Analysis of mutant tRNA gene transcripts in vivo in Saccharomyces cerevisiae by abortive primer extension.

When the primer extension of a synthetic oligonucleotide hybridized to a complementary region of RNA is made in the presence of only three deoxyribonucleosides triphosphates, elongation of the primer stops as soon as the missing nucleotide is needed. This abortive primer extension assay has been adapted to analyse tRNA gene transcripts and has two main advantages. First it is specific and allows the identification of particular tRNA gene products in an homologous system provided the gene bears a point mutation. Second, it is highly sensitive and can be used to complement and confirm results of Northern blot hybridization. This assay should be a useful tool in the further in vivo study of the transcription and processing of particular tRNA genes in the homologous system. In this report the expression of wild-type and mutant yeast Sup4- tyrosine inserting suppressor gene was studied.

A structural analysis of P. polycephalum U1 RNA at the RNA and gene levels. Are there differentially expressed U1 RNA genes in P. polycephalum? U1 RNA evolution.

U1 RNAs were isolated from P. polycephalum microplasmodia nuclei and sequenced. A P. polycephalum gene coding for U1 RNA was also isolated. The coding region of this gene differs at 3 positions compared to the isolated U1 RNA species. Both isolated RNAs and the gene encoded RNA can be folded according to the secondary structure model previously proposed for U1 RNA. Putative regulatory elements very similar to those required for efficient transcription of U RNA genes from vertebrates, in particular, the -200 distal enhancer element, are present in the flanking regions of this gene. The presence of several U1 RNA genes in P. polycephalum was confirmed by Southern blot analysis of genomic DNA. In contrast to yeast S. cerevisiae U1 RNA, P. polycephalum U1 RNAs have a length similar to that of U1 RNAs from higher eukaryotes. Nevertheless, P. polycephalum U1 RNAs probably differ from these RNAs in the 5'-terminal segment supposed to base-pair with the 5'-end of introns. The results are discussed taking into account phylogenetic evolution and functional role of U1 RNA.