Product Of Open Reading Frame Research Articles

Translation products of cauliflower mosaic virus ORF V, the coding region corresponding to the retrovirus pol gene

Open reading frame (ORF) V of cauliflower mosaic virus (CaMV), the candidate for the reverse transcriptase gene, has been expressed in E. coli under control of the PR promoter of bacteriophage lambda either as an N-terminal polypeptide fused to beta-galactosidase or as the total ORF V without fusion. Antibodies against these proteins were used to analyze extracts from CaMV-infected plants by immunoblotting. ORF V-specific polypeptides of 80, 62, 58, 22, and 18 kD apparent molecular weights were detected, with the largest species corresponding to the full length translation product. The 62 and 22 kD species could be assigned to the N-terminus and the remaining two species to the C-terminus of the ORF.

Transcriptional trans-activation by the human papillomavirus type 16 E2 gene product

We identified a conditional transcriptional enhancer in the long control region (LCR) of human papillomavirus type 16 (HPV-16). This conditional enhancer requires activation in trans by a product of the viral early-region open reading frames (ORFs). Primer extension analysis of chloramphenicol acetyltransferase RNA isolated from transiently transfected CV-1 cells demonstrated that trans-activation of the HPV-16 LCR enhancer operated at the transcriptional level. Mutational analysis of the early ORFs demonstrated that the conditional enhancer of the LCR was trans-activated by the product of the E2 ORF. The E2 gene product of bovine papillomavirus type 1, which can trans-activate the conditional enhancer in the bovine papillomavirus type 1 LCR, was also capable of trans-activating the E2-responsive enhancer of HPV-16. The activity of the HPV-16 LCR enhancer was also assayed in two human cervical carcinoma cell lines, HeLa and SiHa, which harbor transcriptionally active, integrated HPV-18 and HPV-16 DNA sequences, respectively. No endogenous E2 or E2-like activity was detected in either cell line.

Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions.

We examined bovine papillomavirus type 1 (BPV-1) DNAs mutated in the E2 open reading frame (ORF) to determine their ability (i) to transform C127 cells and (ii) to remain extrachromosomal in transfected cells. Results obtained with deletion mutants and insertion mutants containing a linker with translational termination codons in all possible reading frames indicated that an E2 ORF gene product(s) is necessary for efficient transformation, as well as viral plasmid replication and maintenance in the context of the full BPV-1 genome. Complementation assays in which mutant BPV-1 DNAs were transfected into cell lines expressing some viral functions from integrated BPV-1 cDNAs demonstrated that the E2 ORF product, when provided in trans, could allow BPV-1 E2 mutants to remain extrachromosomal. The E2 function could also augment transformation of some, but not all, BPV-1 E2 mutants, allowing identification of another region of BPV-1 involved in cellular transformation. It is likely that the role of the BPV-1 E2 product(s) in transformation and plasmid maintenance is indirect. A BPV-1 mutant altered in the E5 ORF is transformation defective and unable to replicate as a stable plasmid in C127 cells.

Expression of the mitochondrial split gene coding for cytochrome oxidase subunit I in S. cerevisiae: RNA splicing pathway.

We have studied the splicing pathway leading to the synthesis of cytochrome oxidase subunit I (COX I) mRNA, by analysing the transcription pattern of several oxi3- splicing deficient mutants located in the first four introns of the gene. The four introns contain long open reading frames (ORFs) in phase with the upstream exons. All the mutations block the excision of the mutated intervening sequence (IVS) from the pre-mRNA, and accumulate characteristic novel polypeptides of sizes which could correspond to the translation products of the intron's ORF. Most of the mutations do not affect the splicing of the following intervening sequences; only in the case of mutations in the aI1 intron is a polar effect observed on the splicing of the second intron, aI2. Our results indicate that the splicing of these two intervening sequences which both belong to the class II of introns described by Michel et al. (1982), is controlled by the activity of the maturases encoded by their respective ORFs and that the translation of the aI2 maturase depends on the previous excision of aI1 IVS. (Moreover, the aI1 maturase, which accumulates in some mutants, can efficiently splice aI2 IVS when the translation of the latter's proper maturase cannot occur).

Dissociation of transforming and trans-activation functions for bovine papillomavirus type 1.

It has been shown that genetic information encoded by the 3' open reading frames (ORFs), E2, E3, E4 and E5, of bovine papillomavirus type 1 (BPV-1), is sufficient to induce cellular transformation of certain mouse cells. The product of the E2 ORF has further been shown to be responsible for the trans-activation of a transcriptional regulatory element located in the noncoding region (NCR) of the BPV-1 genome. To examine whether or not the E2 trans-activation function is encoded by the same gene that encodes the 3' ORF viral transformation function, we have now analysed the expression of the trans-activation function in series of mouse C127 cells transformed by BPV-1 deletion mutants. In addition, using mutated complementary DNA clones generated by the insertion of a premature translational termination linker into different sites of a BPV-1 cDNA clone containing the 3' ORFs intact, we demonstrate that transformation and transcriptional trans-activation functions can be dissociated and that they map respectively to the E5 and E2 ORFs.

Nonreciprocal exchange between alleles of the yeast mitochondrial 21S rRNA gene: Kinetics and the involvement of a double-strand break

A 1.1 kb intron containing an open reading frame (ORF) in one allele ( ω +) of the yeast mitochondrial 21S rRNA gene is nearly quantitatively inserted in crosses into a 21S rRNA allele lacking that Intron ( ω −). We have determined that this nonreciprocal exchange initiates soon after cells fuse to form zygotes and is complete by 10–16 hr after mating. We have discovered a unique in vivo double-strand cut in ω − mitochondrial DNA (mtDNA) at or near the site of intron insertion that is implicated in the process. Markers flanking the intron insertion site are coconverted with frequencies inversely proportional to their distance from that site. There is no net conversion of ω − to ω + in crosses between petites retaining these alleles, nor do we observe the unique double-strand cut in the mtDNA from zygotes of such crosses. The data suggest that a translation product of the intron ORF is required for the double-strand cut and nonreciprocal recombination at ω.

A second cauliflower mosaic virus gene product influences the structure of the viral inclusion body

We have used electron microscopy of thin sections and experiments on isolated viroplasms to compare the properties of four strains of cauliflower mosaic virus (CaMV), three of which were partially or completely deleted in open reading frame (ORF) II. Our results confirm that this gene is required for aphid transmissibility and show that the product of ORF II influences the firmness with which virions are held within the viroplasm. Analysis of the proteins in the viroplasms showed that a mutant with a partial deletion in ORF II produced a protein smaller than the normal ORF product. This smaller protein was non-functional with respect both to aphid transmissibility and properties of the viroplasms.