Molecular and Genetic Analysis of the Snf7 Gene in Saccharomyces Cerevisiae

Mutations in the SNF7 gene of Saccharomyces cerevisiae prevent full derepression of the SUC2 (invertase) gene in response to glucose limitation. We report the molecular cloning of the SNF7 gene by complementation. Sequence analysis predicts that the gene product is a 27-kDa acidic protein. Disruption of the chromosomal locus causes a fewfold decrease in invertase derepression, a growth defect on raffinose, temperature-sensitive growth on glucose, and a sporulation defect in homozygous diploids. Genetic analysis of the interactions of the snf7 null mutation with ssn6 and spt6/ssn20 suppressor mutations distinguished SNF7 from the SNF2, SNF5 and SNF6 genes. The snf7 mutation also behaved differently from mutations in SNF1 and SNF4 in that snf7 ssn6 double mutants displayed a synthetic phenotype of severe temperature sensitivity for growth. We also mapped SNF7 to the right arm of chromosome XII near the centromere.     

Gene-enzyme relationships in the proline biosynthetic pathway of Saccharomyces cerevisiae.

The PRO1, PRO2, and PRO3 genes were isolated by functional complementation of pro1, pro2, and pro3 (proline-requiring) strains of Saccharomyces cerevisiae. Independent clones with overlapping inserts were isolated from S. cerevisiae genomic libraries in YEp24 (2 microns) and YCp50 (CEN) plasmids. The identity of each gene was determined by gene disruption, and Southern hybridization and genetic analyses confirmed that the bona fide genes had been cloned. Plasmids containing each gene were introduced into known bacterial proline auxotrophs, and the ability to restore proline prototrophy was assessed. Interspecies complementation demonstrated that the S. cerevisiae PRO1 gene encoded gamma-glutamyl kinase, PRO2 encoded gamma-glutamyl phosphate reductase, and PRO3 encoded delta 1-pyrroline-5-carboxylate reductase. The presence of the PRO3 gene on a high-copy-number plasmid in S. cerevisiae caused a 20-fold overproduction of delta 1-pyrroline-5-carboxylate reductase. The PRO2 gene mapped on chromosome XV tightly linked to cdc66, and the PRO3 gene was located on the right arm of chromosome V between HIS1 and the centromere.     

A Pleiotropic Phospholipid Biosynthetic Regulatory Mutation in Saccharomyces Cerevisiae Is Allelic to Sin3 (Sdi1, Ume4, Rpd1)

Three mutants were identified in a genetic screen using an INO1-lacZ fusion to detect altered INO1 regulation in Saccharomyces cerevisiae. These strains harbor mutations that render the cell unable to fully repress expression of INO1, the structural gene for inositol-1-phosphate synthase. The Cpe(-) (constitutive phospholipid gene expression) phenotype associated with these mutations segregated 2:2, indicating that it was the result of a single gene mutation. The mutations were shown to be recessive and allelic. A strain carrying the tightest of the three alleles was examined in detail and was found to express the set of co-regulated phospholipid structural genes (INO1, CHO1, CHO2 and OPI3) constitutively. The Cpe(-) mutants also exhibited a pleiotropic defect in sporulation. The mutations were mapped to the right arm of chromosome XV, close to the centromere, where it was discovered that they were allelic to the previously identified regulatory mutation sin3 (sdi1, ume4, rpd1, gam2). A sin3 null mutation failed to complement the mutation conferring the Cpe(-) phenotype. A mutant harboring a sin3 null allele exhibited the same altered INO1 expression pattern observed in strains carrying the Cpe(-) mutations isolated in this study.     

Structural and functional conservation between yeast and human 3-hydroxy-3-methylglutaryl coenzyme A reductases, the rate-limiting enzyme of sterol biosynthesis.

