Isolation and characterization of cold-sensitive mutations at the benA, beta-tubulin, locus of Aspergillus nidulans

Summary We have isolated large numbers of conditionally lethal -tubulin mutations to provide raw material for analyzing the structure and function of tubulin and of microtubules. We have isolated such mutations as intragenic suppressors of benA33, a heat-sensitive (hs^-) -tubulin mutation of Aspergillus nidulans. Among over 2,600 revertants isolated, 126 were cold-sensitive (cs^-). In 41 of 78 cs^- revertants analyzed, cold sensitivity and reversion from hs^- to hs⁺ were due to mutations linked to benA33. In three cases reversion was due to mutations closely linked to benA33 but cold sensitivity was due to a coincidental mutation unlinked to benA33. In the remaining 34 cases reversion was due to mutations unlinked to benA33. Thirty-three of the revertants in which cold sensitivity and reversion were linked to benA33 were sufficiently cold-sensitive to allow us to select for rare recombinants between benA33 and putative suppressors in a revertant x wild-type (wt) cross. We found only one recombinant among 1,000 or more viable progeny from crosses of each of these revertants with a wt strain. Reversion is thus due to a back mutation or very closely linked suppressor in each case. We have analyzed 17 of these 33 revertants with greater precision and have found that, in each case, reversion is due to a suppressor mutation that maps to the right of benA33. The recombination frequencies between benA33 and the suppressors are very low (less than 1.2×10^-4) in all cases. Five of these 33 revertants have been examined microscopically and in each of them nuclear division and nuclear migration are inhibited at a restrictive temperature. We conclude that at least some and perhaps all of these revertants carry intragenic suppressors of benA33 that, in combination with benA33, cause cold sensitivity by inhibiting the functioning of microtubules at low temperatures. Of the 17 suppressors mapped, 11 map to two clusters. These clusters are likely to define regions particularly important to the functioning of the -tubulin molecule.     

Neurospora crassa Cytoplasmic Ribosomes: Isolation and Characterization of a Cold-Sensitive Mutant Defective in Ribosome Biosynthesis

Twenty-seven cold-sensitive mutants of Neurospora crassa were isolated by mutagenesis of wild-type conidia followed by filtration enrichment in complete medium at the nonpermissive temperature (10 C). Zone sedimentation analyses of cytoplasmic ribosomes isolated from the wild-type strain and from 14 of the mutant strains grown at 10 C indicate that one cold-sensitive mutant is defective in ribosome biosynthesis at that temperature: instead of the 2.3:1 mass ratio of 60S:37S ribosomal subunits characteristic of wild type, the mutant strain PJ30201 (called crib-1 for cytoplasmic ribosome biosynthesis) exhibits a mass ratio of approximately 7.2:1. Ribosomal subunits synthesized by strain PJ30201 at 25 C are present in wild-type proportions. The cold-sensitive and ribosomal phenotypes segregate together in tetrads isolated from crosses between strain PJ30201 and the wild type indicating that a single nuclear gene mutation is probably responsible for both mutant phenotypes. The crib-1 locus lies near the centromere in linkage group IV.     

Suppressors of the secY24 mutation: identification and characterization of additional ssy genes in Escherichia coli

We previously reported (Shiba et al., J. Bacteriol. 160:696-701, 1984) the isolation and characterization of the mutation (ssy) that suppresses the protein export defect due to the secY24(Ts) mutation and causes cold-sensitive growth of Escherichia coli. This report describes more systematic isolation of ssy mutations. Among temperature-resistant revertants of the secY24 mutant, 65 mutants were found to be cold sensitive. These cold-sensitive mutations have been classified by genetic mapping. Twenty-two mutations fell into the ssyA class previously described. The remaining mutations were located at five new loci: ssyB at 9.5 min between tsx and lon; ssyD around 3 min; ssyE at 72.5 min near secY; ssyF at 20.5 min within rpsA; and ssyG at 69.0 min near argG. Two predominant classes, ssyA and ssyB, are probably affected in protein synthesis at the elongation step, whereas the ssyF mutant contained an altered form of ribosomal protein S1 (the gene product of rpsA). These cold-sensitive ssy mutations which suppress secY24 may define genes whose function is somehow involved in the secY-dependent protein secretion mechanism. However, the existence of multiple suppressor loci makes it unlikely that all of these genes specify additional components of the export machinery. A delicate balance may exist between the systems for synthesizing and exporting proteins.     

