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At least 145 records · Page 8Linked to original sources
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↗
PubMed2000

A non-stop antisense reading frame in the grp78 gene of Neurospora crassa is homologous to the Achlya klebsiana NAD-gdh gene but is not being transcribed.

A long non-stop reading frame exists on the antisense strand of the grp78 gene (cDNA and genomic DNA) of Neurospora crassa. Computer analysis revealed a strong similarity of the putative antisense protein to the 10th exon of the NAD-dependent glutamate dehydrogenase gene (NAD-gdh) of Achlya klebsiana, which is itself located on the complementary strand of a transcribed hsc70 gene homologue. In Neurospora, no grp78 antisense mRNA was detected by Northern blot and reverse transcription-coupled polymerase chain reaction analyses, indicating that this long reading frame is not being transcribed. Hypotheses for the presence of such unexpressed non-stop reading frames are discussed.

Blotting, Northern↗
PubMed1992

Partite expression of the bovine papillomavirus E1 open reading frame in Escherichia coli.

Six recombinants were constructed which expressed portions of the bovine papillomavirus E1 open reading frame as OmpF/E1/beta-galactosidase tribrid fusion proteins in Escherichia coli. Rabbit sera containing E1-specific antibodies were generated against five of these six fusion proteins (which together constitute 74% of the full-length E1 open reading frame). The individual fusion proteins and their cognate antisera will be useful reagents for defining the structure and function of the BPV E1 protein(s).

Bacterial Outer Membrane Proteins↗
PubMed2006

Simultaneous high-throughput recombinational cloning of open reading frames in closed and open configurations.

Comprehensive open reading frame (ORF) clone collections, ORFeomes, are key components of functional genomics projects. When recombinational cloning systems are used to capture ORFs in master clones, these DNA sequences can be easily transferred into a variety of expression plasmids, each designed for a specific assay. Depending on downstream applications, an ORF is cloned either with or without a stop codon at its original position, referred to as closed or open configuration, respectively. The former is preferred when the encoded protein is produced in its native form or with an amino-terminal tag; the latter is obligatory when the protein is produced as a fusion with a carboxyl-terminal tag. We developed a streamlined protocol for high-throughput, simultaneous cloning of both open and closed ORF entry clones with the Gateway recombinational cloning system. The protocol is straightforward to set up in large-scale ORF cloning projects, and is cost-effective, because the initial ORF amplification and the cloning in a pDONR vector are performed only once to obtain the two ORF configurations. We illustrated its implementation for the isolation and validation of 346 Arabidopsis ORF entry clones.

Arabidopsis↗
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↗
PubMed1997

RNA processing alters open reading frame stoichiometry from the large ATP synthase gene cluster of spinach chloroplasts.

The large ATP synthase gene cluster of spinach chloroplasts is a multigenic cluster that encodes the small ribosomal subunit 2 followed by four ATP synthase subunits. The stoichiometry of the ATP synthase gene products from this cluster changes markedly between transcription and assembly of the complex. The two primary transcripts from this gene cluster undergo a complex series of RNA processing steps. Here we show that the extensive RNA processing that the large ATP synthase gene cluster transcripts undergo results in a substantial change in the stoichiometry of complete open reading frames (ORFs) of the four ATP synthase genes. Processing directly affects the stoichiometry of open reading frames from this gene cluster by intragenic cleavage. It may also affect open reading frame stoichiometry more indirectly, but equally significantly, by cleavage-induced alteration of stability of some of the processed transcripts relative to the others.

Chloroplasts↗
PubMed1996

A leader open reading frame is essential for the expression in Escherichia coli of GC-rich leuB gene of an extreme thermophile, Thermus thermophilus.

To improve expression efficiency of the leuB gene of an extreme thermophile. Thermus thermophilus, in Escherichia coli, the gene was placed under a potent promoter, tac. However, the expression was hardly improved, despite increased transcription. The expression under tac promoter was significantly improved by introducing a leader open reading frame in front of the gene. Similar improvement under a weak promoter, tet, with a leader open reading frame had been described previously. The present results provide evidence that the major limiting step in the expression of a GC-rich thermophile gene in E. coli is translation, and that the addition of a leader open reading frame is more crucial for high level expression of the gene than the use of a potent promoter.

