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PubMed2003

An archaeal homing endonuclease I-PogI cleaves at the insertion site of the neighboring intron, which has no nested open reading frame.

Homing endonucleases (HEs) of the LAGLIDADG family cleave intron/inteinless cognate DNA at, or near, the insertion site (IS) of their own intron/intein. Here, we describe a notable exception to this rule. Two introns, Pog.S1205 (length 32 bp) and Pog.S1213 (664 bp), whose ISs are 8 bp apart, exist within the 16S rRNA gene of the archaeon Pyrobaculum oguniense. Pog.S1213 harbors a nested open reading frame (ORF) encoding a 22 kDa monomeric protein, I-PogI, which contains two LAGLIDADG motifs and has optimal DNA cleavage activity at 90 degrees C. Intriguingly, I-PogI cleaves the Pog.S1205-less substrate DNA in the presence or absence of Pog.S1213. The cleavage site (CS) of I-PogI does not coincide with the IS of Pog.S1213 but with that of Pog.S1205. Thus, I-PogI activity both promotes the homing of its own intron, Pog.S1213, and guarantees co-conversion of the ORF-less intron Pog.S1205.

Amino Acid Motifs↗

PubMed1996

The sequence of 36.8 kb from the left arm of chromosome XIV reveals 24 complete open reading frames: 18 correspond to new genes, one of which encodes a protein similar to the human myotonic dystrophy kinase.

We have determined the complete nucleotide sequence of a 36.8 kb segment from the left arm of chromosome XIV carried by the cosmid 14-11. The sequence encodes the 5' coding region of the PSD1 gene, the 3' coding region of an unknown gene and 24 complete open reading frames, of which 18 correspond to new genes and six (SKO1, SCL41A, YGP1, YCK2, RPC31 and MFA2) have been sequenced previously. Of the 24 new genes, five show significant similarities to sequences present in the databanks. These include elongation factors 2 and the human myotonic dystrophy kinase.

Amino Acid Sequence↗

PubMed1996

Spontaneous and induced mutations in a single open reading frame alter both virulence and avirulence in Xanthomonas campestris pv. vesicatoria avrBs2.

Molecular characterization of the avrBs2 locus from Xanthomonas campestris pv. vesicatoria has revealed that expression of this gene triggers disease resistance in Bs2 pepper (Capsicum annuum) plants and contributes to virulence of the pathogen. Deletion analysis and site-directed mutagenesis established the avrBs2 gene as a 2,190-bp open reading frame encoding a putative 80.1-kDa protein. Two classes of Xanthomonas pathogens evading Bs2 host resistance and displaying reduced fitness were found to be specifically mutated in avrBs2. Members of one class contained a 5-bp insertion, while the second class was distinguished by a divergent 3' region of avrBs2; both mutant classes were complemented in trans by a plasmid-borne copy of avrBs2. A divergent avrBs2 homolog was cloned from the Brassica pathogen X. campestris pv. campestris. The predicted AvrBs2 proteins from the two Xanthomonas pathovars were strongly conserved and had predicted sequence similarity with both Agrobacterium tumefaciens agrocinopine synthase and Escherichia coli UgpQ, two enzymes involved in the synthesis or hydrolysis of phosphodiester linkages. On the basis of homology with agrocinopine synthase and UgpQ and the dual phenotype of avirulence and virulence, several models for the function of AvrBs2 are proposed.

Alleles↗

PubMed2001

A small upstream open reading frame causes inhibition of human major vault protein expression from a ubiquitous mRNA splice variant.

Overexpression of the major vault protein (MVP) has been linked to a multidrug resistance (MDR) phenotype. We describe a ubiquitously expressed MVP mRNA splice variant (long (L)-MVP) differing from the regular isoform (short (S)-MVP) within the 5'-leader. Only L-MVP mRNA contains a small upstream open reading frame which was proven to inhibit in vitro and in vivo MVP expression in cis. L-MVP represented an almost constant portion of total MVP mRNA in diverse normal tissues, but was more variable in malignant cell types. MDR sublines with altered MVP expression displayed changed S-MVP/L-MVP ratios as compared to their drug-sensitive counterparts. Our results suggest alternative splicing as one mechanism for regulation of MVP expression.

