Inositol-Requiring Mutants of SACCHAROMYCES CEREVISIAE

Fifty-two inositol-requiring mutants of Saccharomyces cerevisiae were isolated following mutagenesis with ethyl methanesulfonate. Complementation and tetrad analysis revealed ten major complementation classes, representing ten independently segregating loci (designated ino1 through ino10) which recombined freely with their respective centromeres. Members of any given complementation class segregated as alleles of a single locus. Thirteen complementation subclasses were identified among thirty-six mutants which behaved as alleles of the ino1 locus. The complementation map for these mutants was circular.—Dramatic cell viability losses indicative of unbalanced growth were observed in liquid cultures of representative mutants under conditions of inositol starvation. Investigation of the timing, kinetics, and extent of cell death revealed that losses in cell viability in the range of 2-4 log orders could be prevented by the addition of inositol to the medium or by disruption of protein synthesis with cycloheximide. Mutants defective in nine of the ten loci identified in this study displayed these unusual characteristics. The results suggest an important physiological role for inositol that may be related to its cellular localization and function in membrane phospholipids. The possibility is discussed that inositol deficiency initiates the process of unbalanced growth leading to cell death through the loss of normal assembly, function, or integrity of biomembranes.—Part of this work has been reported in preliminary form (Culbertson and Henry 1974).     

The mechanism of interallelic complementation at the INO1 locus in yeast: Immunological analysis of mutants

Summary The ino1 locus of yeast has been demonstrated to be the structural gene for the repressible enzyme, L-myo-inositol-1-phosphate synthase (Donahue and Henry 1981 a). We have screened a large number of allelic representatives of the ino1 locus for the presence of protein which cross reacts with antibody produced in response to purified wild type inositol-1-phosphate synthase. Approximately 50% of all ino1 representatives screened by immunoprecipitation produce a protein of 62,000 molecular weight, identical in size to the wild type enzyme subunit. These mutants (termed crm⁺) were tested for expression of the 62,000 MW protein under conditions which are repressing for the wild type enzyme (greater than 25 M exogenous inositol). The protein produced by the crm⁺ mutants, like the active enzyme in wild type yeast, is repressed in the presence of high levels of exogenous inositol. In addition, we have reassessed the interallelic complementation pattern observed among mutants at the ino1 locus. The entire pattern of interallelic complementation is temperature sensitive.     

The OPI1 gene of Saccharomyces cerevisiae, a negative regulator of phospholipid biosynthesis, encodes a protein containing polyglutamine tracts and a leucine zipper

In Saccharomyces cerevisiae, recessive mutations at the OPI1 locus result in constitutively derepressed expression of inositol 1-phosphate synthase, the product of the INO1 gene. Many of the other enzymes involved in phospholipid biosynthesis are also expressed at high derepressed levels in opi1 mutants. Thus, the OPI1 gene is believed to encode a negative regulator that is required to repress a whole subset of structural genes encoding for phospholipid biosynthetic enzymes. In this study, the OPI1 gene was mapped to chromosome VIII and cloned. When transformed into an opi1 mutant, the cloned DNA was capable of complementing the mutant phenotype and restoring correct regulation to the INO1 structural gene. Construction of two opi1 disruption alleles and subsequent genetic analysis of strains bearing these alleles confirmed that the cloned DNA was homologous to the genomic OPI1 locus. Furthermore, the OPI1 gene was found to be nonessential to the organism since mutants bearing the null allele were viable and exhibited a phenotype similar to that of previously isolated opi1 mutants. Similar to other opi1 mutants, the opi1 disruption mutants accumulated INO1 mRNA constitutively to a level 2-3-fold higher than that observed in wild-type cells. The cloned OPI1 gene was sequenced, and translation of the open reading frame predicted a protein composed of 404 amino acid residues with a molecular weight of 40,036. The predicted Opi1 protein contained a well defined heptad repeat of leucine residues that has been observed in other regulatory proteins. In addition, the predicted protein contained polyglutamine residue stretches which have also been reported in yeast genes having regulatory functions. Sequencing of opi1 mutant alleles, isolated after chemical mutagenesis, revealed that several were the result of a chain termination mutation located within the largest polyglutamine residue stretch.     