Reduced Dosage of Genes Encoding Ribosomal Protein S18 Suppresses a Mitochondrial Initiation Codon Mutation in Saccharomyces Cerevisiae

A yeast mitochondrial translation initiation codon mutation affecting the gene for cytochrome oxidase subunit III (COX3) was partially suppressed by a spontaneous nuclear mutation. The suppressor mutation also caused cold-sensitive fermentative growth on glucose medium. Suppression and cold sensitivity resulted from inactivation of the gene product of RPS18A, one of two unlinked genes that code the essential cytoplasmic small subunit ribosomal protein termed S18 in yeast. The two S18 genes differ only by 21 silent substitutions in their exons; both are interrupted by a single intron after the 15th codon. Yeast S18 is homologous to the human S11 (70% identical) and the Escherichia coli S17 (35% identical) ribosomal proteins. This highly conserved family of ribosomal proteins has been implicated in maintenance of translational accuracy and is essential for assembly of the small ribosomal subunit. Characterization of the original rps18a-1 missense mutant and rps18aΔ and rps18bΔ null mutants revealed that levels of suppression, cold sensitivity and paromomycin sensitivity all varied directly with a limitation of small ribosomal subunits. The rps18a-1 mutant was most affected, followed by rps18aΔ then rps18bΔ. Mitochondrial mutations that decreased COX3 expression without altering the initiation codon were not suppressed. This allele specificity implicates mitochondrial translation in the mechanism of suppression. We could not detect an epitope-tagged variant of S18 in mitochondria. Thus, it appears that suppression of the mitochondrial translation initiation defect is caused indirectly by reduced levels of cytoplasmic small ribosomal subunits, leading to changes in either cytoplasmic translational accuracy or the relative levels of cytoplasmic translation products.     

A Suppressor of snf1 Mutations Causes Constitutive High-Level Invertase Synthesis in Yeast

The SNF1 gene product of Saccharomyces cerevisiae is required to derepress expression of many glucose-repressible genes, including the SUC2 structural gene for invertase. Strains carrying a recessive snf1 mutation are unable to ferment sucrose. We have isolated 30 partial phenotypic revertants of a snf1 mutant that were able to ferment sucrose. Genetic characterization of these revertants showed that the suppressor mutations were all recessive and defined eight complementation groups, designated ssn1 through ssn8 (suppressor of snf1 ). The revertants were assayed for secreted invertase activity, and although activity was detected in members of each complementation group, only the ssn6 strains contained wild-type levels. Synthesis of secreted invertase in ssn6 strains was found to be constitutive, that is, insensitive to glucose repression; moreover, the ssn6 mutations also conferred constitutivity in a wild-type ( SNF1 ) genetic background and are, therefore, not merely suppressors of snf1 . Pleiotropic defects were observed in ssn6 mutants. Genetic analysis suggested that the ssn6 mutations are allelic to the cyc8 mutation isolated by R. J. Rothstein and F. Sherman, which causes increased production of iso-2-cytochrome c. The data suggest a regulatory function for SSN6 .     

NAM9 nuclear suppressor of mitochondrial ochre mutations in Saccharomyces cerevisiae codes for a protein homologous to S4 ribosomal proteins from chloroplasts, bacteria, and eucaryotes.

