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PubMed1999

Deletion of 24 open reading frames from chromosome XI from Saccharomyces cerevisiae and phenotypic analysis of the deletants.

As a part of the EUROFAN program, 24 open reading frames from Saccharomyces cerevisiae (YKR010c to YKR013w, YKR015c to YKR025w, YKR081c to YKR083c, YKR087c to YKR091w and YKR096w) were disrupted in two genetic backgrounds, FY1679 and W303. Systematic deletions and phenotypic analysis were performed following a hierarchical strategy, the so-called 'mass murder'. Of the 24 genes thus deleted, four are essential, whereas the deletion of 17 did not reveal any significant difference between the parental and mutant strains. Deletions of the remaining three show some growth phenotype; ykr024c mutants grow slowly under any conditions, ykr019c mutants grow slower in a rich medium and ykr082w mutants are temperature sensitive, being unable to germinate at 30 degrees C and above.

Base Sequence↗

PubMed2001

The sequence of a 15 769 bp segment of Pichia anomala identifies the SEC61 and FBP1 genes and five new open reading frames.

We have determined the sequence of a 15 769 bp DNA segment of Pichia anomala. The sequence contains seven complete open reading frames (ORFs) longer than 100 amino acids and a putative tRNA gene. Two of the ORFs code for the well-characterized genes SEC61 (which codes for the core subunit of the ER translocation complex) and FBP1 (encoding fructose-1,6-bisphosphatase). A gene coding for a protein similar to S. cerevisiae YDL054c was found between the two genes. These three genes show a different organization (intermingled triples) in three yeast species: Saccharomyces cerevisiae, Candida albicans and P. anomala. Two out of the four remaining ORFs show weak homology with different proteins from other species and the other two show non-significant similarity with previously sequenced genes. The nucleotide sequence has been submitted to the EMBL database under Accession No. AJ306295.

Amino Acid Sequence↗

PubMedDate not supplied

Comparison of nucleic and amino acid sequences and phylogenetic analysis of open reading frames 3 and 4 of various equine arteritis virus isolates.

The genetic variation in equine arteritis virus (EAV) protein-encoding open reading frames (ORFs) 3 and 4 genes was investigated. Nucleic and deduced amino acid sequences from seven different EAV isolates (one European, one American and five Canadian isolates) and the Arvac vaccine strain were compared with those of Bucyrus reference strain. ORF 3 nucleotide and amino acid sequence identities between these isolates (including the Arvac vaccine strain) and the Bucyrus reference strain ranged from 85.6 to 98.8%, and 85.3 to 98.2%, respectively, whereas ORF 4 nucleotide and amino acid sequence identities ranged from 90.4 to 98.3%, and 90.8 to 97.4%, respectively. Phylogenetic analysis and estimation of genetic distances based on the ORF 3 nucleic acid sequences showed that the European Vienna isolate could be classified into a genetically divergent group from all other isolates and the Arvac vaccine strain. In contrast, the nucleic acid sequences of ORF 4 were found to be less variable, with a closer phylogenetic relationship evident among the EAV isolates and the Arvac vaccine strain.

Amino Acid Sequence↗

PubMed2003

Molecular analysis of human herpesvirus 8 by using single nucleotide polymorphisms in open reading frame 26.

Human herpesvirus 8 (HHV-8) can be classified into distinct subtypes on the basis of sequence polymorphisms in several open reading frames (ORFs). We analyzed the subtypes of HHV-8 in 59 human immunodeficiency virus-infected Japanese patients by using polymorphisms in ORF26 and found that over two-thirds of the HHV-8 isolates fell into major subtype A. We also found that single nucleotide polymorphisms (SNPs) at nucleotide positions 1032 (C-to-A substitution) and 1055 (G-to-T substitution) in HHV-8 ORF26 were correlated with increased susceptibility to Kaposi's sarcoma, compared to the results obtained with HHV-8 with wild-type nucleotides at these positions (P = 0.0106). This observation suggests that molecular heterogeneity of the HHV-8 genome affects the biological properties of HHV-8, resulting in different clinical phenotypes of HHV-8 infection. Since sensitive PCR of ORF26 allowed us to analyze the SNPs by using peripheral blood from HHV-8-infected patients, the ORF26 SNPs will be a potent tool for investigating the pathogenesis of HHV-8 infection.

