In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30°C but not at 20°C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mksl and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30°C and prevent the cells from growing at 8°C. Elimination of M double-stranded RNA from a coldsensitive ski-strain results in the loss of cold sensitivity. ski-[KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-kl] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski-strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI' products.The killer system of Saccharomyces cerevisiae is unique among eucaryotic virus and plasmid systems in the detail in which interactions of viral and host components have been explored and in the large number of host components shown to be involved. This probably reflects the ease with which these aspects can be investigated in S. cerevisiae rather than any difference in the degree to which viral and host functions are intertwined. Numerous examples are known of host restriction of viral growth in mammalian systems, but analysis of the host genes involved is generally not practical. We show here that overproduction of a S. cerevisiae virus, due to a host mutation, results in host pathology, apparently due to over-utilization by the virus of a specific, essential host component.

Ability to secrete the K1 (or K2) toxin protein and immunity to that toxin [the K1 (or K2) killer trait] are determined by a double-stranded (ds) RNA, called M1 (or M2), whose replication and maintenance depend on at least one of the larger (L) ds RNAs and 29 chromosomal genes, called MAK genes (maintenance of killer). The location of the MAK8 gene near TCM1 (trichodermin resistance) on the yeast map suggested the possible identity of these two genes. Of six independently isolated tcml mutants, five were clearly mak-, and the sixth was weakly mak-. In each case, the mak-phenotype and the trichodermin-resistant phenotypes cosegregated in meiosis and showed the expected tight linkage to petl7. The mak-mutations in the trichodermin-resistant strains did not complement mak8-l, indicating that MAK8 and TCM1 are the same gene. The K1 killer double-stranded (ds) RNA genome (M1 ds RNA; 1.5 X 106 daltons) codes'for an 11,000-dalton protein toxin and determines immunity to that toxin (1-4). A second (K2) killerimmunity system is similarly determined by M2 ds 'RNA (1.0 X 106 daltons). M1l is dependent, for its replication and maintenance, on the products of at least 29 chromosomal genes, called MAK genes (for maintenance of killer), as well as on at least one of the larger L ds RNAs (5-12). Ofthese 29 genes, the product of only one is known. SPE2 codes for adenosylmethionine decarboxylase, an enzyme in spermine and spermidine synthesis (13), and cells bearing it lose M ds RNA when not supplied with exogenous polyamines

The K 1 killer virus (or plasmid) of Saccharomyces cerevisiae is a noninfectious double-stranded RNA genome found intracellularly packaged in an icosahedral capsid. This genome codes for a protein toxin and for resistance to that toxin. Defective interfering virus mutants are deletion derivatives of the killer virus double-stranded RNA genome; such mutants are called suppressive. Unlike strains carrying the wild-type genome, strains with these deletion derivatives are neither toxin producers nor toxin resistant. If both the suppressive and the wildtype virus are introduced into the same cell, most progeny become toxin-sensitive nonkillers (J. M. Somers, Genetics 74 :571-579, 1973). Diploids formed by the mating of a killer with a suppressive strain were grown in liquid culture, and RNA was extracted from samples taken up to 41 generations after the mating. The ratio of killer RNA to suppressive RNA decreased with increasing generations; by 41 generations the killer RNA was barely detectable. The copy numbers of the suppressive genome and its parental killer were virtually the same in isogenic strains, as were the growth rates of diploid strains containing either virus alone. Therefore, suppressiveness, not being due to segregation or overgrowth by faster growing segregants, is likely due to preferential replication or maintenance of the suppressive genome. Three suppressive viruses, all derivatives of the same killer virus (T. K. Sweeney et al., Genetics 84: 27-42, 1976), did not coexist stably. The evidence strongly indicates that the largest genome of the three slowly suppressed both of the smaller genomes, showing that larger genomes can suppress smaller ones and that suppression can occur between two suppressives. Of 48 isolates of strains carrying the suppressive viruses, 5 had newly detectable RNA species, all larger than the original suppressive genomes. At least seven genes necessary for maintenance of the wild-type killer virus ( MAK genes) were needed by a suppressive mutant. No effect of ski mutations (affecting regulation of killer virus double-stranded RNA replication) on suppressiveness was observed.

In an mktl host, L-A-HN double-stranded RNA excludes M2 double-stranded RNA at 30 degrees C but not at 20 degrees C. Recessive mutations suppressing the exclusion of M2 by L-A-HN in an mktl host include six ski (superkiller) genes, three of which (ski6, ski7 and ski8) are new genes. The dominant mutations in one gene (MKS50) and recessive mutations in at least two genes (mks1 and mks2) suppress M2 exclusion by L-A-HN but do not show other characteristics of ski mutations and thus define a new class of killer-related chromosomal genes. Mutations in ski2, ski3, ski4, ski6, ski7, and ski8 result in increased M copy number at 30 degrees C and prevent the cells from growing at 8 degrees C. Elimination of M double-stranded RNA from a cold-sensitive ski- strain results in the loss of cold sensitivity. ski- [KIL-sd1] strains lack L-A-HN, carry L-A-E, and have a lower M1 copy number than do ski- [KIL-k1] strains and are only slightly cold sensitive. The LTS5 (=MAK6) product is required both for low temperature growth and for M1 maintenance or replication. We propose that the elevated levels of M in ski- strains divert the host LTS5 product away from the host and to the M replication process. We also suggest that the essential role of L-A in M replication is protection of M double-stranded RNA from the negative influence of SKI+ products.

An Escherichia coli strain carrying an amber mutation (UAG) in rpoC, the gene encoding the beta prime subunit of RNA polymerase, was isolated after mutagenesis with nitrosoguanidine. The mutation was moved into an unmutagenized strain carrying the supD43,74 allele, which encodes a temperature-sensitive su1 amber suppressor, and sue alleles, which enhance the efficiency of the suppressor. In this background, beta prime is not synthesized at high temperature. Suppression of the mutation by the non-temperature-sensitive amber suppressor su1+ yields a protein which is functional at all temperatures examined (30, 37, and 42 degrees C).