The INO2 and INO4 loci of Saccharomyces cerevisiae are pleiotropic regulatory genes.

We isolated a mutant of Saccharomyces cerevisiae defective in the formation of phosphatidylcholine via methylation of phosphatidylethanolamine. The mutant synthesized phosphatidylcholine at a reduced rate and accumulated increased amounts of methylated phospholipid intermediates. It was also found to be auxotrophic for inositol and allelic to an existing series of ino4 mutants. The ino2 and ino4 mutants, originally isolated on the basis of an inositol requirement, are unable to derepress the cytoplasmic enzyme inositol-1-phosphate synthase (myo-inositol-1-phosphate synthase; EC 5.5.1.4). The INO4 and INO2 genes were, thus, previously identified as regulatory genes whose wild-type product is required for expression of the INO1 gene product inositol-1-phosphate synthase (T. Donahue and S. Henry, J. Biol. Chem. 256:7077-7085, 1981). In addition to the identification of a new ino4-allele, further characterization of the existing series of ino4 and ino2 mutants, reported here, demonstrated that they all have a reduced capacity to convert phosphatidylethanolamine to phosphatidylcholine. The pleiotropic phenotype of the ino2 and ino4 mutants described in this paper suggests that the INO2 and INO4 loci are involved in the regulation of phospholipid methylation in the membrane as well as inositol biosynthesis in the cytoplasm.     

Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed.     

Two Recessive Suppressors of SACCHAROMYCES CEREVISIAE CHO1 That Are Unlinked but Fall in the Same Complementation Group

Phenotypic reversion of ethanolamine-requiring Saccharomyces cerevisiae cho1 mutants is predominantly due to recessive mutations at genes unlinked to the chromosome V cho1 locus. The recessive suppressors do not correct the primary cho1 defect in phosphatidylserine synthesis but circumvent it with a novel endogenous supply of ethanolamine. One suppressor (eam1) was previously mapped to chromosome X, and 135 suppressor isolates were identified as eam1 alleles by complementation analysis. Additional meiotic recombination studies have identified a second genetic locus, eam2, that falls in the eam1 complementation group but maps close to the centromere of chromosome IV. Although the normal EAM1 and EAM2 alleles are fully dominant over recessive mutant alleles, their dominance fails in diploids heterozygous for defects in both genes simultaneously. The unusual complementation pattern could be explained by interaction of the gene products in formation of the same enzyme.     

Saccharomyces Cerevisiae Cho2 Mutants Are Deficient in Phospholipid Methylation and Cross-Pathway Regulation of Inositol Synthesis

Five allelic Saccharomyces cerevisiae mutants deficient in the methylation of phosphatidylethanolamine (PE) have been isolated, using two different screening techniques. Biochemical analysis suggested that these mutants define a locus, designated CHO2, that may encode a methyltransferase. Membranes of cho2 mutant cells grown in defined medium contain approximately 10% phosphatidylcholine (PC) and 40-50% PE as compared to wild-type levels of 40-45% PC and 15-20% PE. In spite of this greatly altered phospholipid composition, cho2 mutant cells are viable in defined medium and are not auxotrophic for choline or other phospholipid precursors such as monomethylethanolamine (MME). However, analysis of yeast strains carrying more than one mutation affecting phospholipid biosynthesis indicated that some level of methylated phospholipid is essential for viability. The cho2 locus was shown by tetrad analysis to be unlinked to other loci affecting phospholipid synthesis. Interestingly, cho2 mutants and other mutant strains that produce reduced levels of methylated phospholipids are unable to properly repress synthesis of the cytoplasmic enzyme inositol-1-phosphate synthase. This enzyme was previously shown to be regulated at the level of mRNA abundance in response to inositol and choline in the growth medium. We cloned the CHO2 gene on a 3.6-kb genomic DNA fragment and created a null allele of cho2 by disrupting the CHO2 gene in vivo. The cho2 disruptant, like all other cho2 mutants, is viable, exhibits altered regulation of inositol biosynthesis and is not auxotrophic for choline or MME.     

