Extragenic Suppressors of Saccharomyces Cerevisiae Prp4 Mutations Identify a Negative Regulator of Prp Genes

The PRP4 gene encodes a protein that is a component of the U4/U6 small nuclear ribonucleoprotein particle and is necessary for both spliceosome assembly and pre-mRNA splicing. To identify genes whose products interact with the PRP4 gene or gene product, we isolated second-site suppressors of temperature-sensitive prp4 mutations. We limited ourselves to suppressors with a distinct phenotype, cold sensitivity, to facilitate analysis of mutants. Ten independent recessive suppressors were obtained that identified four complementation groups, spp41, spp42, spp43 and spp44 (suppressor of prp4, numbers 1-4). spp41-spp44 suppress the pre-mRNA splicing defect as well as the temperature-sensitive phenotype of prp4 strains. Each of these spp mutations also suppresses prp3; spp41 and spp42 suppress prp11 as well. Neither spp41 nor spp42 suppresses null alleles of prp3 or prp4, indicating that the suppression does not occur via a bypass mechanism. The spp41 and spp42 mutations are neither allele- nor gene-specific in their pattern of suppression and do not result in a defect in pre-mRNA splicing. Thus the SPP41 and SPP42 gene products are unlikely to participate directly in mRNA splicing or interact directly with Prp3p or Prp4p. Expression of PRP3-lacZ and PRP4-lacZ gene fusions is increased in spp41 strains, suggesting that wild-type Spp41p represses expression of PRP3 and PRP4. SPP41 was cloned and sequenced and found to be essential. spp43 is allelic to the previously identified suppressor srn1, which encodes a negative regulator of gene expression.     

Potential RNA Binding Proteins in Saccharomyces Cerevisiae Identified as Suppressors of Temperature-Sensitive Mutations in Npl3

The NPL3 gene of the yeast Saccharomyces cerevisiae encodes a protein with similarity to heterogeneous nuclear ribonucleoproteins (hnRNPs). Npl3p has been implicated in many nuclear-related events including RNA export, protein import, and rRNA processing. Several temperature-sensitive alleles of NPL3 have been isolated. We now report the sequence of these alleles. For one allele, npl3-1, four complementation groups of suppressors have been isolated. The cognate genes for the two recessive mutants were cloned. One of these is the previously known RNA15, which, like NPL3, also encodes a protein with similarity to the vertebrate hnRNP A/B protein family. The other suppressor corresponds to a newly defined gene we term HRP1, which also encodes a protein with similarity to the hnRNP A/B proteins of vertebrates. Mutations in HRP1 suppress all npl3 temperature-sensitive alleles but do not bypass an npl3 null allele. We show that HRP1 is essential for cell growth and that the corresponding protein is located in the nucleus. The discovery of two hnRNP homologues that can partially suppress the function of Np13p, also an RNA binding protein, will be discussed in terms of the possible roles for Npl3p in RNA metabolism.     

Genetic and Molecular Characterization of Suppressors of Sir4 Mutations in Saccharomyces Cerevisiae

In order to learn more about other proteins that may be involved in repression of HML and HMR in Saccharomyces cerevisiae, extragenic suppressor mutations were identified that could restore repression in cells defective in SIR4, a gene required for function of the silencer elements flanking HML and HMR. These suppressor mutations, which define at least three new genes, SAN1, SAN2 and SAN3, arose at the frequency expected for loss-of-function mutations following mutagenesis. All san mutations were recessive. Suppression by san1 was allele-nonspecific, since san1 could suppress two very different alleles of SIR4, and was locus-specific since san1 was unable to suppress a SIR3 mutation or a variety of mutations conferring auxotrophies. The SAN1 gene was cloned, sequenced, and used to construct a null allele. The null allele had the same phenotype as the EMS-induced mutations and exhibited no pleiotropies of its own. Thus, the SAN1 gene was not essential. SAN1-mediated suppression was neither due to compensatory mutations in interacting proteins, nor to translational missense suppression. SAN1 may act posttranslationally to control the stability or activity of the SIR4 protein.     

Intragenic and Extragenic Suppressors of Temperature Sensitive Mutations in the Replication Initiation Genes dnaD and dnaB of Bacillus subtilis

Background

The Bacillus subtilis genes dnaD and dnaB are essential for the initiation of DNA replication and are required for loading of the replicative helicase at the chromosomal origin of replication oriC. Wild type DnaD and DnaB interact weakly in vitro and this interaction has not been detected in vivo or in yeast two-hybrid assays.

