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Cellular responses to DNA damage and inhibited replication are evolutionarily conserved sets of pathways that are critical to preserving genome stability. To identify new participants in these responses, we undertook a screen for regulators that, when present on a high-copy vector, alter expression of a DNA damage-inducible RNR3-lacZ reporter construct in Saccharomyces cerevisiae. From this screen we isolated a plasmid encoding two closely related paralogs, WTM1 and WTM2, that greatly increases constitutive expression of RNR3-lacZ. Moderate overexpression of both genes together, or high-level expression of WTM2 alone from a constitutive promoter, upregulates RNR3-lacZ in the absence of DNA damage. Overexpressed, tagged Wtm2p is associated with the RNR3 promoter, indicating that this effect is likely direct. Further investigation reveals that Wtm2p and Wtm1p, previously described as regulators of meiotic gene expression and transcriptional silencing, amplify transcriptional induction of RNR3 in response to replication stress and modulate expression of genes encoding other RNR subunits.

Here we build on a previously proposed algorithm to infer direct regulatory relationships using gene-expression profiles from cells in which individual genes are deleted or overexpressed. The updated algorithm can process networks containing feedback loops, incorporate positive and negative regulatory relationships during network reconstruction, and utilize data from double mutants to resolve ambiguous regulatory relationships. When applied to experimental data the reconstruction procedure preferentially retains direct transcription factor-target relationships.

Publisher Summary This chapter discusses cloning regulated yeast genes from a pool of lacZ fusions. Gene fusions have been used during the past decade to study such diverse problems as transcriptional regulation, translational regulation, and protein localization. In prokaryotes, various genetic methods have been employed to create operon and protein fusions. The active, regulated gene fusions constructed by the procedures described below contain a yeast DNA fragment with sequences for the control and initiation of transcription and translation joined to a lacZ fragment from which the 5’ portion including the first few N-terminal amino acid codons of the gene have been deleted. A library of random yeast gene:lacZ fusions in yeast transformants was constructed and screened for regulated gene fusions.

Publisher Summary This chapter discusses cloning regulated yeast genes from a pool of lacZ fusions. Gene fusions have been used during the past decade to study such diverse problems as transcriptional regulation, translational regulation, and protein localization. In prokaryotes, various genetic methods have been employed to create operon and protein fusions. The active, regulated gene fusions constructed by the procedures described below contain a yeast DNA fragment with sequences for the control and initiation of transcription and translation joined to a lacZ fragment from which the 5’ portion including the first few N-terminal amino acid codons of the gene have been deleted. A library of random yeast gene:lacZ fusions in yeast transformants was constructed and screened for regulated gene fusions.

We have identified a locus of HSV-1 which, when mutated, confers resistance to acycloguanosine (ACG); this locus corresponds to the HSV thymidine kinase (TK) gene. ACG-resistant mutants exhibit reduced levels of HSV-specific TK activity, and the lower the TK activity, the greater the degree of ACG-resi stance. Moreover, in a conditionally resistant mutant, KG-111, resistance to ACG at 39° and sensitivity at 34° is associated with thermolabile TK activity. Eleven TK-, ACG-resistant mutants behaved in genetic crosses as alleles of a single locus mapping between the A and G loci on the HSV-1 strain KOS genetic map. Analysis of the fine structure of this locus in now possible with the aid of a recombinant plasmid bearing the KOS TK gene.