The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence

Inositol is utilized by Mycobacterium tuberculosis in the production of its major thiol and of essential cell wall lipoglycans. We have constructed a mutant lacking the gene encoding inositol-1-phosphate synthase (ino1), which catalyses the first committed step in inositol synthesis. This mutant is only viable in the presence of extremely high levels of inositol. Mutant bacteria cultured in inositol-free medium for four weeks showed a reduction in levels of mycothiol, but phosphatidylinositol mannoside, lipomannan and lipoarabinomannan levels were not altered. The ino1 mutant was attenuated in resting macrophages and in SCID mice. We used site-directed mutagenesis to alter four putative active site residues; all four alterations resulted in a loss of activity, and we demonstrated that a D310N mutation caused loss of the active site Zn2+ ion and a conformational change in the NAD+ cofactor.     

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.     

Ty Element-Induced Temperature-Sensitive Mutations of Saccharomyces Cerevisiae

Temperature-sensitive mutants of Saccharomyces cerevisiae were isolated by insertional mutagenesis using the HIS3 marked retrotransposon TyH3HIS3. In such mutants, the TyHIS3 insertions are expected to identify loci which encode genes essential for cell growth at high temperatures but dispensable at low temperatures. Five mutations were isolated and named hit for high temperature growth. The hit1-1 mutation was located on chromosome X and conferred the pet phenotype. Two hit2 mutations, hit2-1 and hit2-2, were located on chromosome III and caused the deletion of the PET18 locus which has been shown to encode a gene required for growth at high temperatures. The hit3-1 mutation was located on chromosome VI and affected the CDC26 gene. The hit4-1 mutation was located on chromosome XIII. These hit mutations were analyzed in an attempt to identify novel genes involved in the heat shock response. The hit1-1 mutation caused a defect in synthesis of a 74-kD heat shock protein. Western blot analysis revealed that the heat shock protein corresponded to the SSC1 protein, a member of the yeast hsp70 family. In the hit1-1 mutant, the TyHIS3 insertion caused a deletion of a 3-kb DNA segment between the delta 1 and delta 4 sequences near the SUP4 locus. The 1031-bp wild-type HIT1 DNA which contained an open reading frame encoding a protein of 164 amino acids and the AGG arginine tRNA gene complemented all hit1-1 mutant phenotypes, indicating that the mutant phenotypes were caused by the deletion of these genes. The pleiotropy of the HIT1 locus was analyzed by constructing a disruption mutation of each gene in vitro and transplacing it to the chromosome. This analysis revealed that the HIT1 gene essential for growth at high temperatures encodes the 164-amino acid protein. The arginine tRNA gene, named HSX1, is essential for growth on a nonfermentable carbon source at high temperatures and for synthesis of the SSC1 heat shock protein.     

Mitotic recombination is responsible for the loss of heterozygosity in cultured murine cell lines.

Heterozygous mammalian cell lines normally express both parental alleles at most autosomal loci. However, mutants can be isolated that fail to express one of the alleles. Using a murine pre-B cell line that is heterozygous for several loci on chromosome 12, including one encoding the cell surface antigen Ly-18, we found that one of the two Ly-18 antigenic forms was lost at a rate of 1.5 x 10(-5) per cell per generation. Molecular analysis revealed that a genetic marker distal to Ly-18 became homozygous. Analysis of the genotype of the mutants at the rDNA cluster, located close to the centromere, strongly suggests that the mutants arose by mitotic recombination within this multicopy locus.     

Mapping and Gene Conversion Studies with the Structural Gene for Iso-1-Cytochrome C in Yeast

We have investigated the order of the four genes cyc1, rad7, SUP4, and cdc8 which form a tightly linked cluster on the right arm of chromosome X in the yeast Saccharomyces cerevisiae. Crossing over and coconversion data from tetrad analysis established the gene order to be centromere-cyc1-rad7-SUP4. Also cdc8 appeared to be distal to SUP4 on the basis of crossovers that were associated with conversion of SUP4. The frequencies of recombination and the occurrence of coconversions suggest that these four genes are contiguous or at least nearly so. Gene-conversion frequencies for several cyc1 alleles were studied, including cyc1-1, a deletion of the whole gene that extends into the rad7 locus. The cyc1-1 deletion was found to be capable of conversion, though at a frequency some fivefold less than the other alleles studied, and both 3:1 and 1:3 events were detected. In general 1:3 and 3:1 conversion events were equally frequent at all loci studied, and approximately 50% of conversions were accompanied by reciprocal recombination for flanking markers. The orientation of the cyc1 gene could not be clearly deduced from the behavior of the distal marker SUP4 in wild-type recombinants that arose from diploids heteroallelic for cyc1 mutations.     

