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At least 991 records · Page 55Linked to original sources
PubMed1990

Functional analysis of a 603 nucleotide open reading frame upstream of the polyhedrin gene of Autographa californica nuclear polyhedrosis virus.

The sequence of the 2000 nucleotides immediately upstream of the polyhedrin gene of the Autographa californica multiple nucleocapsid nuclear polyhedrosis virus has been determined. Comparative analysis of the data identified a 603 nucleotide open reading frame (ORF) separated from the polyhedrin gene coding sequences by 156 nucleotides and in the opposite strand of DNA. Northern hybridization analysis of polyadenylated RNA from infected cells highlighted a 3.7 kb species produced maximally at 12 h post-infection, but not in the presence of cycloheximide. Preliminary nuclease S1 analysis of the 5' end of this RNA suggested that it initiated at a position very close to that of the polyhedrin mRNA start site. Deletion of a portion of the ORF 603 from viruses containing the normal polyhedrin gene and the lacZ gene in lieu of polyhedrin did not affect replication in cell culture or the production of beta-galactosidase protein. A virus which lacked the ORF 603 gene but produced polyhedrin had similar infectivity in Trichoplusia ni larvae compared to the wild-type virus. The chloramphenicol acetyltransferase (CAT) gene was also inserted in lieu of the ORF 603 in a virus containing the lacZ gene instead of the polyhedrin (Ac.CAT.lacZ). Analysis of CAT expression revealed that a maximum level was reached at 16 h p.i. and that transcription was initiated in Ac.CAT.lacZ at the same site as for the normal gene.

Amino Acid Sequence↗
PubMed1989

Open reading frame 5 (ORF5), encoding a ferredoxinlike protein, and nifQ are cotranscribed with nifE, nifN, nifX, and ORF4 in Rhodobacter capsulatus.

DNA sequence analysis of a 1,600-base-pair fragment located downstream of nifENX in nif region A of Rhodobacter capsulatus revealed two additional open reading frames (ORFs): ORF5, encoding a ferredoxinlike protein, and nifQ. The ferredoxinlike gene product contained two cysteine motifs, typical of ferredoxins coordinating two 4Fe-4S clusters, but the distance between these two motifs was unusual for low-molecular-weight ferredoxins. The R. capsulatus nifQ gene product shared a high degree of homology with Klebsiella pneumoniae and Azotobacter vinelandii NifQ, including a typical cysteine motif located in the C-terminal part. nifQ insertion mutants and also an ORF5-nifQ double deletion mutant showed normal diazotrophic growth only in the presence of high concentrations of molybdate. This demonstrated that the gene encoding the ferredoxinlike protein is not essential for nitrogen fixation. No NifA-activated consensus promoter could be found in the intergenic region between nifENX-ORF4 and ORF5-nifQ. Analyses of a nifQ-lacZYA fusion revealed that transcription of nifQ was initiated at a promoter in front of nifE. In contrast to other nitrogen-fixing organisms, R. capsulatus nifE, nifN, nifX, ORF4, ORF5, and nifQ were organized in one transcriptional unit.

Amino Acid Sequence↗
PubMed1989

The mitochondrial plasmid from Neurospora intermedia strain Labelle-1b contains a long open reading frame with blocks of amino acids characteristic of reverse transcriptases and related proteins.

We have determined the DNA sequence of the 4070 base pair mitochondrial plasmid from the Labelle-1b strain of Neurospora intermedia. Analysis of the sequence revealed that the plasmid contains a long open reading frame (ORF) that could encode a protein of up to 1151 amino acids. Codon usage in the long ORF shows no clear relationship to Neurospora mitochondrial genes, nuclear genes, nor to the ORF of a different Neurospora mitochondrial plasmid. The long ORF contains regions of similarity to yeast mitochondrial RNA polymerase as well as blocks of amino acids that are characteristic of reverse transcriptases and the ORFs of certain group II mtDNA introns (Michel and Lang, (1985) Nature 316,641). The plasmid gives rise to specific transcripts, some of which may be unit length, and which carry the information for expression of the long ORF. The genetic organization and content of the plasmid suggest that it is related to mobile genetic elements.

