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PubMed1999

Disruption of six open reading frames on chromosome X of Saccharomyces cerevisiae reveals a cluster of four essential genes.

In this study we report the construction and basic phenotypic analysis of six Saccharomyces cerevisiae deletion mutants. The open reading frames (ORFs) YJL008C (gene symbol CCT8), YJL010C, YJL011C, YJL012C, YJL017W, and YJL020C from chromosome X have been disrupted by integration of deletion cassettes, comprising the bacterial KanMX4 marker gene and terminal long (LFH) or short (SFH) flanking sequences that are homologous to the 5' and 3' untranslated regions of the respective ORFs. For correct disruption of ORF YJL008C, it was necessary to construct a deletion cassette flanked by 300-350 bp long target guide sequences by LFH-PCR. Transformations using ORF YJL008C gene disruption cassettes synthesized by standard SFH-PCR exclusively resulted in false-positive or multiple integration events, probably because seven additional genes homologous to CCT8 exist in the yeast genome. The other five ORFs have been disrupted using cassettes generated by SFH-PCR, comprising terminal homologous regions of approximately 50 bp to each target site. Correct genomic integration of the reporter modules was verified by analytical PCR and Southern hybridization. Deletion of YJL008C, YJL010C, YJL011C, and YJL012C was found to be lethal, as shown by sporulation and tetrad analysis. This result is in contrast to the finding that only 16-20% of the genes in S. cerevisiae are estimated to be essential. The four essential genes described in this work are clustered, while the two other non-essential ORFs are separated by further ORFs. Although the two viable deletion mutants were tested against 60 different inhibitors, heavy metal ions and salts, no phenotype could be detected that co-segregated with the deletion during meiosis.

Amino Acid Sequence↗

PubMed1993

The herpes simplex virus type 1 reactivation function lies outside the latency-associated transcript open reading frame ORF-2.

The latency-associated transcription unit has been shown to be important for in vivo reactivation of herpes simplex virus from the latent state. A recombinant virus was constructed to alter the largest open reading frame in this region. This virus had a wild-type reactivation phenotype, suggesting that herpes simplex virus does not require a protein function from this reading frame for efficient reactivation from latency.

DNA, Recombinant↗

PubMed1994

A 100-kilodalton polypeptide encoded by open reading frame (ORF) 1b of the coronavirus infectious bronchitis virus is processed by ORF 1a products.

The genome-length mRNA (mRNA 1) of the coronavirus infectious bronchitis virus (IBV) contains two large open reading frames (ORFs), 1a and 1b, with the potential to encode polypeptides of 441 and 300 kDa, respectively. The downstream ORF, ORF 1b, is expressed by a ribosomal frameshifting mechanism. In an effort to detect viral polypeptides encoded by ORF 1b in virus-infected cells, immunoprecipitations were carried out with a panel of region-specific antisera. A polypeptide of approximately 100 kDa was precipitated from IBV-infected, but not mock-infected, Vero cells by one of these antisera (V58). Antiserum V58 was raised against a bacterially expressed fusion protein containing polypeptide sequences encoded by ORF 1b nucleotides 14492 to 15520; it recognizes specifically the corresponding in vitro-synthesized target protein. A polypeptide comigrating with the 100,000-molecular-weight protein (100K protein) identified in infected cells was also detected when the IBV sequence from nucleotides 8693 to 16980 was expressed in Vero cells by using a vaccinia virus-T7 expression system. Deletion analysis revealed that the sequence encoding the C terminus of the 100K polypeptide lies close to nucleotide 15120; it may therefore be generated by proteolysis at a potential QS cleavage site encoded by nucleotides 15129 to 15135. In contrast, expression of IBV sequences from nucleotides 10752 to 16980 generated two polypeptides of approximately 62 and 235 kDa, which represent the ORF 1a stop product and the 1a-1b fused product generated by a frameshifting mechanism, respectively, but no processed products were observed. Since the putative picornavirus 3C-like proteinase domain is located in ORF 1a between nucleotides 8937 and 9357, this observation suggests that deletion of the picornavirus 3C-like proteinase domain and surrounding regions abolishes processing of the 1b polyprotein. In addition, the in vitro translation and in vivo transfection studies also indicate that the ORF 1a region between nucleotides 8763 and 10720 contains elements that down-regulate the expression of ORF 1b.

