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PubMed1989

DNA sequence and comparative analyses of the equine herpesvirus type 1 immediate early gene.

The immediate early (IE) proteins of herpesviruses are important regulatory factors which control the expression of genes at the transcriptional level. We report the DNA sequence of the immediate early gene of the alphaherpesvirus equine herpesvirus type 1 (EHV-1). This sequence is shown to be extremely rich in guanine and cytosine, resulting in a highly biased codon usage. The IE gene region possesses 38 open reading frames (ORFs) greater than 300 bp in length, 11 of which have coding regions of at least 100 amino acids (aa) following potential translation initiator codons. The largest ORF consists of 1487 codons (4461 bp) starting with the first ATG and would encode a protein of MW 155,000. TATA and CCAAT sequences as well as several potential cis-acting elements lie upstream to the major ORF. The deduced amino acid sequence for the 155,000 protein has a high degree of homology to the herpes simplex virus type 1 (HSV-1) ICP4 protein and its varicella-zoster virus (VZV) homolog. The regions of the EHV-1 IE protein that are homologous with these proteins correspond to the previously determined pattern of homology between the HSV and VZV IE polypeptides. However, there are are a number of differences within these broadly defined regions. It is therefore expected that this comparative study will facilitate the identification of functionally important residues within the amino acid sequence of IE proteins.

Amino Acid Sequence↗
PubMed1996

The rRNA operons of Mycobacterium smegmatis and Mycobacterium tuberculosis: comparison of promoter elements and of neighbouring upstream genes.

Mycobacterium smegmatis has two rRNA (rrn) operons designated rrnAf and rrnBf. Appropriate restriction fragments of genomic DNA containing sequences immediately upstream from the 16S rRNA genes were cloned. We now report the nucleotide sequence of 552 bp upstream from the 5'-end of the Box AL antitermination element of the leader region of the rrnAf operon. The 5'-end of this segment of DNA was found to comprise 113 codons of an ORF encoding a protein which is significantly similar to UDP-N-acetylglucosamine 1-carboxyvinyl-transferase (EC 2.5.1.7), which is important to cell wall synthesis. A homologous ORF is located immediately upstream from the single rrn (rrnAs) operons of Mycobacterium tuberculosis and Mycobacterium leprae. Primer-extension analysis of the RNA fraction of M. smegmatis revealed four products which were related to transcription start points; the rrnBf operon appears to have a single promoter whereas the rrnAf operon has three (P1, P2 and P3). Analysis of M. tuberculosis RNA revealed two products corresponding to transcripts directed by promoters homologous with P1 and P3 of the rrnAf of M. smegmatis. Thus, the promoter and upstream regions of the rrnAf operon of M. smegmatis and the rrnAs operon of M. tuberculosis are homologous. The presence of P2 in M. smegmatis and its absence from M. tuberculosis is attributable to insertions/deletions of 97 bp.

Amino Acid Sequence↗
PubMed2004

Characterization of regulatory elements within the coat protein (CP) coding region of Tobacco mosaic virus affecting subgenomic transcription and green fluorescent protein expression from the CP subgenomic RNA promoter.

A replicon based on Tobacco mosaic virus that was engineered to express the open reading frame (ORF) of the green fluorescent protein (GFP) gene in place of the native coat protein (CP) gene from a minimal CP subgenomic (sg) RNA promoter was found to accumulate very low levels of GFP. Regulatory regions within the CP ORF were identified that, when presented as untranslated regions flanking the GFP ORF, enhanced or inhibited sg transcription and GFP expression. Full GFP expression from the CP sgRNA promoter required more than the first 20 nt of the CP ORF but not beyond the first 56 nt. Further analysis indicated the presence of an enhancer element between nt +25 and +55 with respect to the CP translation start site. The inclusion of this enhancer sequence upstream of the GFP ORF led to elevated sg transcription and to a 50-fold increase in GFP accumulation in comparison with a minimal CP promoter in which the entire CP ORF was displaced by the GFP ORF. Inclusion of the 3'-terminal 22 nt had a minor positive effect on GFP accumulation, but the addition of extended untranslated sequences from the 3' terminus of the CP ORF downstream of the GFP ORF was basically found to inhibit sg transcription. Secondary structure analysis programs predicted the CP sgRNA promoter to reside within two stable stem-loop structures, which are followed by an enhancer region.