Antigens, Differentiation↗
PubMed1992

Role of an upstream open reading frame in the translation of polycistronic mRNAs in plant cells.

The influence of an upstream small open reading frame (URF) on the translation of two consecutive coding regions on an eukaryotic mRNA was studied. The cis effects of leader length, URF length, the sequences of the URF and neighboring regions, and the trans effects of the Cauliflower mosaic virus transactivator (TAV) were analyzed. Translation efficiency of the immediate downstream open reading frame (ORF) decreased with increasing URF length. Short URFs did not drastically inhibit translation of immediate downstream ORFs but supported far downstream translation in the presence of TAV. In the latter case, the optimal URF length was 30 codons.

Base Sequence↗
PubMed2001

Evidence for a new hepatitis C virus antigen encoded in an overlapping reading frame.

Many viruses have overlapping genes and/or regions in which a nucleic acid signal is embedded in a coding sequence. To search for dual-use regions in the hepatitis C virus (HCV), we developed a facile computer-based sequence analysis method to map dual-use regions in coding sequences. Eight diverse full-length HCV RNA and polyprotein sequences were aligned and analyzed. A cluster of unusually conserved synonymous codons was found in the core-encoding region, indicating a potential overlapping open reading frame (ORF). Four peptides (A1, A2, A3, and A4) representing this alternate reading frame protein (ARFP), two others from the HCV core protein, and one from bovine serum albumin (BSA) were conjugated to BSA and used in western blots to test sera for specific antibodies from 100 chronic HCV patients, 44 healthy controls, and 60 patients with non-HCV liver disease. At a 1:20,000 dilution, specific IgGs to three of the four ARFP peptides were detected in chronic HCV sera. Reactivity to either the A1 or A3 peptides (both ARFP derived) was significantly associated with chronic HCV infection, when compared to non-HCV liver disease serum samples (10/100 versus 1/60; p < 0.025). Antibodies to A4 were not detected in any serum sample. Our western blot assays confirmed the presence of specific antibodies to a new HCV antigen encoded, at least in part, in an alternate reading frame (ARF) overlapping the core-encoding region. Because this novel HCV protein stimulates specific immune responses, it has potential value in diagnostic tests and as a component of vaccines. This protein is predicted to be highly basic and may play a role in HCV replication, pathogenesis, and carcinogenesis.

Alternative Splicing↗
PubMed1995

Sequence and functional analysis of a 7.2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II.

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79.2 kDa (corresponding gene YBR156c), 12.1 kDa (YBR157c), 62.7 kDa (YBR158w) and 38.7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grrl (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains.

Amino Acid Sequence↗
PubMed1995

Varicella-zoster virus open reading frame 1 encodes a membrane protein that is dispensable for growth of VZV in vitro.

Varicella-zoster virus (VZV) encodes 69 unique open reading frames, 5 of which do not have herpes simplex virus type 1 (HSV-1) homologs. One of the 5, ORF1, is predicted to encode a protein of 108 amino acids. We identified a 470-base RNA corresponding to ORF1. To determine whether ORF1 encodes a protein, an 11-amino-acid epitope was inserted in frame after the ninth codon of the ORF1 open reading frame. A recombinant virus carrying this epitope expressed a protein that was immunoprecipitated with monoclonal antibody to the epitope. The ORF1 protein was detected in the membrane of infected cells. The size of ORF1 protein expressed in VZV-infected cells was slightly larger than the size expressed by translation in vitro, suggesting that the protein may undergo post-translational modification in infected cells. Insertion of stop codons immediately before the epitope in the ORF1 gene resulted in a recombinant virus that did not express ORF1 protein and that was not growth impaired in cell culture. Thus, ORF1 encodes a protein that localizes to the membrane of VZV-infected cells and that is dispensable for virus growth in vitro.

Antibodies, Monoclonal↗
PubMed1994

Evidence for a group II intron in Escherichia coli inserted into a highly conserved reading frame associated with mobile DNA sequences.