Base Sequence↗

PubMed1993

Sequencing and analysis of 51.6 kilobases on the left arm of chromosome XI from Saccharomyces cerevisiae reveals 23 open reading frames including the FAS1 gene.

We have sequenced two segments containing a total of 51.6 kb of the left arm from chromosome XI of Saccharomyces cerevisiae. The first segment of 38.5 kb contains 18 open reading frames (ORFs) of more than 100 amino acid residues. Five ORFs encode known yeast genes, including the fatty acid synthase gene (FAS1). Three new yeast genes were discovered with homologies to non-yeast genes and ten new genes without homologies to any known sequences. The second segment of 13 kb contains five ORFs with two known yeast genes and three unknown genes. The sequences from cosmid pUKG041 were obtained entirely with the walking primer strategy resulting in a very low overall sequence redundancy of 2.8 and an average reading length of 443 bases.

Base Sequence↗

PubMed1993

Sequencing and functional analysis of a 32,560 bp segment on the left arm of yeast chromosome II. Identification of 26 open reading frames, including the KIP1 and SEC17 genes.

We report here the DNA sequence of a segment (alpha 1006.13: YBLO5) of chromosome II of Saccharomyces cerevisiae, extending over 32.5 kb. The segment contains 26 open reading frames (ORFs) from YBLO501 to YBLO526. YBL0505 corresponds to the SEC17 gene and YBL0521 to the KIP1 gene. YBL0516 contains an intron, YBL0513 shows homology with the RAT protein phosphatase and YBL0526 contains a zinc-finger motif. Disruption of 14 genes by insertion of a URA3 cassette has been performed and these mutants were analysed for their mating and sporulation ability, and for their growth on different carbon sources. YBL0515 and YBL0526 ORFs seem to be involved in the sporulation process.

Amino Acid Sequence↗

PubMed1990

The herpes simplex virus Us11 open reading frame encodes a sequence-specific RNA-binding protein.

Herpes simplex virus 1- and 2 (HSV-1 and HSV-2)-infected cell extracts but not uninfected cell extracts contain an RNA-binding activity for an in vitro-transcribed sequence from the domains of the HSV-1 US11 and alpha 47 genes. The transcript of this sequence has not been detected in infected cells. The binding is sequence and secondary structure specific and protects approximately 95 nucleotides from RNase digestion. Analyses of HSV-1 x HSV-2 recombinants and HSV-1 deletion mutants mapped the function necessary for activity to the US11 or alpha 47 open reading frame. The alpha 47 gene was excluded, since the RNA-binding activity is a late (gamma 2) function dependent on viral DNA synthesis for its expression. The US11 function is the only viral function required, since translation in rabbit reticulocyte lysate of an in vitro-synthesized US11 mRNA resulted in the appearance of the RNA-binding activity. The product of the US11 open reading frame is associated with the RNA probe-protein complex inasmuch as insertion of a sequence encoding in frame 15 additional amino acids at the C terminus of the US11 protein caused a corresponding decrease in the electrophoretic mobility of the binding complex.

Base Sequence↗

PubMed1996

Analysis of a 62 kb DNA sequence of chromosome X reveals 36 open reading frames and a gene cluster with a counterpart on chromosome XI.

We have sequenced a 61.989 bp stretch located between genes RAD7 and FIP1 of Saccharomyces cerevisiae chromosome X. This stretch contains 36 open reading frames (ORFs) of at least 100 codons. Fourteen of these correspond to sequences previously published as HIT1, CDC8, YAP17, CBF1, NAT1, RPA12, CCT5, TOR1, RFC2, PEM2, CDC11, MIR1, STE18 and GRR1. The proteins deduced from four ORFs (YJR059w, YJR065c, YJR075w, YJR078w) have significant similarity to proteins of known function from yeast or other organisms, including S. cerevisiae serine/threonine-specific protein kinase. Schizosaccharomyces pombe Act2 protein, S. cerevisiae mannosyltransferase OCH1 protein and mouse indoleamine 2,3-dioxygenase, respectively. Four of the remaining 18 ORFs have similarity to proteins with unknown function, six are weakly similar to other known sequences, while another eight exhibit no similarity to any known sequence. In addition, three tRNA genes have been recognized. Three genes clustered within 22 kb (YJR059w, YJR061w and TOR1) have counterparts arranged within 15 kb on the left arm of chromosome XI.

