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PubMed1994

UL69 of human cytomegalovirus, an open reading frame with homology to ICP27 of herpes simplex virus, encodes a transactivator of gene expression.

The UL69 open reading frame of human cytomegalovirus (HCMV) is homologous to the immediate-early protein ICP27 of herpes simplex virus, an essential viral regulatory protein involved in the transition from early to late gene expression. Genes with homology to ICP27 have been detected in all subclasses of herpesviruses so far. While the respective proteins in alpha- and gammaherpesviruses have been defined as trans-regulatory molecules, nothing is known about these genes in betaherpesviruses. This study was therefore undertaken in order to investigate expression from the UL69 gene locus of HCMV. Northern (RNA) blot experiments revealed a complex pattern of transcripts that changed during the time course of the HCMV replicative cycle: two transcripts of 2.7 and 3.5 kb that were regulated differentially could be detected as early as 7 h after infection. However, these transcripts could not be detected in the presence of cycloheximide. Additional, larger transcripts were present exclusively at late times after infection. To analyze protein expression from the UL69 gene region, the UL69 open reading frame was expressed as a histidine-tagged protein in Escherichia coli. A specific antiserum was generated and used to detect the UL69 protein in HCMV-infected cells which revealed its localization within the intranuclear inclusions that are characteristic for HCMV infection. In cotransfection experiments, an HCMV true late promoter could not be activated by UL69, whereas an early promoter and several heterologous promoters were stimulated about 10-fold. Complementation studies showed that the UL69 protein cannot substitute for ICP27 in the context of the HSV infection, suggesting functional differences between these two proteins. In summary, these experiments define a novel regulatory protein encoded by HCMV that is expressed as an early-late gene and appears to exert a broad stimulatory effect on gene expression.

Amino Acid Sequence↗

PubMed1996

The open reading frame of bamboo mosaic potexvirus satellite RNA is not essential for its replication and can be replaced with a bacterial gene.

A satellite RNA of 836 nt depends on the bamboo mosaic potexvirus (BaMV) for its replication and encapsulation. The BaMV satellite RNA (satBaMV) contains a single open reading frame encoding a 20-kDa nonstructural protein. A full-length infectious cDNA clone has been generated downstream of the T7 RNA polymerase promoter. To investigate the role of the 20-kDa protein encoded by satBaMV, satBaMV transcripts containing mutations in the open reading frame were tested for their ability to replicate in barley protoplasts and in Chenopodium quinoa using BaMV RNA as a helper genome. Unlike other large satellite RNAs, mutants in the open reading frame did not block their replication, suggesting that the 20-kDa protein is not essential for satBaMV replication. Precise replacement of the open reading frame with sequences encoding chloramphenicol acetyltransferase resulted in high level expression of chloramphenicol acetyltransferase in infected C. quinoa, indicating that satBaMV is potentially useful as a satellite-based expression vector.

Base Sequence↗

PubMed1986

Nucleotide sequence of a portion of the Autographa californica nuclear polyhedrosis virus genome containing the EcoRI site-rich region (hr5) and an open reading frame just 5' of the p10 gene.

The nucleotide sequence of a 1587 bp region lying within the HindIII-Q fragment of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) DNA has been determined. It begins in the EcoRI-S-EcoRI-X region, continues to the HindIII-P/Q boundary and contains an open reading frame that codes for a polypeptide of 240 amino acids (p26). This open reading frame is also included in the 1100 and 1500 base transcripts previously mapped to this region. The sequence reveals that the 5' ends of the 1100 and 1500 base transcripts are located 20 bp downstream from the end of a putative TATA box (TAATTAAAT) and 19 bp upstream from the translation start codon (ATG) of the p26 open reading frame. The translation termination codon (TAA) falls in the immediate 5' flanking region of the major late p10 gene of AcMNPV, 3 bp downstream from the putative TATA box. The probable polyadenylation site for the 1100 base transcript lies 23 bp downstream from the cap site for the 750 and 2500 base transcripts encoding the p10 protein. The 5' flanking region of the p26 open reading frame contains the EcoRI site-rich region, hr5, whose sequence is included here. The EcoRI site-rich region, hr5, consists of six imperfect tandem repeats of a sequence that includes the EcoRI recognition site. These direct repeats also include many inverted repeats.

