Generation of cDNA clones of the bacteriophage phi 6 segmented dsRNA genome: characterization and expression of L segment clones

Abstract

Bacteriophage phi 6 has three dsRNA genome segments of about 3.0, 4.0, and 6.4 kbp. More than 90% of the segmented phi 6 dsRNA genome has been cloned as subchromosomal cDNA fragments, generated by reverse transcription of denatured polyadenylated dsRNA, RNA removal, annealing, filling, size fractionation, tailing, and insertion at the PstI site of pBR322. All of the large (L) segment is represented by five overlapping fragments, 98% of the small (S) segment is present in three fragments, and 67% of the medium (M) segment is contained in two fragments. Fragments have been aligned in linear arrays by Southern blot hybridization and restriction enzyme analysis. The orientation of the ordered fragments with respect to genomic RNA and phi 6 transcriptional direction was determined by comparison of terminal DNA sequences with RNA sequences at the genomic ends of phi 6 RNA. Expression of L segment clones using both Escherichia coli minicells and T7 polymerase/promoter vectors indicate that the order of known phi 6 genes on the large chromosome is: 5'--gene 7, gene 2, gene 4, gene 1--3'. cDNA complementation of a ts mutant, ts411, has located this mutation in gene 4.