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PubMed2000

The ORF RTL1 transcript of fowl adenovirus type-8 is spliced and truncated at late stages of the virus replication cycle.

Two transcription products were found for the open reading frame (ORF) RTL1 located near the right terminus of the fowl adenovirus type-8 genome. The larger transcript, which was transcribed mostly during the early stage of the virus infection, contains the complete sequence (933 nucleotides) of the predicted ORF from the genomic DNA sequence encoding a 311 amino acid (aa) polypeptide. In contrast, the shorter transcript, which was more predominant at the late stage of the infection, was missing 580 nucleotides (from nucleotide 117 to 696). A premature stop codon was introduced at 210 nucleotides downstream from the start codon and the shorter transcript would encode a 70 aa polypeptide. This observation indicates that the ORF RTL1 may produce two different proteins, which function differently at different stages of the virus infection. Another possibility is that the virus may use alternative splicing as a mechanism to control the expression of the ORF, since the spliced transcript was prematurely terminated at the late stage of the infection.

Alternative Splicing↗
PubMed1988

First committed step of lipid A biosynthesis in Escherichia coli: sequence of the lpxA gene.

The min 4 region of the Escherichia coli genome contains genes (lpxA and lpxB) that encode proteins involved in lipid A biosynthesis. We have determined the sequence of 1,350 base pairs of DNA upstream of the lpxB gene. This fragment of DNA contains the complete coding sequence for the 28.0-kilodalton lpxA gene product and an upstream open reading frame capable of encoding a 17-kilodalton protein (ORF17). In addition there appears to be an additional open reading frame (ORF?) immediately upstream of ORF17. The initiation codon for lpxA is a GUG codon, and the start codon for ORF17 is apparently a UUG codon. The start and stop codons overlap between ORF? and ORF17, ORF17 and lpxA, and lpxA and lpxB. This overlap is suggestive of translational coupling and argues that the genes are cotranscribed. Crowell et al. (D.N. Crowell, W.S. Reznikoff, and C.R.H. Raetz, J. Bacteriol. 169:5727-5734, 1987) and Tomasiewicz and McHenry (H.G. Tomasiewicz and C.S. McHenry, J. Bacteriol. 169:5735-5744, 1987) have demonstrated that there are three similarly overlapping coding regions downstream of lpxB including dnaE, suggesting the existence of a complex operon of at least seven genes: 5'-ORF?-ORF17-lpxA-lpxB-ORF23-dnaE-ORF37-3 '.

Acyltransferases↗
PubMed2001

Nucleotide sequence of a Dianthovirus RNA1-like RNA found in grassy stunt-diseased rice plants.

A Dianthovirus RNA1-like RNA (DR1L RNA, 4486 nucleotides in length) was found in grassy stunt-diseased rice plants together with Rice grassy stunt virus (RGSV). DR1L RNA has characteristics common to Dianthovirus RNA1 such as (1) presence of a GGAUUUUUAG potential shifty-heptanucleotide at the end of the 5'-proximal ORF, which encodes a 35-kDa protein, followed by a 77-nucleotide sequence capable of forming a stem-loop structure for an efficient--1 frameshift to express the downstream region in a 96-kDa putative replicase protein, (2) presence of nearly identical 17-nucleotide sequences in the 5'-terminal region and in a region upstream of an ORF encoding a 28-kDa, putative capsid protein (CP), and (3) near identity of the 3'-terminal 20 nucleotides to those of Dianthovirus RNAs. Western blot analysis using an antiserum against the C-terminal domain of the putative CP and RT-PCR analysis using primers specific to DR1L RNA of fractions after sucrose density gradient centrifugation of RGSV nucleoproteins indicated that DR1L RNA is associated with the 28-kDa putative CP but not with the 36-kDa RGSV CP. Two additional ORFs for 15-kDa and 33-kDa proteins were present in DR1L RNA although their expression in plants and functions are not known.

Amino Acid Sequence↗
PubMed2000

Regulation of -1 ribosomal frameshifting directed by cocksfoot mottle sobemovirus genome.

