Identification and sequence of gene dicB: translation of the division inhibitor from an in-phase internal start.

The dicA1 mutation, located in the replication termination region of Escherichia coli at 34.9 min, confers a temperature-sensitive, division defective phenotype to its hosts. Previous analysis had suggested that dicA codes for a repressor of a nearby division inhibition gene dicB. We show now that gene dicB is part of a complex operon. Five open reading frames (ORFs 1 to 5) preceeded by a promoter sensitive to dicA repression are found within a 1500 bp segment, and are organized into two clusters separated by a long untranslated region. Evidence for expression of these ORFs was obtained from in vitro or in vivo translation of plasmid-coded genes. IPTG-dependent cell filamentation was obtained when either the entire or the C-terminal part of the fourth ORF was placed under control of the lac promoter. In both cases, a 7 KD protein corresponding to translation from an in-frame ATG of ORF4 (dicB) was made. We propose that this C-terminal protein is the division inhibitor synthesized in dicA1 mutants.     

Control of cell division in Escherichia coli. DNA sequence of dicA and of a second gene complementing mutation dicA1, dicC.

A mutation in a gene dicA of Escherichia coli leads to temperature-sensitive cell division, by allowing expression of a nearby division inhibition gene dicB (1). We have now established the sequence of the DicA region and identified DicA as a 15.5 KD protein. A second gene dicC transcribed divergently from dicA and coding for an 8.5 KD protein can also complement mutation dicA1 when provided on a multicopy plasmid.     

Isolation of cell size mutants of a fission yeast by a new selective method: characterization of mutants and implications for division control mechanisms.

Previously known cell size (wee) mutations of fission yeast suppress the mitotic block caused by a defective cdc25 allele. Some 700 revertants of cdc25-22 were obtained after ultraviolet mutagenesis and selection at the restrictive temperature. Most revertants carried the original cdc25 lesion plus a mutation in or very close to the wee1 gene. Two partial wee1 mutations of a new type were found among the revertants. Two new wee mutations mapping at the cdc2 gene (cdc2-w mutants) were also obtained. The various mutations were examined for their effects on cell division size, their efficiency as cdc25 suppressors, and their dominance relations. Full wee1 mutations were found to suppress cdc25 lesions very efficiently, whereas partial wee1 mutations were poor suppressors. The cdc25 suppression ability of cdc2-w mutations was allele specific for cdc2, suggesting bifunctionality of the gene product. The wee1 mutations were recessive for cdc25 suppression; cdc2-w mutations were dominant. A model is proposed for the genetic control of mitotic timing and cell division size, in which the cdc2+ product is needed and is rate limiting for mitosis. The cdc2+ activity is inhibited by the wee1+ product, whereas the cdc25+ product relieves this inhibition.     

A new dispensable genetic locus of the terminus region involved in control of cell division in Escherichia coli

Summary Temperature-sensitive mutants defective in cell division were isolated after localised mutagenesis of the terminus region of the Escherichia coli chromosome. The defective gene in one of these mutants, dicA, was mapped at 34.9 min by linkage with manA and with three physically characterized Tn10 insertions. Temperature-sensitivity conferred by mutation dicA1 in a recA backround was suppressed by the presence of hybrid plasmids carrying the wild-type gene. In addition, the mutation was suppressed either by tranposon inactivation of a nearby gene, dicB, or by deletion of the entire dicA-dicB interval. These results define the dicA-dicB locus as a new dispensable genetic cluster involved in the control of cell division.     

fipB and fipC: two bacterial loci required for morphogenesis of the filamentous bacteriophage f1.

We describe the identification of two mutations in bacterial genes, designated as fipB and fipC, which resulted in temperature-sensitive morphogenesis of bacteriophage f1. These mutations mapped at separate loci but had to be present simultaneously to block f1 production at 41.5 degrees C. One mutation defined the locus fipB at 85.3 min on the Escherichia coli linkage map; the other defined the locus fipC, which mapped very close to rpsL at 73 min. Since these mutations did not appear to affect phage DNA replication, gene expression, or protein localization, they probably interfered with the its life cycle at the level of assembly. fipB mutants were partially deficient in adsorption of bacteriophage lambda, and fipB and fipC mutants leaked beta-lactamase into the medium, suggesting that the mutations affect outer-membrane structure or function.     

Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape

In yeast, mitochondrial division and fusion are highly regulated during growth, mating and sporulation, yet the mechanisms controlling these activities are unknown. Using a novel screen, we isolated mutants in which mitochondria lose their normal structure, and instead form a large network of interconnected tubules. These mutants, which appear defective in mitochondrial division, all carried mutations in DNM1, a dynamin-related protein that localizes to mitochondria. We also isolated mutants containing numerous mitochondrial fragments. These mutants were defective in FZO1, a gene previously shown to be required for mitochondrial fusion. Surprisingly, we found that in dnm1 fzo1 double mutants, normal mitochondrial shape is restored. Induction of Dnm1p expression in dnm1 fzo1 cells caused rapid fragmentation of mitochondria. We propose that dnm1 mutants are defective in the mitochondrial division, an activity antagonistic to fusion. Our results thus suggest that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively.     