Base 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↗
PubMed1999

Analysis of 4-phosphopantetheinylation of polyhydroxybutyrate synthase from Ralstonia eutropha: generation of beta-alanine auxotrophic Tn5 mutants and cloning of the panD gene region.

The postulated posttranslational modification of the polyhydroxybutyrate (PHA) synthase from Ralstonia eutropha by 4-phosphopantetheine was investigated. Four beta-alanine auxotrophic Tn5-induced mutants of R. eutropha HF39 were isolated, and two insertions were mapped in an open reading frame with strong similarity to the panD gene from Escherichia coli, encoding L-aspartate-1-decarboxylase (EC 4.1.1.15), whereas two other insertions were mapped in an open reading frame (ORF) with strong similarity to the NAD(P)+ transhydrogenase (EC 1.6.1.1) alpha 1 subunit, encoded by the pntAA gene from Escherichia coli. The panD gene was cloned by complementation of the panD mutant of R. eutropha Q20. DNA sequencing of the panD gene region (3,312 bp) revealed an ORF of 365 bp, encoding a protein with 63 and 67% amino acid sequence similarity to PanD from E. coli and Bacillus subtilis, respectively. Subcloning of only this ORF into vectors pBBR1MCS-3 and pBluescript KS- led to complementation of the panD mutants of R. eutropha and E. coli SJ16, respectively. panD-encoded L-aspartate-1-decarboxylase was further confirmed by an enzymatic assay. Upstream of panD, an ORF with strong similarity to pntAA from E. coli, encoding NAD(P)+ transhydrogenase subunit alpha 1 was found; downstream of panD, two ORFs with strong similarity to pntAB and pntB, encoding subunits alpha 2 and beta of the NAD(P)+ transhydrogenase, respectively, were identified. Thus, a hitherto undetermined organization of pan and pnt genes was found in R. eutropha. Labeling experiments using one of the R. eutropha panD mutants and [2-14C]beta-alanine provided no evidence that R. eutropha PHA synthase is covalently modified by posttranslational attachment of 4-phosphopantetheine, nor did the E. coli panD mutant exhibit detectable labeling of functional PHA synthase from R. eutropha.

Acyltransferases↗
PubMed2007

Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G.

A cryptic plasmid from Bacillus sphaericus strain LP1-G, designated as pLG, was sequenced and characterized. It was an 11,066bp circular molecule, with G+C content of 37%. The plasmid pLG was predicted to encode 23 putative ORFs, and ORF 21 shared the highest identity with Rep of pGI1 and pBMB9741, members of rolling-circle replication (RCR) pC194-family. Sequence analysis revealed a pC194-type double strand origin (dso) and a single strand origin (sso) like sequence located upstream and downstream of ORF 21, respectively. Moreover, Mung bean nuclease analysis and Southern hybridization confirmed the existence of single stranded DNA (ssDNA) intermediates, indicating that pLG belongs to the RCR pC194-family. Accumulation of multiple ssDNA intermediates in native strain LP1-G and decline of ssDNA and supercoiled DNA in rifampicin-treated strain implied that a special mechanism might be employed by pLG. Furthermore, the copy number of pLG in its original host was determined and about 58 copies of the plasmid exist in each cell. Subcloning and transformation experiments proved that the minimal replicon of pLG was within a 1.6-kb fragment, which was composed of rep gene and dso. These data are a good basis for the understanding of replication mechanisms and genetics of this B. sphaericus plasmid.

Bacillus↗
PubMed1997

Positional preferences of polypurine/polypyrimidine tracts in Saccharomyces cerevisiae genome: implications for cis regulation of gene expression.

The complete genome of the baker's yeast S. cerevisiae was analyzed for the presence of polypurine/polypyrimidine (poly[pu/py]) repeats and their occurrences were classified on the basis of their location within and outside open reading frames (ORFs). The analysis reveals that such sequence motifs are present abundantly both in coding as well as noncoding regions. Clear positional preferences are seen when these tracts occur in noncoding regions. These motifs appear to occur predominantly at a unit nucleosomal length both upstream and downstream of ORFs. Moreover, there is a biased distribution of polypurines in the coding strands when these motifs occur within open reading frames. The significance of the biased distribution is discussed with reference to the occurrence of these motifs in other known mRNA sequences and expressed sequence tags. A model for cis regulation of gene expression is proposed based on the ability of these motifs to form an intermolecular triple helix structure when present within the coding region and/or to modulate nucleosome positioning via enhanced histone affinity when present outside coding regions.