5' Untranslated Regions↗

PubMed1999

Translational control by an upstream open reading frame in the HER-2/neu transcript.

Overexpression of the HER-2 (neu, erbB-2) receptor results in cellular transformation and is associated with a variety of human cancers. Multiple mechanisms, including gene amplification and transcriptional, post-transcriptional, and translational controls contribute to the regulation of HER-2 expression. One of the components of these regulatory mechanisms is a short upstream open reading frame (uORF) in the HER-2 mRNA that represses downstream translation in a variety of cell types. Here we explore the mechanism by which this uORF exerts its inhibitory effect. As judged by comparisons of protein and mRNA abundance and by polysomal distribution analyses, the uORF represses translation of the HER-2 cistron or of a heterologous reporter gene. Despite its conservation among mammalian species, the peptide sequence of the uORF is not required for this inhibitory effect. Rather, the majority of ribosomes that load on the HER-2 mRNA most likely translate the uORF and are then unable to reinitiate at the downstream AUG codon, in part due to the short intercistronic spacing. A minority of ribosomes gain access to the HER-2 initiation codon either by leaky scanning past the upstream AUG codon or by reinitiating after having translated the uORF despite the short intercistronic region. These results suggest that the HER-2 uORF controls synthesis of this oncoprotein by limiting ribosomal access to downstream initiation sites.

Animals↗

PubMed1993

Herpes simplex virus type 1 capsid protein, VP21, originates within the UL26 open reading frame.

The goal of experiments reported here was to identify the genes that encode capsid proteins VP21 and VP24 of herpes simplex virus type 1 (HSV-1). Capsids were isolated from infected cells and the proteins were separated by SDS-PAGE. N-terminal amino acid sequence analysis of partial CNBr digestion products, and of intact VP21, showed that it is encoded within the UL26 open reading frame (ORF) of HSV-1 beginning with codon 248 and probably extending to the end of the ORF (codon 635). Similar analysis of digestion products confirmed that VP24 is specified by codons 1 to 247 at the 5' end of the UL26 ORF. Each of the seven known capsid proteins has now been assigned to an ORF.

Amino Acid Sequence↗

PubMed1989

Gramicidin S biosynthesis operon containing the structural genes grsA and grsB has an open reading frame encoding a protein homologous to fatty acid thioesterases.

The DNA sequence of about 5.9 kilobase pairs (kbp) of the gramicidin S biosynthesis operon (grs) was determined. Three open reading frames were identified; the corresponding genes, called grsT, grsA, and grsB, were found to be organized in one transcriptional unit, not two as previously reported (M. Krause and M. A. Marahiel, J. Bacteriol. 170:4669-4674, 1988). The entire nucleotide sequence of grsA, coding for the 126.663-kilodalton gramicidin S synthetase 1, grsT, encoding a 29.191-kilodalton protein of unknown function, and 732 bp of the 5' end of grsB, encoding the gramicidin S synthetase 2, were determined. A single initiation site of transcription 81 bp upstream of the grsT initiation condon GTG was identified by high-resolution S1 mapping studies. The sequence of the grsA gene product showed a high degree of homology to the tyrocidine synthetase 1 (TycA protein), and that of grsT exhibited a significant degree of homology to vertebrate fatty acid thioesterases.

Amino Acid Sequence↗

PubMed1993

Sequences of 3' end of genome and of 5' end of open reading frame 1a of lactate dehydrogenase-elevating virus and common junction motifs between 5' leader and bodies of seven subgenomic mRNAs.