The pathway of sterol biosynthesis is highly conserved in all eucaryotic cells. We demonstrated structural and functional conservation of the rate-limiting enzyme of the mammalian pathway, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMG-CoA reductase), between the yeast Saccharomyces cerevisiae and humans. The amino acid sequence of the two yeast HMG-CoA reductase isozymes was deduced from DNA sequence analysis of the HMG1 and HMG2 genes. Extensive sequence similarity existed between the region of the mammalian enzyme encoding the active site and the corresponding region of the two yeast isozymes. Moreover, each of the yeast isozymes, like the mammalian enzyme, contained seven potential membrane-spanning domains in the NH2-terminal region of the protein. Expression of cDNA clones encoding either hamster or human HMG-CoA reductase rescued the viability of hmg1 hmg2 yeast cells lacking this enzyme. Thus, mammalian HMG-CoA reductase can provide sufficient catalytic function to replace both yeast isozymes in vivo. The availability of yeast cells whose growth depends on human HMG-CoA reductase may provide a microbial screen to identify new drugs that can modulate cholesterol biosynthesis.     

Dominant and Recessive Suppressors That Restore Glucose Transport in a Yeast Snf3 Mutant

The SNF3 gene of Saccharomyces cerevisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters. To identify genes that are functionally related to SNF3, we selected for suppressors that remedy the growth defect of snf3 mutants on low concentrations of glucose or fructose. We recovered 38 recessive mutations that fall into a single complementation group, designated rgt1 (restores glucose transport). The rgt1 mutations suppress a snf3 null mutation and are not linked to snf3. A naturally occurring rgt1 allele was identified in a laboratory strain. We also selected five dominant suppressors. At least two are tightly linked to one another and are designated RGT2. The RGT2 locus was mapped 38 cM from SNF3 on chromosome IV. Kinetic analysis of glucose uptake showed that the rgt1 and RGT2 suppressors restore glucose-repressible high-affinity glucose transport in a snf3 mutant. These mutations identify genes that may regulate or encode additional glucose transport proteins.     

Ochre Suppression in SALMONELLA TYPHIMURIUM

A bacterial strain was constructed which permitted positive selection for ochre suppressor mutations as well as for the loss of suppressor function. A derivative bearing an ochre suppressor mutation was selected following mutagenesis with N-methyl-N-nitroso-N'-nitroguanidine. The suppressor-bearing strain was treated with nitrous acid to eliminate suppressor function by mutation, and a strain lacking suppressor activity was selected. The selected strain which had lost suppressor function was then subjected to mutagenesis to induce a second suppressor mutation. The alternating sequence (induction of an ochre suppressor mutation → induction of a mutation eliminating ochre suppressor activity) was repeated 29 and one-half times in a single strain. Some of the suppressor mutations were tentatively mapped at four locations on the chromosome. The first suppressor mutation selected maps at about minute 30 on the chromosome. The second suppressor selected maps at approximately minute 60, while the third suppressor maps nearby, possibly as far as minute 72. Among the subsequently selected suppressor mutations, all eleven which were mapped were cotransducible with the gal and nic loci near minute 36 on the chromosome and may represent more than one suppressor gene. Deletions were selected which inactivate two of the ochre suppressor alleles mapping near the gal-nic region, suggesting that one or more such genes are dispensable. Some evidence also suggests that the occurrence of either deletion mutations or transduction-mediated recombination events in the gal-nic region can cause instability of nearby suppressor alleles.     

Feedback regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in Saccharomyces cerevisiae.

In eukaryotic cells all isoprenoids are synthesized from a common precursor, mevalonate. The formation of mevalonate from 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is catalyzed by HMG-CoA reductase and is the first committed step in isoprenoid biosynthesis. In mammalian cells, synthesis of HMG-CoA reductase is subject to feedback regulation at multiple molecular levels. We examined the state of feedback regulation of the synthesis of the HMG-CoA reductase isozyme encoded by the yeast gene HMG1 to examine the generality of this regulatory pattern. In yeast, synthesis of Hmg1p was subject to feedback regulation. This regulation of HMG-CoA reductase synthesis was independent of any change in the level of HMG1 mRNA. Furthermore, regulation of Hmg1p synthesis was keyed to the level of a nonsterol product of the mevalonate pathway. Manipulations of endogenous levels of several isoprenoid intermediates, either pharmacologically or genetically, suggested that mevalonate levels may control the synthesis of Hmg1p through effects on translation.     