Amino Acid Sequence↗

PubMed2003

The two upstream open reading frames of oncogene mdm2 have different translational regulatory properties.

Few details are known of the mechanisms through which multiple upstream open reading frames (uORFs) interact to regulate translation in higher eukaryotes. The predominant transcript of oncogene mdm2 in normal human cells (L-mdm2) contains two upstream open reading frames in its 5' leader. Elimination of these two uORFs raises the translational efficiency of the transcript by over 10-fold in HeLa cells. The 5'-most uORF (uORF1) alone suppresses downstream translational activity by over 5-fold, whereas uORF2 contributes <2-fold to the inhibition by the intact leader. The different activities of the two uORFs do not depend on the nucleotide sequence surrounding the uORFs in the 5' leader, the order of the two uORFs in the 5' leader, or the occurrence of secondary structure or rare codons within the uORFs. Specific features of the amino acid sequence encoded by uORF1 contribute to its stronger suppressive activity, suggesting that it belongs to the class of "sequence-specific" uORFs. The weaker inhibitory activity inherent in uORF2 is potentiated by a sub-optimal nucleotide context surrounding its initiator AUG. The occurrence of two uORFs with differing activities in both the human gene and the mouse orthologue suggests that this pair of elements may play a fundamental role in regulating expression of the mdm2 gene.

5' Untranslated Regions↗

PubMed1995

Construction and genetic analysis of dicistronic polioviruses containing open reading frames for epitopes of human immunodeficiency virus type 1 gp120.

On the basis of previous studies of dicistronic (dc) polioviruses that carried two internal ribosomal entry sites (L. Alexander, H.-H. Lu, and E. Wimmer, Proc. Natl. Acad. Sci. USA 91:1406-1410, 1994; A. Molla, S. K. Jang, A. V. Paul, Q. Reuer, and E. Wimmer, Nature [London] 356:255-257, 1992), we have constructed a variety of dc polioviruses which express foreign genetic elements that were inserted either between two internal ribosomal entry site elements upstream of the poliovirus open reading frame (pPNENPO derivatives) or upstream of the open reading frame for the poliovirus proteinase 2Apro (pDI-E2A derivatives). Surprisingly, the addition of an N-terminal secretory pathway signal sequence to the open reading frame of the inserted foreign sequences (specifying either truncated versions of human immunodeficiency virus type 1 [HIV-1] gp120 or chloramphenicol acetyltransferase) resulted in a null phenotype, whereas removal of the signal sequence led to the production of viable viruses. Constructs that carried a foreign gene with a signal sequence were negative in RNA synthesis, an observation that suggested a very early block in viral replication. The insertion of transmembrane sequences downstream of the leader sequence did not reverse the replication block. Studies of dc polioviruses that encoded the truncated versions of HIV-1 gp120 showed an increase in genetic stability that correlated with a decrease in the size of the insert. A dc construct that contained a minigene encoding the principal neutralization determinant of HIV-1 produced a stable virus that retained the foreign sequence through multiple passages in cultured cells. These data indicate that dc polioviruses have potential as vaccines for the expression of small foreign epitopes.

Amino Acid Sequence↗

PubMed1994

Murine fibroblast growth factor receptor 1 gene generates multiple messenger RNAs containing two open reading frames via alternative splicing.

The arrangement of exons and introns encoding 5'-side of murine fibroblast growth factor (FGF) receptor 1 (FGFR-1) gene was mapped. A large intron with a size of 14 kb was identified between exon 1 and exon 2. In addition, all FGFR-1 subtypes including a unique variant form with 12 amino acids insertion and two amino acids deletion were observed to be able to be generated through alternative splicing. Furthermore, complete sequencing of the 5'-region of FGFR-1 mRNA revealed that a relatively large open reading frame precedes the major open reading frame encoding FGFR-1. These results indicate that FGFR-1 mRNAs are uniquely translated from an internal translation start site.

3T3 Cells↗

PubMed1995

[A new open reading frame in the genome of cyanobacteria Synechocystis sp. PCC 6803].