Methodology/Principal Findings

We isolated second site suppressors of the temperature sensitive phenotypes caused by one dnaD mutation and two different dnaB mutations. Five different intragenic suppressors of the dnaD23ts mutation were identified. One intragenic suppressor was a deletion of two amino acids in DnaD. This deletion caused increased and detectable interaction between the mutant DnaD and wild type DnaB in a yeast two-hybrid assay, similar to the increased interaction caused by a missense mutation in dnaB that is an extragenic suppressor of dnaD23ts. We isolated both intragenic and extragenic suppressors of the two dnaBts alleles. Some of the extragenic suppressors were informational suppressors (missense suppressors) in tRNA genes. These suppressor mutations caused a change in the anticodon of an alanine tRNA so that it would recognize the mutant codon (threonine) in dnaB and likely insert the wild type amino acid (alanine).

Conclusions/Significance

The intragenic suppressors should provide insights into structure-function relationships in DnaD and DnaB, and interactions between DnaD and DnaB. The extragenic suppressors in the tRNA genes have important implications regarding the amount of wild type DnaB needed in the cell. Since missense suppressors are typically inefficient, these findings indicate that production of a small amount of wild type DnaB, in combination with the mutant protein, is sufficient to restore some DnaB function.     

Functional Studies of the Carboxy-Terminal Repeat Domain of Drosophila RNA Polymerase II in Vivo

To understand the in vivo function of the unique and conserved carboxy-terminal repeat domain (CTD) of RNA polymerase II largest subunit (RpII215), we have studied RNA polymerase II biosynthesis, activity and genetic function in Drosophila RpII215 mutants that possessed all (C4), half (W81) or none (IIt) of the CTD repeats. We have discovered that steady-state mRNA levels from transgenes encoding a fully truncated, CTD-less subunit (IIt) are essentially equal to wild-type levels, whereas the levels of the CTD-less subunit itself and the amount of polymerase harboring it (Pol IIT) are significantly lower than wild type. In contrast, for the half-CTD mutant (W81), steady-state mRNA levels are somewhat lower than for wild type or IIt, while W81 subunit and polymerase amounts are much less than wild type. Finally, we have tested genetically the ability of CTD mutants to complement (rescue) partially functional RpII215 alleles and have found that IIt fails to complement whereas W81 complements partially to completely. These results suggest that removal of the entire CTD renders polymerase completely defective in vivo, whereas eliminating half of the CTD results in a polymerase with significant in vivo activity.     

DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain

The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length.     

Extragenic Suppressors of Mutations in the Cytoplasmic C Terminus of Sec63 Define Five Genes in Saccharomyces Cerevisae

Mutations in the SEC63 gene of Saccharomyces cerevisiae affect both nuclear protein localization and translocation of proteins into the endoplasmic reticulum. We now report the isolation of suppressors of sec63-101 (formerly npl1-1), a temperature-sensitive allele of SEC63. Five complementation groups of extragenic mutations, son1-son5 (suppressor of npl1-1), were identified among the recessive suppressors. The son mutations are specific to SEC63, are not bypass suppressors, and are not new alleles of previously identified secretory (SEC61, SEC62, KAR2) or nuclear protein localization genes (NPL3, NPL4, NPL6). son1 mutations show regional specificity of suppression of sec63 alleles. At low temperatures, son1 mutants grow slowly and show partial mislocalization of nuclear antigens. The SON1 gene maps to chromosome IV and encodes a nuclear protein of 531 amino acids that contains two acidic stretches and a putative nuclear localization sequence. We show that son1 mutations suppress sec63-101 by elimination of Son1p function.     

Intragenic Enhancers and Suppressors of Phytoene Desaturase Mutations in Chlamydomonas reinhardtii

Photosynthetic organisms synthesize carotenoids for harvesting light energy, photoprotection, and maintaining the structure and function of photosynthetic membranes. A light-sensitive, phytoene-accumulating mutant, pds1-1, was isolated in Chlamydomonas reinhardtii and found to be genetically linked to the phytoene desaturase (PDS) gene. PDS catalyzes the second step in carotenoid biosynthesis-the conversion of phytoene to ζ-carotene. Decreased accumulation of downstream colored carotenoids suggested that the pds1-1 mutant is leaky for PDS activity. A screen for enhancers of the pds1-1 mutation yielded the pds1-2 allele, which completely lacks PDS activity. A second independent null mutant (pds1-3) was identified using DNA insertional mutagenesis. Both null mutants accumulate only phytoene and no other carotenoids. All three phytoene-accumulating mutants exhibited slower growth rates and reduced plating efficiency compared to wild-type cells and white phytoene synthase mutants. Insight into amino acid residues important for PDS activity was obtained through the characterization of intragenic suppressors of pds1-2. The suppressor mutants fell into three classes: revertants of the pds1-1 point mutation, mutations that changed PDS amino acid residue Pro64 to Phe, and mutations that converted PDS residue Lys90 to Met. Characterization of pds1-2 intragenic suppressors coupled with computational structure prediction of PDS suggest that amino acids at positions 90 and 143 are in close contact in the active PDS enzyme and have important roles in its structural stability and/or activity.     

