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PubMed2000

Termination and peptide release at the upstream open reading frame are required for downstream translation on synthetic shunt-competent mRNA leaders.

We have shown recently that a stable hairpin preceded by a short upstream open reading frame (uORF) promotes nonlinear ribosome migration or ribosome shunt on a synthetic mRNA leader (M. Hemmings-Mieszczak and T. Hohn, RNA 5:1149-1157, 1999). We have now used the model mRNA leader to study further the mechanism of shunting in vivo and in vitro. We show that a full cycle of translation of the uORF, including initiation, elongation, and termination, is a precondition for the ribosome shunt across the stem structure to initiate translation downstream. Specifically, AUG recognition and the proper release of the nascent peptide are necessary and sufficient for shunting. Furthermore, the stop codon context must not impede downstream reinitiation. Translation of the main ORF was inhibited by replacement of the uORF by coding sequences repressing reinitiation but stimulated by the presence of the virus-specific translational transactivator of reinitiation (cauliflower mosaic virus pVI). Our results indicate reinitiation as the mechanism of translation initiation on the synthetic shunt-competent mRNA leader and suggest that uORF-dependent shunting is more prevalent than previously anticipated. Within the above constraints, uORF-dependent shunting is quite tolerant of uORF and stem sequences and operates in systems as diverse as plants and fungi.

5' Untranslated Regions↗

PubMed1993

Yeast open reading frame YCR14C encodes a DNA beta-polymerase-like enzyme.

We have shown by activity gel that overexpression in E. coli of a yeast chromosome 3 open reading frame (ORF) designated YCR14C and bearing homology to mammalian DNA polymerases beta results in a new DNA polymerase in the host cells. The molecular mass of this enzyme corresponded to the YCR14C-predicted 67 kDa protein, and NH2-terminal amino acid sequencing confirmed that the expressed protein was encoded by the yeast ORF. This new yeast DNA polymerase was purified to homogeneity from E.coli. In a fashion similar to that of mammalian beta-polymerases, the purified yeast enzyme exhibited distributive DNA synthesis on DNA substrate with a single-stranded template and processive gap-filling synthesis on a short-gapped DNA substrate. Activity of this yeast beta-polymerase-like enzyme was sensitive to the beta-polymerase inhibitor ddNTP and resistant to both 1 mM NEM and neutralizing antibody to E. coli DNA polymerase I. These results, therefore, indicate that YCR14C encodes a DNA beta-polymerase-like enzyme in yeast, and we name it DNA polymerase IV. Yeast strains harboring a deletion mutation of the pol IV gene are viable, they exhibit no increase in sensitivity to ultraviolet light, ionizing radiation or alkylating agents, and sporulation and spore viability are not affected in the mutant.

Amino Acid Sequence↗

PubMed2005

Role of the varicella-zoster virus gene product encoded by open reading frame 35 in viral replication in vitro and in differentiated human skin and T cells in vivo.

Although genes related to varicella-zoster virus (VZV) open reading frame 35 (ORF35) are conserved in the herpesviruses, information about their contributions to viral replication and pathogenesis is limited. Using a VZV cosmid system, we deleted ORF35 to produce two null mutants, designated rOkaDelta35(#1) and rOkaDelta35(#2), and replaced ORF35 at a nonnative site, generating two rOkaDelta35/35@Avr mutants. ORF35 Flag-tagged recombinants were made by inserting ORF35-Flag at the nonnative Avr site as the only copy of ORF35, yielding rOkaDelta35/35Flag@Avr, or as a second copy, yielding rOka35Flag@Avr. Replication of rOkaDelta35 viruses was diminished in melanoma and Vero cells in a 6-day analysis of growth kinetics. Plaque sizes of rOkaDelta35 mutants were significantly smaller than those of rOka in melanoma cells. Infection of melanoma cells with rOkaDelta35 mutants was associated with disrupted cell fusion and polykaryocyte formation. The small plaque phenotype was not corrected by growth of rOkaDelta35 mutants in melanoma cells expressing the major VZV glycoprotein E, gE. The rOkaDelta35/35@Avr viruses displayed growth kinetics and plaque morphologies that were indistinguishable from those of rOka. Analysis with ORF35-Flag recombinants showed that the ORF35 gene product localized predominantly to the nuclei of infected cells. Evaluations in the SCIDhu mouse model demonstrated that ORF35 was required for efficient VZV infection of skin and T-cell xenografts, although the decrease in infectivity was most significant in skin. These mutagenesis experiments indicated that ORF35 was dispensable for VZV replication, but deleting ORF35 diminished growth in cultured cells and was associated with attenuated VZV infection of differentiated human skin and T cells in vivo.

