| Summary: | K. lactis killer strains carry two cytoplasmic, linear dsDNA plasmids, termed k1 and k2. DNA sequence analysis has revealed the presence of 14 plasmid-encoded ORFs. All genes are transcribed independently and all are preceded by putative promoter elements, the UCS motifs. Plasmid genes are generally not expressed when cloned with their own promoters into yeast nuclear vectors; similarly, cytoplasmic expression of genes carrying nuclear promoters does not occur when they are placed on k1 or k2. To overcome this compartmental incompatibility I have constructed suitable UCS-marker gene fusions for k2 gene disruption analysis. Disruption of the putative DNA (k2ORF2) and RNA (k2ORF6) polymerase structural genes by integration of the the K1TRP1 gene fused to the k1UCS2 element as selection marker, yielded ORF2 and ORF6 deletion plasmids. ORF20 and ORF60 plasmids were unable to displace parental k2 during Trp+ selective growth indicating both genes to be essential for plasmid functionality. The hybrids were reduced in copy numbers relative to k2 with ORF20 more drastically affected than ORF60 plasmids implying direct involvement of ORF2 in plasmid replication and an indirect maintenance function for the ORF6 gene product. Similarly, disruption of k2ORF5 via integration of a k2UCS5-ScLEU2 marker gene yielded ORF5 deletion plasmids, termed rk2. ORF5 appears to be an essential gene for plasmid integrity since rk2 was unable to displace native k2 during Leu+ selective growth. By shuffling ORF5 from k2 onto k1 I have shown this essential gene to be functionally interchangeable between both episomes. Once transferred onto k1, ORF5 was fully able to complement the ORF50 deletion on rk2 in trans. ORF5 is a protein encoding gene as shown by shuffling an in vitro epitope-tagged allele. Western blotting identified a protein (19.5kDa) corresponding to the expected size of the tagged Orf5p product. Expression levels indicate Orf5p to be an abundant k2 product implying structural rather than regulatory function. The gene shuffle has been used to investigate UCS function. By transplacing various ORF5 deletion constructs, and analysing trans-complementation, a 40 bp k2 fragment including the UCS5 motif has been identified as a cis-acting promoter essential for k2 gene function and transcriptional activation of a ScLEU2 reporter gene. Analyses revealed a plasmid-dependent LEU2 transcript distinct in size and regulation from its nuclear counterpart: cytoplasmic, UCS5-driven expression of ScLEU2 was non-repressible by leucine and reduced up to eight fold compared to fully derepressed nuclear K1LEU2 mRNA levels. Thus, k2 and k1 appear to employ a balanced low level expression system. Finally, the k2ORF7 (putative RNApol subunit) and k2ORF5 products were detected by over-expression of epitope-tagged alleles in E. coli and baculovirus systems. Western analysis identified proteins with apparent molecular weights of 18 and 20 kDa, corresponding in size to the predicted products.
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