide cleavage site of its deduced peptide is between codons 24 and 25. Japan sea bass hepcidin-like genomic DNA hepc2 sequence including upstream and downstream regions was composed of two introns and three exons. The cloned 173-bp upstream sequence of Japan sea bass hepcidin-like gene contains putative regulatory elements and several binding motifs for transcription factors. High homologies with hepcidin cDNAs and peptides of white bass (Morone chrysops), human and other fish were shown. Hepc2 of Lateolabrax japonicus is a new member of the hepcidin gene family.

Amino Acid Sequence↗
PubMed2001

Analysis of the Desulfovibrio gigas transcriptional unit containing rubredoxin (rd) and rubredoxin-oxygen oxidoreductase (roo) genes and upstream ORFs.

Rubredoxin-oxygen oxidoreductase, an 86-kDa homodimeric flavoprotein, is the final component of a soluble electron transfer chain that couples NADH oxidation with oxygen reduction to water from the sulfate-reducing bacterium Desulfovibrio gigas. A 4.2-kb fragment of D. gigas chromosomal DNA containing the roo gene and the rubredoxin gene was sequenced. Additional open reading frames designated as ORF-1, ORF-2, and ORF-3 were also identified in this DNA fragment. ORF-1 encodes a protein exhibiting homology to several proteins of the short-chain dehydrogenase/reductase family of enzymes. The N-terminal coenzyme-binding pattern and the active-site pattern characteristic of short chain dehydrogenase/reductase proteins are conserved in ORF-1 product. ORF-2 does not show any significant homology with any known protein, whereas ORF-3 encodes a protein having significant homologies with the branched-chain amino acid transporter AzlC protein family. Northern blot hybridization analysis with rd and roo-specific probes identified a common 1.5-kb transcript, indicating that these two genes are cotranscribed. The transcription start site was identified by primer extension analysis to be a guanidine 87 bp upstream the ATG start codon of rubredoxin. The transcript size indicates that the rd-roo mRNA terminates downstream the roo-coding unit. Putative -10 and -35 regulator regions of a sigma(70)-type promoter, having similarity with E. coli sigma(70) promoter elements, are found upstream the transcription start site. Rubredoxin-oxygen oxidoreductase and rubredoxin genes are shown to be constitutively and abundantly expressed. Using the data available from different prokaryotic genomes, the rubredoxin genomic organization and the first tentative to understand the phylogenetic relationships among the flavoprotein family are reported in this study.

Amino Acid Sequence↗
PubMed1999

The HaeIV restriction modification system of Haemophilus aegyptius is encoded by a single polypeptide.

The HaeIV restriction endonuclease (ENase) belongs to a distinct class of ENases, characterized by its ability to cleave double-stranded DNA on both sides of its recognition sequence, excising a short DNA fragment that includes the recognition sequence. The gene encoding the HaeIV ENase was cloned from Haemophilus aegyptius into pUC19 using a previously described system that does not need the knowledge that a particular ENase is produced by a bacterial strain. DNA sequence analysis of the insert contained on this plasmid identified a single open reading frame (ORF), with the predicted protein having an apparent molecular mass of approximately 110 kDa. The protein encoded by this ORF was purified to homogeneity from Escherichia coli strain ER1944 carrying the haeIVRM gene on a recombinant plasmid under the control of the inducible ara promoter. The protein possessed both ENase and methyltransferase (MTase) activities. Amino acid sequence analysis was able to identify several conserved motifs found in DNA MTases, located in the middle of the protein. The enzyme recognizes the interrupted palindromic sequence 5' GAPyNNNNNPuTC 3', cleaving double-stranded DNA on both strands upstream and downstream of the recognition sequence, releasing an approximately 33 bp fragment. The ENase possessed an absolute requirement only for Mg(+2). ATP had no influence on ENase or MTase activities. The ENase made the first strand cleavage randomly on either side of the recognition sequence, but the second cleavage occurred more slowly. The MTase activity modified symmetrically located adenine residues on both strands within the recognition sequence yielding N6-methyl adenine. Furthermore, the MTase was active as a dimer.

Adenosine Triphosphate↗
PubMed2005

The goose reovirus genome segment encoding the minor outer capsid protein, sigma1/sigmaC, is bicistronic and shares structural similarities with its counterpart in Muscovy duck reovirus.

Reoviruses have recently been shown to be associated with disease in young geese and to be involved in epizooties of severe outcome in Hungary. To assess the genetic variability among these pathogenic goose reoviruses (GRVs), we sequenced the S4 genome segment of five GRV strains isolated from different diseased flocks. We found that the GRV S4 genome segment, consisting of two partially overlapping open reading frames (ORFs), shares substantial structural similarity with its counterpart in muscovy duck reoviruses (DRVs). ORF1 is predicted to encode a polypeptide highly similar to the p10 polypeptide of DRV, and ORF2 supposedly encodes the minor outer capsid protein, sigma1/sigmaC. In one of the five GRV strains examined, we identified a single uracil base insertion close to the middle of ORF2. This insertion resulted in a frameshift and in concomitant acquisition of a termination codon (UAA) a few codons downstream, apparently causing truncation of the C-terminal part of the protein. The functional consequences of this assumed mutation, which would result in loss of more than a half of the protein, have yet to be determined. Nonetheless, the sequence and structural similarities between the genome segment encoding sigmal/sigmaC in GRVs and DRVs suggest that these viruses belong to a species distinct from other established species within subgroup 2 of orthoreoviruses.

