Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE

The umr7–1 mutation, previously identified in a set of mutants that had been selected for defective UV-induced mutagenesis at CAN1, affects other cellular functions, including many of those regulated by the mating-type locus (MAT) in heterothallic Saccharomyces cerevisiae. The recessive umr7–1 allele, mapping approximately 20 cM distal to thr4 on chromosome III, causes clumpy growth in both a and α cells and has no apparent effect on a mating functions. However, α umr7 meiotic segregants fail to express several α-specific functions (e.g., high-frequency conjugation with a strains, secretion of the hormone α-factor and response to the hormone a-factor). In addition, α umr7 cells exhibit some a-specific characteristics, such as the barrier phenotype (Bar⁺) that prevents diffusion of α-factor and an increased mating frequency with α strains. The most striking property of α umr7 strains is their altered morphology, in which mitotic cells develop an asymmetric pear shape, like that of normal a cells induced to form "shmoos" by interaction with α-factor. Some a/α-specific diploid functions are also affected by umr7; instead of polar budding patterns, a/α umr7/umr7 diploids have medial budding like a/a, α/α and haploid strains. Moreover, a/α umr7/umr7 diploids have lost the ability to sporulate and are Bar⁺ like a or a/a strains. Revertant studies indicate that umr7–1 is a single point mutation. The umr7 mutant fails to complement mutants of both tup1 (selected for deoxythymidine monophosphate utilization) and cyc9 (selected for high iso-2-cytochrome c levels), and all three isolates have similar genetic and phenotypic properties. It is suggested that the product of this gene plays some common central role in the complex regulation of the expression of both MAT-dependent and MAT-independent functions.     

Isolation of Saccharomyces cerevisiae mutants constitutive for invertase synthesis.

A new method for detecting invertase activity in Saccharomyces cerevisiae colonies was used to screen for mutants resistant to catabolite repression of invertase. Mutations causing the highest level of derepression were located in two previously identified genes, cyc8 and tup1. Several of the cyc8 mutations, notably cyc8-10 and cyc8-11, were temperature dependent, repressed at 23 degrees C, and derepressed at 37 degrees C. The kinetics of derepression of invertase mRNA in cyc8-10 cells shifted from 23 to 37 degrees C was determined by Northern blots. Invertase mRNA was detectable at 5 min after the shift, with kinetics of accumulation very similar to that of wild-type cells shifted from high-glucose to low-glucose medium. Assays of representative enzymes showed that many but not all glucose-repressible enzymes are derepressed in both cyc8 and tup1 mutants. cyc8 and tup1 appear to be the major negative regulatory genes controlling catabolite repression in yeasts.     

Pathways of ultraviolet mutability in Saccharomyces cerevisiae. III. Genetic analysis and properties of mutants resitant to ultraviolet-induced forward mutation.

Non-allelic mutants of Saccharomyces cerevisiae with reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were assigned to seven distinct genetic loci, each with allele designations umr1-1, umr2-1, …, umr7-1 to indicate UV mutation resistance. Each allele complemented rev1-1, rev2-1, and rev3-1. None conferred a great deal of UV sensitivity. When assayed on yeast extract-peptone-dextrose complex growth agar, umr1, umr3, and umr7 (a mating type) were the most UV-sensitive, with a dose-reduction factor of approximately 1.2 at 10% survival. When assayed on synthetic agar lacking arginine, however, umr3 was the most UV-sensitive (dose-reduction factor of 1.5 at 10% survival). UV revertability of his5-2, lys1-1, and ura4-1 was normal in strains carrying the single genes umr4, umr5, umr6 and umr7; umr1 reduced revertibility of his5-2 and ura4-1 but not lys1-1; umr2 reduced only ura4-1 revertibility; umr3 reduced UV reversion of all three test alleles. Five a/α homozygous umr diploids (except umr1 and umr4) failed to sporulate. One of these, umr7, blocked normal secretion of alpha hormone in α segregants and could not conjugate with a strains. The phenotypes of umr mutants are consistent with the existence of branched UV mutation pathways of different specificity, some of which may function in the single RAD6-dependent error-prone pathway for repair of UV damage. Other possible pathways of action are discussed. It is also suggested that regulatory functions interacting with the mating-type locus or its gene products may play some role in UV mutagenesis or error-prone repair.     

Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.     

