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At least 55 records · Page 3Linked to original sources
PubMed2005

Murine gammaherpesvirus 68 open reading frame 11 encodes a nonessential virion component.

Open reading frame 11 (ORF11) is a conserved gammaherpesvirus gene that remains undefined. We identified the product of murine gammaherpesvirus 68 (MHV-68) ORF11, p43, as a virion component with a predominantly perinuclear distribution in infected cells. MHV-68 lacking p43 grew normally in vitro but showed reduced lytic replication in vivo and a delay in seeding to the spleen. Subsequent latency amplification was normal. Thus, MHV-68 ORF11 encoded a virion component that was important for in vivo lytic replication but dispensable for the establishment of latency.

Animals↗
PubMed1996

The 5' untranslated region of Arabidopsis thaliana calmodulin cDNA is an independent cDNA containing an open reading frame.

A cDNA clone, pAUK1, with an open reading frame (ORF) coding for a hypothetical 164-amino-acid protein was isolated from an Arabidopsis thaliana (L.) Heynh cDNA library. The clone was attached, tail to tail, to the 3' end of A. thaliana hexokinase cDNA. An almost identical sequence had been previously described as the 5' untranslated region (5' UTR) of A. thaliana calmodulin cDNA (ACaM-2). Sequence comparison with three additional A. thaliana truncated cDNA clones which appear in a database (GenBank) supports the conclusion that pAUK1 is identical to the 5' UTR of ACaM-2 and that the 5' UTR of ACaM-2 is an independent cDNA artificially linked to A. thaliana calmodulin cDNA.

Amino Acid Sequence↗
PubMed1993

Structural and functional studies in vitro on the p6 protein from the HIV-1 gag open reading frame.

Protein p6 from HIV-1 gag open reading frame is reported to affect both the final phase of assembly of the viral particle and the early stage of the gag polyprotein maturation in vitro. Two separate hypotheses have been proposed, on only one of these reported effects. We think that both observations may be eventually explained if p6 protein strongly inhibits the HIV-1 proteinase. Protein p6 was synthesised by solid-phase peptide synthesis. Several methods of folding the p6 protein were tested, each resulting in the random structure according to both CD and 1D proton NMR spectra. A uniformly high exposure of NH protons to the solution was confirmed by temperature-dependent NMR spectra and isotope exchange experiments. Thus the p6 protein does not have any rigid conformation in solution. A rigid structure is not formed after further cleavage by HIV-1 proteinase as neither the protein nor its fragments are cleaved by this proteinase. In addition, the p6 protein itself does not act as inhibitor of HIV-1 proteinase. This excludes a direct role of p6 protein and supports the hypothesis that p6 is involved in forming the appropriate structure of gag polyprotein precursor. The role of slowly cleaved tight gag-proteinase in the final stage of maturation may be to slow down maturation of the precursor polyproteins prior to their transport to final location in the membrane.

Amino Acid Sequence↗
PubMed1995

Analysis and mapping of a family of 3'-coterminal transcripts containing coding sequences for human cytomegalovirus open reading frames UL93 through UL99.

Human cytomegalovirus (HCMV) open reading frames (ORFs) UL93 through UL99 are contained within a region of viral genome that is well conserved in all herpesviruses. Previous reports detailing the expression of ORF UL99 (also referred to as the 28-kDa virion phosphoprotein or pp28) indicated that the pattern of transcription proximal to pp28 is extremely complex and involves a number of large overlapping transcripts, none of which have been characterized. We have used an RNA-mapping approach consisting of Northern (RNA) hybridization, RNase protection, and primer extensions to determine the coding capacity of several large-molecular-weight transcripts which overlap the 1.3- and 1.6-kb UL99-specific transcripts. Our results suggest that six differentially regulated transcripts with sizes of 2.6, 4.7, 5.6, 7.3, 9.1, and 10.5 kb, and derived from the same strand of the viral genome overlap, are 3'-coterminal with the smaller UL99-specific transcripts. On the basis of 5'-end mapping via primer extension and RNase protection, we have determined that the 2.6- to 10.5-kb messages initiate upstream of each of the potential ORFs in this region, UL98, UL97, UL96, UL95, UL94, and UL93. By using cycloheximide and ganciclovir [9-(1,3-dihydroxy-2-propoxymethyl)guanine] to block de novo viral protein synthesis and viral DNA replication, respectively, we have determined that the 2.6-, 4.7-, 5.6-, and 7.3-kb messages have characteristics of early or early-late transcripts, whereas the 9.1- and 10.5-kb messages appear to be true late transcripts. The evolutionary conservation of ORFs UL93 through UL99 and their transcriptional regulation in other herpesviruses are discussed.