Amino Acid Sequence↗
PubMed1990

The primary structure and expression of the second open reading frame of the polymerase gene of the coronavirus MHV-A59; a highly conserved polymerase is expressed by an efficient ribosomal frameshifting mechanism.

Sequence analysis of a substantial part of the polymerase gene of the murine coronavirus MHV-A59 revealed the 3' end of an open reading frame (ORF1a) overlapping with a large ORF (ORF1b; 2733 amino acids) which covers the 3' half of the polymerase gene. The expression of ORF1b occurs by a ribosomal frameshifting mechanism since the ORF1a/ORF1b overlapping nucleotide sequence is capable of inducing ribosomal frameshifting in vitro as well as in vivo. A stem-loop structure and a pseudoknot are predicted in the nucleotide sequence involved in ribosomal frameshifting. Comparison of the predicted amino acid sequence of MHV ORF1b with the amino acid sequence deduced from the corresponding gene of the avian coronavirus IBV demonstrated that in contrast to the other viral genes this ORF is extremely conserved. Detailed analysis of the predicted amino acid sequence revealed sequence elements which are conserved in many DNA and RNA polymerases.

Amino Acid Sequence↗
PubMed1989

Expression in Escherichia coli of seven DNA fragments comprising the complete L1 and L2 open reading frames of human papillomavirus type 6b and localization of the 'common antigen' region.

Molecular cloning was used to express human papillomavirus type 6b (HPV-6b) antigens in Escherichia coli. Seven genomic DNA fragments of HPV-6b which together comprise the complete L1 and L2 open reading frames, known to code for capsid proteins, were cloned and expressed in E. coli as both beta-galactosidase and TrpE fusion proteins. Western blots of HPV-6b beta-galactosidase fusion proteins using 'genus-specific' antisera produced by immunization of rabbits with disrupted bovine papillomavirus type 1 (BPV-1) showed that polypeptides encoded by two DNA fragments from the mid portion of L1 of HPV-6b were cross-reactive. Only one of these two polypeptides reacted with antisera raised against disrupted HPV-1, directly demonstrating that this polypeptide contains the papillomavirus 'common antigen'. The cross-reactive region was confirmed by reversing antigen and antibody. Polyclonal antisera were raised against the seven HPV-6b beta-galactosidase fusion proteins and tested against BPV-1 virion proteins on Western blots. Only antiserum against the mid portion of L1 of HPV-6b reacted with the BPV-1 major capsid protein. HPV-6b fusion proteins were also used to test human sera for antibodies reactive in Western blots. Serum samples from 38 patients with documented HPV-6 infections and from 22 presumably uninfected controls were tested. Antibodies were not detected in any of the sera to any of the seven fusion proteins. HPV-6b beta-galactosidase fusion proteins are antigenic and can be used on Western blots to localize immunologically reactive sub-regions of proteins by reacting protein fragments with antisera from immunized animals. However, alternative methods will be required to detect anti-HPV antibodies in human sera.

Adult↗
PubMed2005

An Sp1 response element in the Kaposi's sarcoma-associated herpesvirus open reading frame 50 promoter mediates lytic cycle induction by butyrate.

Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.

Base Sequence↗
PubMed1989

Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames.

Intron mobility in the T-even phages has been demonstrated. Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB. Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF). Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology. The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration. A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance. These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes.

Cloning, Molecular↗
PubMed1996

Sequence analysis of a 14.2 kb fragment of Saccharomyces cerevisiae chromosome XIV that includes the ypt53, tRNALeu and gsr m2 genes and four new open reading frames.

