ABSTRACT
A search was conducted for suppressors of the inositol auxotrophic phenotype of the ino4-8 mutant of yeast. The ino4-8 mutation is a single base pair change that results in substitution of lysine for glutamic acid at position 79 in the bHLH domain of the yeast regulatory protein, Ino4p. Ino4p dimerizes with a second bHLH protein, Ino2p, to form a complex that binds to the promoter of the INO1 gene, activating transcription. Of 31 recessive suppressors of ino4-8 isolated, 29 proved to be alleles of a single locus, identified as REG1, which encodes a regulatory subunit of a protein phosphatase involved in the glucose response pathway. The suppressor mutation, sia1-1, identified as an allele of REG1, caused constitutive INO1 expression and was capable of suppressing the inositol auxotrophy of a second ino4 missense mutant, ino4-26, as well as ino2-419, a missense mutation of INO2. The suppressors analyzed were unable to suppress ino2 and ino4 null mutations, but the reg1 deletion mutation could suppress ino4-8. A deletion mutation in the OPI1 negative regulator was incapable of suppressing ino4-8. The relative roles of the OPI1 and REG1 gene products in control of INO1 expression are discussed.
Subject(s)
Fungal Proteins/physiology , Myo-Inositol-1-Phosphate Synthase/physiology , Phosphoprotein Phosphatases , Repressor Proteins , Saccharomyces cerevisiae Proteins , Trans-Activators , Transcription Factors , Basic Helix-Loop-Helix Transcription Factors , Blotting, Northern , Cell Division , DNA-Binding Proteins , Diploidy , Gene Expression , Genes, Reporter , Genotype , Mutagenesis , Mutation, Missense , Phenotype , Point Mutation , Protein Phosphatase 1 , Saccharomyces cerevisiae/genetics , Suppression, Genetic , Time Factors , beta-Galactosidase/metabolismABSTRACT
The PRP31 gene encodes a factor essential for the splicing of pre-mRNA in Saccharomyces cerevisiae. Cell extracts derived from a prp31-1 strain fail to form mature spliceosomes upon heat inactivation, although commitment complexes and prespliceosome complexes are detected under these conditions. Coimmunoprecipitation experiments indicate that Prp31p is associated both with the U4/U6 x U5 tri-snRNP and, independently, with the prespliceosome prior to assembly of the tri-snRNP into the splicing complex. Nondenaturing gel electrophoresis and glycerol gradient analyses demonstrate that while Prp31p may play a role in maintaining the assembly or stability of tri-snRNPs, functional protein is not essential for the formation of U4/U6 or U4/U6 x U5 snRNPs. These results suggest that Prp31p is involved in recruiting the U4/U6 x U5 tri-snRNP to prespliceosome complexes or in stabilizing these interactions.
Subject(s)
Fungal Proteins/metabolism , RNA Splicing , Ribonucleoprotein, U4-U6 Small Nuclear/ultrastructure , Ribonucleoprotein, U5 Small Nuclear/ultrastructure , Saccharomyces cerevisiae Proteins , Spliceosomes/ultrastructure , Macromolecular Substances , Nucleic Acid Precursors/metabolism , RNA, Fungal/metabolism , RNA, Messenger/metabolism , RNA, Small Nuclear/metabolism , Ribonucleoprotein, U4-U6 Small Nuclear/metabolism , Ribonucleoprotein, U5 Small Nuclear/metabolism , Saccharomyces cerevisiae , Spliceosomes/metabolismABSTRACT
The pre-mRNA splicing factor Prp31p was identified in a screen of temperature-sensitive yeast strains for those exhibiting a splicing defect upon shift to the non- permissive temperature. The wild-type PRP31 gene was cloned and shown to be essential for cell viability. The PRP31 gene is predicted to encode a 60 kDa polypeptide. No similarities with other known splicing factors or motifs indicative of protein-protein or RNA-protein interaction domains are discernible in the predicted amino acid sequence. A PRP31 allele bearing a triple repeat of the hemagglutinin epitope has been generated. The tagged protein is functional in vivo and a single polypeptide species of the predicted size was detected by Western analysis with proteins from yeast cell extracts. Functional Prp31p is required for the processing of pre-mRNA species both in vivo and in vitro, indicating that the protein is directly involved in the splicing pathway.
Subject(s)
Fungal Proteins/genetics , Fungal Proteins/metabolism , Genes, Fungal , RNA Precursors/metabolism , RNA, Fungal/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Cloning, Molecular , Mutation , RNA Processing, Post-Transcriptional , RNA Splicing/genetics , Restriction Mapping , TemperatureABSTRACT
Cholesteryl ester transfer protein (CETP) has been shown to transfer cholesteryl esters among plasma lipoproteins. However, when reconstituted into phosphatidylcholine liposomes, the 74,000 protein mediates above non-specific values the transfer of HDL bound [3H]cholesterol into the artificial membrane system. Employing the known cDNA sequence of CETP, we synthesized a series of oligonucleotides with specific sequences for different regions of CETP and RNA isolated from tissues known to be producers of CETP (liver), and tissues not design to synthesize and secrete CETP (ovary, lung, intestine and heart). Hybridization experiments showed that independently of the type of tissue tested CETP sequences were found. It is suggested that a membrane form of CETP might have important repercussions locally.
