S1-sensitive sites in the supercoiled double-stranded form of tomato golden mosaic virus DNA component B: identification of regions of potential alternative secondary structure and regulatory function

Abstract

The sensitivity of the supercoiled double-stranded form of the DNA of tomato golden mosaic virus (TGMV), a geminivirus, to the single-strand specific enzyme S1 nuclease has been demonstrated. Specific S1 cleavage sites were identified in TGMV DNA component B by cloning into the single-strand bacteriophage vector M13 mp8 and sequencing of the inserted DNA. Analysis of the DNA sequence at the sites of S1 sensitivity in TGMV DNA component B revealed several possible regions of alternative secondary structure which were clustered in an intergenic region upstream of the starts of the two major open reading frames which are in opposite orientations. This region contains putative transcriptional promoter and modulatory sequences and a possible replication origin. The extreme S1 sensitivity of the supercoiled form of TGMV DNA component A precluded its cloning under the conditions employed for selective cleavage of DNA component B.