Construction of industrial baker's yeast strains able to assimilate maltose under catabolite repression conditions

Spore progeny from an industrial baker's yeast strain were mutagenized with UV and mutants resistant to 2-deoxyglucose isolated. One of these mutants (10a12–13) showed high levels of maltase (-glucosidase) and external invertase, and assimilated maltose when growing under catabolite repression conditions. This mutant was not allelic to any of the catabolite repression mutants tested cat4, cat80, cid1, cyc8, hex2, hxk2 and tup1. Mutant 10a12–13 was crossed with appropriate strains to construct hybrids that were also able to assimilate maltose in the presence of glucose. These hybrids may be useful in fermentation processes where both glucose and maltose are present.     

Transductional construction of aspartase-hyperproducing strain of Escherichia coli B

Two aspartase-overproducing mutants of Escherichia coli B were characterized. Strain EAPc7 had a mutation enhancing aspartase formation in the region of aspartase gene. This mutation did not affect catabolite repression by aspartase. Strain EAPc244 showed a high cAMP content and an elevated adenylate cyclase activity. This mutation was closely linked to the ilv locus and caused the release of catabolite repression for various catabolite repression-sensitive enzymes, resulting in overproduction of adenylate cyclase. This mutation was transduced to an Ile⁻ strain derived from strain EAPc7 using the Ile⁺ selective marker. The constructed strain AT202, having the above 2 mutations, produced about 3-fold and 18-fold more aspartase than did the 2 parent strains and the wild-type strain, respectively, when cultured in the medium used for industrial production of aspartase. Strain AT202 maintained stably high aspartase activity after 30 cell generations. On the other hand, in E. coli K-12 harboring the aspA⁺ recombinant plasmid pYT471 (pBR322-aspA⁺), the activity decreased to the E. coli K-12 level. Hence, strain AT202 is more advantageous for industrial production of l-aspartic acid than cells harboring the aspA⁺-recombinant plasmid pYT471.     

Catabolite repression of the lac operon. Separate repression of two enzymes

1. Catabolite repression of β-galactosidase and of thiogalactoside transacetylase was studied in several strains of Escherichia coli K 12, in an attempt to show whether a single site within the structural genes of the lac operon co-ordinately controls translational repression for the two enzymes. In all experiments the rate of synthesis of the enzymes was compared in glycerol–minimal medium and in glucose–minimal medium. 2. In a wild-type strain, glucose repressed the synthesis of the two enzymes equally. 3. The possibility that repression was co-ordinate was investigated by studies of mutant strains that carry deletions in the genes for β-galactosidase or galactoside permease or both. In all of the strains with deletions, the repression of thiogalactoside transacetylase persisted, and it is concluded that there is no part of the structural gene for β-galactosidase that is essential for catabolite repression of thiogalactoside transacetylase. 4. Subculture of one strain through several transfers in rich medium greatly increased its susceptibility to catabolite repression by glucose. It is concluded that unknown features of the genotype can markedly affect sensitivity to catabolite repression. 5. These results make it clear that one cannot draw valid conclusions about the effect of known genotypic differences on catabolite repression from a comparison of two separate strains; to study the effect of a particular genetic change in a lac operon it is necessary to construct a partially diploid strain so that catabolite repression suffered by one lac operon can be compared with that suffered by another. 6. Four such partial diploids were constructed. In all of them catabolite repression of β-galactosidase synthesized by one operon was equal in extent to catabolite repression of thiogalactoside transacetylase synthesized by the other. 7. Taken together, these results suggest that catabolite repression of β-galactosidase and thiogalactoside transacetylase is separate but equal.     

Formation of composite iso-cytochromes c by recombination between non-allelic genes of yeast

We present evidence that two non-allelic genes, located on two non-homologous chromosomes in the yeast Saccharomyces cerevisiae, recombine and in this process generate new composite genes containing portions of both genes. The two genes CYC1 and CYC7 encode, respectively, iso-1-cytochrome c and iso-2-cytochrome c; CYC1 is located on the right arm of chromosome X and CYC7 is located on the left arm of chromosome V. The coding regions of CYC1 and CYC7 and the corresponding iso-1-cytochrome c and iso-2-cytochrome c are approximately 80% homologous. Composite genes were uncovered among revertants of certain but not all cyc1 mutants lacking iso-1-cytochrome c; composite genes were observed in most revertants from the low-reverting strains cyc1-11, cyc1-136 and cyc1-158, and in low proportions of the revertants from the typically reverting strains cyc1-94 and cyc1-156. Protein analysis of 14 composite iso-cytochromes c and DNA sequencing of five composite genes indicated that recombinational events produced replacements of central portions of the cyc1 gene with a corresponding segment from the wild-type CYC7⁺ gene. The replacements varied in length from 13% to 61% of the translated portion of the CYC1 locus. The formation of composite genes occurred spontaneously at very low frequencies and at low but enhanced frequencies after treatments with mutagens including ultraviolet light, ethylmethane sulfonate, methylmethane sulfonate and nitrous acid. Genetic tests indicated that composite genes are formed mitotically by a conversion-like event in which the wild-type CYC1⁺ allele remains intact. Recombination between non-allelic genes can lead to identical sequences at different loci and to diverse composite genes. These results support the indirect evidence from other eukaryotic systems that non-allelic genes with extensive but not complete homology recombine during evolution.     