AIDS-Related Opportunistic Infections↗

PubMed1996

Characterization of a promoter in the E7 open reading frame of human papillomavirus type 11.

A human papillomavirus type 11 (HPV-11) early gene promoter, PE1--E4, in the E7 open reading frame and involved in the transcription of E1--E4 and E1--E4, L1 mRNAs was characterized by chloramphenicol acetyl transferase (CAT) assay, gel-shift assay, in vitro transcription, and primer extension. By CAT assay with CAT-expression plasmids containing various portions of the HPV-11 genome from nucleotide position (nt) 235 to nt 743, we identified a 112-basepair (bp) sequence (nt 632-743) that had high promoter activity and a 188-bp upstream sequence from nt 395 to nt 572 that strongly suppressed activity. The 112-bp sequence contained a 7-bp repeat (7-bp Rep) with high homology to the Sp1-binding motif (BM) in the E6 promoter of HPV-11 but lacked an identifiable TATA-box. A cellular protein other than Sp1 bound to the 7-bp Rep. Removal of a 21-bp segment (nt 632-652) containing the 7-bp Rep significantly reduced activity. By in vitro transcription assay and primer extension, one of the transcription start sites was mapped to near nt 719. These results suggested that a cellular protein that binds to the 7-bp Rep may act as an activator for PE1--E4.

DNA-Binding Proteins↗

PubMedDate not supplied

Open reading frames of turnip crinkle virus involved in satellite symptom expression and incompatibility with Arabidopsis thaliana ecotype Dijon.

Carmoviruses are single-stranded, single component RNA viruses that include turnip crinkle virus (TCV) and the recently discovered cardamine chlorotic fleck virus (CCFV). Full-length, biologically active cDNAs were constructed for the TCV-M isolate and the Blue Lake isolate of CCFV. Using chimeric viruses constructed between isolates of TCV that produce mild or severe symptoms when coinoculated with a virulent satellite RNA, a Glu residue at position 1,144 in the polymerase open reading frame was identified as being involved in satellite-mediated symptom expression. To analyze viral determinants involved in resistance, chimeric viruses with precisely exchanged open reading frames were produced between TCV, which does not infect the Arabidopsis thaliana ecotype Dijon (Di-0), and CCFV, which can infect Di-0, TCV with the coat protein of CCFV was able to systemically infect Di-0 although whole plant hybridizations revealed that the hybrid virus spread more slowly than either of the two parental viruses. These results indicate that the two parental viruses. These results indicate that the coat protein is an important viral determinant in the resistance of Di-0 to TCV.

Amino Acid Sequence↗

PubMed2003

Protein products of the open reading frames encoding nonstructural proteins of human astrovirus serotype 8.

Human astroviruses have a positive-strand RNA genome, which contains three open reading frames (ORF1a, ORF1b, and ORF2). The genomic RNA is translated into two nonstructural polyproteins, nsp1a and nsp1ab, that contain sequences derived from ORF1a and from both ORF1a and ORF1b, respectively. Proteins nsp1a and nsp1ab are thought to be proteolytically processed to yield the viral proteins implicated in the replication of the virus genome; however, the intermediate and final products of this processing have been poorly characterized. To identify the cleavage products of the nonstructural polyproteins of a human astrovirus serotype 8 strain, antisera to selected recombinant proteins were produced and were used to analyze the viral proteins synthesized in astrovirus-infected Caco-2 cells and in cells transfected with recombinant plasmids expressing the ORF1a and ORF1b polyproteins. Pulse-chase experiments identified proteins of approximately 145, 88, 85, and 75 kDa as cleavage intermediates during the polyprotein processing. In addition, these experiments and kinetic analysis of the synthesis of the viral proteins identified polypeptides of 57, 20, and 19 kDa, as well as two products of around 27 kDa, as final cleavage products, with the 57-kDa polypeptide most probably being the virus RNA polymerase and the two approximately 27-kDa products being the viral protease. Based on the differential reactivities of the astrovirus proteins with the various antisera used, the individual polypeptides detected were mapped to the virus ORF1a and ORF1b regions.