A new open reading frame ORF242, coding for a 26.47-kDa polypeptide, was found in a DNA fragment of the cyanobacterium Synechocystis 6803, transforming a photosynthetic mutant to photoautotrophy and having homology with plant chloroplast DNA. In the 5' flanking region of ORF242, consensus sequences characteristic of a functioning gene were found. One copy of ORF242 is present in the Synechocystis 6803 genome. Insertion inactivation of ORF242 does not lead to a decrease in photosynthetic activity in cells of cyanobacteria but may influence the ratio between active complexes of photosystems I and II.

Amino Acid Sequence↗

PubMed2001

Disruption and phenotypic analysis of six open reading frames from chromosome VII of Saccharomyces cerevisiae reveals one essential gene.

Six open reading frames (ORFs) located on chromosome VII of Saccharomyces cerevisiae (YGR205w, YGR210c, YGR211w, YGR241c, YGR243w and YGR244c) were disrupted in two different genetic backgrounds using short-flanking homology (SFH) gene replacement. Sporulation and tetrad analysis showed that YGR211w, recently identified as the yeast ZPR1 gene, is an essential gene. The other five genes are non-essential, and no phenotypes could be associated to their inactivation. Two of these genes have recently been further characterized: YGR241c (YAP1802) encodes a yeast adaptor protein and YGR244c (LSC2) encodes the beta-subunit of the succinyl-CoA ligase. For each ORF, a replacement cassette with long flanking regions homologous to the target locus was cloned in pUG7, and the cognate wild-type gene was cloned in pRS416.

Chromosomes, Fungal↗

PubMed1990

naf, a trans-regulating negative-acting factor encoded within the mouse mammary tumor virus open reading frame region.

The mouse mammary tumor virus (MMTV) long terminal repeat (LTR) open reading frame (ORF) encodes a negative acting factor (naf). In our test system, naf mediates its effect in trans on another MMTV provirus in which the 5' LTR has been replaced by that of Rous sarcoma virus. naf effects are evidenced at the level of transcriptional initiation rather than as reduced mRNA stability. The introduction of a premature termination codon into the MMTV LTR-encoded ORF abolishes the transcriptional down regulation localizing naf within the ORF. In addition, sequences in the gag/pol genes between +320 and +646 and between +3626 and +4590 relative to the site of transcription initiation are also involved in the MMTV-mediated transcriptional down regulation.

Amino Acid Sequence↗

PubMed1986

Characterization of a member of the highly repeated long interspersed rat DNA family with long open reading frames.

A highly repetitive long interspersed sequence from rat DNA has been isolated and partly characterized. This sequence comprises at least a 1300 base-pair and a 2400 base-pair EcoRI fragment and probably additional elements. The 2400 base-pair segment has been analyzed in detail. It appears to be part of the chromosomal DNA in rat cells. The 2400 base-pair repeat is likely to be distributed over several regions in the rat genome. The 2400 base-pair segment has been cloned, mapped for restriction sites, and part of its nucleotide sequence has been determined. The 2400 base-pair sequence is a member of a typical highly repetitive long interspersed sequence with high copy number and restriction site polymorphism. There are sequence homologies to mouse and human DNA. A striking homology has been detected to the flanking sequences of a repetitive mouse DNA sequence that has been described to be located adjacent to one of the kappa-immunoglobulin variable genes. Elements in the 2400 base-pair rat repeat are transcribed in cells from most rat organs and from several continuous rat cell lines. This RNA from rat cell lines was found polyadenylated or not polyadenylated. The nucleotide sequence of parts of the 2400 base-pair DNA segment revealed open reading frames for polypeptide sequences. Such open reading frames have been detected in two different segments of the 2400 base-pair DNA repeat. Open reading frames exist in the two complementary strands in the same DNA segment. The hypothetical polypeptide whose sequence has been determined in toto has a length of 190 amino acid residues and is enriched in hydrophobic amino acids, reminiscent of the amino acid composition in membrane proteins. Hence, it is conceivable that the 2400 base-pair repeat sequence from rat DNA, at least in part, encodes messenger RNAs that might be translated into functional proteins.

Amino Acid Sequence↗

PubMed1998

Adenovirus type 5 E4 open reading frame 4 protein induces apoptosis in transformed cells.