As part of the EU yeast genome program, a fragment of 14,262 bp from the left arm of Saccharomyces cerevisiae chromosome XIV has been sequenced. This fragment corresponds to cosmid 14-14b and is located roughly 130 kb from the centromere. It contains four new open reading frames which encode potential proteins of more than 99 amino acids, as well as the ypt53, tRNALeu and gsr moffenes. The putative protein N2212 is similar to the ribosomal protein S7 from humans. N2215 contains several predicted transmembrane elements. N2231 contains regions which are rich in acidic, as well as basic, residues which could from alpha-helical structures. Similar regions are found in a variety of proteins including glutamic acid rich protein, trichohyalin, caldesmon, Tb-29 and several cytoskeleton-interacting proteins.

Amino Acid Sequence↗
PubMed1994

Isolation of a maize cDNA encoding a protein with extensive similarity to an inhibitor of protein kinase C and a cyanobacterial open reading frame.

A full-length cDNA clone, Mz2-12, with a predicted amino acid sequence showing extensive similarity to the sequence of a protein inhibitor of protein kinase C, purified from bovine brain, has been isolated from maize. The sequence of Mz2-12 is also similar to an open reading frame of unknown function on a cyanobacterial dicistronic message. The extensive similarity of the three protein sequences identifies a novel class of evolutionarily conserved proteins.

Amino Acid Sequence↗
PubMed1988

An adenovirus mRNA which encodes a 14,700-Mr protein that maps to the last open reading frame of region E3 is expressed during infection.

The E3 regions of adenovirus types 2 and 5, respectively, are known to synthesize proteins of 19,000 Mr (19K) and 11.6K, but information regarding the identity and characterization of other potential E3 proteins encoded by the six remaining open reading frames (ORFs) is lacking. In this study, we show that the last ORF of region E3, which encodes a 14.7K protein, is expressed in adenovirus-infected cells. This information was largely derived from analysis of an E3 deletion mutant (H2dl801) in which an extensive deletion (1,939 base pairs) was found to eliminate all ORFs except for two proteins of 12.5K and 14.7K. The 14.7K protein was translated from RNA isolated from H2dl801-infected cells that had been hybridization selected to E3 DNA; hybridization-selected RNA from wild-type adenovirus type 5-infected cells translated both the 19K and the 14.7K proteins. Moreover, an antiserum directed against a bacterial 14.7K fusion protein (A. E. Tollefson and W. S. M. Wold, J. Virol. 62:33-39, 1988) immunoprecipitated the 14.7K translation product synthesized by wild-type and mutant H2dl801 adenovirus mRNAs.

Adenoviruses, Human↗
PubMed2002

Restoration of a functional open reading frame by homologous recombination between two adenoviral vectors.

In this study, we examined the ability of adenoviral (Ad) vectors to undergo homologous recombination. The lacZ gene was divided between two parental, first-generation vectors such that neither encoded a functional product but both shared 494 bp in common. The open reading frame could only be restored by homologous recombination. We observed beta-galactosidase activity only upon co-infection of both parental vectors and after the onset of viral DNA replication, creating a delay in expression of 24-36 hours in HeLa cells. At peak efficiency, this recombination vector system resulted in beta-galactosidase activity levels 100x above background and just 18x less than a conventional, first-generation vector in HeLa cells. After recombination, the resultant progeny vector genomes containing reconstituted expression cassettes were devoid of all viral genes and contained two packaging signals. These progeny genomes were efficiently packaged, could be separated from their parental vectors based on their lighter buoyant densities in CsCl gradients, and were subsequently used as functional gene transfer vectors. This novel recombination vector system should be useful for transferring large transgenes (because the carrying capacity of two Ad vectors can be exploited) or expressing any cytotoxic or Ad replication inhibitory protein (because the parental vectors exhibit no background expression).

Adenoviridae↗
PubMed1986

Euglena gracilis chloroplast DNA: analysis of a 1.6 kb intron of the psb C gene containing an open reading frame of 458 codons.