Isolation and Characterization of Omnipotent Suppressors in the Yeast Saccharomyces Cerevisiae

Approximately 290 omnipotent suppressors, which enhance translational misreading, were isolated in strains of the yeast Saccharomyces cerevisiae containing the psi+ extrachromosomal determinant. The suppressors could be assigned to 8 classes by their pattern of suppression of five nutritional markers. The suppressors were further distinguished by differences in growth on paromomycin medium, hypertonic medium, low temperatures (10 degrees), nonfermentable carbon sources, alpha-aminoadipic acid medium, and by their dominance and recessiveness. Genetic analysis of 12 representative suppressors resulted in the assignment of these suppressors to 6 different loci, including the three previously described loci SUP35 (chromosome IV), SUP45 (chromosome II) and SUP46 (chromosome II), as well as three new loci SUP42 (chromosome IV), SUP43 (chromosome XV) and SUP44 (chromosome VII). Suppressors belonging to the same locus had a wide range of different phenotypes. Differences between alleles of the same locus and similarities between alleles of different loci suggest that the omnipotent suppressors encode proteins that effect different functions and that altered forms of each of the proteins can effect the same function.     

Instability of a Plasmid-Borne Inverted Repeat in Saccharomyces Cerevisiae

Inverted repeated DNA sequences are common in both prokaryotes and eukaryotes. We found that a plasmid-borne 94 base-pair inverted repeat (a perfect palindrome of 47 bp) containing a poly GT sequence is unstable in S. cerevisiae, with a minimal deletion frequency of about 10(-4)/mitotic division. Ten independent deletions had identical end points. Sequence analysis indicated that all deletions were the result of a DNA polymerase slippage event (or a recombination event) involving a 5-bp repeat (5' CGACG 3') that flanked the inverted repeat. The deletion rate and the types of deletions were unaffected by the rad52 mutation. Strains with the pms1 mutation had a 10-fold elevated frequency of instability of the inverted repeat. The types of sequence alterations observed in the pms1 background, however, were different than those seen in either the wild-type or rad52 genetic backgrounds.     

Suppressors of a Gpa1 Mutation Cause Sterility in Saccharomyces Cerevisiae

The Saccharomyces cerevisiae GPA1 gene encodes a protein highly homologous to the α subunit of mammalian G proteins and is essential for haploid cell growth. We have selected 77 mutants able to suppress the lethality resulting from disruption of GPA1 (gpa1::HIS3). Two strains bearing either of two recessive mutations, sgp1 and sgp2, in combination with the disruption mutation, showed a cell type nonspecific sterile phenotype, yet expressed the major α-factor gene (MFα1) as judged by the ability to express a MFα1-lacZ fusion gene. The sgp1 mutation was closely linked to gpa1::HIS3 and probably occurred at the GPA1 locus. The sgp2 mutation was not linked to GPA1 and was different from the previously identified cell type nonspecific sterile mutations (ste4, ste5, ste7, ste11 and ste12). sgp2 GPA1 cells showed a fertile phenotype, indicating that the mating defect caused by sgp2 is associated with the loss of GPA1 function. While expression of a FUS1-lacZ fusion gene was induced in wild-type cells by the addition of α-factor, mutants bearing sgp1 or sgp2 as well as gpa1::HIS3 constitutively expressed FUS1-lacZ. These observations suggest that GPA1 (SGP1) and SGP2 are involved in mating factor-mediated signal transduction, which causes both cell cycle arrest in the late G(1) phase and induction of genes necessary for mating such as FUS1.     

Nonsense Mutations in Essential Genes of Saccharomyces Cerevisiae

A new method for isolating nonsense mutations in essential yeast genes has been used to develop a collection of 115 ochre mutations that define 94 complementation groups. The mutants are isolated in a genetic background that includes an ochre suppressor on a metastable plasmid and a suppressible colony-color marker on a chromosome. When the parental strain is plated on a rich medium, the colonies display a pattern of red, plasmid-free sectors on a white background. Mutants containing an ochre mutation in any essential yeast gene give rise to nonsectoring, white colonies, since cell growth is dependent on the presence of the plasmid-borne suppressor. Analysis of the data suggests that mutations are being recovered from a pool of approximately 250 genes.