Caco-2 Cells↗

PubMed2003

Open reading frame sso2387 from the archaeon Sulfolobus solfataricus encodes a polypeptide with protein-serine kinase activity.

The predicted polypeptide product of open reading frame sso2387 from the archaeon Sulfolobus solfataricus, SsoPK2, displayed several of the sequence features conserved among the members of the "eukaryotic" protein kinase superfamily. sso2387 was cloned, and its polypeptide product was expressed in Escherichia coli. The recombinant protein, rSsoPK2, was recovered in insoluble aggregates that could be dispersed by using high concentrations (5 M) of urea. The solubilized polypeptide displayed the ability to phosphorylate itself as well as several exogenous proteins, including mixed histones, casein, bovine serum albumin, and reduced carboxyamidomethylated and maleylated lysozyme, on serine residues. The source of this activity resided in that portion of the protein displaying homology to the catalytic domain of eukaryotic protein kinases. By use of mass spectrometry, the sites of autophosphorylation were found to be located in two areas, one immediately N terminal to the region corresponding to subdomain I of eukaryotic protein kinases, and the second N terminal to the presumed activation loop located between subdomains VII and VIII. Autophosphorylation of rSsoPK2 could be uncoupled from the phosphorylation of exogenous proteins by manipulation of the temperature or mutagenic alteration of the enzyme. Autophosphorylation was detected only at temperatures >or=60 degrees C, whereas phosphorylation of exogenous proteins was detectable at 37 degrees C. Similarly, replacement of one of the potential sites of autophosphorylation, Ser(548), with alanine blocked autophosphorylation but not phosphorylation of an exogenous protein, casein.

Amino Acid Sequence↗

PubMed1996

The UL3 open reading frame of herpes simplex virus type 1 codes for a phosphoprotein.

Based on sequence analysis, the protein encoded by the UL3 open reading frame (ORF) of herpes simplex virus type 1 (HSV-1) was predicted to contain an N glycosylation site and to be a glycoprotein. To determine if this prediction was correct, we cloned and expressed the DNA encoding the complete sequence of the UL3 ORF in a baculovirus expression system. Western blotting was done using polyclonal antibody raised against synthetic UL3 peptides. Two major baculovirus-UL3 expressed protein bands with apparent molecular weights of 30 kDa and 31 kDa, and two minor protein bands with apparent molecular weights of 29 kDa and 33 kDa were detected. None of the expressed UL3 protein species were susceptible to tunicamycin treatment, suggesting that they were not N-linked glycosylated. Cell fractionation studies indicated that the UL3 protein was localized in the cytoplasmic and nuclear portion of the cells, rather than the cell membrane, again suggesting a lack of glycosylation. In contrast, the baculovirus expressed UL3 protein was phosphorylated as judged by 32Pi-labeling. Immunoprecipitation followed by SDS-PAGE demonstrated a single 32Pi-labeled UL3 related band with an apparent molecular weight of 33 kDa, indicating that the UL3 protein was a phosphoprotein. Antibodies produced in mice vaccinated with baculovirus-UL3 protein reacted with two UL3 related HSV-1 bands on Western blots. These protein bands had apparent molecular weights of 27 and 33 kDa and presumably represent the unphosphorylated and phosphorylated forms of UL3.

Animals↗

PubMed2005

The protein phosphatases of Synechocystis sp. strain PCC 6803: open reading frames sll1033 and sll1387 encode enzymes that exhibit both protein-serine and protein-tyrosine phosphatase activity in vitro.

