RAD4 gene of Saccharomyces cerevisiae: molecular cloning and partial characterization of a gene that is inactivated in Escherichia coli.

In contrast to other Saccharomyces cerevisiae RAD genes involved in nucleotide excision repair of DNA, the RAD4 gene could not be isolated by screening a yeast genomic library for recombinant plasmids which complement the UV sensitivity of rad4 mutants (Pure et al., J. Mol. Biol. 183:31-42, 1985). We therefore attempted to walk to RAD4 from the neighboring SPT2 gene and obtained an integrating derivative of a plasmid isolated by Roeder et al. (Mol. Cell. Biol. 5:1543-1553, 1985) which contains a 4-kilobase fragment of yeast DNA including a mutant allele of SPT2. When integrated into several different rad4 mutant strains, this plasmid (pR169) complements UV sensitivity at a frequency of approximately 10%. However, a centromeric plasmid containing rescued sequences which include flanking yeast DNA no longer complements the phenotype of rad4 mutants. Complementing activity was restored by in vivo repair of a defined gap in the centromeric plasmid. The repaired plasmid fully complements the UV sensitivity of all rad4 mutants tested when isolated directly from yeast cells, but when this plasmid is propagated in Escherichia coli complementing activity is lost. We have mapped the physical location of the RAD4 gene by insertional mutagenesis and by transcript mapping. The gene is approximately 2.3 kilobases in size and is located immediately upstream of the SPT2 gene. Both genes are transcribed in the same direction. RAD4 is not an essential gene, and no increased transcription of this gene is observed in cells exposed to the DNA-damaging agent 4-nitroquinoline-1-oxide. The site of inactivation of RAD4 in a particular plasmid propagated in E. coli was localized to a 100-base-pair region by gene disruption and gap repair experiments. In addition, we have identified the approximate locations of the chromosomal rad4-2, rad4-3, and rad4-4 mutations.

Partial suppression of an ochre mutation in Saccharomyces cerevisiae by multicopy plasmids containing a normal yeast tRNAGln gene.

We screened a yeast genomic library for recombinant DNA plasmids that complemented the ultraviolet (u.v.) sensitivity of a strain of Saccharomyces cerevisiae designated rad4-3 that is defective in excision repair of DNA. A multicopy plasmid (pNF4000) with a 9.4 X 10(3) base-pair yeast DNA insert partially complemented the u.v. sensitivity of rad4-3, but not of two other rad4 allelic mutants (rad4-2 and rad4-4), or of other u.v.-sensitive rad mutants. The yeast insert was analyzed by restriction mapping, DNA-DNA hybridization, DNA-tRNA hybridization and DNA sequencing. This analysis revealed the presence of a normal tRNAGln gene, a yeast sigma element situated 5' to the transfer RNA gene, a Ty element and a solo delta element. Deletion analysis of pNF4000 showed that the tRNAGln gene is required for partial complementation of the u.v. sensitivity of rad4-3. Furthermore, a multicopy plasmid containing a tRNAGln gene derived from a different region of the yeast genome also partially complemented the u.v. sensitivity of rad4-3. The rad4-3 mutation is suppressed following transformation with a plasmid containing the known ochre suppressor SUP11-o, indicating that it is an ochre mutation. We therefore conclude that when expressed in sufficient quantity, normal tRNAGln (which usually decodes the sense codon CAA) can weakly suppress the nonsense ochre codon UAA, and suggest that this represents an example of wobble occurring at the first rather than at the third position of the codon.

Changes in Wheat Leaf Polysomal Messenger RNA Populations during the Early Stages of Rust Infection: EFFECTS OF CHLORAMPHENICOL AND LINCOMYCIN ON CELL-FREE TRANSLATION BY POLYSOMES FROM HEALTHY AND INFECTED LEAVES.

Polysomes isolated from a susceptible variety of wheat leaves (cultivar W2691) and those inoculated with the wheat stem rust fungus (f. sp. tritici, race 126-ANZ-6, 7) were incubated in a cell-free protein-synthesizing system. Under these conditions, different size classes of polypeptides, ranging in molecular weight from 10,000 to 80,000, are radiolabeled. Using double-isotope labeling technique, we show that some discrete size classes of polypeptides are synthesized in significantly greater quantitites by polysomes from inoculated leaves compared to the corresponding size classes synthesized by polysomes from healthy leaves. These results confirm our previous observation that there are significant changes in the wheat leaf polysomal messenger RNA populations at 3 days after inoculation with the rust fungus.The effects of the organelle-specific inhibitors of protein synthesis, chloramphenicol and lincomycin, on in vitro polysomal messenger RNA translation were investigated. The polypeptides synthesized by polysomes from healthy and inoculated leaves in the presence of chloramphenicol were compared. The results show that, even in the presence of this antibiotic, the polysomes from inoculated leaves synthesize greater quantities of some size classes of polypeptides. These data indicate that changes in polysomal messenger RNA populations involve, at least in part, cytoplasmic messenger RNA.