Repeated Family of Genes Controlling Maltose Fermentation in Saccharomyces carlsbergensis

Maltose fermentation in Saccharomyces spp. requires the presence of any one of five unlinked genes: MAL1, MAL2, MAL3, MAL4, or MAL6. Although the genes are functionally equivalent, their natures and relationships to each other are not known. At least three proteins are necessary for maltose fermentation: maltase, maltose permease, and a regulatory protein. The MAL genes may code for one or more of these proteins. Recently a DNA fragment containing a maltase structural gene has been cloned from a MAL6 strain, CB11, to produce plasmid pMAL9-26. We have conducted genetic and physical analyses of strain CB11. The genetic analysis has demonstrated the presence of two cryptic MAL genes in CB11, MAL1g and MAL3g (linked to MAL1 and to MAL3, respectively), in addition to the MAL6 locus. The physical analysis, which used a subclone of plasmid pMAL9-26 as a probe, detected three HindIII genomic fragments with homology to the probe. Each fragment was shown to be linked to one of the MAL loci genetically demonstrated to be present in CB11. Our results indicate that the cloned maltase structural gene in plasmid pMAL9-26 is linked to MAL6. Since the MAL6 locus has previously been shown to contain a regulatory gene, the MAL6 locus must be a complex locus containing at least two of the factors needed for maltose fermentation: the structural gene for maltase and the maltase regulatory protein. The absence of other fragments which hybridize to the MAL6-derived probe shows that either MAL2 and MAL4 are not related to MAL6, or the DNA corresponding to these genes is absent from the MAL6 strain CB11.     

Genes Affecting the Expression of Cytochrome c in Yeast: Genetic Mapping and Genetic Interactions

The four mutant genes, cyc2, cyc3, cyc8 and cyc9, that affect the levels of the two iso-cytochromes c in the yeast Saccharomyces cerevisiae have been characterized and mapped. Both cyc2 and cyc3 lower the amount of iso-1-cytochrome c and iso-2-cytochrome c; whereas, cyc8 and cyc9 increase the amount of iso-2-cytochrome c. The cyc2, cyc3, cyc8 and cyc9 genes are located, respectively, on chromosomes XV, I, II and III, and are, therefore, unlinked to each other and unlinked to CYC1, the structural gene of iso-1-cytochrome c and to CYC7, the structural gene of iso-2-cytochrome c. While some cyc3 mutants are completely or almost completely deficient in cyotchromes c, none of the cyc2 mutants contained less than 10% of parental level of cytochrome c even though over one-half of the mutants contain UAA or UAG nonsense mutations. Thus, it appears as if a complete block of the cyc2 gene product still allows the formation of a residual fraction of cytochrome c. The cyc2 and cyc3 mutant genes cause deficiencies even in the presence of CYC7, cyc8 and cyc9, which normally cause overproduction of iso-2-cytochrome c. We suggest that cyc2 and cyc3 may be involved with the regulation or maturation of the iso-cytochromes c. In addition to having high levels of iso-2-cytochromes c, the cyc8 and cyc9 mutants are associated with flocculent cells and other abnormal phenotypes. The cyc9 mutant was shown to be allelic with the tup1 mutant and to share its properties, which include the ability to utilize exogenous dTMP, a characteristic flocculent morphology, the lack of sporulation of homozygous diploids and low frequency of mating and abnormally shaped cells of alpha strains. The diverse abnormalities suggest that cyc8 and cyc9 are not simple regulatory mutants controlling iso-2-cytochrome c.     

Alleviation of glucose repression of maltose metabolism by MIG1 disruption in Saccharomyces cerevisiae.