Amino Acid Sequence↗

PubMed1993

Varicella-zoster virus (VZV) open reading frame 61 protein transactivates VZV gene promoters and enhances the infectivity of VZV DNA.

The varicella-zoster virus (VZV) open reading frame 61 (ORF61) protein is the homolog of herpes simplex virus type 1 (HSV-1) ICP0. Both genes are located in similar parts of the genome, their predicted products share a cysteine-rich motif, and cell lines expressing VZV ORF61 are able to complement an HSV-1 ICP0 deletion mutant (H. Moriuchi, M. Moriuchi, H. A. Smith, S. E. Straus, and J. I. Cohen, J. Virol. 66:7303-7308, 1992). In transient expression assays, HSV-1 ICP0 is a transactivator alone and transactivates in synergy with another viral transactivator, ICP4. However, VZV ORF61 represses the activation by VZV-encoded proteins ORF62 (the homolog of ICP4) and ORF4. To further characterize the function of VZV ORF61 and its role(s) in regulation of viral gene expression, we performed transient expression assays using target promoters from VZV, HSV-1, and unrelated viruses. In the absence of other viral activators, VZV ORF61 transactivated most promoters tested. In addition, a cell line stably expressing VZV ORF61 complemented the HSV-1 mutant in 1814, which lacks the transactivating function of VP16. The cell line expressing VZV ORF61 enhanced the infectivity of HSV-1 virion DNA. Moreover, transient expression of VZV ORF61 also enhanced the infectivity of VZV DNA. These results indicate that VZV ORF61 can stimulate expression of HSV-1 and VZV genes at an early stage in the viral replicative cycle and that ORF61 has an important role in VZV gene regulation.

Animals↗

PubMed1997

Rice tungro bacilliform virus open reading frames II and III are translated from polycistronic pregenomic RNA by leaky scanning.

Posttranscriptional components of the gene expression mechanism of rice tungro bacilliform virus (RTBV) were studied in transiently transfected protoplasts. RTBV translates several open reading frames from a polycistronic mRNA by leaky scanning. This mechanism is supported by the particular sequence features of the corresponding genome region and does not require a virus-encoded transactivator.

Badnavirus↗

PubMed1998

A 9359 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII includes the FOL2 and YTA7 genes and three unknown open reading frames.

In the framework of the EU programme for systematic sequencing of the Saccharomyces cervisiae genome we determined the sequence of a 9359 bp fragment of the right arm of chromosome VII. Five open reading frames (ORFs) of at least 300 nucleotides were found in this region. YGR267c encodes a protein with significant similarity to the enzyme GTP-cyclohydrolase I, that controls the first step in the biosynthetic pathway leading to various pterins and shows a high degree of sequence conservation from bacteria to mammals. We have recently demonstrated (Nardese et al., 1996) that YGR267c corresponds to the FOL2 gene, previously localized in the same chromosomal region by genetic mapping. The protein deduced from YGR270w belongs to the superfamily of putative ATPases associated with diverse cellular activities. It corresponds to the YTA7 gene, a member of a set of yeast genes coding for putative ATPases with high similarity to constituents of the 26S protease. The three ORFs YGR266w, YGR268c and YGR269w encode putative products of unknown function, with neither significant similarity to proteins in databases nor recognizable domains. YGR268c and YGR269w are partially overlapping ORFs: YGR268c seems to correspond to a real gene. whereas YGR269w is probably a fortuitous ORF.

Adenosine Triphosphatases↗

PubMed2000

Organization and expression of a Thermus thermophilus arginine cluster: presence of unidentified open reading frames and absence of a Shine-Dalgarno sequence.