Functional expression of human HMG-CoA reductase in Saccharomyces cerevisiae: a system to analyse normal and mutated versions of the enzyme in the context of statin treatment

Aims:  Statins – inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase – are known to reduce blood cholesterol levels. In this paper, we present a Saccharomyces cerevisiae expression system, which enables quick evaluation of the sensitivity of the wild-type and/or mutant forms of human HMG-CoA reductase towards statins or other drugs.
Methods and results:  We analysed the sequence of the HMG-CoA reductase gene in DNA extracted from blood samples of 16 patients with cardiovascular disorders. We applied the yeast system to examine the sensitivity of the wild-type and mutated versions of the hHMG-CoA reductase to different types of statins.
Conclusion:  The yeast and mammalian HMG-CoA reductases demonstrate structural and functional conservation, and expression of human HMG-CoA reductase in yeast complements the lethal phenotype of strains lacking the HMG1 and HMG2 genes.
Significance and Impact of the Study:  These data indicate that a yeast expression system can serve to study the influence of selected mutations in human HMG-CoA reductase on the sensitivity of the enzyme to commonly prescribed statins. Our results suggest that this model system is suitable for the development and selection of lipid-lowering drugs as well as for the examination of DNA sequence variations in the context of statin therapy.     

Mapping of the genes of some components of the electron transport chain (complex I) on the X chromosome of mammals

This paper describes genetic mapping studies with several respiration-deficient mutants of Chinese hamster fibroblasts which have a defect in complex I of the electron transport chain (NADH-coenzyme Q reductase). The mutations associated with two different complementation groups map on the X chromosome. In two cases (G14 and G20) karyotypic and isozyme analyses in hybrids have shown that a gene(s) on the mouse X chromosome complements the mutation(s) in the hamster cell mutant(s). A cosegregation analysis in hybrid cells has shown the corresponding genes to be linked to the HPRT genes (hamster-mouse hybrids of G14, and hamster-hamster hybrids for G14 and G20). By the same method the defective gene in a third mutant (G4) was also shown to be X-linked. A mutation representing a third complementation group (G11) was shown to be on an autosomal gene. These results provide an explanation for our observation that cells with recessive mutations in complementation groups I and II can be selected at relatively high frequencies.     

Mutations in the EXT1 and EXT2 genes in hereditary multiple exostoses.

Hereditary multiple exostoses (EXT; MIM 133700) is an autosomal dominant bone disorder characterized by the presence of multiple benign cartilage-capped tumors (exostoses). Besides suffering complications caused by the pressure of these exostoses on the surrounding tissues, EXT patients are at an increased risk for malignant chondrosarcoma, which may develop from an exostosis. EXT is genetically heterogeneous, and three loci have been identified so far: EXT1, on chromosome 8q23-q24; EXT2, on 11p11-p12; and EXT3, on the short arm of chromosome 19. The EXT1 and EXT2 genes were cloned recently, and they were shown to be homologous. We have now analyzed the EXT1 and EXT2 genes, in 26 EXT families originating from nine countries, to identify the underlying disease-causing mutation. Of the 26 families, 10 families had an EXT1 mutation, and 10 had an EXT2 mutation. Twelve of these mutations have never been described before. In addition, we have reviewed all EXT1 and EXT2 mutations reported so far, to determine the nature, frequency, and distribution of mutations that cause EXT. From this analysis, we conclude that mutations in either the EXT1 or the EXT2 gene are responsible for the majority of EXT cases. Most of the mutations in EXT1 and EXT2 cause premature termination of the EXT proteins, whereas missense mutations are rare. The development is thus mainly due to loss of function of the EXT genes, consistent with the hypothesis that the EXT genes have a tumor- suppressor function.     

Genetic Analysis of Saccharomyces Cerevisiae Chromosome I: On the Role of Mutagen Specificity in Delimiting the Set of Genes Identifiable Using Temperature-Sensitive-Lethal Mutations

In a previous attempt to identify as many as possible of the essential genes on Saccharomyces cerevisiae chromosome I, temperature-sensitive (Ts(-)) lethal mutations that had been induced by ethyl methanesulfonate or nitrosoguanidine were analyzed. Thirty-two independently isolated mutations that mapped to chromosome I identified only three complementation groups, all of which had been known previously. In contrast, molecular analyses of segments of the chromosome have suggested the presence of numerous additional essential genes. In order to assess the degree to which problems of mutagen specificity had limited the set of genes detected using Ts(-) lethal mutations, we isolated a new set of such mutations after mutagenesis with UV or nitrogen mustard. Surprisingly, of 21 independently isolated mutations that mapped to chromosome I, 17 were again in the same three complementation groups as identified previously, and two of the remaining four mutations were apparently in a known gene involved in cysteine biosynthesis. Of the remaining two mutations, one was in one of the essential genes identified in the molecular analyses, and the other was too leaky to be mapped. These results suggest that only a minority of the essential genes in yeast can be identified using Ts(-) lethal mutations, regardless of the mutagen used, and thus emphasize the need to use multiple genetic strategies in the investigation of cellular processes.     