Adenovirus type 5 E4 open reading frame 4 (E4orf4) protein has been previously shown to counteract transactivation of the junB and c-fos genes by cyclic AMP plus E1A protein and to interact with protein phosphatase 2A (PP2A). Here, we show that the wild-type E4orf4 protein induces apoptosis in the E1A-expressing 293 cells, in NIH 3T3 cells transformed with v-Ras, and in the lung carcinoma cell line H1299. The induction of apoptosis is not accompanied by enhanced levels of p53 in 293 cells and occurs in the absence of p53 in H1299 cells, indicating involvement of a p53-independent pathway. A mutant E4orf4 protein that had lost the ability to induce apoptosis also lost its ability to bind PP2A. We suggest that E4orf4 antagonizes continuous signals to proliferate, like those given by E1A or v-Ras, and that the conflicting signals lead to the induction of cell death.

3T3 Cells↗

PubMed1998

An Arabidopsis cDNA encodes an apparent polyprotein of two non-identical thylakoid membrane proteins that are associated with photosystem II and homologous to algal ycf32 open reading frames.

We have characterised an Arabidopsis thaliana cDNA homologous to the ycf32 open reading frames present in the Synechocystis genome and the plastid genomes of several eukaryotic algae. The predicted protein is also homologous to a novel protein reported to be associated with photosystem II. The protein is synthesised as a 23 kDa precursor with an N-terminal presequence that appears to be bipartite in structure, and the protein is targeted into the thylakoid membrane of pea chloroplasts. Although the Ycf32 presequence contains an apparent signal peptide, we find that this protein is not imported by either of the standard Sec- or deltapH-dependent pathways. The mature protein is also unusual in two respects. First, there are two distinct, non-identical copies of typical single-span Ycf32 sequences in the Arabidopsis sequence, separated by an additional hydrophobic region. Secondly, the imported protein runs as a doublet of 6 kDa and 7 kDa polypeptides whereas the mature protein is predicted to be 14 kDa. We speculate that the protein undergoes further maturation once inserted into the thylakoid membrane to yield two separate Ycf32-like polypeptides.

Amino Acid Sequence↗

PubMed1993

Effects of the open reading frames in the Rous sarcoma virus leader RNA on translation.

Three short open reading frames (ORFs) reside in the 5' leader of Rous sarcoma virus (RSV) and are conserved in all avian sarcoma-leukosis retroviruses. Both extensions of the lengths of the ORFs and alterations in their initiation codons affect viral replication and gene expression. To determine whether the effects on viral replication were due to translational regulation mediated by the ORFs, we examined translation following mutation of the initiation and termination codons of each of the three ORFs. We found that the ORFs marginally enhanced downstream gene expression. Moreover, repression of downstream gene translation was proportional to the lengths of the elongated ORFs and depended on the initiation contexts of the AUG codons. Although the ORFs play a major role in viral activities, their effects on translation were relatively minor. Rather, the ORFs may affect the fate of unspliced avian retroviral RNA in chronically infected cells by participating in the sorting of viral RNA for either translation or encapsidation into virions.

Animals↗

PubMed2001

Open reading frame III of borna disease virus encodes a nonglycosylated matrix protein.

The open reading frame III of Borna disease virus (BDV) codes for a protein with a mass of 16 kDa, named p16 or BDV-M. p16 was described as an N-glycosylated protein in several previous publications and therefore was termed gp18, although the amino acid sequence of p16 does not contain any regular consensus sequence for N glycosylation. We examined glycosylation of p16 and studied its membrane topology using antisera raised against peptides, which comprise the N and the C termini. Neither an N- nor a C-terminal peptide is cleaved from p16 during maturation. Neither deglycosylation of p16 by endoglycosidases nor binding of lectin to p16 was detectable. Introduction of typical N-glycosylation sites at the proposed sites of p16 failed in carbohydrate attachment. Flotation experiments with membranes of BDV-infected cells on density gradients revealed that p16 is not an integral membrane protein, since it can be dissociated from membranes. Our experimental data strongly suggest that p16 is a typical nonglycosylated matrix protein associated at the inner surface of the viral membrane, as is true for homologous proteins of other members of the Mononegavirales order.