The open reading frames (ORFs) encoding two potential protein-serine/threonine phosphatases from the cyanobacterium Synechocystis sp. strain PCC 6803 were cloned and their protein products expressed in Escherichia coli cells. The product of ORF sll1033, SynPPM3, is a homologue of the PPM family of protein-serine/threonine phosphatases found in all eukaryotes as well as many members of the Bacteria. Surprisingly, the recombinant protein phosphatase dephosphorylated phosphotyrosine- as well as phosphoserine-containing proteins in vitro. While kinetic analyses indicate that the enzyme was more efficient at dephosphorylating the latter, replacement of Asp608 by asparagine enhanced activity toward a phosphotyrosine-containing protein fourfold. The product of ORF sll1387, SynPPP1, is the sole homolog of the PPP family of protein phosphatases encoded by the genome of Synechocystis sp. strain PCC 6803. Like many other bacterial PPPs, the enzyme dephosphorylated phosphoserine- and phosphotyrosine-containing proteins with comparable efficiencies. However, while previously described PPPs from prokaryotic organisms required the addition of exogenous metal ion cofactors, such as Mg2+ or Mn2+, for activity, recombinantly produced SynPPP1 displayed near-maximal activity in the absence of added metals. Inductively coupled plasma mass spectrometry indicated that recombinant SynPPP1 contained significant quantities, 0.32 to 0.44 mol/mole total, of Mg and Mn. In this respect, the cyanobacterial enzyme resembled eukaryotic members of the PPP family, which are metalloproteins. mRNA encoding SynPPP1 or SynPPM3 could be detected in cells grown under many, but not all, environmental conditions.

Amino Acid Sequence↗

PubMed1999

Transcriptional analysis of human herpesvirus-8 open reading frames 71, 72, 73, K14, and 74 in a primary effusion lymphoma cell line.

We examined the transcription and splicing of open reading frames (ORFs) 71 (K13)-74 of human herpesvirus-8 (HHV-8) in the primary effusion lymphoma cell line BCP-1 (latently infected with HHV-8), using a combination of NORTHERN blot analysis, RT-PCR, and rapid amplification of cDNA ends (PCR-RACE). The three genes encoded by ORFs 71, 72, and 73 [viral FLICE inhibitory protein (v-FLIP), v-cyclin, latent nuclear antigen (LNA)] are transcribed from a common transcription start site in BCP-1 cells uninduced (latent) or induced (lytic) with n-butyrate. The resulting transcript is spliced to yield a 5.32-kb message encoding LNA, v-cyclin, and v-FLIP and a 1.7-kb bicistronic message encoding v-cyclin and v-FLIP. The two genes encoded by ORFs K14 and 74 (v-Ox2 and v-GPCR) are transcribed as a 2.7-kb bicistronic transcript that is induced with n-butyrate. A small (149-bp) intron is spliced from the intragenic noncoding region immediately before the v-GPCR initiating codon. Examination of sequence elements in the promoter of the LNA/v-cyclin/v-FLIP operon revealed TAATGARAT and Octamer binding motifs characteristic of herpesvirus immediate-early genes. Sequence elements in the v-Ox2/v-GPCR promoter included AP1 and Zta-like (EBV Zebra transactivator) binding motifs consistent with the n-butyrate induction of this operon.

Base Sequence↗

PubMed1999

Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and Is essential for virus replication.