We report the genetic characterization, molecular cloning, and sequencing of a novel nuclear suppressor, the NAM9 gene from Saccharomyces cerevisiae, which acts on mutations of mitochondrial DNA. The strain NAM9-1 was isolated as a respiration-competent revertant of a mitochondrial mit mutant which carries the V25 ochre mutation in the oxi1 gene. Genetic characterization of the NAM9-1 mutation has shown that it is a nuclear dominant omnipotent suppressor alleviating several mutations in all four mitochondrial genes tested and has suggested its informational, and probably ribosomal, character. The NAM9 gene was cloned by transformation of the recipient oxi1-V25 mutant to respiration competence by using a gene bank from the NAM9-1 rho o strain. Orthogonal-field alternation gel electrophoresis analysis and genetic mapping localized the NAM9 gene on the right arm of chromosome XIV. Sequence analysis of the NAM9 gene showed that it encodes a basic protein of 485 amino acids with a presequence that could target the protein to the mitochondrial matrix. The N-terminal sequence of 200 amino acids of the deduced NAM9 product strongly resembles the S4 ribosomal proteins from chloroplasts and bacteria. Significant although less extensive similarity was found with ribosomal cytoplasmic proteins from lower eucaryotes, including S. cerevisiae. Chromosomal inactivation of the NAM9+ gene is not lethal to the cell but leads to respiration deficiency and loss of mitochondrial DNA integrity. We conclude that the NAM9 gene product is a mitochondrial ribosomal counterpart of S4 ribosomal proteins found in other systems and that the suppressor acts through decreasing the fidelity of translation.     

A nuclear mutant of Saccharomyces cerevisiae deficient in mitochondrial DNA replication and polymerase activity

We have isolated a thermosensitive mutant which is transformed into a population of cells devoid of mitochondrial DNA (rho 0 cells) at 35 degrees C and is deficient in mitochondrial (mt) DNA polymerase activity. A single recessive nuclear mutation (mip1) is responsible for rho 0 phenotype and mtDNA polymerase deficiency in vitro. At 25 degrees C (or 30 degrees C) a dominant suppressor mutation (SUP) masks the deficiency in vivo. The meiotic segregants (mip1 sup) which do not harbor the suppressor have a rho 0 phenotype both at 25 and 35 degrees C. They have no mtDNA polymerase activity, in contrast with MIP rho 0 mutants of mitochondrial inheritance which do exhibit mtDNA polymerase activity. In the thermosensitive mutant (mip1 SUP), the replication of mtDNA observed in vivo at 30 degrees C is completely abolished at 35 degrees C. In the meiotic segregants (mip1 sup), no mtDNA replication takes place at 30 and 35 degrees C. The synthesis of nuclear DNA is not affected. DNA polymerases may have replicative and/or repair activity. There is no evidence that mip mutants are deficient in mtDNA repair. In contrast the MIP gene product is strictly required for the replication of mtDNA and for the expression of the mtDNA polymerase activity. This enzyme might be the replicase of mtDNA.     

Cold-Sensitive Cell-Division-Cycle Mutants of Yeast: Isolation, Properties, and Pseudoreversion Studies

We isolated 18 independent recessive cold-sensitive cell-division-cycle (cdc) mutants of Saccharomyces cerevisiae, in nine complementation groups. Terminal phenotypes exhibited include medial nuclear division, cytokinesis, and a previously undescribed terminal phenotype consisting of cells with a single small bud and an undivided nucleus. Four of the cold-sensitive mutants proved to be alleles of CDC11, while the remaining mutants defined at least six new cell-division-cycle genes: CDC44, CDC45, CDC48, CDC49, CDC50 and CDC51.—Spontaneous revertants from cold-sensitivity of four of the medial nuclear division cs cdc mutants were screened for simultaneous acquisition of a temperature-sensitive phenotype. The temperature-sensitive revertants of four different cs cdc mutants carried single new mutations, called Sup/Ts to denote their dual phenotype: suppression of the cold-sensitivity and concomitant conditional lethality at 37°. Many of the Sup/Ts mutations exhibited a cell-division-cycle terminal phenotype at the high temperature, and they defined two new cdc genes (CDC46 and CDC47). Two cold-sensitive medial nuclear division cdc mutants representing two different cdc genes were suppressed by different Sup/Ts alleles of another gene which also bears a medial nuclear division function (CDC46). In addition, the cold-sensitive medial nuclear division cdc mutant csH80 was suppressed by a Sup/Ts mutation yielding an unbudded terminal phenotype with an undivided nucleus at the high temperature. This mutation was an allele of CDC32. These results suggest a pattern of interaction among cdc gene products and indicate that cdc gene proteins might act in the cell cycle as complex specific functional assemblies.     

