The Canonical Locus of rI
Katherine L. Gailbreath, Patrick J. Paddison, Julia H. Tracy, Burton S. Guttman and Elizabeth Kutter, The Evergreen State College
Genes rI, rII, and rIII were key in defining the early T4 maps. Nonetheless, the locus of the rI gene has proven difficult to determine precisely. This difficulty can be largely attributed to its presumed location in the nrdC.-tk region, which contains no essential genes, so conditional-lethal mutants cannot be used for precise mapping. however, complementation tests and deletion mapping of rI+ and rI mutants place the rI locus between nrdC.11 and tk. Additionally, at the 1994 T4 conference John Drake and colleagues estimated the size of the rI gene to be approximately 1300 bp based on the frequency of proflavin-induced rI mutants relative to rII, rIII, and rV mutants. Armed with this knowledge, we have attempted to locate the rI gene. Using several purported rI mutants, nine open reading frames counter clockwise to tk were sequenced and compared to wild-type T4 and the Genbank T4 sequence of this region, starting nrdC.11 and tk.-9, the only ORFs in the presumed rI region that approximate 1300 bp and have a sufficient number of potential amber mutation sites.
The results were somewhat surprising. All five classic rI mutants tested (r48, r52, r53, r57, & r58) show alterations in the tk.-2 open reading frame, which maps between 59483 and 59193 in the T4 genome. this open reading frame is only 291 nucleotides in length and presumably encodes a 97-amino-acid protein with a molecular weight of 11,125 Da. tk.-2 is the second ORF in a cluster of three downstream from a consensus sequence late promoter. Both r48 and r52 contain a deletion of one adenine in a run of five adenines in wild-type, which truncates the reading frame; r53 and r58 carry an insertion of an AT pair, which also stops the reading frame early. A G to C conversion, which substitutes an arginine for a glycine, is apparent in r57.
However, no mutations in tk.-2 (or in reading frames tk.-7 through tk.-1) are found in purported rI amber mutants am47-22 and am48-23. Yet, am47-22 and am48-23 do not appear to complement with r48 in lysis tests, usually indicating a mutation in the same gene. Thus it remains to be seen whether rI is polycistronic. Our findings suggest that tk.-2 is the canonical locus of rI.
Now that most of the genes involved in lysis inhibition have been identified, many interesting questions arise about the mechanism of lysis inhibition, which up to now has been shrouded in mystery. We are currently investigating some of these questions and hope to have some molecular evidence soon.
Return to top.