A long open reading frame (ORF) closely linked to the Cephalosporium acremonium gene cefEF was identified by DNA sequencing. The cefEF gene encodes the enzyme involved in cephalosporin C (CPC) biosynthesis known as expandase/hydroxylase. Complementation of a C. acremonium cefG mutant, as well as expression of the gene in Aspergillus niger, showed this ORF to be the cefG gene, encoding cephalosporin C acetyltransferase, which catalyzes the last step in CPC biosynthesis. Analysis of transformants containing additional copies of this gene showed that a direct relationship exists between cefG copy number, cefG message levels, and CPC titers. This gene encodes an enzyme for what may be a rate-limiting step in CPC production.
Five previously unmapped frameshift suppressor genes have been located on the yeast genetic map. In addition, we have further characterized the map positions of two suppressors whose approximate locations were determined in an earlier study. These results represent the completion of genetic mapping studies on all 25 of the known frameshift suppressor genes in yeast.—The approximate location of each suppressor gene was initially determined through the use of a set of mapping strains containing 61 signal markers distributed throughout the yeast genome. Standard meiotic linkage was assayed in crosses between strains carrying the suppressors and the mapping strains. Subsequent to these approximate linkage determinations, each suppressor gene was more precisely located in multi-point crosses. The implications of these mapping results for the genomic distribution of frameshift suppressor genes, which include both glycine and proline tRNA genes, are discussed.
To evaluate the role of exon domains in tRNA splicing, the anti-codon stem of proline pre-tRNAUGG from Saccharomyces cerevisiae was altered by site-directed mutagenesis of the suf8 gene. Sixteen alleles were constructed that encode mutant pre-tRNAs containing all possible base combinations in the last base pair of the anticodon stem adjacent to the anticodon loop (positions 31 and 39). The altered pre-tRNAs were screened by using an in vitro endonucleolytic cleavage assay to determine whether perturbations in secondary structure affect the intron excision reaction. The pre-tRNAs were cleaved efficiently whenever secondary structure in the anticodon stem was maintained through standard base pairing or G.U interactions. However, most of the pre-tRNAs with disrupted secondary structure were poor substrates for intron excision. We also determined the extent to which the suf8 alleles produce functional products in vivo. Each allele was integrated in one to three copies into a yeast chromosome or introduced on a high-copy-number plasmid by transformation. The formation of a functional product was assayed by the ability of each allele to suppress the +1 frameshift mutation his4-713 through four-base codon reading, as shown previously for the SUF8-1 suppressor allele. We found that alleles containing any standard base pair or G.U pair at position 31/39 in the anticodon stem failed to suppress his4-713. We could not assess in vivo splicing with these alleles because the tRNA products, even if they are made, would be expected to read a normal triplet rather than a quadruplet codon. However, all of the alleles that contained a disrupted base pair at position 31/ 39 in the anticodon stem altered the structure of the tRNA in a manner that caused frameshift suppression. Suppression indicated that splicing must have occurred to some extent in vivo even though most of the suppression alleles produced pre-tRNAs that were cleaved with low efficiency or not at all in vitro. These results have important implications for the interpretation of in vitro cleavage assays in general and for the potential use of suppressors to select mutations that affects tRNA splicing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.