Suppression of carboxy-terminal truncations of the yeast mitochondrial mRNA-specific translational activator PET122 by mutations in two new genes, MRP17 and PET127

Summary The PET122 protein is one of three Saccharomyces cerevisiae nuclear gene products required specifically to activate translation of the mitochondrially coded COX3 mRNA. We have previously observed that mutations which remove the carboxy-terminal region of PET122 block translation of the COX3 mRNA but can be suppressed by unlinked nuclear mutations in several genes, two of which have been shown to code for proteins of the small subunit of mitochondrial ribosomes. Here we describe and map two more new genes identified as allele-specific suppressors that compensate for carboxy-terminal truncation of PET122. One of these genes, MRP17, is essential for the expression of all mitochondrial genes and encodes a protein of M_r 17343. The MRP17 protein is a component of the small ribosomal subunit in mitochondria, as demonstrated by the fact that a missense mutation, mrp17-1, predicted to cause a charge change indeed alters the charge of a mitochondrial ribosomal protein of the expected size. In addition, mrp17-1, in combination with some mutations affecting another mitochondrial ribosomal protein, caused a synthetic defective phenotype. These findings are consistent with a model in which PET122 functionally interacts with the ribosomal small subunit. The second new suppressor gene described here, PET127, encodes a protein too large (M_r 95900) to be a ribosomal protein and appears to operate by a different mechanism. PET127 is not absolutely required for mitochondrial gene expression and allele-specific suppression of pet122 mutations results from the loss of PET127 function: a pet127 deletion exhibited the same recessive suppressor activity as the original suppressor mutation. These findings suggest the possibility that PET127 could be a novel component of the mitochondrial translation system with a role in promoting accuracy of translational initiation.     

Respiration-deficient Chinese hamster cell mutants: genetic characterization.

We present here genetic experiments with a series of Chinese hamster cell mutants defective in oxidative energy metabolism. The mutations were all shown to be recessive in intraspecies hybrids. Thirty-five mutants were sorted into eight complementation groups, but one of these mutants failed to complement representatives of two distinct complementation groups. The possibility was raised that this is a cell carrying two mutations or a deletion. Because of the greatly different frequencies with which such mutants could be isolated from two different Chinese hamster cell lines, CCL16 (DON) and V79, the stability of representatives from each cell line was examined, and it was found that revertants could be obtained after treatment with mutagens, while spontaneous revertants appeared at unmeasurable or extremely low frequencies, with one exception. The mutant with a very noticeable frequency of spontaneous reversion was defective in mitochondrial protein synthesis, and the question arose whether the mutation was on the mitochondrial genome. A detailed fluctuation analysis of reversion rate and comparison with rates for other mutations was consistent with a nuclear mutation. This conclusion was supported by experiments involving fusions with cytoplasts.     

Era and RbfA have overlapping function in ribosome biogenesis in Escherichia coli

A cold-shock protein, RbfA (ribosome-binding factor A), is essential for cell growth at low temperature. In an rbfA-deletion strain, 30S and 50S ribosomal subunits increase relative to 70S monosomes with concomitant accumulation of a precursor 16S rRNA (17S rRNA). Recently, we have reported that overexpression of Era, an essential GTP-binding protein, suppresses not only the cold-sensitive cell growth but also defective ribosome biogenesis in the rbfA-deletion strain. Here, in order to elucidate how RbfA and Era functionally overlap, we characterized a cold-sensitive Era mutant (a point mutation at the Glu-200 to Lys; E200K) which shows a similar phenotype as the rbfA-deletion strain; accumulation of free ribosome subunits and 17S rRNA. To examine the effect of E200K in the rbfA-deletion strain, we constructed an E200K-inducible expression system. Interestingly, unlike wild-type Era, overexpression of Era(E200K) protein in the rbfA-deletion strain severely inhibited cell growth even at permissive temperature with further concomitant reduction of 16S rRNA. Purified Era(E200K) protein binds to 30S ribosomal subunits in a nucleotide-dependent manner like wild-type Era and retains both GTPase and autophosphorylation activities. Furthermore, we isolated spontaneous revertants of the E200K mutant. These revertants partially suppressed the accumulation of 17S rRNA. All the spontaneous mutations were found to result in higher Era(E200K) expression. These results suggest that the Era(E200K) protein has an impaired function in ribosome biogenesis without losing its ribosome binding activity. The severe growth defect caused by E200K in the rbfA-deletion strain may be due to competition between intrinsic wild-type Era and overexpressed Era(E200K) for binding to 30S ribosomal subunits. We propose that Era and RbfA have an overlapping function that is essential for ribosome biogenesis, and that RbfA becomes dispensable only at high temperatures because Era can complement its function only at higher temperatures.     