Chromosome Mapping↗
PubMed1999

The Saccharomyces cerevisiae protein YJR043C (Pol32) interacts with the catalytic subunit of DNA polymerase alpha and is required for cell cycle progression in G2/M.

We have analysed the YJR043c gene of Saccharomyces cerevisiae, previously identified by systematic sequencing. The deletion mutant (yjr043cdelta) shows slow growth at low temperature (15 degrees C), while at 30 degrees C and 37 degrees C the growth rate of mutant cells is only moderately affected. At permissive and nonpermissive temperatures, mutant cells were larger and showed a high proportion of large-budded cells with a single duplicated nucleus at or beyond the bud neck and a short spindle. This phenotype was even more striking at low temperature, the mutant cells becoming dumbbell shaped. All these phenotypes suggest a role for YJR043C in cell cycle progression in G2/M phase. In two-hybrid assays, the YJR043c gene product specifically interacted with Pol1, the catalytic subunit of DNA polymerase alpha. The pol1-1 /yjr043cdelta double mutant showed a more severe growth defect than the pol1-1 single mutant at permissive temperature. Centromeric plasmid loss rate elevated in yjr043cdelta. Analysis of the sequence upstream of the YJR043c ORF revealed the presence of an MluI motif (ACGCGT), a sequence associated with many genes involved in DNA replication in budding yeast. The cell cycle phenotype of the yjr043cdelta mutant, the evidence for genetic interaction with Pol1, the presence of an MluI motif upstream and the elevated rate of CEN plasmid loss in mutants all support a function for YJR043C in DNA replication.

Catalytic Domain↗
PubMed1998

The Lotus japonicus LjNOD70 nodulin gene encodes a protein with similarities to transporters.

A novel nodule-specific gene, LjNOD70, associated with late stages in Lotus japonicus nodule development and/or functioning was characterized. The LjNOD70 gene is a member of a small family of closely related L. japonicus genes. Two major mRNA species corresponding to the LjNOD70 gene were identified in nodules and shown to be the result of a mechanism resembling alternative splicing. The longer, presumably unspliced, mRNA species was shown to contain a single open reading frame (ORF), encoding a polytopic hydrophobic protein, LjN70, with a predicted molecular mass of 70 kDa. The second, presumably spliced, mRNA species was shown to be less abundant in nodules. The absence of the presumptive 'intron' was found to divide the reading frame into an upstream and a downstream ORF encoding the partial N- and C-terminal regions of the LjN70 protein, respectively. The predicted amino acid sequence of nodulin LjN70 revealed structural features characteristic of transport proteins, and was found to share similarity with the oxalate/formate exchange protein of Oxalobacter formigenes. Therefore, we postulate that the L. japonicus LjNOD70 gene family encodes nodule-specific transport proteins, which may have evolved as a result of exon-intron shuffling.

Alternative Splicing↗
PubMed2000

Identification and transcriptional characterization of the gene encoding the stress-response sigma factor sigma(H) in streptomyces coelicolor A3(2).

We cloned a new gene, sigH, encoding an alternative sigma factor in Streptomyces coelicolor A3(2). The deduced protein of 354 amino acids with an M(r) of 39486 showed greatest similarity to the sporulation sigma factor (sigma(F)) of S. coelicolor, general stress-response sigma(B) of Bacillus subtilis, and stationary-phase stress-response sigma(F) of Mycobacterium tuberculosis. Sequence analysis of the upstream region revealed an ORF encoding a protein (UshX) similar to several anti-sigma factors, and short ORF (UshY) containing zinc-finger DNA binding motif. Transcriptional analysis revealed that all three genes are located on the same polycistronic transcript in order ushY, ushX, and sigH. Expression of the operon was directed by four promoters differentially expressed in the course of differentiation. The first (P1) constitutive promoter was located upstream of ushY. The other three promoters (P2, P3, and P4) were located upstream of ushX, and were differentially induced after various stress conditions. The magnitude of the induction was greatest after osmotic stress and heat shock.

