Yeast regulatory gene PPR1. II. Chromosomal localization, meiotic map, suppressibility, dominance/recessivity and dosage effect

The Saccharomyces cerevisiae gene PPR1 encodes a positive regulator of the expression of the two unlinked structural genes URA1 and URA3. The gene has been mapped to a position 6.5 cM from the centromere of chromosome XII. Uninducible alleles have been selected and used to establish a meiotic map. Suppressible alleles have been identified. The sequencing of a suppressible allele confirms the nonsense nature of the mutation as well as the reading frame deduced from the nucleotide sequence. No evidence of intracistronic complementation was found, and enzymatic analysis of leaky mutants did not reveal any mutations dissociating regulation of URA1 from that of URA3. Three in vitro-constructed deletions of PPR1 have been integrated at the chromosomal locus, giving strains with a completely negative phenotype. These deletion mutants display the wild-type basal level of URA1 and URA3 expression and show a semi-dominant phenotype in heteroallelic ppr1+/ppr1-delta diploids. Amplifying PPR1 by introduction into yeast on a multicopy vector increases the induction factor of URA1 and URA3 expression. These results show that the extent of regulation of the two structural genes is dependent on the concentration of the active PPR1 protein.     

A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5'-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.     

High-efficiency transformation of Pichia stipitis based on its URA3 gene and a homologous autonomous replication sequence, ARS2.

This paper describes the first high-efficiency transformation system for the xylose-fermenting yeast Pichia stipitis. The system includes integrating and autonomously replicating plasmids based on the gene for orotidine-5'-phosphate decarboxylase (URA3) and an autonomous replicating sequence (ARS) element (ARS2) isolated from P. stipitis CBS 6054. Ura- auxotrophs were obtained by selecting for resistance to 5-fluoroorotic acid and were identified as ura3 mutants by transformation with P. stipitis URA3. P. stipitis URA3 was cloned by its homology to Saccharomyces cerevisiae URA3, with which it is 69% identical in the coding region. P. stipitis ARS elements were cloned functionally through plasmid rescue. These sequences confer autonomous replication when cloned into vectors bearing the P. stipitis URA3 gene. P. stipitis ARS2 has features similar to those of the consensus ARS of S. cerevisiae and other ARS elements. Circular plasmids bearing the P. stipitis URA3 gene with various amounts of flanking sequences produced 600 to 8,600 Ura+ transformants per micrograms of DNA by electroporation. Most transformants obtained with circular vectors arose without integration of vector sequences. One vector yielded 5,200 to 12,500 Ura+ transformants per micrograms of DNA after it was linearized at various restriction enzyme sites within the P. stipitis URA3 insert. Transformants arising from linearized vectors produced stable integrants, and integration events were site specific for the genomic ura3 in 20% of the transformants examined. Plasmids bearing the P. stipitis URA3 gene and ARS2 element produced more than 30,000 transformants per micrograms of plasmid DNA. Autonomously replicating plasmids were stable for at least 50 generations in selection medium and were present at an average of 10 copies per nucleus.     

A genome wide study in fission yeast reveals nine PPR proteins that regulate mitochondrial gene expression

Pentatricopeptide repeat (PPR) proteins are particularly numerous in plant mitochondria and chloroplasts, where they are involved in different steps of RNA metabolism, probably due to the repeated 35 amino acid PPR motifs that are thought to mediate interactions with RNA. In non-photosynthetic eukaryotes only a handful of PPR proteins exist, for example the human LRPPRC, which is involved in a mitochondrial disease. We have conducted a systematic study of the PPR proteins in the fission yeast Schizosaccharomyces pombe and identified, in addition to the mitochondrial RNA polymerase, eight proteins all of which localized to the mitochondria, and showed some association with the membrane. The absence of all but one of these PPR proteins leads to a respiratory deficiency and modified patterns of steady state mt-mRNAs or newly synthesized mitochondrial proteins. Some cause a general defect, whereas others affect specific mitochondrial RNAs, either coding or non-coding: cox1, cox2, cox3, 15S rRNA, atp9 or atp6, sometimes leading to secondary defects. Interestingly, the two possible homologs of LRPPRC, ppr4 and ppr5, play opposite roles in the expression of the cox1 mt-mRNA, ppr4 being the first mRNA-specific translational activator identified in S. pombe, whereas ppr5 appears to be a general negative regulator of mitochondrial translation.     

Sequence analysis of the URA1 gene encoding orotidine-5'-monophosphate decarboxylase of Schizophyllum commune

The URA1 gene (encoding orotidine-5'-monophosphate decarboxylase) of the basidiomycete fungus Schizophyllum commune was mapped to a 1.4-kb BglI-BamHI fragment of two independent phage lambda clones previously isolated from a Schizophyllum genomic library. The fragment was identified by its ability to complement Schizophyllum ura1 mutants via transformation. The complete nucleotide sequence of the fragment containing the URA1 gene was determined. Sequence analysis revealed that the coding region of the URA1 gene encompasses a polypeptide of 279 amino acids (aa) interrupted by two small introns. The deduced aa sequence corresponds to 30.3 kDa and is substantially similar to the sequences of analogous polypeptides from other organisms. No canonical 5'-TATA sequence nor 3'-AATAAA polyadenylation signal are evident in the flanking regions of the URA1 gene.     

Histone gene organization of fission yeast: a common upstream sequence.

Histone genes of the fission yeast Schizosaccharomyces pombe were cloned from Charon 4A and cosmid gene libraries by hybridization, and their nucleotide sequences were determined. The genome of S. pombe has a single, isolated H2A, a pair of H2A-H2B and three pairs of H3-H4 (one H2B, two H2A and three each of H3 and H4). This non-assorted histone gene organization is distinct from that of the budding yeast which has two pairs of H2A-H2B and H3-H4. The predicted amino acid sequences of S. pombe histone H2As, H3s and H4s were identical except for three residue changes in H2As. Compared with those os S. cerevisiae and human, variable residues were clustered near the NH2- and COOH-terminal regions of H2A and H2B. Sequence homologies to the two organisms were roughly the same in H2A (79-83%), H3 (92-93%) and H4 (91%), but differed in H2B (82% to S. cerevisiae and 68% to human). The coding sequences in pairs of S. pombe histone genes were divergently directed. A 17-bp long highly homologous sequence (AACCCT box) that had internal 6-bp direct repeats was present in the intergene spacer sequences or in the 5' upstream region of all the cloned histone genes. A possible regulatory role of the common upstream sequence for histone gene expression is discussed.