The MIG1 gene was disrupted in a haploid laboratory strain (B224) and in an industrial polyploid strain (DGI 342) of Saccharomyces cerevisiae. The alleviation of glucose repression of the expression of MAL genes and alleviation of glucose control of maltose metabolism were investigated in batch cultivations on glucose-maltose mixtures. In the MIG1-disrupted haploid strain, glucose repression was partly alleviated; i.e., maltose metabolism was initiated at higher glucose concentrations than in the corresponding wild-type strain. In contrast, the polyploid delta mig1 strain exhibited an even more stringent glucose control of maltose metabolism than the corresponding wild-type strain, which could be explained by a more rigid catabolite inactivation of maltose permease, affecting the uptake of maltose. Growth on the glucose-sucrose mixture showed that the polypoid delta mig1 strain was relieved of glucose repression of the SUC genes. The disruption of MIG1 was shown to bring about pleiotropic effects, manifested in changes in the pattern of secreted metabolites and in the specific growth rate.     

A carbon catabolite repression mutant of Saccharomyces cerevisiae with elevated hexokinase activity: Evidence for regulatory control of hexokinase PII synthesis

Summary Mutants were investigated that had elevated hexokinase activity and had been isolated previously as resistant to carbon catabolite repression (Zimmermann and Scheel 1977). They were allele tested with mutant strains of Lobo and Maitra (1977), which had defects in one or more of the genes coding for glucokinase and unspecific hexokinases. It was shown, that the mutation abolishing carbon catabolite repression had occured in a gene that was not allelic to any of the structural genes coding for hexokinases. This indicated that a regulatory defect was responsible for elevated hexokinase activity. This agreed with observations that hexokinase activities were like wild-type during growth on non-fermentable carbon sources in hex2 mutants. Recombination between the mutant allele hex2 and mutant alleles hxk1 and hxk2, coding for hexokinase PI and PII respectively, clearly demonstrated that only hexokinase PII was elevated in hex2 mutants. When hex2 mutant cells grown on YEP ethanol were shifted to YEP glucose media, hexokinase activity increased after 30min. This increase depended on de novo protein synthesis. hex2 mutants provide evidence, that carbon catabolite repression and synthesis of hexokinase PII are under common regulatory control.     

Catabolite repression of β-galactosidase synthesis in Escherichia coli

1. Repression by glucose of β-galactosidase synthesis is spontaneously reversible in all strains of Escherichia coli examined long before the glucose has all been consumed. The extent of recovery and the time necessary for reversal differ among various strains. Other inducible enzymes show similar effects. 2. This transient effect of glucose repression is observed in constitutive (i⁻) and permease-less (y⁻) cells as well as in the corresponding i⁺ and y⁺ strains. 3. Repression is exerted by several rapidly metabolizable substrates (galactose, ribose and ribonucleosides) but not by non-metabolized or poorly metabolized compounds (2-deoxyglucose, 2-deoxyribose, phenyl thio-β-galactoside and 2-deoxyribonucleosides). 4. The transient repression with glucose is observed in inducible cells supplied with a powerful inducer of β-galactosidase synthesis (e.g. isopropyl thio-β-galactoside) but not with a weak inducer (lactose); in the latter instance glucose repression is permanent. Diauxic growth on glucose plus lactose can be abolished by including isopropyl thio-β-galactoside in the medium. 5. In some strains phosphate starvation increases catabolite repression; in others it relieves it. Adenine starvation in an adenine-requiring mutant also relieves catabolite repression by glycerol but not that by glucose. Restoration of phosphate or adenine to cells starved of these nutrients causes a pronounced temporary repression. Alkaline-phosphatase synthesis is not affected by the availability of adenine. 6. During periods of transient repression of induced enzyme synthesis the differential rate of RNA synthesis, measured by labelled uracil incorporation in 2min. pulses, shows a temporary rise. 7. The differential rate of uracil incorporation into RNA falls during exponential growth of batch cultures of E. coli. This is equally true for uracil-requiring and non-requiring strains. The fall in the rate of incorporation has been shown to be due to a real fall in the rate of RNA synthesis. The significance of the changes in the rate of RNA synthesis is discussed. 8. A partial model of catabolite repression is presented with suggestions for determining the chemical identification of the catabolite co-repressor itself.     

