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PubMed1996

The UL2 open reading frame of bovine herpesvirus 1 encodes a uracil-DNA glycosylase.

Sequence analysis within the unique long segment of the bovine herpesvirus 1 (BHV-1) genome previously identified an open reading frame (ORF), designated UL2, whose deduced polypeptide of 204 amino acids contained a consensus uracil-DNA glycosylase (UDGase) signature sequence. To determine whether the BHV-1 UL2 ORF product has UDGase activity, we positioned the UL2 sequence down-stream of the T7 promoter on the vector pET-28b(+) and expressed it in Escherichia coli. Upon induction with isopropyl beta-D-thiogalactopyranoside these cells produced a 23-kDa protein, the molecular mass of which was in accordance with the prediction from the nucleotide sequence. A one-step purification procedure using nickel-chelating affinity chromatography resulted in a homogeneous preparation of this protein, which displayed specific UDGase activity in an in vitro enzyme assay. These results provide evidence that the BHV-1 UL2 gene does encode a UDGase.

Amino Acid Sequence↗

PubMed1997

Targeted inactivation of a tobacco intron-containing open reading frame reveals a novel chloroplast-encoded photosystem I-related gene.

The chloroplast genome of all higher plants encodes, in its large single-copy region, a conserved open reading frame of unknown function (ycf3), which is split by two group II introns and undergoes RNA editing in monocotyledonous plants. To elucidate the function of ycf3 we have deleted the reading frame from the tobacco plastid genome by biolistic transformation. We show here that homoplasmic Deltaycf3 plants display a photosynthetically incompetent phenotype. Molecular analyses indicate that this phenotype is not due to a defect in any of the general functions of the plastid genetic apparatus. Instead, the mutant plants specifically lack detectable amounts of all photosystem I (PSI) subunits analyzed. In contrast, at least under low light conditions, photosystem II subunits are still present and assemble into a physiologically active complex. Faithful transcription of photosystem I genes as well as correct mRNA processing and efficient transcript loading with ribosomes in the Deltaycf3 plants suggest a posttranslational cause of the PSI-defective phenotype. We therefore propose that ycf3 encodes an essential protein for the assembly and/or stability of functional PSI units. This study provides a first example for the suitability of reverse genetics approaches to complete our picture of the coding capacity of higher plant chloroplast genomes.

Chloroplasts↗

PubMed1991

Nucleotide and amino acid sequence variation in the L1 and E7 open reading frames of human papillomavirus type 6 and type 16.

Human papillomavirus (HPV) type 6 and type 16 DNA sequence variants were found by partially sequencing the L1 and E7 open reading frames, using templates generated with the polymerase chain reaction. Identical variants were found in patients from widely separated locations, such as the United States, the Philippines, and India. The same sequence variants of HPV 16 were found in women with invasive cervical carcinoma and in women with no evidence of disease. Variation in the predicted amino acid sequences of the HPV 16 L1 and E7 proteins was found. A single nucleotide change at position 6433 was found in about 50% of the HPV 16 DNAs, resulting in a change in predicted amino acid sequence from threonine to alanine at the equivalent position in the L1 protein. Predicted amino acid changes were found in the HPV 16 E7 proteins at amino acid positions 28, 29, and 47. Variation at these positions could affect known properties of the E7 protein, including binding to the retinoblastoma protein.

Amino Acid Sequence↗

PubMed1994

Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.

Complex oncoviruses contain, in addition to the classical retroviral genes (gag, pol, and env), a region (X) located between the envelope sequences and the 3' long terminal repeat. The X region contains two genes, tax and rex, whose protein products are involved in transcriptional and posttranscriptional regulation of viral expression. In addition to these activators, the bovine leukemia virus (BLV) and the human T-cell leukemia virus (HTLV) contain alternative open reading frames (R3 and G4 for BLV; p30, p13, and p12 for HTLV). As a virus/animal model for HTLV-induced leukemogenesis, BLV provirus can be injected intradermally into sheep, where it induced B-lymphocyte transformation. Deletion of the R3 and G4 sequences from an infectious and tumorigenic BLV provirus greatly impaired the in vivo propagation of the viruses as demonstrated by DNA polymerase chain reaction, RNA blots, structural-protein ELISA, and immunofluorescence analysis. Our results show that the alternative open reading frames are required for maintaining high virus loads during the course of persistent infection in vivo. Thus, R3 and G4 are candidates for antiviral drug development. Furthermore, viruses with a deletion in these sequences should be tested as live attenuated vaccines.