Base Sequence↗

PubMed2003

Two nonoverlapping domains on the Norwalk virus open reading frame 3 (ORF3) protein are involved in the formation of the phosphorylated 35K protein and in ORF3-capsid protein interactions.

Expression of the Norwalk virus open reading frame 3 (ORF3) in Spodoptera frugiperda (Sf9) cells yields two major forms, the predicted 23,000-molecular-weight (23K) form and a larger 35K form. The 23K form is able to interact with the ORF2 capsid protein and be incorporated into virus-like particles. In this paper, we provide mass spectrometry evidence that both the 23K and 35K forms are composed only of the ORF3 protein. Two-dimensional gel electrophoresis and phosphatase treatment showed that the 35K form results solely from phosphorylation and that the 35K band is composed of several different phosphorylated forms with distinct isoelectric points. Furthermore, we analyzed deletion and point mutants of the ORF3 protein. Mutants that lacked the C-terminal 33 amino acids (ORF3(1-179), ORF3(1-152), and ORF3(1-107)) no longer produced the 35K form. An N-terminal truncation mutant (ORF3(51-212)) and a site-directed mutant (ORF3(T201V)) were capable of producing the larger form, which was converted to the smaller form by treatment with protein phosphatase. These data suggest that the region between amino acids 180 and 212 is phosphorylated, and mass spectrometry showed that amino acids Arg196 to Arg211 are not phosphorylated; thus, phosphorylation of the serine-threonine-rich region from Thr181 to Ser193 must be involved in the generation of the 35K form. Studies of the interaction between the ORF2 protein and full-length and mutated ORF3 proteins showed that the full-length ORF3 protein (ORF3(FL)), ORF3(1-179), ORF3(1-152), and ORF3(51-212) interacted with the ORF2 protein, while an ORF3(1-107) protein did not. These results indicate that the region of the ORF3 protein between amino acids 108 and 152 is responsible for interaction with the ORF2 protein.

Amino Acid Sequence↗

PubMed1992

Identification of a protein encoded in the EB-viral open reading frame BMRF2.

Using monospecific rabbit sera against a peptide derived from a potential antigenic region of the Epstein-Barr viral amino acid sequence encoded in the open reading frame BMRF2 we could identify a protein-complex of 53/55 kDa in chemically induced B95-8, P3HR1 and Raji cell lines. This protein could be shown to be membrane-associated, as predicted by previous computer analysis of the secondary structure and hydrophilicity pattern, and may be a member of EBV-induced membrane proteins in lytically infected cells.

Amino Acid Sequence↗

PubMed1995

The sequence of an 11.1 kb DNA fragment between ADH4 and ADE5 on the left arm of chromosome VII reveals the presence of eight open reading frames.

The complete sequence of a 11, 132 bp segment located on the left arm of chromosome VII of Saccharomyces cerevisiae has been determined and analysed. Eight open reading frames (ORFs) of at least 100 amino acids were identified. Five show similarities to known amino acid sequences. Another ORF encoding the chromosome segregation protein CSE1 is not entirely located in our sequenced fragment and is incomplete at its C-terminus. The two remaining ORFs do not display similarities to known sequences.

Adenosine Deaminase↗

PubMed1996

DNA sequence analysis of a 10 624 bp fragment of the left arm of chromosome XV from Saccharomyces cerevisiae reveals a RNA binding protein, a mitochondrial protein, two ribosomal proteins and two new open reading frames.

We have determined the sequence of a 10624 bp DNA segment located in the left arm of chromosome XV of Saccharomyces cerevisiae. The sequence contains eight open reading frames (ORFs) longer than 100 amino acids. Two of them do not present significant homology with sequences found in the databases. The product of ORF o0553 is identical to the protein encoded by the gene SMF1. Internal to it there is another ORF, o0555 that is apparently expressed. The proteins encoded by ORFs o0559 and o0565 are identical to ribosomal proteins S19.e and L18 respectively. ORF o0550 encodes a protein with an RNA binding signature including RNP motifs and stretches rich in asparagine, glutamine and arginine.

Base Sequence↗

PubMed2000

Characterization of transcripts expressed from human herpesvirus 6A strain GS immediate-early region B U16-U17 open reading frames.

