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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.

The mammalian 70K protein, a component of the U1 small nuclear ribonucleoprotein involved in pre-mRNA splicing, interacts with a number of proteins important for regulating constitutive and alternative splicing. Similar proteins that interact with the yeast homolog of the 70K protein, Snp1p, have yet to be identified. We used the two-hybrid system to find four U1-Snp1 associating (Usa) proteins. Two of these proteins physically associate with Snp1p as assayed by coimmunoprecipitation. One is Prp8p, a known, essential spliceosomal component. This interaction suggests some novel functions for Snp1p and the U1 small nuclear ribonucleoprotein late in spliceosome development. The other, Exo84p, is a conserved subunit of the exocyst, an eight-protein complex functioning in secretion. We show here that Exo84p is also involved in pre-mRNA splicing. A temperature-sensitive exo84 mutation caused increased ratios of pre-mRNA to mRNA for the Rpl30 and actin transcripts in cells incubated at the non-permissive temperature. The mutation also led to a defect in splicing and prespliceosome formation in vitro; an indication that Exo84p has a direct role in splicing. The results elucidate a surprising link between splicing and secretion.

The removal of introns from the primary transcript occurs by RNA splicing. Three major types of RNAs: tRNA, rRNA and mRNA, are known to contain introns. There are two general questions concerning any splicing mechanism: first, how is the precise recognition and alignment of the splice junctions achieved in introns whose lengths vary from a few hundred nucleotides to several thousands of nucleotides and, second, what are the biochemical mechanisms of the cleavage and ligation reactions.

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.

copia-specific RNA was isolated from Drosophila melanogaster tissue culture cells by hybridization of cytoplasmic polyadenylylated RNA to copia DNA immobilized on cellulose. The purified RNA was translated in reticulocyte lysates. One major polypeptide of approximately 51,000 daltons was synthesized in addition to several others between 18,000 and 38,000 daltons. The 51,000-dalton polypeptide and several of the others are encoded by mRNAs of about 2000 nucleotides. The approximate locations on the copia element of the coding sequences for the 51,000-dalton polypeptide and several other proteins were determined by hybrid-arrested translation with copia restriction fragments. The relative abundance of copia-specific RNA was determined at various stages of the Drosophila life cycle. The level of copia-specific RNA is modulated during development of the organism, with the highest level occurring during the larval stages.

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.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.