The polyprotein of Cocksfoot mottle virus (CfMV) is encoded by two overlapping open reading frames (ORFs). The putative replicase of CfMV is produced as a part of the polyprotein from ORF2b by the -1 ribosomal frameshifting mechanism. The signals leading to -1 ribosomal frameshifting directed by CfMV RNA are the slippery heptamer UUUAAAC and a stem-loop structure starting seven nucleotides downstream from the heptamer. We studied the effect of different parts of the CfMV genome on the -1 ribosomal frameshifting efficiency using a wheat germ extract transcription/translation system. A point mutation in the slippery heptamer and a mutation deleting the stem-loop structure prevented frameshifting. Seventy nucleotides of CfMV sequence, including the slippery sequence and the stem-loop structure, was found to act as a minimal region for frameshifting. Interestingly, a termination codon introduced into the -1-frame 27 nucleotides downstream of the stem-loop structure increased frameshift efficiency threefold, while a similarly located termination codon in the 0-frame had no effect. Even fourfold to fivefold efficiencies were observed when the polyprotein encoding ORFs were fused together, which led simultaneously to the formation of a termination codon downstream of the frameshift signal. Possible reasons underlying these observations are discussed.

Base Sequence↗
PubMed2001

Expression and localization of LEF-11 in Autographa californica nucleopolyhedrovirus-infected Sf9 cells.

The Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) lef-11 gene was found previously to be necessary to support optimal levels of transient expression from an AcMNPV late promoter. The lef-11 gene is unusual in that it overlaps both upstream (orf38) and downstream (pp31) genes. In this study, the expression and cellular localization of LEF-11 were examined. The lef-11 transcripts were detected from 4 to 36 h post-infection (p.i.). The 1.5 kb lef-11 mRNA initiates 196 nt upstream of the lef-11 translation initiation codon, within the upstream orf38 gene. This relatively long 5' upstream region encodes a potential small upstream open reading frame (ORF) of 58 amino acids that overlaps the lef-11 ORF. The 3' end of the lef-11 mRNA was mapped as co-terminal with mRNAs from the downstream pp31 gene. Using affinity purified anti-LEF-11 antibodies, levels of LEF-11 expression were found to be maximal between approximately 8 and 24 h p.i., although LEF-11 could be detected as late as 72 h p.i. Using immunofluorescence microscopy, it was determined that LEF-11 localized to dense regions of infected cell nuclei, consistent with its role as a possible late transcription factor.

Amino Acid Sequence↗
PubMed2001

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA.

Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

DNA, Chloroplast↗
PubMed1989

Identification of p74, a gene essential for virulence of baculovirus occlusion bodies.

DNA sequencing of the HindIII-P fragment of the baculovirus Autographa californica nuclear polyhedrosis virus downstream of a major late protein, p10, revealed the presence of an open reading frame (ORF) 1935 nucleotides in length and in opposite polarity to p10. The gene product is considered essential for virus virulence in Trichoplusia ni larvae since infection with occlusion bodies from a mutant, Ac228z, in which portions of adjacent carboxy-termini from peptides p74 and p10 were deleted, failed to kill larvae, whereas virus with deletions in p10 alone were as infectious to larvae as wild-type virus. The ORF has the potential to code for a polypeptide of 645 amino acid residues (Mr 73,819) and was designated p74. Time course analysis of RNA from infected cells using primer extension assays suggested that the gene's promoter was weak and was most active at 16-20 hr postinfection. The transcription initiation site of the RNA was located at -90/-91 bases upstream of the start codon. The p74 gene was cloned into a baculovirus expression vector and a recombinant virus was produced which overexpressed the p74 protein.

Amino Acid Sequence↗
PubMed1998

A method for the identification of promoters recognized by RNA polymerase containing a particular sigma factor: cloning of a developmentally regulated promoter and corresponding gene directed by the Streptomyces aureofaciens sigma factor RpoZ.

