Identification of the structural gene for the S9 ribosomal protein in the fungus Podospora anserina: a new protein involved in the control of translational accuracy

Summary AS9-1 was isolated as a mutation restoring growth in a strain carrying the ribosomal mutation su12-1. The AS9-1 mutation confers a weak antisuppressor effect and a low level of resistance to paromomycin. Two-dimensional polyacrylamide gel electrophoresis patterns of the ribosomal proteins from AS9-1 strains show an altered S9 protein which is more basic than the wild-type form. The presence of the two forms of the protein (wild-type and mutant) in heterocaryotic strains strongly suggests that AS9 is the structural gene for the ribosomal protein S9.     

At least seven ribosomal proteins are involved in the control of translational accuracy in a eukaryotic organism

In the filamentous fungus Podospora anserina, ribosomal proteins of 60 mutants impaired in the control of translational fidelity have been submitted to electrophoretic analysis. The "four corners" system combining four different two-dimensional polyacrylamide gel electrophoretic systems has been used. An altered electrophoretic pattern has been observed for 12 mutants. In mutants su3, su12 and su11 (decreased translational fidelity), proteins S1, S7 and S8, respectively, are altered. For AS mutants (increased translational fidelity), proteins S9, S12 and S19, respectively, are altered in AS9, AS1 and AS6 mutants, and protein S29 is lacking in AS3 mutants. The data suggest that five of these genes (at least) are the structural genes for the relevant proteins (su3:S1, su12:S7, AS1:S12, AS6:S19, AS9:S9), while the AS3 gene may code for a modifying enzyme.     

Different alterations of the ribosomal protein S7 lead to opposite effects on translational fidelity in the fungus Podospora anserina

In the fungus Podospora anserina, the su12-1 mutation was previously found to decrease translational accuracy and to alter the ribosomal protein S7. The mutant protein is more basic than the wild type. Among the revertants of the two ribosomal mutations su12-1 and su12-2, 29 contained a second mutation very closely linked to su12. Biochemical analysis of these revertants by functional poly(U) tests and electrophoretical study of the ribosomes led to two conclusions. First, some revertant strains contain new mutant forms of S7. This suggests that su12 is the structural gene for the ribosomal protein S7. Second, the su12-2 revertants display antisuppressor properties in vivo and in vitro (i.e. increased translational accuracy). The electrophoretical patterns of their ribosomal proteins show new, more acidic, forms of S7. Therefore, su12 can be mutated towards either a lower or a greater translational accuracy corresponding to two opposite modifications of the global charge of the ribosomal protein S7. A more acidic form than wild type leads to increased accuracy and a more basic form to decreased accuracy.     

Mutations affecting translational fidelity in the eucaryote Podospora anserina: Characterization of two ribosomal restrictive mutations

Summary Fifty-nine mutations that restrict suppressor efficiency were selected in the fungus Podospra anserina using four different screening methods. Previous genetic analysis has shown that these antisuppressors lie in six loci and that they could be similar to ribosomal restrictive mutations known in Escherichia coli. The present study deals with the response of two of them, AS1-1 and AS6-1, to paromomycin and low temperature both in vivo and in vitro. The data demonstrate that ribosomes of the mutant and double-mutant strains are equally resistant to the ambiguity effect of paromomycin. These data are the first demonstration of mutations that increase translational fidelity in a eucaryotic organism.     

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

Macrolide and aminoglycoside antibiotic resistance mutations in the Bacillus subtilis ribosome resulting in temperature-sensitive sporulation

Summary Mutants of Bacillus subtilis resistant to various macrolide antibiotics have been isolated and characterized with respect to their sporulation phenotype and the electrophoretic mobility of their ribosomal proteins (r-proteins). Two types of major alterations of r-protein L17, one probably due to a small deletion, are found among mutants exhibiting high-level macrolide resistance. These mutants are all temperature-sensitive for sporulation (Spo^ts). Low-level resistance to some macrolides is found to be associated with minor alterations in r-protein L17. These mutations do not cause a defective sporulation phenotype. All of the macrolide resistance mutations map at the same locus within the Str-Spc region of the B. subtilis chromosome. Hence, changes in a single ribosomal protein can result in different sporulation phenotypes.Mutants resistant to the aminoglycoside antibiotics neomycin and kanamycin have been isolated. Approximately 5% of these are Spo^ts. Representative mutations, neo 162 and kan25, cause concomitant drug resistance and sporulation temperature-sensitivity and map as single-site lesions in the Str-Spc region of the chromosome. Strains bearing neo162 or kan25 are equally cross-resistant to several aminoglycoside antibiotics but show no resistance to streptomycin or spectinomycin. These mutations define a new B. subtilis drug resistance locus at which mutation can cause defective sporulation.     