Amino Acid Sequence↗
PubMed1996

Identification and cloning of centaurin-alpha. A novel phosphatidylinositol 3,4,5-trisphosphate-binding protein from rat brain.

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia.

The complete genome sequences are reported here of two field isolates of bovine coronavirus (BCoV), which were isolated from respiratory and intestinal samples of the same animal experiencing fatal pneumonia during a bovine shipping fever epizootic. Both genomes contained 31028 nucleotides and included 13 open reading frames (ORFs) flanked by 5'- and 3'-untranslated regions (UTRs). ORF1a and ORF1b encode replicative polyproteins pp1a and pp1ab, respectively, that contain all of the putative functional domains documented previously for the closest relative, mouse hepatitis virus. The genomes of the BCoV isolates differed in 107 positions, scattered throughout the genome except the 5'-UTR. Differences in 25 positions were non-synonymous and were located in all proteins except pp1b. Six replicase mutations were identified within or immediately downstream of the predicted largest pp1a-derived protein, p195/p210. Single amino acid changes within p195/p210 as well as within the S glycoprotein might contribute to the different phenotypes of the BCoV isolates.

Amino Acid Sequence↗
PubMed1996

Gene expression in the cos region of the Streptomyces temperate actinophage phi C31.

A transcription map of a 5.12 kb region containing the cos ends of actinophage phi C31 was determined using RNA prepared from induced and uninduced cultures of the temperature-sensitive lysogen, Streptomyces coelicolor A3(2) (phi C31cts1). In induced cultures, RNA synthesis was detected only late in the lytic cycle. A late operon initiated downstream of the int gene on the right-hand end of the genome traversed the cos ends and extended at least 3.6 kb along the left-hand end. Shorter, possibly processed, mRNAs were also present. The map was superimposed on the DNA sequence of 2.8 kb of the region, part of which had been determined previously and part of which is presented here. The late-expressed transcripts contained a tRNA(Thr)-like gene and four ORFs (1, 2, 3 and 5) detected on the basis of codon bias. Analysis of the putative protein products showed that one of the ORFs could encode a lysis protein and at least one may be involved in DNA maturation. Transcription mapping of RNA from uninduced cultures demonstrated a 620 nt transcript, xRNA1, of ORF6. So far this is the only gene in phi C31 found to be expressed right to left with respect to the standard map of phi C31; its function during lysogenic growth could not be deduced from database searches.

Amino Acid Sequence↗
PubMed2001

Structural and functional analysis of the phosphonoacetate hydrolase (phnA) gene region in Pseudomonas fluorescens 23F.

The Pseudomonas fluorescens 23F phosphonoacetate hydrolase gene (phnA) encodes a novel carbon-phosphorus bond cleavage enzyme whose expression is independent of the phosphate status of the cell. Analysis of the regions adjacent to the phosphonoacetate hydrolase structural gene (phnA) indicated the presence of five open reading frames (ORFs). These include one (phnR) whose putative product shows high levels of homology to the LysR family of positive transcriptional regulators. Its presence was shown to be necessary for induction of the hydrolase activity. 2-Phosphonopropionate was found to be an inducer (and poor substrate) for phosphonoacetate hydrolase. Unlike phosphonoacetate, which is also an inducer of phosphonoacetate hydrolase, entry of 2-phosphonopropionate into cells appeared to be dependent on the presence of a gene (phnB) that lies immediately downstream of phnA and whose putative product shows homology to the glycerol-3-phosphate transporter. RNA analysis revealed transcripts for the phnAB and phnR operons, which are transcribed divergently; the resulting mRNAs overlapped by 29 nucleotide bases at their 5' ends. Transcripts of phnAB were detected only in cells grown in the presence of phosphonoacetate, whereas transcripts of phnR were observed in cells grown under both induced and uninduced conditions. The expression of three additional genes found in the phnA region did not appear necessary for the degradation of phosphonoacetate and 2-phosphonopropionate by either Pseudomonas putida or Escherichia coli cells.

Alkaline Phosphatase↗
PubMed2002

A -1 ribosomal frameshift element that requires base pairing across four kilobases suggests a mechanism of regulating ribosome and replicase traffic on a viral RNA.

Programmed -1 ribosomal frameshifting is necessary for translation of the polymerase genes of many viruses. In addition to the consensus elements in the mRNA around the frameshift site, we found previously that frameshifting on Barley yellow dwarf virus RNA requires viral sequence located four kilobases downstream. By using dual luciferase reporter constructs, we now show that a predicted loop in the far downstream frameshift element must base pair to a bulge in a bulged stem loop adjacent to the frameshift site. Introduction of either two or six base mismatches in either the bulge or the far downstream loop abolished frameshifting, whereas mutations in both sites that restored base pairing reestablished frameshifting. Likewise, disruption of this base pairing abolished viral RNA replication in plant cells, and restoration of base pairing completely reestablished virus replication. We propose a model in which Barley yellow dwarf virus uses this and another long-distance base-pairing event required for cap-independent translation to allow the replicase copying from the 3' end to shut off translation of upstream ORFs and free the RNA of ribosomes to allow unimpeded replication. This would be a means of solving the "problem," common to positive strand RNA viruses, of competition between ribosomes and replicase for the same RNA template.

3' Untranslated Regions↗