Base Sequence↗
PubMed2005

Dissection of a novel nuclear localization signal in open reading frame 29 of varicella-zoster virus.

Open reading frame 29 (ORF29) of varicella-zoster virus (VZV) encodes a 120-kDa single-stranded DNA binding protein (ORF29p) that is not packaged in the virion and is expressed during latency. During lytic infection, ORF29p is localized primarily to infected cell nuclei. In contrast, ORF29p is found exclusively in the cytoplasm in neurons of the dorsal root ganglia obtained at autopsy from seropositive latently infected patients. ORF29p accumulates in the nuclei of neurons in dorsal root ganglia obtained at autopsy from patients with active zoster. The localization of this protein is, therefore, tightly correlated with the proposed VZV lytic/latent switch. In this report, we have investigated the nuclear import mechanism of ORF29p. We identified a novel nuclear targeting domain bounded by amino acids 9 to 154 of ORF29p that functions independent of other VZV-encoded factors. In vitro import assays in digitonin-permeabilized HeLa cells reveal that ORF29p is transported into the nucleus by a Ran-, karyopherin alpha- and beta-dependent mechanism. These data are further supported by the demonstration that a glutathione S-transferase-karyopherin alpha fusion interacts with ORF29p, but not with a protein containing a point mutation in its nuclear localization signal (NLS). Therefore, the region of ORF29p responsible for its nuclear targeting is also involved in the association with karyopherin alpha. As a result of this interaction, this noncanonical NLS appears to hijack the classical cellular nuclear import machinery. Elucidation of the mechanisms governing ORF29p nuclear targeting could shed light on the VZV reactivation process.

Cell Line↗
PubMed1996

A [2Fe-2S] protein encoded by an open reading frame upstream of the Escherichia coli bacterioferritin gene.

An open reading frame located upstream of the bacterioferritin gene in Escherichia coli encodes a hypothetical 64-residue protein [Andrews, S.C., Harrison, P.C., & Guest, J.R. (1989) J. Bacteriol. 171, 3940-3947)]. The spacing of the four cysteine residues in this hypothetical protein is identical to that in a region of NIFU, a [2Fe-2S] protein found in nitrogen-fixing bacteria [Fu, W., Jack, R.F., Morgan, T.V., Dean, D.R., & Johnson, M.K. (1994) Biochemistry 33, 13455-13463)]. The NIFU-like E. coli gene was cloned and overexpressed with a C-terminal "His tag" in E. coli using the T7 RNA polymerase/promoter system, and the protein was purified by metal-chelate affinity chromatography. UV-vis absorption and EPR spectra together with iron and amino acid analyses conclusively established that this NIFU-like E. coli protein contains one [2Fe-2S] cluster which can exist in at least two oxidation levels: +2 for the as-purified protein, and +1 for dithionite-reduced protein. Size-exclusion chromatography established that this His-tagged [2Fe-2S] protein is monomeric in solution. Affinity chromatography demonstrated specific complex formation between bacterioferritin (Bfr) and this NIFU-like [2Fe-2S] protein, which is dubbed Bfd. An open reading frame encoding a homologous Bfd is located near a Bfr gene in at least one other bacterium. Bfd may, therefore, constitute a general redox and/or regulatory component participating in the iron storage or mobilization functions of Bfr.

Amino Acid Sequence↗
PubMed1994

Open reading frames in the antisense strands of genes coding for glycolytic enzymes in Saccharomyces cerevisiae.