Animals↗

PubMed2002

Effective structure of a leader open reading frame for enhancing the expression of GC-rich genes.

To overexpress broad kinds of GC-rich genes in Escherichia coli, we examined how the structures of leader open reading frames (leader ORFs) affect the expression of GC-rich genes, such as polA, trpA, and trpB, from Thermus thermophilus. When a leader ORF overlapped with the polA-initiation codon by 1 bp in the TGATG motif, gene expression increased by more than 3-fold compared to when a leader ORF was several-bp distant from the initiation codon. A 4-bp overlap with the ATGA motif was more effective than a 1-bp overlap with the TGATG motif. When a 4-bp overlapping leader ORF was placed in front of the successive trpB and trpA genes, the trpA gene was poorly expressed whereas the trpB gene was overexpressed. Mutation analysis revealed that the expression of the trpA gene was strongly enhanced by replacing G and C in the translation termination region of the leader ORF with A and T. In contrast, other mutations, such as alterations between synonymous codons in the trpA-coding region, produced diminished gene expression. Using the most effective leader ORF obtained from these results, new expression vectors were constructed.

Amino Acids↗

PubMed1995

Inactivation of a Synechocystis sp strain PCC 6803 gene with homology to conserved chloroplast open reading frame 184 increases the photosystem II-to-photosystem I ratio.

A gene of the unicellular cyanobacterium Synechocystis sp strain PCC 6803 that is homologous to the conserved chloroplast open reading frame orf184 has been cloned and sequenced. The nucleotide sequence of the gene predicts a protein of 184 amino acids with a calculated molecular mass of 21.5 kD and two membrane-spanning regions. Amino acid sequence analysis showed 46 to 37% homology of the cyanobacterial orf184 with tobacco orf184, rice orf185, liverwort orf184, and Euglena gracilis orf206 sequences. Two orf184-specific mutants of Synechocystis sp PCC 6803 were constructed by insertion mutagenesis. Cells of mutants showed growth characteristics similar to those of the wild type. Their pigment composition was distinctly different from the wild type, as indicated by an increase in the phycocyanin-to-chlorophyll ratio. In addition, mutants also had a two- to threefold increase in photosynthetic electron transfer rates as well as in photosystem II-to-photosystem I ratio-a phenomenon hitherto not reported for mutants with altered photosynthetic characteristics. The observed alterations in the orf184-specific mutants provide strong evidence for a functional role of the orf184 gene product in photosynthetic processes.

Amino Acid Sequence↗

PubMed1992

The complete sequence of K3B, a 7.9 kb fragment between PGK1 and CRY1 on chromosome III, reveals the presence of seven open reading frames.

We have determined the nucleotide sequence of a segment from chromosome III of Saccharomyces cerevisiae extending over 7.9 kb between the PGK1 and CRY1 loci. The fragment contains seven open reading frames, YCR241, YCR242, YCR243, YCR244, YCR245, YCR246 and YCR247, of more than 70 codons. The study of the effects of a global disruption of YCR242, YCR243, YCR244, YCR245 and YCR247 shows that they are not essential for growth and division.

Amino Acid Sequence↗

PubMed2001

The regulation of ornithine decarboxylase gene expression by sucrose and small upstream open reading frame in tomato (Lycopersicon esculentum Mill).

We identified a near-full-length cDNA clone encoding ornithine decarboxylase (ODC) from tomato (Lycopersicon esculentum Mill). It contained a small upstream open reading frame (uORF) within its 5' untranslated region. An in vitro translation assay demonstrated that the uORF repressed expression of downstream ORF. Neither nucleotide nor predicted peptide sequence of the uORF was responsible for the repression. The presence of upstream AUG codon was shown to be responsible. ODC expression appeared to be organ specific. The ODC gene was expressed in roots, hypocotyls and sink leaves but not in source leaves. ODC transcripts were observed in apical meristem of primary roots, and were distributed in cells of cortex layer preferentially. ODC expression responded immediately to sucrose availability via the sucrose-specific pathway independent of hexokinase. Sucrose induction of ODC gene was seen in roots, hypocotyls and flowers but not in mature leaves. Moreover, only the root apical meristem responded to sucrose availability. These observations indicate that the spatial pattern of ODC expression is closely associated with cell proliferation and that sucrose sensing plays a major role in the spatial pattern of ODC expression. Also, the differential regulation of ODC and arginine decarboxylase gene expression by factors modulating plant growth suggests that they would have different physiological roles in plant development.

