We developed a genetic selection system based on nonsense suppression in Saccharomyces cerevisiae to identify mutations in proteins involved in transcription initiation by RNA polymerase III. A SUP4 tRNATyr internal promoter mutation (A53T61) that was unable to suppress ochre mutations in vivo and was incapable of binding TFIIIC in vitro was used as the target for selection of trans-acting compensatory mutations. We identified two such mutations in the same gene, which we named TAPI (for transcription activation protein). The level of the SUP4A53T61 transcript was threefold higher in the tap)-) mutant than in the wild type. The tap)-) mutant strain was also temperature sensitive for growth. The thermosensitive character cosegregated with the restorer of suppression activity, as shown by meiotic linkage analysis and coreversion of the two traits. At 1 to 2 h after a shift to the restrictive temperature, RNA synthesis was strongly inhibited in the tap)-) mutant, preceding any effect upon protein synthesis or growth. A marked decrease in tRNA and 5S rRNA synthesis was seen, and shortly after that, rRNA synthesis was inhibited. By complementation of the ts-growth defect, we cloned the wild-type TAP) gene. It is essential for yeast growth. We show in the accompanying report

The sequence of 2965 nucleotides 5' of the human epsilon-globin gene has been completed. It includes two Alu family repeats present in an inverted configuration. Only the one located farthest from the gene was active as template for RNA polymerase III in a transcription system prepared from nuclei of Xenopus laevis oocytes. This selective transcription may be explained by the lack of homology of the first 45 nucleotides of the non transcribed repeat with other members of the Alu family. In fact this region includes one of the homology blocks described for other RNA polymerase III templates.

Saccharomyces cerevisiae uses G protein-coupled receptors for signal transduction. We show that a fusion protein between the alpha-factor receptor (Ste2) and the Galpha subunit (Gpa1) transduces the signal efficiently in yeast cells devoid of the endogeneous STE2 and GPA1 genes. To evaluate the function of different domains of Galpha, a chimera between the N-terminal region of yeast Gpa1 and the C-terminal region of rat Gsalpha has been constructed. This chimeric Gpa1-Gsalpha is capable of restoring viability to haploid gpa1Delta cells, but signal transduction is prevented. This is consistent with evidence showing that the C-terminus of the homologous Galpha is required for receptor-G protein recognition. Surprisingly, a fusion protein between Ste2 and Gpa1-Gsalpha is able to transduce the signal efficiently. It appears, therefore, that the C-terminus of Galpha is mainly responsible for bringing the G protein into the close proximity of the receptor's intracellular domains, thus ensuring efficient coupling, rather than having a particular role in transmitting the signal. To confirm this conclusion, we show that two proteins interacting with each other (such as Snf1 and Snf4, or Ras and Raf), each of them fused either to the receptor or to the chimeric Galpha, allow efficient signal transduction.

Despite advances in next generation sequencing technologies, determining the genetic basis of ocular disease remains a major challenge due to the limited access and prohibitive cost of human forward genetics. Thus, less than 4,000 genes currently have available phenotype information for any organ system. Here we report the ophthalmic findings from the International Mouse Phenotyping Consortium, a large-scale functional genetic screen with the goal of generating and phenotyping a null mutant for every mouse gene. Of 4364 genes evaluated, 347 were identified to influence ocular phenotypes, 75% of which are entirely novel in ocular pathology. This discovery greatly increases the current number of genes known to contribute to ophthalmic disease, and it is likely that many of the genes will subsequently prove to be important in human ocular development and disease.

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