Cold-sensitive growth of a mutant of Escherichia coli with an altered ribosomal protein S8: analysis of revertants.

Summary 26 cold-resistant revertants of a cold-sensitiveEscherichia coli mutant with an altered ribosomal protein S8 were analyzed for their ribosomal protein pattern by two-dimensional polyacrylamide gel electrophoresis. It was found that 16 of them had acquired the apparent wild-type form of protein S8, one exhibits a more strongly altered S8 than the original mutant and two revertants regained the wild-type form of S8 and, in addition, possess alterations in protein L30. The ribosomes of the residual revertants showed no detectable difference from those of the parental S8 mutant.The mutation leading to the more strongly altered S8 was genetically not separable from the primary S8 mutation; this indicates that both mutations are very close to each other or at the same site. The structural gene for ribosomal protein L30 was mapped relative to two other ribosomal protein genes (for proteins S5 and S8) by the aid of one of the L30 mutants: The relative order obtained is:aroE....rpmD(L30)....rpsE(S5)....rpsH(S8)....THe L30 mutation impairs growth and ribosomal assembly at 20°C and is therefore the first example of a mutant with a defined 50S alteration that has (partial) cold-sensitive ribosome assembly. A double mutant was constructed which possesses both the S8 and the L30 mutations. It was found that the L30 mutation had a slight antagonistic effect on the growth inhibition caused by the S8 mutation. Thus the L30 mutants might have possibly arisen from the original S8 mutants first as S8/L30 double mutants which was followed by the loss of the original S8 lesion.     

A suppressor of temperature-sensitive rna mutations that affect mRNA metabolism in Saccharomyces cerevisiae.

We have isolated a dominant suppressor of rna mutation (SRN1) that relieves the temperature-sensitive inhibition of mRNA synthesis of ribosomal protein genes in the yeast Saccharomyces cerevisiae. The suppressor was selected for its ability to alleviate simultaneously the temperature-sensitive growth phenotypes of rna2 and rna6. Several independently isolated suppressors appeared to be recessive lethal mutations. One suppressor, SRN1, was recovered as viable in haploid strains. SRN1 can suppress rna2, rna3, rna4, rna5, rna6, and rna8 singly or in pairs, although some combinations of rna mutations are less well suppressed than others. The suppressor allows strains with rna mutations to grow at 34 degrees C but is unable to suppress at 37 degrees C; however, SRN1 does not, by itself, prevent growth at 37 degrees C. In addition, SRN1 suppresses the rna1 mutation which affects general mRNA levels and also leads to the accumulation of precursor tRNA for those tRNAs that have intervening sequences. SRN1 can suppress the rna1 mutation as well as the rna1 rna2 double mutation at 34 degrees C. The suppressor does not affect the temperature-sensitive growth of two unrelated temperature-sensitive mutations, cdc4 and cdc7.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