REV7, a new gene concerned with UV mutagenesis in yeast

Summary Three allelic mutations of a new yeast gene, which we have named REV7, have been isolated by testing 313 methyl methane sulfonate sensitive mutants for UV-induced reversion of a lys2 allele. Rev7 mutants are markedly deficient with respect to UV-induced reversion of lys2, are slightly sensitive to UV and appear to be in the RAD6 epistasis group for UV survival. Rev7-1, which is probably an amber mutation, does not appear to affect sporulation in homozygous diploids. The REV7 gene is located about 12 cM distal to HIS5 on chromosome IX.     

Changes in regulation of ribosome synthesis during different stages of the life cycle of Saccharomyces cerevisiae.

Summary Diploid strains of Saccharomyces cerevisiae, each homozygous for one of the temperature sensitive mutations rna2, rna4, rna6 or rna8, are temperature sensitive for ribosome synthesis during vegetative growth, but are not inhibited for ribosomal synthesis at the restrictive temperature under sporulation conditions. The continued ribosome biosynthesis at the restrictive temperature (34° C) during sporulation includes de novo synthesis of both ribosomal RNA and ribosomal proteins. This lack of inhibition of ribosome biosynthesis is found even when cells committed to complete sporulation are returned to vegetative growth medium. The ribosomes synthesized at 34° C are apparently functional, as they are found in polyribosomes. Although the rna mutants do not regulate ribosome synthesis during sporulation, all of these diploid strains fail to complete sporulation at 34° C. The cells are arrested after the second meiotic nuclear division but before ascus formation. The failure to complete sporulation at the restrictive temperature and the inhibition of ribosome biosynthesis during growth are caused by the same mutation, because revertants selected for temperature independent growth were also able to sporulate at 34° C.     

Homologous and heterologous expression of a ribosomal protein gene in Podospora anserina requires an intron

Although the role of introns in eucaryotic nuclear genes has been much debated, it remains underinvestigated in fungi. The AS1 gene of Podospora anserina contains three introns and encodes a ribosomal protein (S12) belonging to the well-conserved bacterial S19 family. We attempted to complement the highly pleiotropic mutation AS1-4 with a cDNA encoding the homologous human (S15) protein (rig gene) under the control of the AS1 promoter. In a control experiment, the AS1 ⁺ cDNA was unable to complement fully the AS1-4 mutation. It was assumed that the AS1 cDNA was not well expressed and that the AS1 gene needed intron(s) to be efficiently expressed. Addition of the first intron of the AS1 gene to the AS1 and rig cDNAs did indeed allow complementation of all the phenotypic defects of the AS1-4 mutation. These data lead to two main conclusions. First, the human S15 ribosomal protein is functional in Podospora. Second, full expression of the Podospora AS1 gene requires at least one intron. Received: 26 April 1996 / Accepted: 22 August 1996     

ScoC Mediates Catabolite Repression of Sporulation in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Sporulation in Bacillus subtilis can be triggered by carbon catabolite limitation. Conversely, carbon source excess can repress the production of extracellular enzymes, motility, and sporulation. Recent studies have implicated a pH-sensing mechanism, involving AbrB, the TCA cycle, Spo0K, and Ï^H in controlling the catabolite repression of sporulation gene expression. In an accompanying paper, we demonstrate that the AbrB-dependent pH-sensing mechanism may not be the only means by which carbon catabolites affect sporulation. In the studies reported here, we have examined the molecular basis underlying the catabolite repression phenotype of mutations in the hpr (scoC), rpoD (crsA47), and spo0A (rvtA11) loci. Loss of function mutations in hpr (scoC) restored sporulation gene expression and sporulation in the presence of excess catabolite(s), suggesting that Hpr (ScoC) has a pivotal role in mediating catabolite repression. Moreover, hpr gene expression increased substantially in the presence of excess catabolite(s), further supporting the involvement of Hpr (ScoC) in the carbon catabolite response system. We suggest that alterations in the phosphorelay response to catabolites may be one mechanism by which catabolite-resistant mutants such as crsA and rvtA are able to sporulate in the presence of excess glucoseReceived: 12 November 2002 / Accepted: 13 December 2002     

cdc7-1 A temperature sensitive cell-cycle mutant which interferes with induced mutagenesis in Saccharomyces cerevisiae

