THE DNA AND RNA-POLYMERASE GENES OF YEAST PLASMID PGKL2 ARE ESSENTIALLOCI FOR PLASMID INTEGRITY AND MAINTENANCE

Novel recombinant plasmids derived from the Kluyveromyces lactis kille r plasmid k2 have been constructed to study plasmid biology and gene f unction. in vivo recombination between native resident k2 and suitable disruption vectors, employing the KITRP1 gene fused to a plasmid prom oter as selection marker, yielded ORF2 and ORF6 deletion plasmids at h igh frequencies. As judged from Southern hybridization and plasmid res triction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6 , respectively, carry deletions in their putative DNA (ORF2) and RNA ( ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for TRP1 over 350 generations o f growth did not favour maintenance of hybrids over wild-type k2. Thus , neither rk2/2 nor rk2/6 was fully functional and able to displace pa rental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy n umber relative to k2 with rk2/2 more drastically affected than rk2/6 i mplying a direct involvement of the ORF2 product in plasmid replicatio n and an indirect maintenance function for the ORF6 gene product.