Gene Interactions Affecting Muscle Organization in CAENORHABDITIS ELEGANS

Revertants of unc-15(e73)I, a paralyzed mutant with an altered muscle paramyosin, include six dominant and two recessive intragenic unc-15 revertants, two new alleles of the previously identified suppressor gene, sup-3 V, and a new suppressor designated sup-19(m210)V. The recessive intragenic unc-15 revertants exhibit novel alterations in paramyosin paracrystal structure and distribution, and these alterations are modified by interaction with unc-82(e1220)IV, another mutation that affects paramyosin. A strain containing both unc-15 and a mutation in sup-3 V that restores movement was mutagenized, and paralyzed mutants resembling unc-15 were isolated. Twenty mutations that interfere with suppression were divided into three classes (nonmuscle, sus-1, and mutations within sup-3) based on phenotype, genetic map position and dominance. The nonmuscle mutations include dumpy and uncoordinated types that have no obvious direct effect on muscle organization. Two recessive mutations define a new gene, sus-1 III. These mutations modify the unc-15(e73) phenotype to produce a severely paralyzed, dystrophic double mutant that is not suppressed by sup-3. Five semidominant, intragenic sup-3 antisuppressor mutations, one of which occurred spontaneously, restore the wild-type sup-3 phenotype of nonsuppression. However, reversion of these mutants generated no new suppressor alleles of sup-3, suggesting that the sup-3 antisuppressor alleles are not wild type but may be null alleles.     

Nuclear and mitochondrial revertants of a mitochondrial mutant with a defect in the ATP synthetase complex

Summary Yeast strain 990 carries a mutation mapping to the oli1 locus of the mitochondrial genome, the gene encoding ATPase subunit 9. DNA sequence analysis indicated a substitution of valine for alanine at residue 22 of the protein. The strain failed to grow on nonfermentable carbon sources such as glycerol at low temperature (20°C). At 28°C the strain grew on nonfermentable carbon sources and was resistant to the antibiotic oligomycin. ATPase activity in mitochondria isolated from 990 was reduced relative to the wild-type strain from which it was derived, but the residual activity was oligomycin resistant. Subunit 9 (the DCCD-binding proteolipid) from the mutant strain exhibited reduced mobility in SDS-polyacrylamide gels relative to the wild-type proteolipid. Ten revertant strains of 990 were analyzed. All restored the ability to grow on glycerol at 20°C. Mitotic segregation data showed that eight of the ten revertants were attributable to mitochondrial genetic events and two were caused by nuclear events since they appeared to be recessive nuclear suppressors. These nuclear mutations retained partial resistance to oligomycin and did not alter the electrophoretic behavior of subunit 9 or any other ATPase subunit. When mitochondrial DNA from each of the revertant strains was hybridized with an oligonucleotide probe covering the oli1 mutation, seven of the mitochondrial revertants were found to be true revertants and one a second mutation at the site of the original 990 mutation. The oli1 gene from this strain contained a substitution of glycine for valine at residue 22. The proteolipid isolated from this strain had increased electrophoretic mobility relative to the wild-type proteolipid.Abbreviations DCCD dicyclohexylcarbodiimide - SDS sodium dodecyl sulfate - PMSF phenylmethylsulfonyl fluoride - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonate - SMP submitochondrial particles - mit^- mitochondrial point mutant     

The Lethal(1)tw-6(cs) Mutation of Drosophila Melanogaster Is a Dominant Antimorphic Allele of Nod and Is Associated with a Single Base Change in the Putative Atp-Binding Domain

The l(1)TW-6cs mutation is a cold-sensitive recessive lethal mutation in Drosophila melanogaster, that affects both meiotic and mitotic chromosome segregation. We report the isolation of three revertants of this mutation. All three revert both the meiotic and mitotic effects as well as the cold sensitivity, demonstrating that all three phenotypes are due to a single lesion. We further show that these revertants fail to complement an amorphic allele of the nod (no distributive disjunction) locus, which encodes a kinesin-like protein. These experiments demonstrate that l(1)TW-6cs is an antimorphic allele of nod, and we rename it nodDTW. Sequencing of the nod locus on a nodDTW-bearing chromosome reveals a single base change in the putative ATP-binding region of the motor domain of nod. Recessive, loss-of-function mutations at the nod locus specifically disrupt the segregation of nonexchange chromosomes in female meiosis. We demonstrate that, at 23.5 degrees, the meiotic defects in nodDTW/+ females are similar to those observed in nod/nod females; that is, the segregation of nonexchange chromosomes is abnormal. However, in nodDTW/nodDTW females, or in nodDTW/+ females at 18 degrees, we observe a more severe meiotic defect that apparently affects the segregation of both exchange and nonexchange chromosomes. In addition, nodDTW homozygotes and hemizygous males have previously been shown to exhibit mitotic defects including somatic chromosome breakage and loss. We propose that the defective protein encoded by the nodDTW allele interferes with proper chromosome movement during both meiosis and mitosis, perhaps by binding irreversibly to microtubules.     