The sequences of the 3'-terminal 3.7 kb of the genome and of a 1.7 kb 5' end cDNA clone of one isolate of lactate dehydrogenase-elevating virus (LDV) are reported. The 3' end sequence encodes six major independent open reading frames (ORFs 2 to 7), which are overlapping by between one and 130 nucleotides. Each ORF is expressed at the 5' end of one of six 3'-coterminal subgenomic mRNAs (mRNAs 2 to 7, respectively; 3.5 to 0.8 kb). The smallest mRNA, mRNA 7, encodes the nucleocapsid protein, VP1; mRNA 6 probably encodes the non-glycosylated envelope protein, VP2; and mRNAs 2 to 5 encode proteins of 26.0K, 21.5K, 19.2K and 22.4K, respectively, each possessing several potential N-glycosylation sites and membrane-spanning segments. About 72% of the LDV genome segment carrying ORFs 2 to 7 exhibits about 50% or higher nucleotide identity with the corresponding genome segment of swine infertility and respiratory syndrome (Lelystad) virus (LV), whereas only limited similarity is observed in discontinuous regions of the same corresponding genome segments of LDV and equine arteritis virus (EAV). EAV and LV belong to the same new group of positive-strand RNA viruses as LDV. One additional subgenomic mRNA of about 4 kb is produced in LDV- but not in EAV- or LV-infected cells. The 5' end of this mRNA (1-1) carries a continuous coding sequence. The N-terminal 80 amino acids of the predicted product exhibit about 50% identity with segments in the ORF 1b proteins of both EAV and LV. These segments are located 117 to 150 amino acids upstream of the C termini of the ORF 1b proteins of these viruses. The 5' end cDNA clone contains part of a 5' leader associated with all seven subgenomic mRNAs and the 5' end of ORF 1a. The junctions between the 5' leader and the bodies of all seven subgenomic mRNAs have been determined. Only a single junction sequence was detected for each mRNA. Linkage occurs between a 5' UAUAACC 3' sequence at the 3' end of the leader and only partially identical segments specified downstream in the genome preceding ORFs 2 to 7. The generated junctions differ for different subgenomic mRNAs but possess the consensus sequence 5' U(A/G)(U/A)AACC 3'. In mRNA 7, the UA in positions 1 and 2 are derived from the leader, but a G in position 2 in mRNAs 1-1, 3 and 4 and an A in position 3 in mRNA 6 seem to be specified by the 3' genomic sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed2000

A rapid phenotypic assay for detection of acyclovir-resistant varicella-zoster virus with mutations in the thymidine kinase open reading frame.

Susceptibility assays by cell culture methods are time-consuming and are particularly difficult to perform with varicella-zoster virus (VZV). To overcome this limitation, we have adapted a functional test of the viral thymidine kinase (TK) in TK-deficient (tdk mutant) bacteria to detect ACV-resistant VZV in clinical samples. After PCR amplification, the complete viral TK open reading frame (ORF) is purified from PCR primers, digested with two restriction enzymes, and ligated in an oriented fashion into a bacterial expression vector. The ligation products are then used to transform tdk mutant bacteria. After transformation, an aliquot of the bacteria is plated onto a plate with minimal medium containing (i) ampicillin to select for plasmids carrying the viral TK ORF and (ii) isopropyl beta-D-thiogalactopyranoside (IPTG) to induce its expression. An identical aliquot of bacteria is also plated onto a medium containing, in addition to the components described above, 5-fluorodeoxyuridine (FUdR). Compared to the number of transformants on FUdR-free medium, the number of colonies carrying TK derived from susceptible strains was reduced by 86%, on average, in the presence of FUdR. In contrast, the number of transformants carrying TK from resistant strains with a mutant TK were reduced by only 4%, on average, on FUdR-containing plates. We have assessed the validity of this assay with cell culture isolates and several clinical samples including two cerebrospinal fluid samples from which no virus could be isolated. This colony reduction assay allowed the correct identification of the TK phenotype of each VZV isolate tested and can be completed within 3 days of receipt of the sample.

Acyclovir↗

PubMed1993

Characterization of a temperature-sensitive mutant of the UL15 open reading frame of herpes simplex virus 1.