The distribution of group II introns in the living world is an important aspect of the hypothesis which postulates their evolutionary relation to the nuclear spliceosome. As an alternative to the restricted experimental approaches towards their identification we devised a strategy to recognize group II introns in sequence data. By this approach we identified a locus on a plasmid in the bacterium Escherichia coli. Modelling of the derived RNA secondary structure reveals the presence of perfectly conserved domains V and VI as typical features of group II introns. An intron internal reading frame upstream of domain V is homologous to group II intron encoded maturases. A reading frame downstream of the predicted 3'-splice site is highly similar to a small polypeptide encoded in the central part of the Agrobacterium tumefaciens T-DNA. With the TBLASTN algorithm a set of plasmid-borne insertion sequences in Agrobacteria and Rhizobia and surprisingly also in a Yersinia pseudotuberculosis strain was identified which contain this highly conserved reading frame.

Amino Acid Sequence↗
PubMed1993

Chicken repeat 1 elements contain a pol-like open reading frame and belong to the non-long terminal repeat class of retrotransposons.

Chicken genomes contain approximately 30,000 chicken repeat 1 (CR1) elements scattered among single-copy sequences, but no information has yet been presented to account for how these elements could have dispersed. The fact that CR1 elements have common (although atypical) 3' ends and variable 5' truncations suggested to us that they might belong to the class of non-long terminal repeat retrotransposons that encode reverse transcriptases. From an analysis of unusually large CR1 elements, we now provide evidence for the presence of such a reverse transcriptase open reading frame. CR1 elements are distantly related to previously described non-long terminal repeat retrotransposons; however, we find that frog and torpedo ray genomes contain dispersed open reading frame segments that have > 50% identity to the CR1 open reading frame. This result suggests that CR1-like elements exist in several vertebrate classes that have evolved independently for approximately 400 million years.

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↗
PubMed2006

A peptide-loaded dendritic cell based cytotoxic T-lymphocyte (CTL) vaccination strategy using peptides that span SIV Tat, Rev, and Env overlapping reading frames.

CTL based vaccine strategies in the macaque model of AIDS have shown promise in slowing the progression to disease. However, rapid CTL escape viruses can emerge rendering such vaccination useless. We hypothesized that such escape is made more difficult if the immunizing CTL epitope falls within a region of the virus that has a high density of overlapping reading frames which encode several viral proteins. To test this hypothesis, we immunized macaques using a peptide-loaded dendritic cell approach employing epitopes in the second coding exon of SIV Tat which spans reading frames for both Env and Rev. We report here that autologous dendritic cells, loaded with SIV peptides from Tat, Rev, and Env, induced a distinct cellular immune response measurable ex vivo. However, conclusive in vivo control of a challenge inoculation of SIVmac239 was not observed suggesting that CTL epitopes within densely overlapping reading frames are also subject to escape mutations.

Animals↗
PubMed1985

Polymorphism of the 3' open reading frame of the virus associated with the acquired immune deficiency syndrome, human T-lymphotropic virus type III.

The genome of the virus associated with the acquired immune deficiency syndrome (AIDS), human T-lymphotropic virus type III (HTLV-III), includes two open reading frames, not found in other retroviruses. One of these, designated 3' open reading frame (3'orf) is 648 base pairs (bp) in length, and overlaps with the 3' long terminal repeat (LTR) sequences. Sequences of additional HTLV-III clones were determined in order to estimate the level and location of variation within 3'orf, to gain some insight into the function of its protein product. Newly determined sequences are reported for 3'orf of two unintegrated clones of HTLV-III and three cDNA clones made from virion RNA derived from the same cell line infected with pooled blood samples of different patients with AIDS or AIDS-related complex symptoms (ARC). In addition, sequences for 3'orf were derived from an unintegrated viral clone derived from a different cell line infected with a distinct isolate from a single patient. These sequences are compared to those previously reported for six other viral clones. Sequences of 3'orf differ among clones by 1.1-10.4% bp and 2.4-17.0% of predicted amino acids. This represents significantly greater sequence variation than is found in the entire genome on average. Moreover, a functional proviral clone has a termination codon at amino acid residue 124 of this open reading frame. This raises questions concerning the structure, and regulation of expression of the protein encoded by 3'orf.

Acquired Immunodeficiency Syndrome↗