Control of inositol biosynthesis in Saccharomyces cerevisiae; inositol-phosphate synthetase mutants.

Inositol-requiring mutants of Saacharomyces cerevisiae were tested in cell extracts for the ability to convert glucose-6-phosphate to inositol-phosphate (IP synthetase) and inositol (IP phosphatase). Mutants representing any one of 10 unlinked loci conferring the inositol requirement were unable to synthesize either compound in an assay with glucose-6-phosphate as the substrate. These results indicate that the mutants lack IP synthetase activity and that at least 10 genes control the conversion of glucose-6-phosphate to inositol-phosphate. In addition, a mutation known to be unlinked with the ino1 locus interacts with a leaky ino1 allele and may play a role in the regulation of IP synthetase. This mutation causes a 47% reduction in wild-type IP synthetase activity and, when combined in a haploid strain with the leaky ino1 allele, it reduced IP synthetase activity to a level below that which is growth supporting. Wild-type and IP synthetase-deficient strains were tested for reduced nicotinamide adenine dinucleotide (NADH) accumulation, since NAD+ is required in the conversion of glucose-6-phosphate to inositol. No detectable accumulation of NADH was observed in the wild-type strain, presumably because the NADH generated is rapidly oxidized during subsequent partial reactions of IP synthetase. Mutants representing three different loci accumulate NADH and may, therefore, lack the NADH-mediated reductase activity of IP synthetase. Other mutants tested fail to accumulate NADH and may, therefore, lack the NAD+-mediated oxidase activity of IP synthetase. Phospholipid synthesis was studied by 32P pulse labeling in one mutant under conditions of inositol supplementation and starvation. Starved cells incorporate 32P into phospholipids normally for 2 h, followed by a period in which the rate of phosphatidylinositol synthesis decreases and the rate of phosphatidylcholine synthesis increases. After 5 to 6 h starvation, all cellular phospholipid synthesis ceases.     

Murine retroviral restriction genes Fv-4 and Akvr-1 are alleles of a single locus.

The two murine retroviral restriction genes, Fv-4 and Akvr-1, are very similar in their effects, distributions, ranges of action, and phenotypes. Akvr-1 has been shown to segregate independently in backcrosses with a variety of retroviral restriction loci, including Fv-1, Fv-2, Ril-1, and Ril-2. An allelism test cross of FRG (Fv-4R) X LCRR (Akvr-1R) hybrids mated to AKR mice failed to produce any viremic offspring. These results suggested that Akvr-1R and Fv-4R are alleles of a single locus, Fv-4, on mouse chromosome 12.     

Suppressors of the ras2 Mutation of SACCHAROMYCES CEREVISIAE

Saccharomyces cerevisiae contains two members of the ras gene family. Strains with disruptions of the RAS2 gene fail to grow efficiently on nonfermentable carbon sources. This growth defect can be suppressed by extragenic mutations called sra. We have isolated 79 independent suppressor mutations, 68 of which have been assigned to one of five loci. Eleven additional dominant mutations have not been assigned to a specific locus. Some sra1 and SRA4 and all SRA3 mutations were RAS independent, allowing growth of yeast cells that lack a functional RAS gene. Mutations in sra1, SRA3, SRA4 and sra6 are linked to his6, ino1, met3 and ade6, respectively. Some sra mutants have pleiotropic phenotypes that affect glycogen accumulation, sporulation, viability, respiratory capacity and suppression of two cell-division-cycle mutations, cdc25 and cdc35. The proposed functions of many of the suppressor genes are consistent with the model in which RAS activates adenylate cyclase.