Attachment Sites, Microbiological↗

PubMed2000

In vitro translation of the upstream open reading frame in the mammalian mRNA encoding S-adenosylmethionine decarboxylase.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a polyamine-responsive element that suppresses translation of the associated downstream cistron in vivo. In this paper, we provide the first direct evidence of peptide synthesis from the S-adenosylmethionine decarboxylase uORF using an in vitro translation system. We examine both the influence of cation concentration on peptide synthesis and the effect of altering the uORF sequence on peptide synthesis. Synthesis of wild type and altered peptides was similar at all concentrations of magnesium tested. In contrast, synthesis of the wild type peptide was more sensitive than that of altered peptides to elevated concentrations of the naturally occurring polyamines, spermidine and spermine, as well as several polyamine analogs. The sensitivity of in vitro synthesis to spermidine was influenced by both the amino acid sequence and the length of the peptide product of the uORF. Findings from the present study correlate with the effects of the uORF and polyamines on translation of a downstream cistron in vivo and support the hypothesis that polyamines and the structure of the nascent peptide create a rate-limiting step in uORF translation, perhaps through a ribosome stalling mechanism.

Adenosylmethionine Decarboxylase↗

PubMed2003

Selecting open reading frames from DNA.

We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the beta-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the beta-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems.

Antibodies, Monoclonal↗

PubMed2000

African swine fever virus EP153R open reading frame encodes a glycoprotein involved in the hemadsorption of infected cells.

The open reading frame EP153R, located within the EcoRI E' fragment of the African swine fever (ASF) virus genome, is predicted to encode a membrane protein of 153 amino acids that presents significant homology to the N-terminal region of several CD44 molecules. EP153R contains multiple putative sites for N-glycosylation, phosphorylation, and myristoylation, a central transmembrane region, a C-type animal lectin-like domain, and a cell attachment sequence. Transcription of EP153R takes place at both early and late times during the virus infection. The disruption of the gene, achieved by insertion of the marker gene LacZ within EP153R, did not change either the in vitro virus growth rate or the virus-sensitive/resistant condition of up to 17 established cell lines, but abrogated the hemadsorption phenomenon induced in ASF virus-infected cells. As the sequence and expression of the ASF virus protein pEP402R, a CD2 homolog responsible for the adhesion of erythrocytes to susceptible cells, was unaffected in cultures infected with the EP153R deletion mutant, we conclude that the gene EP153R is needed to induce and/or maintain the interaction between the viral CD2 homolog and its corresponding cell receptor.

African Swine Fever Virus↗

PubMed1990

Proteins encoded by the bovine papillomavirus E1 open reading frame: expression in heterologous systems and in virally transformed cells.

The E1 open reading frame (ORF) of bovine papillomavirus type 1 is required for the persistence of viral genomes as multicopy plasmid molecules in transformed rodent fibroblasts. E1 has been reported to contain two separate complementation groups (M and R, corresponding to N- and C-terminal domains, respectively) which regulate viral replication. However, E1 behaves as a single gene with respect to cell transformation and viral transcription. We examined the proteins translated from the entire ORF by using three antisera raised against E1 peptide or bacterial fusion proteins. The capacity of the whole ORF to encode a 72-kDa protein was demonstrated by translation of synthetic RNA in a reticulocyte lysate system, by microinjection of RNA into Xenopus oocytes, and by expression in recombinant baculoviruses and vaccinia viruses. In eucaryotic cells, this protein was found to be phosphorylated and targeted to the cell nucleus. In vitro translation also produced shorter peptides, containing only the E1 C-terminal domain, because of internal translation starts on the third and fourth methionine codons within E1 ORF. On the other hand, mammalian cells infected by vaccinia E1 recombinant virus contained additional larger E1 phosphoproteins (transient 85-kDa and stable 88-kDa species), likely representing processed forms of the 72-kDa species. The E1 72-kDa nuclear phosphoprotein was detected in bovine papillomavirus type 1-transformed cells. We report the biochemical characteristics of full-sized and truncated E1 proteins: (i) the C-terminal half of E1 ORF contains a phosphorylation site(s); (ii) the full-sized E1, but not the C-terminal protein, binds DNA, without indication for recognition of defined sequences, and critical determinants for this activity are likely confined to an N-terminal domain of the protein; (iii) covalent affinity labeling experiments performed on vaccinia virus-encoded E1 proteins with an ATP analog confirmed our previous observation of sequence similarities between the E1 C-terminal domain and the ATPase domain of simian virus 40 large T antigen.

Animals↗