The Effects of Three Different MAL Loci on the Regulation of Maltase Synthesis in Yeast

Inbred haploid strains of Saccharomyces cerevisiae carrying MAL1, MAL2 or MAL6 in a common background have been crossed to each other and to strains carrying no active MAL loci. The kinetics of maltase induction and the induced maltase levels have been examined in the inbred strains and in haploid segregants of the crosses. Differences have been found in the kinetics of induction and induced maltase levels that segregate with the different MAL loci. In the strains tested, the relative rates of maltase induction were MAL2>MAL6>>MAL1; the relative induced maltase levels were MAL2>MAL6~MAL1. These results indicate that MAL1, MAL2 and MAL6 are (or include) regulatory genes that control the accumulation of the enzymes of maltose fermentation.     

Mutants of Saccharomyces cerevisiae That Incorporate Deoxythymidine-5′-Monophosphate Into Deoxyribonucleic Acid In Vivo

Spontaneous mutants of Saccharomyces cerevisiae able to incorporate deoxythymidine-5′-monophosphate (dTMP) into deoxyribonucleic acid (DNA) have been selected based on their ability to grow in the presence of aminopterin and sulfanilamide if dTMP is present. Essentially all mutants (called tup) selected in this way required dTMP for growth in the presence of the two drugs, but none required dTMP in the absence of the drugs. Neither thymine nor thymidine would satisfy this requirement. Equimolar amounts of ³²P- and ³H-base-labeled dTMP were incorporated by the mutants into alkali-stable, deoxyribonuclease-sensitive material. In the presence of aminopterin and sulfanilamide, this incorporation was sufficient to account for a substantial proportion of the thymine residues in the cellular DNA, whereas in the absence of the drugs only about 40% as much of the thymine residues originated from the medium. Of 29 mutants examined, all were recessive and 17 showed 2:2 segregation in crosses with a wild-type strain. The lesions in these mutants fell into four complementation groups: one (tup1) occurs on chromosome III; another (tup3) is on chromosome II; and a third (tup4) was centromere linked. Strains of the genotype α tup1 mated with lower than normal efficiency with a strains, but with higher than normal efficiency with α strains. Strains of genotype a/α tup1/tup1 failed to sporulate, whereas homozygous diploids for tup2, tup3, or tup4 sporulated normally, as did a/α tup1/+ strains.     

Serine substitutions caused by an ochre suppressor in yeast.

The suppressor SUQ5 in yeast can cause the production of approximately 10 to 20% of the normal amount of iso-l-cytochrome c when coupled to the ochre (UAA) mutants cyc1–2 and cyc1–72. The iso-l-cytochromes c contain residues of serine at positions that correspond to the sites of the ochre codons. SUQ5 is efficient only in strains having the non-Mendelian factor ψ⁺, although the low amount of suppressed iso-l-cytochrome c from a ψ⁻SUQ5 cyc1–72 strain was also shown to contain serine at the ochre site. Thus SUQ5 differs from the eight other characterized suppressors of UAA in yeast, which were previously shown to insert residues of tyrosine at ochre sites (Gilmore et al., 1971) and which are only effective in strains haying the non-Mendelian factor ψ⁻, since they generally cause inviability in the ψ⁺ state. Like the tyrosine-inserting suppressors, SUQ5 can also act on another ochre allele cyc1–9, but with a very low efficiency of approximately 0.4%, while it does not appear to act at all on amber (UAG) mutants. SUQ5 was found to be 6.4 cM (centiMorgans) from tyr7 on chromosome XVI. It is suggested that the gene product of SUQ5 is serine tRNA.     

Fermentative production of dipicolinic acid by Penicilium citreoviride from sugarcane molasses

In Penicillium citreoviride strain 3114, dipicolinic acid (DPA) synthesis is inhibited by Ca⁺⁺ ions and susceptible to catabolite repression, making it unsuitable for fermentation in sugarcane molasses. A mutant, 27133-dpa-Ca-14, was derived through stepwise mutation and selection to produce DPA in the presence of 1000 ppm Ca⁺⁺ and also to be resistant to catabolite repression. With this mutant, higher product concentrations (36 g DPA/l) could be reached without prior removal of Ca⁺⁺ from the molasses. The DPA yields increased by about four times (0.4 g DPA/g glucose consumed) and productivity by two and a half-times (3.0 g DPA/l.d) compared with that of the parent strain 3114. Higher product yields (0.58–0.59 g DPA/g glucose consumed) were obtained in a multiple stage fermentation system. DPA was recovered through sepration by ion exchange chromatography followed by concentration and crystallization.