Lethal Mutations Flanking the 68c Glue Gene Cluster on Chromosome 3 of DROSOPHILA MELANOGASTER

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.     

Mutations affecting gyrase in Haemophilus influenzae.

Mutants separately resistant to novobiocin, coumermycin, nalidixic acid, and oxolinic acid contained gyrase activity as measured in vitro that was resistant to the antibiotics, indicating that the mutations represented structural alterations of the enzyme. One Novr mutant contained an altered B subunit of the enzyme, as judged by the ability of a plasmid, pNov1, containing the mutation to complement a temperature-sensitive gyrase B mutation in Escherichia coli and to cause novobiocin resistance in that strain. Three other Novr mutations did not confer antibiotic resistance to the gyrase but appeared to increase the amount of active enzyme in the cell. One of these, novB1, could only act in cis, whereas a new mutation, novC, could act in trans. An RNA polymerase mutation partially substituted for the novB1 mutation, suggesting that novB1 may be a mutation in a promoter region for the B subunit gene. Growth responses of strains containing various combinations of mutations on plasmids or on the chromosome indicated that low-level resistance to novobiocin or coumermycin may have resulted from multiple copies of wild-type genes coding for the gyrase B subunit, whereas high-level resistance required a structural change in the gyrase B gene and was also dependent on alteration in a regulatory region. When there was mismatch at the novB locus, with the novB1 mutation either on a plasmid or the chromosome, and the corresponding wild-type gene present in trans, chromosome to plasmid recombination during transformation was much higher than when the genes matched, probably because plasmid to chromosome recombination, eliminating the plasmid, was inhibited by the mismatch.     

CtIP Mutations Cause Seckel and Jawad Syndromes

Seckel syndrome is a recessively inherited dwarfism disorder characterized by microcephaly and a unique head profile. Genetically, it constitutes a heterogeneous condition, with several loci mapped (SCKL1-5) but only three disease genes identified: the ATR, CENPJ, and CEP152 genes that control cellular responses to DNA damage. We previously mapped a Seckel syndrome locus to chromosome 18p11.31-q11.2 (SCKL2). Here, we report two mutations in the CtIP (RBBP8) gene within this locus that result in expression of C-terminally truncated forms of CtIP. We propose that these mutations are the molecular cause of the disease observed in the previously described SCKL2 family and in an additional unrelated family diagnosed with a similar form of congenital microcephaly termed Jawad syndrome. While an exonic frameshift mutation was found in the Jawad family, the SCKL2 family carries a splicing mutation that yields a dominant-negative form of CtIP. Further characterization of cell lines derived from the SCKL2 family revealed defective DNA damage induced formation of single-stranded DNA, a critical co-factor for ATR activation. Accordingly, SCKL2 cells present a lowered apoptopic threshold and hypersensitivity to DNA damage. Notably, over-expression of a comparable truncated CtIP variant in non-Seckel cells recapitulates SCKL2 cellular phenotypes in a dose-dependent manner. This work thus identifies CtIP as a disease gene for Seckel and Jawad syndromes and defines a new type of genetic disease mechanism in which a dominant negative mutation yields a recessively inherited disorder.     

Mapping CDC Mutations in the Yeast S. Cerevisiae by RAD52-Mediated Chromosome Loss

Using the chromosome loss-mapping method of Schild and Mortimer, I have mapped several new temperature-sensitive mutations that define five CDC genes. Modified procedures were used to facilitate mapping temperature-sensitive mutations in general, and these modifications are discussed. The mutations were assigned to specific chromosomes by chromosome loss procedures, and linkage relationships were determined subsequently by standard tetrad analysis. Four of the mutations define new loci. The fifth mutation, cdc63-1, is shown to be allelic to previously known mutations in the PRT1 gene.