We have developed a method for the identification of promoters recognized by a particular sigma factor of RNA polymerase, based on a two-compatible plasmid system in Escherichia coli (Ec). Using the method, a DNA fragment containing the promoter, PREN40, recognized by sporulation-specific Streptomyces aureofaciens (Sa) sigma factor RpoZ, was cloned. High-resolution S1 nuclease mapping using RNA prepared from Ec, and Sa from various developmental stages has shown a high degree of similarity of PREN40 to consensus sequence of flagellar and chemotaxis promoters. The promoter was induced at the time of aerial mycelium formation, and was off in the Sa strain with the rpoZ-disrupted gene. A promoter-bearing DNA fragment was inserted into the promoter-probe plasmid pARC1 to give expression patterns consistent with the results of direct RNA analysis. The region downstream of the promoter was cloned in Sa. Sequence analysis revealed an open reading frame (ORF) of 283 amino acids (Mr 30006), encoding a highly basic (pI 12.35) protein with high percentage of serine, threonine and alanine (41.8%).

Alanine↗
PubMed1998

Unconventional organization of the division and cell wall gene cluster of Streptococcus pneumoniae.

The genes responsible for cell wall biosynthesis and cell division (dcw genes) were identified and sequenced in Streptococcus pneumoniae. The genetic organization of the dcw cluster in Streptococcus pneumoniae differed significantly from the clusters of other bacteria reported to date. In particular, the genes corresponding to the 2 min region of the Escherichia coli chromosome were found distributed in three genetically separate regions of the Streptococcus pneumoniae chromosome. The first region contained the expected ftsA and ftsZ cell division genes at one end and pbp2b, ddl and murF at the other end. The murD, murG and divIB genes, always found located upstream of ftsA, were found in a second region separated from the first. A third region contained the yllC, yllD, pbp2x and mraY genes. The chromosomal region downstream of ftsZ was also sequenced and characterized. In Streptococcus pneumoniae this region contains four ORFs, all of unknown function, and an ORF encoding the Bacillus subtilis DivIVA homologue. The gene order and the organization of this region was found to be conserved in Staphylococcus aureus, Streptococcus pyogenes and Bacillus subtilis, raising the possibility that previously unidentified loci may also be involved in division.

Bacterial Proteins↗
PubMed1995

Determination of a 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome.

A 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome, located 306 kb downstream from the zero point of the physical map, was determined. Twenty-one putative ORFs were identified: two ORFs (Orf1 and Orf20) were consistent with the nucleotide and amino acid sequence of B. subtilis OrfJ, whose function is not known, and Pcp, respectively; four were found to display significant similarities to known proteins in data banks, i.e. 5-keto-3-deoxyglucarate dehydratase (Orf2), aldehyde dehydrogenase (Orf3) and two glucarate dehydratases (Orf4 and 5); three had considerable similarity to the sensor-regulator proteins of bacterial two-component signal transduction systems (Orf1, 11 and 12); two had considerably high levels of similarity to parts of known proteins (Orf13 and 19); and five showed low levels of similarity to known proteins in the data banks. The remaining six ORFs showed no similarity to known proteins.

Aldehyde Dehydrogenase↗
PubMed1995

An operon encoding aspartokinase and purine phosphoribosyltransferase in Thermus flavus.

The nucleotide sequence of a 1.1 kb XhoI-HindIII fragment downstream of the malate dehydrogenase (mdh) gene of Thermus flavus revealed the presence of an ORF and an incomplete ORF lacking its NH2-terminal portion, in the opposite orientation to that of the mdh gene. These two genes overlapped with each other, sharing two base pairs, suggesting that these genes are co-transcribed in a single mRNA. One ORF (termed gpt) encoded a protein of 154 amino acids showing significant amino acid sequence similarity to purine phosphoribosyltransferases, such as xanthine-guanine phosphoribosyltransferase of Escherichia coli and human hypoxanthine phosphoribosyltransferase. Cloning and sequencing of the upstream region of the gpt gene, together with sequence comparison of the gene product encoded by the region upstream of gpt, suggested that the upstream ORF encoded two in-frame overlapping aspartokinase genes, askA, encoding the alpha-subunit of 405 amino acids, and askB, encoding the beta-subunit of 161 amino acids, which was part of the 3' portion of askA. Consistent with the sequence data, the askAB and the gpt genes conferred the heat-stable enzyme activities of aspartokinase and phosphoribosyltransferase, respectively, on E. coli. Preliminary characterization of these enzymes produced in E. coli is described.

