Summary auto-generated
This paper describes the successful molecular cloning of genes from a human rotavirus isolate collected from a patient with diarrhea. The researchers used a novel strategy combining reverse transcription of denatured double-stranded RNA, cDNA synthesis, size separation via alkaline agarose gel electrophoresis, and cloning into the pBR322 plasmid vector. The key innovation was separating full-length cDNA before hybridization to avoid cloning incomplete gene copies. Four individual clones were successfully isolated and characterized: one containing gene 6 (1360 bp insert), one containing gene 7, 8, or 9 (1140 bp), and two containing genes 10 and 11 (780 bp and 660 bp respectively). The clones were identified through hybridization to bovine rotavirus genomic segments, and restriction maps were generated. The orientation of inserts relative to viral mRNA was determined using 3'-end-specific probes. This work established methods for cloning rotavirus genes, providing a foundation for understanding viral structure, function, and antigenic diversity, with potential applications for developing rotavirus vaccines through expression of cloned genes in bacteria.
Key findings
- Four human rotavirus genes were successfully cloned into pBR322 plasmid vectors from a field isolate, representing complete or near-complete copies of genes 6, 7-8-9, 10, and 11.
- The cloning strategy involved polyadenylation of double-stranded RNA, reverse transcription, size fractionation of cDNA by alkaline agarose gel electrophoresis, and hybridization before insertion into the vector, successfully producing plasmids with size-appropriate inserts.
- Molecular identification of the clones was confirmed through hybridization of 32P-labeled plasmids to bovine rotavirus genome segments immobilized on paper.
- The relative orientation of cloned genes was determined using 3'-end-specific probes, enabling identification of which DNA strand corresponds to the viral mRNA sense strand.
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Abstract
The genes of a field isolate of human rotavirus were cloned into pBR322. The strategy used involved polyadenylation of denatured double- stranded (ds) genomic RNA, followed by cDNA synthesis using reverse transcriptase. Oligo(dC) tails were added to the 3' end of the single- stranded cDNA and then separated by alkaline agarose electrophoresis. Sized cDNA of both polarities were allowed to hybridize and inserted into the PstI site of pBR322. Transformations done with sized cDNA always resulted in the selection of hybrid plasmids carrying inserts with a size representative of the original cDNA. Four individual clones were selected for preliminary characterization. One clone has an insert of 1360 base pairs (bp) and corresponds to gene 6. The second clone has an insert of 1140 bp and corresponds to one of the genes in the triplet 7-8-9. The other two genes, with inserts of 780 and 660 bp, were identified as corresponding to dsRNA segments 10 and 11.