The open reading frame (ORF) 1b-encoded part of the equine arteritis virus (EAV) replicase is expressed by ribosomal frameshifting during genome translation, which results in the production of an ORF1ab fusion protein (345 kDa). Four ORF1b-encoded processing products, nsp9 (p80), nsp10 (p50), nsp11 (p26), and nsp12 (p12), have previously been identified in EAV-infected cells (L. C. van Dinten, A. L. M. Wassenaar, A. E. Gorbalenya, W. J. M. Spaan, and E. J. Snijder, J. Virol. 70:6625-6633, 1996). In the present study, the generation of these four nonstructural proteins was shown to be mediated by the nsp4 serine protease, which is the main viral protease (E. J. Snijder, A. L. M. Wassenaar, L. C. van Dinten, W. J. M. Spaan, and A. E. Gorbalenya, J. Biol. Chem. 271:4864-4871, 1996). Mutagenesis of candidate cleavage sites revealed that Glu-2370/Ser, Gln-2837/Ser, and Glu-3056/Gly are the probable nsp9/10, nsp10/11, and nsp11/12 junctions, respectively. Mutations which abolished ORF1b protein processing were introduced into a recently developed infectious cDNA clone (L. C. van Dinten, J. A. den Boon, A. L. M. Wassenaar, W. J. M. Spaan, and E. J. Snijder, Proc. Natl. Acad. Sci. USA 94:991-997, 1997). An analysis of these mutants showed that the selective blockage of ORF1b processing affected different stages of EAV reproduction. In particular, the mutant with the nsp10/11 cleavage site mutation Gln-2837-->Pro displayed an unusual phenotype, since it was still capable of RNA synthesis but was incapable of producing infectious virus.

Amino Acid Sequence↗

PubMed1989

Identification and characterization of an L1 family sequence with a very long open reading frame in the third intron of the human thymidylate synthase gene.

A long L1 repetitive sequence (3.6 kilobase pairs) was found in the third intron of the human thymidylate synthase gene. This L1 family sequence is unique in that it possesses the longest open reading frame (1.7 kilobase pairs) of all L1 family members identified in sequences associated with specific genes that have been cloned thus far. Furthermore, the amino acid sequence deduced from the open reading frame of the L1 sequence was found to be highly homologous (90%) to that encoded by a known human teratocarcinoma L1 RNA species, and to contain several blocks of sequences homologous to ones in RNA-dependent DNA polymerases of various origins.

Amino Acid Sequence↗

PubMed1996

A novel ribozyme target site located in the HIV-1 nef open reading frame.

We have tested the sequence UUC CAG UCA GAC CU, at position 9016--9029 within the HIV-1(SF2) nef open reading frame, for accessibility to antisense and hammerhead ribozyme attack. The accessibility was first studied using a phosphorothioate-modified 14-nt DNA oligo (complementary to the nef9016--9029 site). A dose-dependent repression of HIV-1(SF2) growth was observed in human peripheral blood mononuclear cells after exogenous administration of the oligo to the cell culture medium. A hammerhead ribozyme with a 6+7-nt antisense specificity for the nef9016--9029 site (hhRz.nef9016--9029) was constructed and transfected into the human T-cell line HUT78. Again, a dose-dependent repression of virus growth was observed when different individual clones expressing hhRz.nef9016--9029 were infected with HIV-1(SF2). A complete abrogation of virus production was observed after infection with a low (0.5 TCID50) HIV-1 titer. Increasing doses (2.5 and 12.5 TCID50) of HIV-1 virus yielded a low production (10(3)-fold reduced) of virus particles in most cases; but a complete, or close to complete, abrogation was observed even in individual cultures infected with the highest dose. Presence of proviral pol and gag sequences in hhRz.nef9016--9029-expressing HUT78 clones was assayed, using PCR. Interestingly, since no pol and gag PCR products could be detected, the results strongly indicated that the hammerhead ribozyme was already acting on the infecting HIV RNA before its reverse transcription and integration as proviral DNA. In summary, the results obtained in this study support the nef9016--9029 site as a strong new candidate for ribozymal gene therapy against HIV-1 infection.

Base Sequence↗

PubMed1985

Tobacco chloroplast tRNA(UUU) gene contains a 2.5-kilobase-pair intron: An open reading frame and a conserved boundary sequence in the intron.