Amino Acid Sequence↗
PubMed1988

Characterization of a second gene involved in bacterio-opsin gene expression in a halophilic archaebacterium.

Southern blot analysis and nucleotide sequencing of DNA from three bacterio-opsin-deficient mutants of the archaebacterium Halobacterium halobium (M86, W105, and W109) revealed that they each contain an alteration in a region 2,000 to 3,800 base pairs (bp) upstream of the bacterio-opsin gene (bop). Nucleotide sequence analysis of this region, which is also located downstream of the previously characterized brp gene, revealed that it contains an open reading frame (ORF) of 2,022 bp. This 2,022-bp ORF has a start codon which overlaps the stop codon of the brp gene and is read in the same direction. The ORF could encode an acidic protein of 73,334 daltons (674 amino acids) with a predicted secondary structure typical of a soluble protein. Bop mutant M86 contains a 1,883-bp deletion extending from bp 351 of the ORF, to 197 bp beyond the stop codon. Mutant W105 has an ISH2 element integrated at bp 1239 of the ORF, and mutant W109 has an ISH26 element integrated at bp 1889. Our results suggest that the ORF is a gene (designated bat for bacterio-opsin activator gene) involved in bop gene expression.

Amino Acid Sequence↗
PubMed1992

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.

Amino Acid Sequence↗
PubMed2001

An element downstream of the transcription start site is required for activation of Bombyx mori nucleopolyhedrovirus bro-c promoter.

Bombyx mori nucleopolyhedrovirus (BmNPV) contains five baculovirus repeated ORF (bro) genes. We have previously reported that all of these genes (bro-a, b, c, d and e) are transcribed as early genes and require viral factor(s) for their expression. In this study, we investigated the mechanism of promoter activation of the bro-c gene. Transient expression assays using genomic libraries of BmNPV indicated that the baculoviral trans-regulator IE-1 is responsible for activating the bro-c promoter. To identify essential site(s) for promoter activation, mutations were introduced to the promoter region of bro-c. Interestingly, it was shown that the pentanucleotide sequence CACGC located 30 nucleotides downstream of the RNA start site was essential for bro-c promoter activation. In addition, the RNA start site and the spacing between the RNA start site and CACGC were also required for promoter activation. By introducing a CACGC sequence into the corresponding region of the bro-b promoter, which is not normally trans-activated by IE-1, we demonstrated that this pentanucleotide motif has the ability to confer trans-activation by IE-1 on a promoter. Gel retardation experiments also showed a sequence-specific DNA binding protein induced by baculovirus infection interacts with the CACGC motif.

Animals↗
PubMed1992

Rice dwarf phytoreovirus segment S12 transcript is tricistronic in vitro.

Sequence analysis revealed that rice dwarf phytoreovirus segment S12 is 1066 nucleotides long with a small out-of-phase, overlapping open reading frame (ORF) as well as a major ORF. The large ORF (positions 42 to 980) encodes 312 amino acids, while the small one (bases 313 to 591) encodes 92 amino acids with an additional in-frame AUG codon (positions 337-339) 24 nucleotides downstream from the first one. Transcripts from a full-length cDNA directed the in vitro synthesis of three polypeptides of 33 (considered to be translated from the long ORF), 8, and 7 kDa. Alteration of each of the two ATG codons on the small ORF demonstrated their involvement in the generation of the 8- and 7-kDa polypeptides. Although it is still unknown whether these proteins are expressed in vivo, the small ORF is shown to be conserved in S9s of two other members of the genus Phytoreovirus, rice gall dwarf virus and wound tumor virus, suggesting its common, important function.