Several gene fragments of human herpesvirus 6 (HHV-6) have been shown to activate the human immunodeficiency virus (HIV) type 1 long terminal repeat (LTR). An open reading frame (ORF) designated B701 (Y. Geng, B. Chandran, S. F. Josephs, and C. Wood, J. Virol. 66:1564-1570, 1992), found within a 22-kb HHV-6A strain GS [HHV-6A(GS)] genomic fragment and a 3.8-kb SalI subfragment, was shown to activate the HIV LTR. B701, also known as HHV-6 U16, is located in the immediate-early B (IE-B) region of the genome. The sequence of the 3.8-kb genomic fragment of HHV-6A(GS) is nearly identical to the published sequence of HHV-6A strain U1102, with minor differences. The HHV-6A(GS) B701 ORF (U16) was used to screen an HHV-6A(GS) cDNA library, and two different but overlapping cDNAs were identified. These cDNAs represent differently spliced transcripts ending at different polyadenylation signals. The ORFs included in the cDNAs are positionally homologous to the human cytomegalovirus (HCMV) UL36 ORF. The ORF in one cDNA was generated by splicing together in frame ORFs U17 and U16, and the second cDNA included ORFs U16 and U15. A third differentially spliced cDNA (U16+), was identified by 5' rapid amplification of cDNA ends. The predicted protein was identical to the U16 portion of the U17/U16 spliced gene product but did not include the U17 portion. 5'-extension analyses of the mRNAs demonstrated that at least two potential transcription initiation sites were used to express the transcripts encoding U17 and U16 gene products. Single-stranded U16 and U17 gene-specific RNA probes hybridized with at least five RNA species from infected cells and demonstrated that the expression of these transcripts was differentially regulated. The U17/U16 spliced gene products were expressed at IE times after infection, but a multiply spliced gene product encoded by U16 was expressed as a late gene. The U17/U16 and the U16+ gene products transactivated the HIV LTR. Thus, while there are similarities to the HCMV UL36-UL38 gene family, some of the IE-B U17/U16 transcripts are unique to HHV-6.

Base Sequence↗

PubMed1997

Sequence analysis of a 10.5 kb DNA fragment from the yeast chromosome VII reveals the presence of three new open reading frames and of a tRNAThr gene.

We report the sequence analysis of a 10,531 bp DNA of Saccharomyces cerevisiae chromosome VII. This sequence contains five complete open reading frames (ORFs) potentially encoding proteins longer than 100 amino acids and incomplete ORF encoding for the 3' part of the GCN5 gene (Georgakopoulos and Thireos, 1992). ORFs G9160 and G9155 correspond to the genes ENO1 (Holland et al. 1981) and PUP2 (Gergatsou et al., 1992) respectively. ORF G9165 codes for a protein which shares significant homology with known proteins present in databases (see below). The translated sequence of ORF G9170 shows 88% identity to the 6-phosphogluconate dehydrogenase encoded by the gene 6PGD from S. cerevisiae present in the SwissProt data library (P38720). This indicates that G9170 might code for a second 6-phosphogluconate dehydrogenase. ORF G9175 codes for a putative new member of the mitochondrial carrier family. A hypothetical tRNAThr (TGT) is also present in position 6842-6913.

Chromosomes, Fungal↗

PubMed1986

Long interspersed L1 repeats in rabbit DNA are homologous to L1 repeats of rodents and primates in an open-reading-frame region.

Determination of the DNA sequence of a cluster of repetitive elements located 3' to the rabbit beta-1 globin gene shows that previously identified B, E, and D repeats are linked to form a long repeat. The B and E regions are homologous to the L1 long interspersed repeats of primates and rodents. Thus L1 repeats are widely dispersed in several different mammalian orders. The sequence similarity among L1 repeats from three species is limited to a long segment that contains extensive open reading frames. This conserved region is followed by a highly divergent segment that corresponds to a 3' untranslated region. The conservation of sequences in the open-reading-frame region coupled with the divergence of the untranslated region supports the hypothesis that the L1 repeats are derived from transcripts of a functional gene that encodes a protein.

Animals↗

PubMed1999

Only the non-glycosylated fraction of hepatitis E virus capsid (open reading frame 2) protein is stable in mammalian cells.