Summary The mutant cdc7-1 is shown here to block UV induced reversion of six different auxotrophic mutations and forward mutations at several genes concerned with adenine biosynthesis in Saccharomyces cerevisiae. Chemical mutagenesis is also drastically reduced. In its effect on mutagenesis cdc7-1 resembles rad6-1. However, in contrast to rad6-1, cdc7-1 does not affect sporulation or mitotic recombination neither is it sensitive to the antifolate drug trimethoprim. It appears to fall in the same epistatic group as rad6-1. Possible explanations for its action are briefly considered.     

Physiological suppression of Bacillus subtilis conditional sporulation phenotypes: RNA polymerase and ribosomal mutations

Summary The temperature-sensitive sporulation phenotype (Spo^ts) of Bacillus subtilis RNA polymerase, ribosomal and protein synthesis elongation factor G mutations can be corrected by supplementing the growth medium with carbohydrates such as ribose or glycerol, or with synthetic lipids such as Tween 40. The data suggest that these mutations affect a single common aspect of developmental cell function. It is proposed that these lesions prevent sporulation by disturbing the regulation of sporulating cell metabolic balance.     

Characterization of Usher syndrome type I gene mutations in an Usher syndrome patient population

Usher syndrome type I (USH1), the most severe form of this syndrome, is characterized by profound congenital sensorineural deafness, vestibular dysfunction, and retinitis pigmentosa. At least seven USH1 loci, USH1A-G, have been mapped to the chromosome regions 14q32, 11q13.5, 11p15, 10q21-q22, 21q21, 10q21-q22, and 17q24-25, respectively. Mutations in five genes, including MYO7A, USH1C, CDH23, PCDH15 and SANS, have been shown to be the cause of Usher syndrome type 1B, type 1C, type 1D, type 1F and type 1G, respectively. In the present study, we carried out a systematic mutation screening of these genes in USH1 patients from USA and from UK. We identified a total of 27 different mutations; of these, 19 are novel, including nine missense, two nonsense, four deletions, one insertion and three splicing defects. Approximatelly 35–39% of the observed mutations involved the USH1B and USH1D genes, followed by 11% for USH1F and 7% for USH1C in non-Acadian alleles and 7% for USH1G. Two of the 12 MYO7A mutations, R666X and IVS40-1G>T accounted for 38% of the mutations at that locus. A 193delC mutation accounted for 26% of CDH23 (USH1D) mutations, confirming its high frequency. The most common PCDH15 (USH1F) mutation in this study, 5601-5603delAAC, accounts for 33% of mutant alleles. Interestingly, a novel SANS mutation, W38X, was observed only in the USA cohort. The present study suggests that mutations in MYO7A and CDH23 are the two major components of causes for USH1, while PCDH15, USH1C, and SANS are less frequent causes.X.-M. Ouyang and D. Yan contributed equally to this work     

Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

Isolation and characterization of recessive sporeless mutants in the basidiomycete Coprinus cinereus

Summary Twenty-four sporeless mutants were isolated from an Amut Bmut strain (mutant in the incompatibility factors) of the basidiomycete Coprinus cinereus. All the sporeless mutations were recessive to the wild type. These mutants and a previously isolated recessive sporeless strain, N2-7 (Kanda and Ishikawa 1986) were crossed with a wildtype strain. An F1 random spore analysis indicated that sporulation deficiencies in these mutants were caused by single nuclear gene mutations. These mutations were all complementary to each other, thus twenty-five sporulation genes were identified. Five of them were linked to the A incompatibility factor. Cytological observations classified these mutants into the following four types according to the stage of the blockage: (1) meiosis stopped at meta-anaphase I; (2) meiosis was completed, but further basidial development did not occur; (3) basidial development stopped at the sterigma stage; (4) basidial development stopped at the prespore stage.     