Amino acid substitutions in subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Sequence analysis of a series of revertants of an oli1 mit- mutant carrying an amino acid substitution in the hydrophilic loop of subunit 9

This work concerns a biochemical genetic study of subunit 9 of the mitochondrial ATPase complex of Saccharomyces cerevisiae. Subunit 9, encoded by the mitochondrial oli1 gene, contains a hydrophilic loop connecting two transmembrane stems. In one particular oli1 mit- mutant 2422, the substitution of a positively charged amino acid in this loop (Arg39----Met) renders the ATPase complex non-functional. A series of 20 revertants, selected for their ability to grow on nonfermentable substrates, has been isolated from mutant 2422. The results of DNA sequence analysis of the oli1 gene in each revertant have led to the recognition of three groups of revertants. Class I revertants have undergone a same-site reversion event: the mutant Met39 is replaced either by arginine (as in wild-type) or lysine. Class II revertants maintain the mutant Met39 residue, but have undergone a second-site reversion event (Asn35----Lys). Two revertants showing an oligomycin-resistant phenotype carry this same second-site reversion in the loop region together with a further amino acid substitution in either of the two membrane-spanning segments of subunit 9 (either Gly23----Ser or Leu53----Phe). Class III revertants contain subunit 9 with the original mutant 2422 sequence, and additionally carry a recessive nuclear suppressor, demonstrated to represent a single gene. The results on the revertants in classes I and II indicate that there is a strict requirement for a positively charged residue in the hydrophilic loop close to the boundary of the lipid bilayer. The precise location of this positive charge is less stringent; in functional ATPase complexes it can be found at either residue 39 or 35. This charged residue is possibly required to interact with some other component of the mitochondrial ATPase complex. These findings, together with hydropathy plots of subunit 9 polypeptides from normal, mutant and revertant strains, led to the conclusion that the hydrophilic loop in normal subunit 9 extends further than previously suggested, with the boundary of the N-terminal membrane-embedded stem lying at residue 34. The possibility is raised that the observed suppression of the 2422 mutant phenotype in class III revertants is manifested through an accommodating change in a nuclear-encoded subunit of the ATPase complex.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

Genetic and Molecular Analysis of the Soe1 Gene: A Trna(3)(glu) Missense Suppressor of Yeast Cdc8 Mutations

The CDC8 gene of Saccharomyces cerevisiae encodes deoxythymidylate (dTMP) kinase and is required for nuclear and mitochondrial DNA replication in both the mitotic and meiotic cell cycles. All cdc8 temperature-sensitive mutants are partially defective in meiotic and mitochondrial functions at the permissive temperature. In a study of revertants of temperature-sensitive cdc8 mutants, the SOE201 and SOE1 mutants were isolated. The SOE201 mutant is a disome of chromosome X to which the cdc8 gene maps. Using the chromosome X aneuploids to vary cdc8 gene dosage, we demonstrate that different levels of dTMP kinase activity are required for mitotic, meiotic or mitochondrial DNA replication. The SOE1 mutant contains a dominant suppressor that suppresses five different cdc8 alleles but does not suppress a complete cdc8 deletion. The SOE1 gene is located less than 1.5 cM from the CYH2 gene on chromosome VII and is adjacent to the TSM437-CYH2 region, with the gene order being SOE1-TSM437-CYH2. SOE1 is an inefficient suppressor that can neither suppress the cdc8 hypomorphic phenotype nor restore dTMP kinase activity in vitro. SOE1 is a single C to T mutation in the anticodon of a tRNA(3Glu) gene and thereby, produces a missense suppressor tRNA capable of recognizing AAA lysine codons. We propose that the resultant lysine to glutamate change stabilizes thermo-labile dTMP kinase molecules in the cell.