The UL15 gene of herpes simplex virus 1 consists of two exons and is highly conserved among the herpesviruses sequenced to date. Other than its homology to a phage protein involved in the packaging of DNA, nothing is known of its function. This report concerns the isolation of a temperature-sensitive mutant with a mutation mapping in the UL15 open reading frame. Cells infected with the parent, mutant, and rescued viruses all make DNA at the nonpermissive temperature. Direct analyses of the DNA and electron microscopic studies indicate that although viral DNA is made, it is not packaged into capsids present in nuclei. These studies suggest that UL15 may be involved in the packaging of viral DNA.

Animals↗

PubMed1991

A superantigen encoded in the open reading frame of the 3' long terminal repeat of mouse mammary tumour virus.

Mice express a collection of superantigens, which bind to class II major histocompatibility proteins and interact with T cells bearing particular V beta chains as part of their alpha beta receptors. These superantigens have been suggested to be encoded by exogenous or endogenous mouse mammary tumour viruses. One such superantigen is now shown to be encoded in the open reading frame of the long terminal repeat of a mammary tumour virus, a gene of previously unknown function.

Amino Acid Sequence↗

PubMed2003

Simultaneous cloning of open reading frames into several different expression vectors.

Genomic sequencing has enabled the prediction of thousands of genes, most of which either cannot be assigned a function or can be only broadly categorized on the basis of sequence alone. High-throughput strategies for elucidating protein function are of high priority, and numerous approaches are being developed. Many of these approaches require the cloning of open reading frames (ORFs) into expression vectors that enable the encoded proteins to be tested for biological and biochemical activities. Typically, more than one type of vector must be employed, as different experiments require different conditions of protein production. Here we show that it is possible to simultaneously transfer a single ORF from a source vector to four target vectors using a commercially available in vitro recombination system. To test the approach, we constructed new vectors for expression of fusion proteins in yeast, including vectors for the LexA two-hybrid system. We show that individual ORFs can be efficiently transferred to four different vectors in a single in vitro reaction. The resulting expression plasmids can be separated using prototrophic markers specific to each vector. Using this system to produce multiple expression constructs simultaneously could greatly facilitate high-throughput subcloning and proteomic studies.

Bacterial Proteins↗

PubMed1991

The MAT locus revisited within a 9.8 kb fragment of chromosome III containing BUD5 and two new open reading frames.

This paper reports the DNA sequence of a segment of 9.8 kb of the chromosome III. The sequenced DNA contains the MAT alpha locus. The new sequence of the MAT alpha locus differs from the previously reported sequence by six modifications in the W segment. We have found the same modifications in the HML locus. The corrected sequence contains, in HML, an open reading frame (ORF) of 190 codons which ends at the border between the W segment and the flanking DNA. In the MAT locus, this ORF extends in the flanking DNA up to 538 codons. This ORF corresponds to a gene independently identified as BUD5 (Chant et al., 1991). This gene presents homologies with the exchange factors SDC25 and CDC25. A large ORF of 1399 codons is found on the opposite side of MAT alpha (toward the telomere). This ORF corresponds to a new gene YCR724. Next to this gene is a small ORF, YCR725, of 127 codons. The localization of this fragment on chromosome III, originally supposed to be distal from the MAT locus based on genetic distance, illustrates variation in recombination frequency along the chromosome and suggests the existence of hot spots of recombination between MAT and the THR4 locus.

Base Sequence↗

PubMed2002

An upstream open reading frame impedes translation of the huntingtin gene.

Expansion of a CAG tract within the huntingtin gene, leading to the production of a protein with an expanded polyglutamine tract, is responsible for Huntington's disease. We show here that the 5' untranslated region (UTR) of the huntingtin gene plays an important role in controlling the synthesis of huntingtin. In particular, the 5' UTR contains an upstream open reading frame (uORF) encoding a 21 amino acid peptide. We demonstrate that the presence of this uORF negatively influences expression from the huntingtin mRNA. Our results suggest a role for the uORF in limiting ribosomal access to downstream initiation sites. Mechanisms involving the post-transcriptional regulation of huntingtin are not well understood, and this may be an important way of regulating huntingtin protein levels.