5' Untranslated Regions↗

PubMed1996

Vaccinia virus A17L open reading frame encodes an essential component of nascent viral membranes that is required to initiate morphogenesis.

We generated an antiserum to the predicted C-terminal peptide of the A17L open reading frame (ORF), which encodes a 23-kDa polypeptide with hydrophobic regions characteristic of membrane proteins. Immuno-electron microscopy of infected cells indicated that the A17L protein is intimately associated with the earliest characteristic viral membranes, even those formed in the presence of the drug rifampin. To study the role of the A17L protein in morphogenesis, we constructed recombinant vaccinia viruses in which the endogenous A17L ORF was deleted and a copy of the ORF under the control of the bacteriophage T7 RNA polymerase and the Escherichia coli lac repressor was inserted into an alternative site in the vaccinia virus genome. Growth of these recombinant viruses was entirely dependent on the induction of A17L expression by isopropyl-beta-D-thiogalactopyranoside. Electron microscopic examination of cells infected in the absence of inducer revealed the accumulation of large, well-demarcated electron-dense aggregates but no characteristic membrane-associated viral structures. Viral late protein synthesis occurred under these conditions, although the maturational proteolytic processing of structural proteins was inhibited. We conclude that the product of the A17L gene is an essential component of the immature viral membrane and has an early function in viral morphogenesis.

Animals↗

PubMed2003

The largest open reading frame (pks12) in the Mycobacterium tuberculosis genome is involved in pathogenesis and dimycocerosyl phthiocerol synthesis.

The cell wall lipids in Mycobacterium tuberculosis are probably involved in pathogenesis. The largest open reading frame in the genome of M. tuberculosis H37Rv, pks12, is unique in that it encodes two sets of domains needed to produce fatty acids. A pks12-disrupted mutant was produced, and disruption was confirmed by both PCR analysis and Southern blotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis showed that a 430-kDa protein band present in the wild type was missing in the mutant. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MS) and liquid chromatography (LC)-MS analysis of tryptic peptides showed that 54 peptides distributed throughout this protein matched the pks12-encoded sequence. Biochemical analysis using [1-(14)C]propionate as the radiotracer showed that the pks12 mutant was deficient in the synthesis of dimycocerosyl phthiocerol (DIM). SDS-PAGE, immunoblot analysis of proteins, and analysis of fatty acids showed that the mutant can produce mycocerosic acids. Thus, the pks12 gene is probably involved in the synthesis of phthiocerol, the diol required for DIM synthesis. Growth of the pks12 mutant was attenuated in mouse alveolar macrophage cell line MH-S, and the virulence of the mutant in vivo was highly attenuated in a murine model. Thus, pks12 probably participates in DIM production and its expression is involved in pathogenesis.

Animals↗

PubMed1995

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) are encoded by the carboxyterminal portion of viral open reading frame 3.