The nucleotide sequence of a tRNA(Lys)(UUU) gene on tobacco (Nicotiana tabacum) chloroplast DNA has been determined. This gene is located 215 base pairs upstream from the gene for the 32,000-dalton thylakoid membrane protein on the same DNA strand and has a 2526-base-pair intron in the anticodon loop. The intron boundary sequence does not follow the G-U/A-G rule but is similar to those of tobacco chloroplast split genes for tRNA(Gly)(UCC) and ribosomal proteins L2 and S12. The intron contains one major open reading frame of 509 codons. The codon usage in the open reading frame resembles those observed in the genes for tobacco chloroplast proteins so far analyzed. The primary transcript of this tRNA gene is 2.7 kilobases long.

Journal Article↗

PubMed1991

Identification, cloning, and sequencing of a fragment of Amsacta moorei entomopoxvirus DNA containing the spheroidin gene and three vaccinia virus-related open reading frames.

Entomopoxvirus virions are frequently contained within crystalline occlusion bodies, which are composed of primarily a single protein, spheroidin, which is analogous to the polyhedrin protein of baculovirus. The spheroidin gene of Amsacta moorei entomopoxvirus was identified following the microsequencing of polypeptides generated from cyanogen bromide treatment of spheroidin and the subsequent synthesis of oligonucleotide hybridization probes. DNA sequencing of a 6.8-kb region of DNA containing the spheroidin gene showed that the spheroidin protein is derived from a 3.0-kb open reading frame potentially encoding a protein of 115 kDa. Three copies of the heptanucleotide, TTTTTNT, a sequence associated with early gene transcription in the vertebrate poxviruses, and four in-frame translational termination signals were found within 60 bp upstream of the putative spheroidin gene promoter (TAAATG). The spheroidin gene promoter region contains the sequence TAAATG, which is found in many late promoters of the vertebrate poxviruses and which serves as the site of transcriptional initiation, as shown by primer extension. Primer extension experiments also showed that spheroidin gene transcripts contain 5' poly(A) sequences typical of vertebrate poxvirus late transcripts. The 92 bases upstream of the initiating TAAATG are unusually A + T rich and contain only 7 G or C residues. An analysis of open reading frames around the spheroidin gene suggests that the colinear core of "essential genes" typical of the vertebrate poxviruses is absent in A. moorei entomopoxvirus.

Amino Acid Sequence↗

PubMed1993

The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames.

A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.

Amino Acid Sequence↗

PubMed2005

Purified recombinant hypothetical protein coded by open reading frame Rv1885c of Mycobacterium tuberculosis exhibits a monofunctional AroQ class of periplasmic chorismate mutase activity.

Naturally occurring variants of the enzyme chorismate mutase are known to exist that exhibit diversity in enzyme structure, regulatory properties, and association with other proteins. Chorismate mutase was not annotated in the initial genome sequence of Mycobacterium tuberculosis (Mtb) because of low sequence similarity between known chorismate mutases. Recombinant protein coded by open reading frame Rv1885c of Mtb exhibited chorismate mutase activity in vitro. Biochemical and biophysical characterization of the recombinant protein suggests its resemblance to the AroQ class of chorismate mutases, prototype examples of which include the Escherichia coli and yeast chorismate mutases. We also demonstrate that unlike the corresponding proteins of E. coli, Mtb chorismate mutase does not have any associated prephenate dehydratase or dehydrogenase activity, indicating its monofunctional nature. The Rv1885c-encoded chorismate mutase showed allosteric regulation by pathway-specific as well as cross-pathway-specific ligands, as evident from proteolytic cleavage protection and enzyme assays. The predicted N-terminal signal sequence of Mtb chorismate mutase was capable of functioning as one in E. coli, suggesting that Mtb chorismate mutase belongs to the AroQ class of chorismate mutases. It was evident that Rv1885c may not be the only enzyme with chorismate mutase enzyme function within Mtb, based on our observation of the presence of chorismate mutase activity displayed by another hypothetical protein coded by open reading frame Rv0948c, a novel instance of the existence of two monofunctional chorismate mutases ever reported in any pathogenic bacterium.

Allosteric Regulation↗