Amino Acid Sequence↗
PubMed1989

Sequence organization and gene expression of pGD1, a plasmid found in a wild isolate of Dictyostelium.

We have determined the complete nucleotide sequence of pDG1, a plasmid found in a wild isolate of Dictyostelium. The 4439-bp long pDG1 contains only one, 2718-bases-long, open reading frame (ORF) and nearly perfect inverted repeats of 551 bp and 552 bp. Northern-blot analysis showed that only one 2.7-kb poly (A)+ RNA transcript was expressed at a maximum level, 2 h (early aggregation stage) after the onset of development. The expression of this transcript was suppressed by the addition of cAMP. In the upstream region of the ORF, there are several putative consensus sequences, e.g. (1) TGACTTAGAA-AAATT which is a putative site for cleavage by topoisomerase I, and (2) TGACGACA which may be a cAMP-responsive element, found in several genes that are regulated by cAMP at the level of transcription. A possible mechanism of the partitioning of pDG1 into daughter cells is discussed.

Amino Acid Sequence↗
PubMed1987

Expression of the hepatitis B virus X gene in mammalian cells.

The hepatitis B virus (HBV) DNA contains an open reading frame (ORF) designated X, which has the capacity to encode a protein of 16,560 Da (subtype adw). Such a protein has not been identified in either HBV particles or infected human livers, and therefore its role in the viral life cycle remains unknown. We report here the expression of the HBV X ORF in cultured cells using recombinant vectors. A protein of 16 kDa was identified by means of an antiserum prepared against a synthetic peptide and with human antisera from hepatitis B patients as well as those with hepatocellular carcinoma. Cell fractionation and immunofluorescence studies suggest a probable association with cytoskeletal components. Our studies further located a promoter sequence upstream of the X ORF, which directs the transcription of a 0.7- to 0.8-kilobase X-specific RNA in transfected human hepatoma cells.

Acetyltransferases↗
PubMed2026

Recent advances for the pharmaceutical production of highly attenuated poxviruses as viral vector platforms.

INTRODUCTION: Highly attenuated poxviruses serve as potent viral vectors, oncolytic agents, and therapeutic vaccines. They can accommodate and stably maintain a large genomic payload of foreign inserts. Their limited replication in human cells provides an excellent safety profile, but it concomitantly necessitates higher doses of infectious particles for full therapeutic efficacy. AREAS COVERED: We review recent advances in bioprocesses for the pharmaceutical production of poxvirus-based vectors, focusing mainly on the vaccinia virus and the Orf virus. These include upstream processing using highly permissive cell substrates, optimized feeding strategies, and a virus phenotype that facilitates downstream processing. The study explores ongoing challenges and identifies strategies to adapt the downstream process to intensified upstream processes in order to achieve an economic end-to-end production. EXPERT OPINION: For notably increased virus yields of up to 2 log after amplification, we propose to replace classic adsorption chromatography by a collective and continuous purification platform for separating the virus from process-related impurities. Filtration operations facilitate process scalability while reducing volumes, which is beneficial for a flow-through polishing to meet pharmaceutical quality attributes. Combined with artificial intelligence modeling, these advancements alleviate financial pressures on healthcare systems and accelerate the production of novel vaccine candidates for clinical use.

Humans↗
PubMed2003

Cloning and characterization of the glycogen branching enzyme gene existing in tandem with the glycogen debranching enzyme from Pectobacterium chrysanthemi PY35.

The glycogen branching enzyme gene (glgB) from Pectobacterium chrysanthemi PY35 was cloned, sequenced, and expressed in Escherichia coli. The glgB gene consisted of an open reading frame of 2196bp encoding a protein of 731 amino acids (calculated molecular weight of 83,859Da). The glgB gene is upstream of glgX and the ORF starts the ATG initiation codon and ends with the TGA stop codon at 2bp upstream of glgX. The enzyme was 43-69% sequence identical with other glycogen branching enzymes. The enzyme is the most similar to GlgB of E. coli and contained the four regions conserved among the alpha-amylase family. The glycogen branching enzyme (GlgB) was purified and the molecular weight of the enzyme was estimated to be 84kDa by SDS-PAGE. The glycogen branching enzyme was optimally active at pH 7 and 30 degrees C.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1998

Nucleotide sequence of the inter-structural gene region of feline infectious peritonitis virus.

