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Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

ABSTRACT: The complete nucleotide sequence of turnip yellow mosaic virus (TYMV) genomic RNA has been determined on a set of overlapping cDNA clones using a sequential sequencing strategy. The RNA is 6318 nucleotides long, excluding the cap structure. The genome organization deduced from the sequence confirms previous results of in vitro translation. A novel open reading frame (ORF) putatively encoding a Pro-rich and very basic 69K (K = kilodalton) protein is detected at the 5' end of the genome. It is initiated at the first AUG codon on the RNA and overlaps the major ORF that encodes the non structural 206K (previously referred to as 195K) protein of TYMV; its function is unknown. Several amino acid consensus sequences already described among plant and animal viruses are also found in the TYMV-encoded polypeptides. A comparison with other viruses whose RNA sequence is known leads to the conclusion that TYMV belongs to the "Sindbis-like" supergroup of viruses and could be related to Semliki forest virus.

SUBMITTER: Morch MD

PROVIDER: S-EPMC336854 | biostudies-other | 1988 Jul

REPOSITORIES: biostudies-other

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Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

Morch M D MD Boyer J C JC Haenni A L AL

Nucleic acids research 19880701 13

The complete nucleotide sequence of turnip yellow mosaic virus (TYMV) genomic RNA has been determined on a set of overlapping cDNA clones using a sequential sequencing strategy. The RNA is 6318 nucleotides long, excluding the cap structure. The genome organization deduced from the sequence confirms previous results of in vitro translation. A novel open reading frame (ORF) putatively encoding a Pro-rich and very basic 69K (K = kilodalton) protein is detected at the 5' end of the genome. It is ini ...[more]

PMID: 3399388

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Project description:BackgroundIn order to carry out experimental gene annotation, DNA encoding open reading frames (ORFs) derived from real genes (termed "genic") in the correct frame is required. When genes are correctly assigned, isolation of genic DNA for functional annotation can be carried out by PCR. However, not all genes are correctly assigned, and even when correctly assigned, gene products are often incorrectly folded when expressed in heterologous hosts. This is a problem that can sometimes be overcome by the expression of protein fragments encoding domains, rather than full-length proteins. One possible method to isolate DNA encoding such domains would to "filter" complex DNA (cDNA libraries, genomic and metagenomic DNA) for gene fragments that confer a selectable phenotype relying on correct folding, with all such domains present in a complex DNA sample, termed the "domainome".ResultsIn this paper we discuss the preparation of diverse genic ORF libraries from randomly fragmented genomic DNA using ß-lactamase to filter out the open reading frames. By cloning DNA fragments between leader sequences and the mature ß-lactamase gene, colonies can be selected for resistance to ampicillin, conferred by correct folding of the lactamase gene. Our experiments demonstrate that the majority of surviving colonies contain genic open reading frames, suggesting that ß-lactamase is acting as a selectable folding reporter. Furthermore, different leaders (Sec, TAT and SRP), normally translocating different protein classes, filter different genic fragment subsets, indicating that their use increases the fraction of the "domainone" that is accessible.ConclusionsThe availability of ORF libraries, obtained with the filtering method described here, combined with screening methods such as phage display and protein-protein interaction studies, or with protein structure determination projects, can lead to the identification and structural determination of functional genic ORFs. ORF libraries represent, moreover, a useful tool to proceed towards high-throughput functional annotation of newly sequenced genomes.

Dataset Information

Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

Publications

Overlapping open reading frames revealed by complete nucleotide sequencing of turnip yellow mosaic virus genomic RNA.

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