5' Untranslated Regions↗

PubMed1998

Comparative sequence analysis of open reading frames 2 to 7 of the modified live vaccine virus and other North American isolates of the porcine reproductive and respiratory syndrome virus.

To elucidate changes associated with the attenuated virulence in a modified live porcine reproductive and respiratory syndrome (PRRS) vaccine (Boehringer Ingelheim Animal Health, St. Joseph, MO), derived from an American prototype ATCC virus VR-2332, nucleotide sequence of 3' genome covering open reading frames (ORFs) 2 to 7 coding regions from the vaccine virus was determined by RT-PCR with two overlapping fragments. Comparisons showed 98 base changes (94 substitutions, 3 deletions, and 1 addition) out of 3318 nucleotides between the vaccine virus and its parental virus. There were 15, 26, 17, 29, 9, and 6 base substitutions in ORFs 2, 3, 4, 5, 6, and 7, respectively, resulting in 5, 13, 8, 13, 2, and 3 amino acid (a.a.) substitutions in their deduced proteins, respectively. Most of these a.a. substitutions were also present in 17 known virulent/wild type PRRS virus isolates from North America. However, there were 1, 4, 1, and 1 unique a.a. substitutions in the vaccine virus ORFs 2, 3, 4, and 5 deduced proteins, respectively. These unique amino substitutions may be responsible for the attenuated virulence in the vaccine virus.

Amino Acid Sequence↗

PubMed1995

The relationship between eukaryotic translation and mRNA stability. A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNA degradation in the yeast Saccharomyces cerevisiae.

A new strategy was developed to study the relationship between the translation and degradation of a specific mRNA in the yeast Saccharomyces cerevisiae. A series of 5'-untranslated regions (UTR) was combined with the cat gene from the bacterial transposon Tn9, allowing us to test the influence of upstream open reading frames (uORFs) on translation and mRNA stability. The 5'-UTR sequences were designed so that the minimum possible sequence alteration, a single nucleotide substitution, could be used to create a 7-codon ORF upstream of the cat gene. The uORF was translated efficiently, but at the same time inhibited translation of the cat ORF and destabilized the cat mRNA. Investigations of various derivatives of the 5'-UTR indicated that cat translation was primarily attributable to leaky scanning of ribosomes past the uORF rather than to reinitiation. Therefore, these data directly demonstrate destabilization of a specific mRNA linked to changes in translational initiation on the same transcript. In contrast to the previously proposed nonsense-mediated mRNA decay pathway, destabilization was not triggered by premature translational termination in the main ORF and was not discernibly dependent upon a reinitiation-driven mechanism. This suggests the existence of an as yet not described pathway of translation-linked mRNA degradation.

Base Sequence↗

PubMed1998

Nineteen baculovirus open reading frames, including LEF-12, support late gene expression.

A set of 18 plasmid subclones of the Autographa californica nuclear polyhedrosis virus genome, each containing an identified late expression factor gene (lef), supports expression from a late viral promoter in transient expression assays in the SF-21 cell line derived from Spodoptera frugiperda. We have constructed a further set of plasmids in which each lef open reading frame (ORF) is controlled by the Drosophila melanogaster heat shock protein 70 (hsp70) promoter and epitope tagged. Failure of this set of plasmids to support transient late gene expression, and the inability of the p47 ORF to replace the p47-containing plasmid supplied in the lef plasmid library, led to the identification of a 19th late expression factor gene (lef-12) located adjacent to the p47 gene. The sequence of lef-12 is predicted to encode a protein of 21 kDa with no homology to any previously identified protein. The set of 19 hsp70-controlled lef ORFs (HSEpiHis lef library) supports transient expression from a late viral promoter. lef-12 did not affect expression from an early baculovirus promoter. In TN-368 cells, which are also permissive for virus replication, lef-12 provided a stimulatory effect but did not appear to be essential.

Amino Acid Sequence↗

PubMed1994

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene.

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.

Amino Acid Sequence↗