Hepatitis E virus (HEV) is a non-enveloped, positive-strand RNA virus, with the genome encoding three open reading frames (ORFs) of which ORF 2 directs translation of the capsid protein, PORF2. Following pulse-labelling and cell fractionation of PORF2 expressed in mammalian cells using the Semliki Forest virus replicon, the capsid protein was detected as three major species of 78 (PORF2), 82 and 86 kDa, with P82 and P86 being N-glycosylated (gPORF2 and ggPORF2, respectively). Although gPORF2 and ggPORF2 species represented 79% of total PORF2 after 20 min metabolic labelling and were largely membrane-associated, the glycosylated PORF2 species were much less stable than non-glycosylated PORF2, which was present in the cytosol and represented the major product accumulated in the cell. In the absence of detectable surface expression or export of PORF2, this suggests that glycosylated ORF 2 proteins may not be intermediates in HEV capsid assembly.

Animals↗

PubMed1995

A 29.425 kb segment on the left arm of yeast chromosome XV contains more than twice as many unknown as known open reading frames.

The nucleotide sequence of a 29.425 kb fragment localized on the left arm of chromosome XV from Saccharomyces cerevisiae has been determined. The sequence contains 13 open reading frames (ORFs) of which four encode the known genes ADH1, COQ3, MSH2 and RCF4. Predictions are made concerning the functions of the unknown ORFs. Some of the ORFs contain sequences similar to expressed sequence tags (EST) found in the database made available by TIGR. In particular, the highly expressed ADH1 gene is represented in this database by no less than 20 EST sequences. Two ARS sequences and a putative functional GCN4 motif have also been detected. One ORF (O0953) containing nine putative transmembrane segments is similar to a hypothetical membrane protein of Arabidopsis thaliana. Characteristic features of the other ORFs include ATP/GTP binding sites, a fungal Zn(2)-Cys(6) binuclear centre, an endoplasmic reticulum targeting sequence, a beta-transducin repeat signature and in two instances, good similarity to the prokaryotic lipoprotein signal peptide motif.

Amino Acid Sequence↗

PubMed2005

Large-scale, multi-genome analysis of alternate open reading frames in bacteria and archaea.

Analysis of over 300,000 annotated genes in 105 bacterial and archaeal genomes reveals an unexpectedly high frequency of large (>300 nucleotides) alternate open reading frames (ORFs). Especially notable is the very high frequency of alternate ORFs in frames +3 and -1 (where the annotated gene is defined as frame +1). The occurrence of alternate ORFs is correlated with genomic G+C content and is strongly influenced by synonymous codon usage bias. The frequency of alternate ORFs in frame -1 is also influenced by the occurrence of codons encoding leucine and serine in frame +1. Although some alternate ORFs have been shown to encode proteins, many others are probably not expressed because they lack appropriate signals for transcription and translation. These latter can be mis-annotated by automatic gene finding programs leading to errors in public databases. Especially prone to mis-annotation is frame -1, because it exhibits a potential codon usage and theoretical capacity to encode proteins with an amino acid composition most similar to real genes. Some alternate ORFs are conserved across bacterial or archaeal species, and can give rise to misannotated "conserved hypothetical" genes, while others are unique to a genome and are misidentified as "hypothetical orphan" genes, contributing significantly to the orphan gene paradox.

Algorithms↗

PubMed2001

Kaposi's Sarcoma-associated herpesvirus open reading frame 50 stimulates the transcriptional activity of STAT3.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an important pathogen in Kaposi's sarcoma and abnormal lymphoproliferation. KSHV open reading frame 50 (ORF50), a homolog of the Epstein-Barr virus immediate-early gene product RTA, activates early and late gene transcription in the KSHV lytic cycle, and its expression is closely correlated with KSHV-related diseases. ORF50 interacts with the cellular proteins CBP and histone deacetylase and represses p53-induced apoptosis through a CBP-related mechanism. We show here that KSHV ORF50 also interacts with STAT3. ORF50 stimulated transcription of STAT-driven reporter genes, and interleukin-6 and v-Src further activated this stimulating effect of ORF50. Physical association of STAT3 and ORF50 required the carboxyl-terminal transactivation domain of ORF50 and multiple regions within STAT3. ORF50 recruited STAT3 to the nucleus and induced the dimerization of STAT3 monomers in the absence of STAT3 phosphorylation. We show here that KSHV ORF50 activates STAT3-mediated transcription through direct interaction without mediating tyrosine phosphorylation.