We retrieved a 1.6 kbp intron separating two exons of the psb C gene which codes for the 44 kDa reaction center protein of photosystem II. This intron is 3 to 4 times the size of all previously sequenced Euglena gracilis chloroplast introns. It contains an open reading frame of 458 codons potentially coding for a basic protein of 54 kDa of yet unknown function. The intron boundaries follow consensus sequences established for chloroplast introns related to class II and nuclear pre-mRNA introns. Its 3'-terminal segment has structural features similar to class II mitochondrial introns with an invariant base A as possible branch point for lariat formation.

Amino Acid Sequence↗
PubMed1990

Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC7120.

A 1.8 kb transcript corresponding to a region of the Anabaena 7120 chromosome 4 kb downstream of the nifHDK operon appears 12-18 h after heterocyst induction. The DNA corresponding to this transcript was sequenced and found to contain two open reading frames, designated ORF 1 and ORF 2. Two polypeptides, of 30 kDa and 13 kDa, encoded by these ORFs were expressed in Escherichia coli. An apparent start site for the transcript, detected by S1 nuclease protection, was located 42 bp upstream of the ATG start codon of ORF 1. ORF 2 shows strong sequence similarity to ORF 6 in the nif gene region of Azotobacter vinelandii. ORF 1 was interrupted using a 1.4 kb neomycin resistance cassette and the resulting mutant grew very slowly on medium lacking combined nitrogen. The mutant had 45% of wild-type acetylene reduction activity, which could be complemented by a 2.8 kb EcoRI fragment of wild-type Anabaena DNA containing only ORF 1 and ORF 2. Thus, one or both of these ORFs is required for efficient nitrogen fixation in Anabaena.

Amino Acid Sequence↗
PubMed1985

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. IV. Transcription of the oriC region and expression of DNA gyrase genes and other open reading frames.

We have determined nucleotide sequence of some 10,000 base pairs (bp) in the oriC region of the Bacillus subtilis chromosome. Initiation sites of transcription from this region were determined in vivo by the S1-mapping method. Five major initiation sites were found in the leader sequences of five open reading frames (ORF) deduced from the nucleotide sequence. The sixth site is located inside the ORF323("recF"). Putative promoters were found for each transcript. Function of these promoters was demonstrated in Escherichia coli by the Maxi-cell method using appropriate fragments cloned in pBR vectors. Based on these results, genes in 10,000 bp oriC region are divided into 4 transcriptional units. GyrB composes one unit with two other ORFs, while gyrA constitutes a single unit by itself. The promoters for ORF446("dnaA") and ORF378("dnaN") are located within the putative signal sequences for oriC. Transcription from these promoters is dependent on a dna-initiation gene, dnaB.

Bacillus subtilis↗
PubMed2025

High-quality peptide evidence for annotating non-canonical open reading frames as human proteins.

A major scientific drive is to characterize the protein-coding genome as it provides the primary basis for the study of human health. But the fundamental question remains: what has been missed in prior genomic analyses? Over the past decade, the translation of non-canonical open reading frames (ncORFs) has been observed across human cell types and disease states, with major implications for proteomics, genomics, and clinical science. However, the impact of ncORFs has been limited by the absence of a large-scale understanding of their contribution to the human proteome. Here, we report the collaborative efforts of stakeholders in proteomics, immunopeptidomics, Ribo-seq ORF discovery, and gene annotation, to produce a consensus landscape of protein-level evidence for ncORFs. We show that at least 25% of a set of 7,264 ncORFs give rise to translated gene products, yielding over 3,000 peptides in a pan-proteome analysis encompassing 3.8 billion mass spectra from 95,520 experiments. With these data, we developed an annotation framework for ncORFs and created public tools for researchers through GENCODE and PeptideAtlas. This work will provide a platform to advance ncORF-derived proteins in biomedical discovery and, beyond humans, diverse animals and plants where ncORFs are similarly observed.