Open reading frames longer than 300 bases were observed in the antisense strands of the genes coding for the glycolytic enzymes phosphoglucose isomerase, phosphoglycerate mutase, pyruvate kinase and alcohol dehydrogenase I. The open reading frames on both strands are in codon register. It has been suggested that proteins coded in codon register by complementary DNA strands can bind to each other. Consequently, it was interesting to investigate whether the open reading frames in the antisense strands of glycolytic enzyme genes are functional. We used oligonucleotide-directed mutagenesis of the PGI1 phosphoglucose isomerase gene to introduce pairs of closely spaced base substitutions that resulted in stop codons in one strand and only silent replacements in the other. Introduction of the two stop codons into the PGI1 sense strand caused the same physiological defects as already observed for pgil deletion mutants. No detectable effects were caused by the two stop codons in the antisense strand. A deletion that removed a section from -31 bp to +109 bp of the PGI1 gene but left 83 bases of the 3' region beyond the antisense open reading frame had the same phenotype as a deletion removing both reading frames. A similar pair of deletions of the PYK1 gene and its antisense reading frame showed identical defects. Our own Northern experiments and those reported by other authors using double-stranded probes detected only one transcript for each gene. These observations indicate that the antisense reading frames are not functional. On the other hand, evidence is provided to show that the rather long reading frames in the antisense strands of these glycolytic enzyme genes could arise from the strongly selective codon usage in highly expressed yeast genes, which reduces the frequency of stop codons in the antisense strand.

Base Sequence↗
PubMed1994

Inferring genes from open reading frames.

One expects that in DNA without protein coding function, stop codons (which constitute three of the 64 possible codons) should occur frequently in all reading frames, and that a long open reading frame (ORF) can be interpreted as a sign for the existence of a gene. We make a beginning on introducing quantitative measures of confidence into this inference--taking Saccharomyces cerevisiae as a sample case--and show that some common assumptions can reasonably be questioned. In particular we show that statistical support for the biological function of shorter ORFs listed as putative genes in recent papers is in fact very weak. This is an issue of practical as well as theoretical interest, since researching the function of a putative gene is difficult and expensive.

Base Composition↗
PubMed1988

Hepatitis B virus particles contain a polypeptide encoded by the largest open reading frame: a putative reverse transcriptase.

A segment of the largest open reading frame of hepatitis B virus (HBV) was inserted into an open reading frame vector directing the expression in Escherichia coli of a fusion molecule containing 143 HBV-encoded amino acids. The fusion protein was used to generate antiserum which served in immunoblots to identify a polypeptide with a molecular mass of 65 kilodaltons in HBV particles. Because of the small number of molecules in virus particles, unambiguous detection required the development of a highly sensitive immunoblot procedure.

3,3'-Diaminobenzidine↗
PubMed2000

Coupling of open reading frames by translational bypassing.

Translational bypassing joins the information found within two disparate open reading frames into a single polypeptide chain. The underlying mechanism centers on the decoding properties of peptidyl-transfer RNA (tRNA) and involves three stages: take-off, scanning, and landing. In take-off, the peptidyl-tRNA/messenger RNA (mRNA) complex in the P site of the ribosome dissociates, and the mRNA begins to move through the ribosome. In scanning, the peptidyl-tRNA probes the mRNA sliding through the decoding center. In landing, the peptidyl-tRNA re-pairs with a codon with which it can form a stable interaction. Although few examples of genes are known that rely on translational bypassing to couple open reading frames, ribosomes appear to have an innate capacity for bypassing. This suggests that the strategy of translational bypassing may be more common than presently appreciated. The best characterized example of this phenomenon is T4 gene 60, in which a complex set of signals stimulates bypassing of 50 nucleotides between the two open reading frames. In this review, we focus on the bypassing mechanism of gene 60 in terms of take-off, scanning, and landing.

Amino Acid Sequence↗
PubMed2006

Evidence for conservation and selection of upstream open reading frames suggests probable encoding of bioactive peptides.