Antigenic differences between European and American isolates of porcine reproductive and respiratory syndrome virus (PRRSV) were revealed by serologic analysis of a recombinant protein derived from PRRSV open reading frame 3 (ORF 3). The hydrophilic carboxyterminal 199 amino acids encoded by the ORF 3 of a European (Lelystad) isolate of PRRSV were expressed as a recombinant fusion protein (BP03-P) in a baculovirus gene expression system. Sera from gnotobiotic swine exposed to prototypic reference European and American isolates of PRRSV and sera from conventionally reared European and American swine convalescing from naturally acquired PRRSV infections were used to characterize the BP03-P protein. Sera from gnotobiotic and conventionally reared swine exposed to European isolates of PRRSV were significantly more reactive (P < 0.01) with BP03-P than were the corresponding American PRRSV antisera using the indirect immunoperoxidase monolayer assay (IPMA). Prototypic European, but not American, PRRSV antisera also recognized BP03-P using western immunoblotting and radioimmunoprecipitation assay (RIPA) procedures. However, gnotobiotically derived antiserum to an atypical American-origin PRRSV was reactive with BP03-P by both IPMA and western immunoblot. Despite a predicted potential for N-linked glycosylation, studies with tunicamycin and peptide-N-glycosidase F (PNGase F) indicated that BP03-P was not N-glycosylated in either insect cell cultures or Trichoplusia ni larvae infected with the recombinant baculovirus. Sera from rabbits inoculated with BP03-P failed to neutralize both the European (Lelystad) and American (ATCC VR-2332) reference isolates of PRRSV and did not react by IPMA with PRRSV-infected cell cultures. Taken together, the data suggest that the carboxyterminal portion of PRRSV ORF 3 encodes a non-neutralizing viral peptide that is partially responsible for the serologic differences noted between European and most American isolates of PRRSV.

Animals↗

PubMed2005

The open reading frame VI product of Cauliflower mosaic virus is a nucleocytoplasmic protein: its N terminus mediates its nuclear export and formation of electron-dense viroplasms.

The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replication and assembly occur. In this study, the mechanism involved in viroplasm formation was investigated by in vitro and in vivo experiments. Far protein gel blot assays using a collection of P6 deletion mutants demonstrated that the N-terminal alpha-helix of P6 mediates interaction between P6 molecules. Transient expression in tobacco (Nicotiana tabacum) BY-2 cells of full-length P6 and P6 mutants fused to enhanced green fluorescent protein revealed that viroplasms are formed at the periphery of the nucleus and that the N-terminal domain of P6 is an important determinant in this process. Finally, this study led to the unexpected finding that P6 is a nucleocytoplasmic shuttle protein and that its nuclear export is mediated by a Leu-rich sequence that is part of the alpha-helix domain implicated in viroplasm formation. The discovery that P6 can localize to the nucleus opens new prospects for understanding yet unknown roles of this viral protein in the course of the CaMV infection cycle.

Active Transport, Cell Nucleus↗

PubMed1996

The sequence of 23 kb surrounding the SNF3 locus on the left arm of yeast chromosome IV reveals the location of five known genes and characterizes at least six new open reading frames including putative genes for ribosomal protein L35 and a sugar transport protein.

The nucleotide sequence of 22,846 bp of the left arm of chromosome IV is described. Twelve open reading frames (ORFs) greater than 100 triplets were detected, one of which extends into an adjacent cosmid. Two of the ORFs may contain an intron. One of these is an L35 ribosomal protein gene. Five ORFs (D1204, D1214, D1219, D1234 and D1244) encode previously sequenced genes (MGT1, SHM1, ASF2, SNF3 and ARF2, respectively). The nucleotide sequence of a sixth ORF (D1229) is quite similar to the WEB1 gene, which appeared in the DNA databases shortly after finishing the sequence reported here. It is not clear whether or not WEB1 and D1229 represent one and the same gene. The co-linearity of the reported DNA sequences with the genome of strains from Saccharomyces cerevisiae subspecies carlsbergensis, sake and diastaticus was assessed by comparative PCR with overlapping primer sets.

Chromosome Mapping↗

PubMed1994

The chloroplast ycf8 open reading frame encodes a photosystem II polypeptide which maintains photosynthetic activity under adverse growth conditions.

We have engineered and analyzed a chloroplast mutant of Chlamydomonas reinhardtii that lacks ycf8, the chloroplast open reading frame 8, which is highly conserved in location and predicted amino acid sequence in land plants and C.reinhardtii. The ycf8 sequence was replaced with the aadA cassette which confers resistance to spectinomycin when expressed in the chloroplast. Although the mutant is able to grow phototrophically, photosystem II function and cell growth are impaired under stress conditions such as high light intensity and diminished chloroplast protein synthesis induced by spectinomycin. Use of an antibody generated against the ycf8 product has revealed that this hydrophobic polypeptide is associated with photosystem II, based on its severely reduced levels in various photosystem II-deficient mutants and on its copurification with photosystem II. This protein, therefore, appears to be (i) a novel photosystem II subunit and (ii) required for maintaining optimal photosystem II activity under adverse growth conditions.