Escape from Premature Death Due to Nuclear Mutations inPodospora anserina:Repeal versus Respite

Premature death has been defined as a growth stoppage linked to the accumulation of specific deletions of the mitochondrial genome (mtDNA) inPodospora anserina. This occurs only in strains carrying theAS1-4mutation which lies in a gene encoding a cytosolic ribosomal protein. Here we describe the isolation and genetic characterization of 10 nuclear mutations which either delay the appearance of this syndrome (respite from premature death) or cause a switch to the classical senescence process (repeal of premature death). These mutations lie in at least six genes. Some cause defects at the levels of ascospore germination, growth rates, and/or sensitivity toward inhibitors of protein syntheses. All modify the onset of senescence in wild-type (AS1⁺) strains. The role played by these genes is discussed with respect to the control of diseases due to mtDNA rearrangements in filamentous fungi.     

Recombination and mutagenesis in rad6 mutants of Saccharomyces cerevisiae: Evidence for multiple functions of the RAD6 gene

Summary The rad6-1 and rad6-3 mutants are highly UV sensitive and show an increase in spontaneous and UV induced mitotic heteroallelic recombination in diploids. Both rad6 mutants are proficient in spontaneous and UV induced unequal sister chromatid recombination in the reiterated ribosomal DNA sequence and are deficient in UV induced mutagenesis. In contrast to the above effects where both mutants appear similar, rad6-1 mutants are deficient in sporulation and meiotic recombination whereas rad6-3 mutants are proficient. The differential effects of these mutations indicate that the RAD6 gene is multifunctional. The possible role of the RAD6 gene in error prone excision repair of UV damage during the G1 phase of the cell cycle in addition to its role in postreplication repair is discussed.     

Genetic analysis of the non-sporulating phenotype of brewer's yeasts

The ploidies and sporulation abilities of six brewer's yeasts were examined. One (YB11-1) out of the six was triploid and sporulating, another (IFO2031) was haploid, and the others (IFO1167, IFO2003, S341 and YB3-7) were diploid and non-sporulating. The five non-sporulating strains did not have the premeiotic DNA synthesis. Their non-sporulating phenotypes were genetically analyzed by examining the sporulation abilities of hybrids between brewer's yeasts and standard genetic strains of Saccharomyces cerevisiae. All non-sporulating brewer's yeasts complemented 32 sporulation-deficient mutations (spoT–spoT23, spo1–spo5, spo7, spo8, spo10, and spo11). Hybrids between brewer's yeasts and haploid or diploid strains homozygous for the mating-type locus had poor or no sporualtion. On the contrary, hybrids between brewer's yeasts and diploid strains heterozygous for the mating-type locus sporulated at a high frequency. These results indicated that the non-sporulating phenotype of brewer's yeasts was caused by a deficiency of the mating-type genes rather than by mutations of sporulation genes. The Southern hybridization probed with the MATa gene showed polymorphisms in mating-type genes of brewer's yeasts.     

Characteristics of pulse-labeled ribonucleic acid in relation to medium pH and acetate concentration during meiosis of yeast

Summary Cellular impermeability associated with sporulating cells of Saccharomyces cerevisiae is caused by a rapid increase in the medium pH. Three factors have been identified as being important in regulating the rise in medium pH: 1) the cell density, 2) the potassium acetate concentration of the sporulation medium, and 3) and initial pH below 6.0. Sporulation conditions were established for strain 4579 which resulted in optimum uptake of ³H-adenine at T₇, a period when the cells would be normally impermeable. Pulse-labeled polysomal RNA was characterized at T₄ in naturally permeable cells of strain SK-1 and impermeable cells which required manipulation of the medium pH to facilitate uptake. Transfer ribonucleic acid (RNA), poly A-containing RNA and ribosomal RNA were synthesized in both cultures during the 20 min pulse. Furthermore, the rate of ribosomal RNA synthesis and processing into functional ribosomes approached the rate reported for vegetative cells. Initial sporulation conditions which caused a prolonged delay in the rise in medium pH adversely affected the kinetics of appearance and number of ascospores. The affect was shown to be on meiotic events since a reduction of sporulation was always accompanied by a reduction in the amount of intragenic recombination.