BACKGROUND: Approximately 40% of mammalian mRNA sequences contain AUG trinucleotides upstream of the main coding sequence, with a quarter of these AUGs demarcating open reading frames of 20 or more codons. In order to investigate whether these open reading frames may encode functional peptides, we have carried out a comparative genomic analysis of human and mouse mRNA 'untranslated regions' using sequences from the RefSeq mRNA sequence database. RESULTS: We have identified over 200 upstream open reading frames which are strongly conserved between the human and mouse genomes. Consensus sequences associated with efficient initiation of translation are overrepresented at the AUG trinucleotides of these upstream open reading frames, while comparative analysis of their DNA and putative peptide sequences shows evidence of purifying selection. CONCLUSION: The occurrence of a large number of conserved upstream open reading frames, in association with features consistent with protein translation, strongly suggests evolutionary maintenance of the coding sequence and indicates probable functional expression of the peptides encoded within these upstream open reading frames.

3' Untranslated Regions↗
PubMed1992

Translational modulation in hepatitis B virus preS-S open reading frame expression.

A series of hepatitis B virus open reading frame (ORF) preS-S variants, including mutants in which the relative order of the in-frame start codons (AUG1, AUG2 and AUG3) and nearby sequences had been altered, was expressed both in vivo (in HepG2 hepatoblastoma cells) and in vitro (by T7 promoter-driven transcription followed by translation in a rabbit reticulocyte lysate). The ratio of the synthesis of the large, middle (M) and major (S) proteins or their chimeric counterparts was analysed to study the translational regulation of ORF expression. As expected on the basis of the ribosome scanning model, the AUG sequence context was found to be a prominent factor in determining the different translational behaviour of the two preS-S-specific mRNAs of 2.4 kb (predominantly translated from AUG1) and 2.1 kb (which includes AUG2 and/or AUG3 and can be translated from either). Results from both experimental systems suggested that initiation at internal AUGs in the 2.4 kb RNA is possible. In experiments in vitro, preS-S mutants bearing lesions in a region 5' to AUG2, which has been implicated in AUG2/AUG3 cis repression, showed no increase in the utilization of internal AUGs. In addition, the chimeric envelope polypeptides produced in transfected HepG2 cells in this study were informative with respect to preS-mediated endoplasmic retention: replacement of the preS2 N terminus with that from preS1 generated a chimeric M protein that was glycosylated within the putative preS1 retention sequence ad was not secreted. Thus, the preS1 retention sequence most likely acts inside the lumen of endoplasmic reticulum and its function is insensitive to glycosylation. A similar element might be active at the N terminus of M protein.

Base Sequence↗
PubMed1994

Detailed analysis of a family of RNAs transcribed from varicella-zoster virus open reading frame 21 during replication.

The expression of VZV open reading frame (ORF) 21 during viral replication was examined at the RNA level. Three distinct mRNAs homologous to ORF 21, 3.6, 3.0, and 2.4 kb in size, were detected by Northern blot hybridization. Sequence analysis of eight ORF 21 cDNAs indicated that the corresponding RNAs were 3' coterminal and unspliced. The three largest cDNAs contained the entire ORF 21 coding sequence. Two upstream alternative polyadenylation signals located within ORF 21 were not utilized. Sequence analysis of the site of polyadenylation suggests a virus-specific cis-acting element is present in these transcripts.

Amino Acid Sequence↗
PubMed1994

Antisense overlapping open reading frames in genes from bacteria to humans.

Long Open Reading Frames (ORFs) in antisense DNA strands have been reported in the literature as being rare events. However, an extensive analysis of the GenBank database revealed that a substantial number of genes from several species contain an in-phase ORF in the antisense strand, that overlaps entirely the coding sequence of the sense strand, or even extends beyond. The findings described in this paper show that this is a frequent, non-random phenomenon, which is primarily dependent on codon usage, and to a lesser extent on gene size and GC content. Examination of the sequence database for several prokaryotic and eukaryotic organisms, demonstrates that coding sequences with in-phase, 100% overlapping antisense ORFs are present in every genome studied so far.