A group of genes regulated by arginine was found clustered in the order argF-ORF1-argC-argJ-ORF4 between other, as yet uncharacterized, open reading frames (ORFs). Transcription starts were identified immediately upstream from argF and ORF4. Arginine repressed transcription that was initiated at argF but induced transcription of ORF4. The functions of ORF1 and ORF4 are unknown, but analysis of the sequence of ORF4 suggests that it is a membrane protein, possibly involved in transport of arginine or a related metabolite. Mobility shift and DNase I footprinting have revealed specific binding of pure Escherichia coli ArgR to the promoter region of Thermus thermophilus argF. These results suggest that argF transcription is controlled by a repressor homologous to those characterized in enteric bacteria and bacilli. Thermus argF mRNA is devoid of Shine-Dalgarno (SD) sequences. However, downstream from the ATG start codon of argF and many other Thermus genes (with or without an SD box), sequences were found to be complementary to nucleotides 1392 to 1409 of Thermus 16S rRNA, suggesting that an mRNA-rRNA base pairing in this region is important for correct translation initiation.

Amino Acid Sequence↗

PubMed1999

Disruption of six unknown open reading frames from Saccharomyces cerevisiae reveals two genes involved in vacuolar morphogenesis and one gene required for sporulation.

In this report we describe the construction and basic phenotypic analysis of deletion mutants in six open reading frames (ORFs) of unknown function from the yeast Saccharomyces cerevisiae. Using the dominant kanMX marker and polymerase chain reaction (PCR) methods, deletion cassettes were constructed for five ORFs (YNL099c, YNL100w, YNL101w, YNL106c and YNL242w) located on chromosome XIV and one ORF (YOR109w) located on chromosome XV. The recovery of viable haploid deletant strains among the meiotic products of heterozygous deletants for each ORF demonstrated that none of the analysed ORFs was essential. With the exception of YNL242w, no alterations in growth characteristics or mating and sporulation efficiencies associated with deletion of the ORFs were observed. Homozygous diploid ynl242w delta cells obtained in three different genetic backgrounds were unable to sporulate, indicating that the product of this ORF is required for sporulation. Complementation of the sporulation defect by the cognate gene clone confirmed this observation. YNL106c and YOR109w are very similar and show strong sequence homology with a mammalian phosphatidylinositol-phosphate 5-phosphatase, synaptojanin, known to be involved in synaptic vesicle cycling. Strains bearing single and double deletions of YNL106c and YOR109w were seen to display abnormal vacuolar morphologies of varying degrees. Complementation tests indicated that YNL106c and YOR109w are redundant genes.

Gene Deletion↗

PubMed1992

Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein.

Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.

Amino Acid Sequence↗

PubMed2005

Chimeric recombinant human metapneumoviruses with the nucleoprotein or phosphoprotein open reading frame replaced by that of avian metapneumovirus exhibit improved growth in vitro and attenuation in vivo.

Chimeric versions of recombinant human metapneumovirus (HMPV) were generated by replacing the nucleoprotein (N) or phosphoprotein (P) open reading frame with its counterpart from the closely related avian metapneumovirus (AMPV) subgroup C. In Vero cells, AMPV replicated to an approximately 100-fold-higher titer than HMPV. Surprisingly, the N and P chimeric viruses replicated to a peak titer that was 11- and 25-fold higher, respectively, than that of parental HMPV. The basis for this effect is not known but was not due to obvious changes in the efficiency of gene expression. AMPV and the N and P chimeras were evaluated for replication, immunogenicity, and protective efficacy in hamsters. AMPV was attenuated compared to HMPV in this mammalian host on day 5 postinfection, but not on day 3, and only in the nasal turbinates. In contrast, the N and P chimeras were reduced approximately 100-fold in both the upper and lower respiratory tract on day 3 postinfection, although there was little difference by day 5. The N and P chimeras induced a high level of neutralizing serum antibodies and protective efficacy against HMPV; AMPV was only weakly immunogenic and protective against HMPV challenge, reflecting antigenic differences. In African green monkeys immunized intranasally and intratracheally, the mean peak titer of the P chimera was reduced 100- and 1,000-fold in the upper and lower respiratory tracts, whereas the N chimera was reduced only 10-fold in the lower respiratory tract. Both chimeras were comparable to wild-type HMPV in immunogenicity and protective efficacy. Thus, the P chimera is a promising live HMPV vaccine candidate that paradoxically combines improved growth in vitro with attenuation in vivo.