The sequence of the region located between the S and M glycoprotein genes of the 79-1146 strain of feline infectious peritonitis virus (FIPV) is presented. The inter-structural gene region encodes 3 open reading frames (ORFs), termed ORFs 3a, 3b and 4, with nucleotide sequences conforming to the minimum conserved transcription signal upstream of each. An additional ORF, 3x, partially overlaps the 3' end of ORF 3a. The FIPV interstructural gene region is identical in length when compared to the Insavc-1 strain of canine coronavirus (CCV) but differs from various strains of transmissible gastroenteritis virus (TGEV) by the presence of deletions and insertions. The sizes of ORF 3a and 4 are conserved in FIPV, TGEV and CCV. However, as with CCV, the FIPV ORF 3b is truncated in comparison with TGEV.

Animals↗
PubMed1998

Shotgun cloning and characterization of the thymidylate synthase-encoding gene from Mycobacterium bovis BCG.

The shotgun cloning of a Mycobacterium bovis BCG (BCG) genome into pBluescript SK (+) successfully yielded a 0.9 kbp fragment, confirming the ability of Escherichia coli thyA mutant MH2702 to grow in a thymine-depleted medium. This DNA fragment contained a gene homologous to the thymidylate synthase (TS)-encoding genes (thyA) of other organisms. An inverted repeat sequence and open reading frame (ORF) were observed at the upstream region of the thyA. A computer analysis revealed that the protein encoded by this ORF possessed a structure unique for a DNA binding protein.

Amino Acid Sequence↗
PubMed1998

A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus.

We cloned several genes encoding an Na+/H+ antiporter of Staphylococcus aureus from chromosomal DNA by using an Escherichia coli mutant, lacking all of the major Na+/H+ antiporters, as the host. E. coli cells harboring plasmids for the cloned genes were able to grow in medium containing 0.2 M NaCl (or 10 mM LiCl). Host cells without the plasmids were unable to grow under the same conditions. Na+/H+ antiport activity was detected in membrane vesicles prepared from transformants. We determined the nucleotide sequence of the cloned 7-kbp region. We found that seven open reading frames (ORFs) were necessary for antiporter function. A promoter-like sequence was found in the upstream region from the first ORF. One inverted repeat followed by a T-cluster, which may function as a terminator, was found in the downstream region from the seventh ORF. Neither terminator-like nor promoter-like sequences were found between the ORFs. Thus, it seems that the seven ORFs comprise an operon and that the Na+/H+ antiporter consists of seven kinds of subunits, suggesting that this is a novel type of multisubunit Na+/H+ antiporter. Hydropathy analysis of the deduced amino acid sequences of the seven ORFs suggested that all of the proteins are hydrophobic. As a result of a homology search, we found that components of the respiratory chain showed sequence similarity with putative subunits of the Na+/H+ antiporter. We observed a large Na+ extrusion activity, driven by respiration in E. coli cells harboring the plasmid carrying the genes. The Na+ extrusion was sensitive to an H+ conductor, supporting the idea that the system is not a respiratory Na+ pump but an Na+/H+ antiporter. Introduction of the plasmid into E. coli mutant cells, which were unable to grow under alkaline conditions, enabled the cells to grow under such conditions.

Amino Acid Sequence↗
PubMed1993

Analysis of the promoter sequences of the UL9 gene of herpes simplex virus type 1.

The origin-binding protein, encoded by the UL9 gene of herpes simplex virus-1 (HSV-1), has the properties of an initiator of DNA replication. Since onset of lytic replication of the virus can be critically controlled by availability of the initiator protein, we have analyzed the sequences upstream of the UL9 gene in a monkey kidney cell line (Vero). In this communication, we report the presence of three UL9 transcripts and multiple transcription initiation sites of the UL9 gene in HSV-1-infected cells. The presence of multiple transcription initiation sites for the UL9 gene is consistent with the absence of a TATA element 5'- to the UL9 ORF. A 395-bp upstream sequence encompassing all UL9 transcription initiation sites was active in expressing a reporter gene and was induced by viral immediate-early proteins. This result is consistent with the earlier observation that immediate-early proteins of HSV-1 induce viral early genes.

Animals↗