Animals↗

PubMed2001

Comparison of DNA sequence and transactivation activity of open reading frame 62 of Oka varicella vaccine and its parental viruses.

When nucleotide sequences of Oka vaccine and its parental viruses of varicella-zoster virus (VZV) were compared in 5 open reading frames (ORFs) including glycoprotein C (gC) and 4 immediate-early genes, mutations were detected only in gene 62 which is one of the immediate-early genes. Compared with its parental virus, the vaccine virus contained 15 nucleotide substitutions. With the differentiation method using the simplified restriction-enzyme fragment length polymorphism analysis by Nae I and Bss HII, which was established based on the sequence analysis data in this study, the Oka vaccine virus could be distinguished from its parental virus. Studies of the regulatory activities of the ORF62 gene product (IE62) in a transient assay indicate the IE62 of the parental virus had a stronger transactivational activity than that of the vaccine virus against immediate-early, early and late gene promoters. These data suggest that gene 62 might have an important role for attenuation of VZV. This is the first report in which many substitutions of nucleotides in gene 62 of Oka vaccine virus was found, compared with that of Oka parental virus.

Cell Line↗

PubMed1996

Open reading frame P--a herpes simplex virus gene repressed during productive infection encodes a protein that binds a splicing factor and reduces synthesis of viral proteins made from spliced mRNA.

The open reading frame P (ORF P) is located in the domain and on the DNA strand of the herpes simplex virus 1 transcribed during latent infection. ORF P is not expressed in productively infected cells as a consequence of repression by the binding of the major viral regulatory protein to its high-affinity binding site. In cells infected with a mutant virus carrying a derepressed gene, ORF P protein is extensively posttranslationally processed. We report that ORF P interacts with a component of the splicing factor SF2/ASF, pulls down a component of the SM antigens, and colocalizes with splicing factors in nuclei of infected cells. The hypothesis that ORF P protein may act to regulate viral gene expression, particularly in situations such as latently infected sensory neurons in which the major regulatory protein is not expressed, is supported by the evidence that in cells infected with a mutant in which the ORF P gene was derepressed, the products of the regulatory genes alpha 0 and alpha 22 are reduced in amounts early in infection but recover late in infection. The proteins encoded by these genes are made from spliced mRNAs, and the extent of recovery of these proteins late in infection correlates with the extent of accumulation of post-translationally processed forms of ORF P protein.

Carcinoma, Hepatocellular↗

PubMed1996

The sequence of a 17,933 bp segment of Saccharomyces cerevisiae chromosome XIV contains the RHO2, TOP2, MKT1 and END3 genes and five new open reading frames.

We report the DNA sequence of a 17,933 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Analysis of the sequence reveals the presence of ten open reading frames (ORFs) larger than 100 codons. Four of these were previously identified as genes RHO2, TOP2, MKT1 and END3. Additionally, the NH2 end coding region of PMS1 is found in the 3' end of the sequence. No significant homology to any known protein has been found for the other five ORFs.

Amino Acid Sequence↗

PubMed1990

Control and expression of 3' open reading frames in clover yellow mosaic virus.

The genomic RNA of clover yellow mosaic virus (CYMV) contains at least seven open reading frames (ORFs) which are organized in a more elaborate array than in other sequenced members of the potexvirus group. We have investigated the strategy by which ORFs located in the 3' region of CYMV's genomic RNA are differentially expressed by correlating the location of the 5' termini of the two abundant viral subgenomic RNAs with their coding potential. We have mapped the 5' termini of the subgenomic RNAs precisely to the nucleotide level and have shown that both are capped. The larger 2.1-kb subgenomic RNA encodes as its 5' ORF a 25-kDa polypeptide, whose function is unknown. The smaller 1.0-kb subgenomic RNA can encode only the 23-kDa coat protein. All four ORFs in the 3' 1095 residues of CYMV are efficiently expressed in vitro, but of these only coat protein, which can be expressed from a subgenomic RNA, is detectable in CYMV infected tissue. For this reason, we believe that expression of ORFs in the 3' one-third of CYMV RNA are controlled at the transcriptional level.

Base Sequence↗