Animals↗

PubMed1997

Amino-terminal epitopes are exposed when full-length open reading frame 2 of hepatitis E virus is expressed in Escherichia coli, but carboxy-terminal epitopes are masked.

We constructed a panel of overlapping and non-overlapping fragments of cDNA derived from open reading frame 2 (ORF2) of hepatitis E virus (HEV) and fused to the gene encoding glutathione S-transferase (GST), from which proteins were expressed in Escherichia coli. IgG-specific immunoreactivity against each protein was measured by Western immunoblotting using sera from experimentally infected Rhesus macaques (Macaca mulatta) or from HEV-infected patients. Under these conditions, full-length ORF2 protein (GST-ORF2) was strongly reactive with acute-phase sera from either macaques or patients, but was poorly reactive with convalescent sera. Recombinant protein GST-ORF2.3, representing amino acids 1-110 of the 660 encoded by ORF2, demonstrated a pattern of reactivity largely indistinguishable from the full-length protein. Conversely, GST-ORF2.1, representing amino acids 394-660 of the ORF2 protein was strongly reactive with both acute- and convalescent-phase sera. Extension of GST-ORF2.1 towards the N-terminus led to a progressive loss of convalescent-phase reactivity, apparent with as few as 20 additional HEV-specific amino acids. Deletion of 40 or more amino acids from the N-terminus of ORF2.1 also led to reduced convalescent-phase reactivity, however a protein representing this "reactive" region, containing amino acids 394-473, was poorly reactive, suggesting that the convalescent-reactive epitopes are conformational. Expression of full-length ORF2 protein in E. coli therefore masks the convalescent-reactive epitopes within the C-terminal part of the protein, without affecting N-terminal, acute-reactive epitopes.

Animals↗

PubMed2004

The use of Open Reading frame ESTs (ORESTES) for analysis of the honey bee transcriptome.

BACKGROUND: The ongoing efforts to sequence the honey bee genome require additional initiatives to define its transcriptome. Towards this end, we employed the Open Reading frame ESTs (ORESTES) strategy to generate profiles for the life cycle of Apis mellifera workers. RESULTS: Of the 5,021 ORESTES, 35.2% matched with previously deposited Apis ESTs. The analysis of the remaining sequences defined a set of putative orthologs whose majority had their best-match hits with Anopheles and Drosophila genes. CAP3 assembly of the Apis ORESTES with the already existing 15,500 Apis ESTs generated 3,408 contigs. BLASTX comparison of these contigs with protein sets of organisms representing distinct phylogenetic clades revealed a total of 1,629 contigs that Apis mellifera shares with different taxa. Most (41%) represent genes that are in common to all taxa, another 21% are shared between metazoans (Bilateria), and 16% are shared only within the Insecta clade. A set of 23 putative genes presented a best match with human genes, many of which encode factors related to cell signaling/signal transduction. 1,779 contigs (52%) did not match any known sequence. Applying a correction factor deduced from a parallel analysis performed with Drosophila melanogaster ORESTES, we estimate that approximately half of these no-match ESTs contigs (22%) should represent Apis-specific genes. CONCLUSIONS: The versatile and cost-efficient ORESTES approach produced minilibraries for honey bee life cycle stages. Such information on central gene regions contributes to genome annotation and also lends itself to cross-transcriptome comparisons to reveal evolutionary trends in insect genomes.

Animals↗

PubMed1993

Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence.

The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated.

Alleles↗

PubMed1990

The cytochrome oxidase subunit III gene in sunflower mitochondria is cotranscribed with an open reading frame conserved in higher plants.

The gene encoding subunit III of cytochrome oxidase (COXIII) has been identified in the sunflower mitochondrial genome. The COXIII coding region is located 570 bp downstream of a 477 bp open reading frame (ORFB). Sequence comparisons and hybridization experiments show that ORFB sequences are conserved in other plant mitochondrial genomes. Nucleotide and amino acid sequence comparisons suggest that RNA editing is required in sunflower mitochondria to synthesize a functional COXIII polypeptide.

Amino Acid Sequence↗