GENCODE↗
PubMed1984

Genes for alkaline protease and neutral protease from Bacillus amyloliquefaciens contain a large open reading frame between the regions coding for signal sequence and mature protein.

The genes for alkaline protease (apr[BamP]) and neutral protease (npr[BamP]) from Bacillus amyloliquefaciens have been isolated and expressed in Bacillus subtilis. The DNA sequences of apr[BamP] and npr[BamP] revealed, in each case, the presence of a large open reading frame. The inferred amino acid sequence of either gene contained a signal sequence and an additional polypeptide sequence ('pro' sequence) preceding the mature protein. Based on DNA sequence, the start point of translation has been identified as amino acid residue - 107 for apr[BamP] and -221 for npr[BamP]. To demonstrate that the start point of translation of apr[BamP] in vivo is probably at codon -107, codon -103 (AAA) was changed to an ochre (TAA) by site-directed mutagenesis. Alkaline protease was produced from this ochre mutant derivative of apr[BamP] only when the host strain was Su+. The presence of a pro sequence may be common to all of the secreted proteases from bacilli.

Amino Acid Sequence↗
PubMed1989

The double-stranded RNA genome of yeast virus L-A encodes its own putative RNA polymerase by fusing two open reading frames.

The L-A double-stranded RNA virus of Saccharomyces cerevisiae encodes its major coat protein (80 kDa) and a minor single-stranded RNA binding protein (180 kDa) that has immunological cross-reactivity with the major coat protein. The sequence of L-A cDNA clones revealed two open reading frames (ORF), ORF1 and ORF2. These two reading frames overlap by 130 base pairs and ORF2 is in the -1 reading frame with respect to ORF1. Although the major coat protein of the viral particles is encoded by ORF1, the 180-kDa protein is derived from the entire double-stranded RNA genome by fusing ORF1 and ORF2, probably by a -1 translational frameshift. Within the overlapping region is a sequence similar to that producing a -1 frameshift by "simultaneous slippage" in retroviruses. The coding sequence of ORF2 shows a pattern characteristic of viral RNA-dependent RNA polymerases of icosahedral (+)-strand RNA viruses. Thus, the 180-kDa protein is analogous to gag-pol fusion proteins.

Amino Acid Sequence↗
PubMed2000

Nuclear accumulation of IE62, the varicella-zoster virus (VZV) major transcriptional regulatory protein, is inhibited by phosphorylation mediated by the VZV open reading frame 66 protein kinase.

IE62, the major transcriptional activator protein encoded by varicella-zoster virus (VZV), locates to the nucleus when expressed in transfected cells. We show here that cytoplasmic forms of IE62 accumulate in transfected and VZV-infected cells as the result of the protein kinase activity associated with VZV open reading frame 66 (ORF66). Expression of the ORF66 protein kinase but not the VZV ORF47 protein kinase impaired the ability of coexpressed IE62 to transactivate promoter-reporter constructs. IE62 that was coexpressed with the ORF66 protein accumulated predominantly in the cytoplasm, whereas the normal nuclear localization of other proteins was not affected by the ORF66 protein. In cells infected with VZV, IE62 accumulated in the cytoplasm at late times of infection, whereas in cells infected with a VZV recombinant unable to express ORF66 protein (ROka66S), IE62 was completely nuclear. Point mutations introduced into the predicted serine/threonine catalytic domain and ATP binding domain of ORF66 abrogated its ability to influence IE62 nuclear localization, indicating that the protein kinase activity was required. The region of IE62 that was targeted by ORF66 was mapped to amino acids 602 to 733. IE62 peptides containing this region were specifically phosphorylated in cells coexpressing the ORF66 protein kinase and in cells infected with wild-type VZV but were not phosphorylated in cells infected with ROka66S. We conclude that the ORF66 protein kinase phosphorylates IE62 to induce its cytoplasmic accumulation, most likely by inhibiting IE62 nuclear import.

Amino Acid Sequence↗