1. First- and second-generation crosses between killer, neutral and sensitive strains of yeast have been carried out in all combinations.2. The results of this analysis indicated that the killer character is under the control of two types of cytoplasmic genetic determinant. One type, (k), determines killing, and the other, (n), neutrality. The absence, (o), of both types of determinants confers the sensitive phenotype.3. That both types of cytoplasmic determinant require the same dominant nuclear allele, M, for their maintenance has been indicated in two ways. First, both types are lost when the nuclear genotype is changed from M to m. Secondly, cells of genotype m(k) or m(n), which have been shown to occur among the first formed cells arising from spores of Mm(k) and Mm(n) diploids respectively, are unable to maintain their cytoplasmic determinants. On the other hand, spore cultures of M(k) and M(n) genotype derived from these same diploide continue to maintain the determinants.4. Thus genotype of killer cells is M(k), of neutrals M(n), and of sensitivities either M(o) or m(o).5. Cells maintaining both types of cytoplasmic determinant (i.e. of genotype M(k)(n) or M−(k)(n)) have been obtained by appropriate crosses, and shown to be of killer phenotype.6. Alternative hypotheses to account for the results of this genetic analysis have been discussed.

Membrane-associated gene products of shock-sensitive bacterial transport operons are often difficult to detect. A 4.5-kilobase DNA fragment, known to completely encode the Salmonella typhimurium tctI operon, was cloned in both orientations behind the T7 phage promoter phi 10 and expressed by using the T7 polymerase-promoter system of Tabor and Richardson (S. Tabor and C. C. Richardson, Proc. Natl. Acad. Sci. USA 82:1074-1078, 1985). Under these conditions, five proteins were clearly demonstrated. One DNA strand was shown to encode the periplasmic (29,000-Mr) C protein (as a 31,000-Mr precursor), a 19,000-Mr protein, and a 40,000- to 45,000-Mr protein which ran as a diffuse band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The opposite strand carried the information for two additional proteins of 29,000 and 14,000 Mr. By Tn5 mutagenesis, subcloning of Tn5 insertions, and subcloning of various deletion mutants it was shown that the tctI system is divergently transcribed. The periplasmic binding protein (C protein) is the first product of one operon, followed by the 19,000-Mr and 45,000-Mr integral inner membrane proteins. On the opposite strand only the 29,000-Mr protein was essential for tctI function, and it was found to be weakly attached to the inner membrane. Thus tctI encodes four proteins, one periplasmic, two integral, and one peripheral to the cytoplasmic membrane, with the genes arranged as tctA tctB tctC tctD.

When killer and neutral strains of Saccharomyces cerevisiae are crossed the resulting diploid clones possess a killer phenotype and when spored yield a complete range of tetrad ratios.The combined results of analysing tetrads and vegetative cells of diploid clones derived from two different neutral × killer crosses (K1 × N1 and K2 × N1) demonstrate that the range of tetrad ratios can be accounted for by the occurrence of somatic segregation of killer (k) cytoplasmic determinants prior to sporulation. Such results support the genetic model for the inheritance of the killer character in yeast already proposed (Somers & Bevan, 1969).During the course of these studies a correlation was found between the strengths of the killer phenotypes of diploid colonies and the proportions of killer spore cultures obtained after sporulation of their cells.

Tricarboxylates are transported into Salmonella typhimurium by a binding protein-dependent transport system known as TctI. Genetically, it comprises three structural genes, tctCBA, as well as a fourth gene of unknown function (tctD), which is transcribed divergently from tctC (K. A. Widenhorn, J. M. Somers, and W. W. Kay, J. Bacteriol. 170:3223-3227, 1988). Deletions in tctD strongly reduced expression of tctC or of tctC-lacZ transcriptional fusions; however, expression was restored when tctD was present in trans. Expression of tctD-lacZ transcriptional fusions was strongly repressed in the presence of D-glucose but could be alleviated by the addition of cyclic AMP. Furthermore, transcription of tctD was found not to be autogenously regulated. Thus, tctD is considered to be regulated by catabolite repression and encodes a transcriptional activator of tctCBA expression. From the DNA sequence of tctD, the predicted gene product was hydrophilic and shared distinct homologies with other globally regulated transcriptional activators such as OmpR and NtrC.

Yeast killer factor proteins bind to cells of both sensitive and killer-producing strains, although the latter are immune to killer action. Spheroplasts prepared from sensitive cells bind less than 1% of the killer bound to whole cells, but remain fully sensitive to killer. This finding and those obtained from binding studies of partially purified, radioactive killer protein suggest that most of the toxins remain bound to the yeast cell wall and do not function further in the killing process. A killer-resistant mutant R1s was isolated from a sensitive