Cloning and expression of DNA sequences associated with the killer trait of Paramecium tetraurelia stock 47

Abstract

We have presented direct evidence that at least one of the traits associated with killing of paramecia by kappa particles is determined by an extrachromosomal genetic element. Plasmid DNA was isolated from Caedibacter taeniospiralis 47 (commonly known as 47 kappa), which is an obligate cytoplasmic endosymbiont of Paramecium tetraurelia. Fragments of pKAP47 DNA generated by Pst I digestion were inserted into pBR328 and then introduced into Escherichia coli 294 by transformation. Clones carrying recombinant plasmids were screened for toxicity toward sensitive strains of paramecia or for the ability to produce R bodies. None of the clones appeared to be toxic. However, three clones were found to have the ability to produce R bodies, which are proteinaceous ribbons (10-20 microns long, 0.5 microns wide, and 13 nm thick) rolled up inside the cell to form a hollow cylinder about 0.5 microns in diameter and 0.5 microns long. Each of these clones carry plasmids that contain the Pst I B fragment from pKAP47. Subclones of one of the recombinant plasmids, pBQ51, were constructed to determine the approximate location of DNA sequences necessary for R-body synthesis. The left-hand boundary of the required sequences was found to occur within a 600-base-pair region, and the location of the right-hand boundary was determined to occur within a 700-base-pair region. The minimum and maximum sizes of sequences required for R-body synthesis are between 1,300 and 2,600 base pairs.