Amino Acid Sequence↗
PubMed1991

Alternate promoter selection within a human cytomegalovirus immediate-early and early transcription unit (UL119-115) defines true late transcripts containing open reading frames for putative viral glycoproteins.

The human cytomegalovirus open reading frames (ORFs) UL119 through UL115 (UL119-115) are located downstream of the immediate-early 1 and 2 transcription units. The promoter upstream of UL119 is active at all times after infection and drives the synthesis of a spliced 3.1-kb mRNA. The viral mRNA initiates in UL119, contains UL119-117 and UL116, and terminates just downstream of UL115. True late transcripts that are detected only after viral DNA synthesis originate from this transcription unit. True late mRNAs of 2.1 kb, containing ORFs UL116 and UL115, and 1.2 kb, containing ORF UL115 only, are synthesized. The true late viral mRNAs are 3' coterminal with the 3.1-kb mRNA. This transcription unit is an example of late promoters nested within an immediate-early-early transcription unit. The gene products of UL119-117, UL116, and UL115 are predicted to be glycoproteins. Efficient expression of the downstream ORFs at late times after infection may be related to alternate promoter usage and downstream cap site selection.

Amino Acid Sequence↗
PubMed2005

Comparative analysis of the prion protein open reading frame nucleotide sequences in peacock and parakeet.

The open reading frame of peacock and parakeet prion protein (PrP) genes was cloned and sequenced. The peacock and parakeet PrP genes consisted of 833 and 866 nucleotides encoding 266 and 277 amino acids, respectively (GenBank Accession numbers AY365065 and AY365066). Sequence analysis showed that the peacock and parakeet PrP genes had 93.67% homology to each other, 94.04% and 99.64% homology to the chicken PrP gene and 46.0% and 42.1% similarity to the mammalian PrP genes, respectively. The structural features of all known mammalian and avian PrPs, including N-terminal signal peptides, tandem repeats, conserved hydrophobic region, disulfide bridges and glycoinositol phospholipid anchor, were also found in peacock and parakeet PrPs. The parakeet and peacock PrPs, however, differed in the hexarepeat region, with the peacock having six and the parakeet having seven hexarepeats. The phylogenetic analysis showed that the PrP genes in 52 species were clustered into 2 distinct lineages, the avian and the mammalian. The peacock and parakeet PrP genes belonged to the same lineage but the peacock PrP was sub-classed with the pigeon PrP and the parakeet PrP was sub-classed with the duck and chicken PrPs. The present work added two more species data to the collection of the PrP genes and supported the previous findings that the PrP genes are highly conserved across species.

Amino Acid Sequence↗
PubMed2006

Functional genomics of genes with small open reading frames (sORFs) in S. cerevisiae.

Genes with small open reading frames (sORFs; <100 amino acids) represent an untapped source of important biology. sORFs largely escaped analysis because they were difficult to predict computationally and less likely to be targeted by genetic screens. Thus, the substantial number of sORFs and their potential importance have only recently become clear. To investigate sORF function, we undertook the first functional studies of sORFs in any system, using the model eukaryote Saccharomyces cerevisiae. Based on independent experimental approaches and computational analyses, evidence exists for 299 sORFs in the S. cerevisiae genome, representing approximately 5% of the annotated ORFs. We determined that a similar percentage of sORFs are annotated in other eukaryotes, including humans, and 184 of the S. cerevisiae sORFs exhibit similarity with ORFs in other organisms. To investigate sORF function, we constructed a collection of gene-deletion mutants of 140 newly identified sORFs, each of which contains a strain-specific "molecular barcode," bringing the total number of sORF deletion strains to 247. Phenotypic analyses of the new gene-deletion strains identified 22 sORFs required for haploid growth, growth at high temperature, growth in the presence of a nonfermentable carbon source, or growth in the presence of DNA damage and replication-arrest agents. We provide a collection of sORF deletion strains that can be integrated into the existing deletion collection as a resource for the yeast community for elucidating gene function. Moreover, our analyses of the S. cerevisiae sORFs establish that sORFs are conserved across eukaryotes and have important biological functions.

Conserved Sequence↗