3T3 Cells↗

PubMed1999

Deletion of six open reading frames from the left arm of chromosome IV of Saccharomyces cerevisiae.

The construction of six deletion mutants of Saccharomyces cerevisiae and their basic phenotypic characterization are described. Open reading frames YDL148c, YDL109c, YDL021w, YDL019c, YDL018c and YDL015c from the left arm of chromosome IV were deleted using a polymerase chain reaction (PCR)-based disruption technique, introducing the kanMX4 resistance marker into the respective genes. Gene replacement cassettes (pYORCs) for use in other strain backgrounds were cloned by PCR using DNA templates from haploid or diploid deletion mutants, and inserted into episomal plasmids. Cognate clones of all six ORFs were obtained by gap repair. Deletions were carried out in diploid cells and, after sporulation, yielded four viable spores for clones disrupted in YDL109c, YDL021w, YDL019c and YDL018c. Spores harbouring disruptions in ORFs YDL148c and YDL015c germinated but underwent only a few divisions before ceasing growth, suggesting that the respective genes are essential for vegetative growth on YPD complete media. The other deletion mutants grew like wild-type at different temperatures and on different carbon sources. A brief computational analysis of the six ORFs studied in this work is presented.

Carbon↗

PubMed1995

Phylogenetic analysis of open reading frame 5 of field isolates of equine arteritis virus and identification of conserved and nonconserved regions in the GL envelope glycoprotein.

The variation and phylogenetic relationship of open reading frame 5 (ORF5) of 3 different laboratory strains of the original prototype Bucyrus strain of equine arteritis virus (EAV), the modified live virus vaccine (ARVAC, Fort Dodge Laboratories), and 18 field isolates of EAV from North America and Europe were determined by comparison of their gene sequences. The viruses differed from the published sequence by between 3 (99.6% homology) and 94 (87.8%) nucleotides and by between 3 (98.8%) and 24 (90.6%) amino acids. The field isolates differed from each other by between 2 (99.7%) and 110 (85.7%) nucleotides and by between 1 (99.6%) and 26 (89.8%) amino acids. Comparison of the nucleotide sequences of these viruses indicates that although they are very closely related, the ORF5 of each virus is distinct. The ORF5 of EAV encodes the GL envelope glycoprotein which expresses the neutralization determinants of the virus. Comparative analysis of the deduced amino acid sequence of the GL protein of the viruses identified three distinct variable regions (V1 [aa 61-121], V2 [141-178], and V3 [aa 202-222]), a putative signal sequence (S [aa 1-18]), and four conserved regions (C1 [aa 19-60], C2 [aa 122-140], C3 [aa 179-201], and C4 [aa 223-255]). Amino acid substitutions in the V1 region of the GL protein of EAV field isolates had significant effects on the predicted hydrophobicity and secondary structure of the protein, which is potentially important because this region contains a major neutralization site. Estimation of genetic distances and phylogenetic tree analysis of these viruses identified four distinct groups of EAV isolates, including two North American (NA1 and NA2) and two European (E1 and E2) groups. The sequence data obtained from individual European and North American isolates suggest movement of viruses between the two continents.

Amino Acid Sequence↗

PubMed1997

Differential translational initiation of lbp mRNA is caused by a 5' upstream open reading frame.

Expression of the luciferin-binding protein (LBP) from Gonyaulax polyedra is regulated by the circadian clock at the translational level. Here we report that in vitro translation of lbp mRNA results in the synthesis of two LBP variants of different sizes, which is shown to be due to translational initiation at different in-frame AUG codons on lbp mRNA. Differential initiation is caused by a small open reading frame (ORF, situated in the 5' untranslated region of lbp mRNA), which gives rise to a leaky scanning mechanism. In Gonyaulax, only one of these variants, which is produced by initiation from the first AUG of the lbp ORF, exhibits a circadian rhythm and is far more abundant during night phase.

Amino Acid Sequence↗

PubMed1995

The complete sequence of a 9000 bp fragment of the right arm of Saccharomyces cerevisiae chromosome VII contains four previously unknown open reading frames.

We report the sequence of a 9000 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete previously unknown open reading frames, which were named G7587, G7589, G7591 and G7594 following standard rules for provisional nomenclature. Outstanding features of some of these proteins were the homology of the putative protein coded by G7589 with proteins involved in transcription regulation and the transmembrane domains predicted in the putative protein coded by G7591.

Amino Acid Sequence↗