This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mindich, L Articles by Bamford, D Search for Related Content PubMed PubMed Citation Articles by Mindich, L Articles by Bamford, D

cDNA cloning of portions of the bacteriophage phi 6 genome.

ABSTRACT

Phage phi 6 has a genome consisting of three pieces of double-stranded RNA. Single-stranded RNA was prepared from phi 6 nucleocapsids by in vitro transcription with the phage RNA polymerase. These transcripts were polyadenylated and used as templates for the preparation of cDNA copies. The resulting DNA was cloned into the PstI restriction nuclease site of plasmid pBR322. Insert-bearing plasmids were annealed to phi 6 RNA to assign the inserts to their proper segments. In this way we identified inserts corresponding to the large, medium, and small segments. Two large overlapping inserts of the small segment constitute the complete complement of the segment as determined by the sequence analysis of the DNA. In vitro coupled transcription and translation showed that the small segment inserts were able to direct the synthesis of the four known genes in the small segment. Two overlapping inserts in the medium segment constitute the entire segment and were shown to direct the in vitro synthesis of two of the three known proteins of the medium segment. Several inserts bearing about one-third the complement of the large segment were also isolated, and one of these directed the synthesis of a peptide that resembles protein P1. Restriction endonuclease maps were prepared for the inserts, and by in vitro synthesis it was possible to refine the genetic map of phi 6. A chimeric plasmid was constructed that combines plasmids pUC8 and RSF1010. Inserts placed on this plasmid were transformed to Pseudomonas phaseolicola, the natural host of phage phi 6. It was possible to refine further the genetic map by complementation of nonsense mutants of phi 6 with the cDNA.

This article has been cited by other articles:

• Makeyev, E. V., Bamford, D. H. (2004). Evolutionary Potential of an RNA Virus. J. Virol. 78: 2114-2120 [Abstract] [Full Text]  
• Mindich, L. (1999). Precise Packaging of the Three Genomic Segments of the Double-Stranded-RNA Bacteriophage phi 6. Microbiol. Mol. Biol. Rev. 63: 149-160 [Abstract] [Full Text]  
• Paatero, A. O., Mindich, L., Bamford, D. H. (1998). Mutational Analysis of the Role of Nucleoside Triphosphatase P4 in the Assembly of the RNA Polymerase Complex of Bacteriophage phi 6. J. Virol. 72: 10058-10065 [Abstract] [Full Text]