Amino Acid Sequence↗
PubMed1995

Four additional genes in the sigB operon of Bacillus subtilis that control activity of the general stress factor sigma B in response to environmental signals.

sigma B of the gram-positive bacterium Bacillus subtilis is an alternative transcription factor activated by a variety of environmental stresses, including the stress imposed upon entry into the stationary growth phase. Previous reports have shown that this stationary-phase activation is enhanced when cells are grown in rich medium containing glucose and glutamine. The sigma B structural gene, sigB, lies in an operon with three other genes whose products have been shown to control sigma B activity in response to environmental stress. However, none of these is sufficient to explain the enhanced stationary-phase activation of sigma B in response to glucose. We show here that the four genes previously identified in the sigB operon constitute the downstream half of an eight-gene operon. The complete sigB operon is preceded by a sigma A-like promoter (PA) and has the order PA-orfR-orfS-orfT-orfU-PB-rsbV-rsbW-sig B-rsbX, where rsb stands for regulator of sigma-B and the previously identified sigma B-dependent promoter (PB) is an internal promoter preceding the downstream four-gene cluster. Although the genes downstream of PB were also transcribed by polymerase activity originating at PA, this transcription into the downstream cluster was not essential for normal induction of a sigma B-dependent ctc-lacZ fusion. However, deletion of all four upstream open reading frames was found to interfere with induction of the ctc-lacZ fusion in response to glucose. Additional deletion analysis and complementation studies showed that orfU was required for full glucose induction of sigma B-dependent genes. orfU encodes a trans-acting, positive factor with significant sequence identity to the RsbX negative regulator of sigma B. On the basis of these results, we rename orfU as rsbU to symbolize the regulatory role of its product.

Adaptation, Physiological↗
PubMed2004

Role of a nosX homolog in Streptococcus gordonii in aerobic growth and biofilm formation.

Oral streptococci such as Streptococcus gordonii are facultative anaerobes that initiate biofilm formation on tooth surfaces. An isolated S. gordonii::Tn917-lac biofilm-defective mutant contained a transposon insertion in an open reading frame (ORF) encoding a homolog of NosX of Ralstonia eutropha, a putative maturation factor of nitrous oxide reductase. Located downstream are two genes, qor1 and qor2, predicted to encode two putative NADPH quinone oxidoreductases. These three genes are cotranscribed, forming a putative oxidative stress response (osr) operon in S. gordonii. Inactivation of nosX, qor1, or qor2 resulted in biofilm-defective phenotypes. Expression of nosX, measured by the beta-galactosidase activity of the nosX::Tn917-lac mutant, was growth-phase dependent and enhanced when grown under aerobic conditions or in the presence of paraquat. Real-time reverse transcription-PCR revealed that nosX-specific mRNA levels were increased approximately 8.4 and 3.5 fold in biofilm-derived cells grown on plastic and glass, respectively, when compared to planktonic cells. Expression of nosX increased 19.9 fold in cells grown under aerated aerobic conditions and 4.7 fold in cells grown under static aerobic conditions. Two ORFs immediately adjacent to the osr operon encode a putative NADH oxidase (Nox) and a putative thiol-specific antioxidant enzyme (AhpC, for alkyl hydroperoxide peroxidase C). Expression of nox and ahpC was also significantly increased in cells grown under aerated and static aerobic conditions when compared to anaerobic conditions. In addition, nox expression was increased in biofilm cells compared to planktonic cells. These genes may be part of an island that deals with oxidoreductive response, some of which may be important in S. gordonii biofilm formation.

Amino Acid Sequence↗
PubMed2003

A DNA region of Torulaspora delbrueckii containing the HIS3 gene: sequence, gene order and evolution.

We cloned a genomic DNA fragment of the yeast Torulaspora delbrueckii by complementation of a Saccharomyces cerevisiae his3 mutant strain. DNA sequence analysis revealed that the fragment contained two complete ORFs, which share a high similarity with S. cerevisiae His3p and Mrp51p, respectively. The cloned TdHIS3 gene fully complemented the his3 mutation of S. cerevisiae, confirming that it encodes for the imidazoleglycerol-phosphate dehydrate of T. delbrueckii. Two additional ORFs, with a high homology to S. cerevisiae PET56 and DED1 genes, were mapped upstream and downstream from TdHIS3 and TdMRP51, respectively. This genetic organization is analogous to that previously found in Saccharomyces kluyveri and Zygosaccharomyces rouxii. The evolutionary significance of gene order in this chromosomal region is analysed and discussed.

Amino Acid Sequence↗