Clustering of null mutations in the EcoRI endonuclease

EcoRI endonuclease mutants were isolated in a methylase-deficient background following in vitro hydroxylamine mutagenesis of plasmid pKG2 (Kuhn et al.: Gene 44:253-263, 1986). Mutants which survived high-level endonuclease expression (IPTG induction) were termed null mutants. Sixty-two of 121 null mutants tested by Western blot contained normal levels of endonuclease cross-reacting protein. The complete endonuclease gene was sequenced for 27 null mutants. This group was found to consist of 20 single base-change missense mutations, 6 double mutations, and 1 triple mutation. Ten of the 20 single mutations were clustered between residues 139 and 144. When examined with respect to the structure of the EcoRI-DNA complex (McClarin et al.: Science 234:1526-1541, 1986), these alterations were found to fall predominantly into two classes: substitutions at the protein-DNA interface or substitutions at the protein-protein (dimer) interface. Protein from several of the mutants was purified and sized by using HPLC. Wild-type EcoRI endonuclease and protein from three of the DNA interface mutations (Ala139----Thr, Gly140----Ser, Arg203----Gln) appeared to be dimeric, while protein from subunit interface mutations (Glu144----Lys, Glu152----Lys, Gly210----Arg) migrated as monomers.     

Mating-defective ste mutations are suppressed by cell division cycle start mutations in Saccharomyces cerevisiae.

Temperature-sensitive mutants which arrest in the G1 phase of the cell cycle have been described for the yeast Saccharomyces cerevisiae. One class of these mutants (carrying cdc28, cdc36, cdc37, or cdc39) forms a shmoo morphology at restrictive temperature, characteristic of mating pheromone-arrested wild-type cells. Therefore, one hypothesis to explain the control of cell division by mating factors states that mating pheromones arrest wild-type cells by inactivating one or more of these CDC gene products. A class of mutants (carrying ste4, ste5, ste7, ste11, or ste12) which is insensitive to mating pheromone and sterile has also been described. One possible function of the STE gene products is the inactivation of the CDC gene products in the presence of a mating pheromone. A model incorporating these two hypotheses predicts that such STE gene products will not be required for mating in strains carrying an appropriate cdc lesion. This prediction was tested by assaying the mating abilities of double mutants for all of the pairwise combinations of cdc and ste mutations. Lesions in either cdc36 or cdc39 suppressed the mating defect due to ste4 and ste5. Allele specificity was observed in the suppression of both ste4 and ste5. The results indicate that the CDC36, CDC39, STE4, and STE5 gene products interact functionally or physically or both in the regulation of cell division mediated by the presence or absence of mating pheromones. The cdc36 and cdc39 mutations did not suppress ste7, ste11, or ste12. Lesions in cdc28 or cdc37 did not suppress any of the ste mutations. Other models of CDC and STE gene action which predicted that some of the cdc and ste mutations would be alleles of the same locus were tested. None of the cdc mutations was allelic to the ste mutations and, therefore, these models were eliminated.     

Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis

Membrane trafficking via targeted exocytosis to the Saccharomyces cerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4Δ or elm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.     

Regulation of beta-glucuronidase synthesis in Escherichia coli K-12: pleiotropic constitutive mutations affecting uxu and uidA expression.

Among the beta-glucuronidase (UID)-constitutive mutants obtained by growth on methyl-beta-D-galacturonide, some strains are also derepressed for the two enzymes of the uxu operon: mannonate oxidoreductase (MOR) and mannonate hydrolyase (HLM). By conjugation and transduction experiments, two distinct constitutive mutations were separated in each pleiotropic mutant strain. One of them was specific for uidA gene expression and was characterized as affecting either uidO or uidR sites. The second type of mutation was mapped close to the uxu operon and was found to be responsible for the pleiotropic effect revealed in the primary mutants: after separation such a mutation still fully derepresses MOR and HLM synthesis but weakly derepresses UID synthesis. The pleiotropic effect of this mutation was maintained even though the activity of the structural genes was altered. This rules out the occurrence of an internal derepressing interaction between these enzymes. In merodiploid strains, uxu-linked constitutive mutations were recessive to the wild-type allele, suggesting that these mutations could affect a regulatory gene. The uxuR gene is probably a specific regulatory gene for a very close operon, uxu. Moreover, it has a weak effect on uidA expression. Thus, UID synthesis would be negatively controlled through the activity of two repressor molecules that are synthesized by two distinct regulatory genes, uidR and uxuR. These two repressing factors are antagonized, respectively, by phenyl-thio-beta-D-glucuronide and mannonic amide and could cooperate in a unique repression/induction control over uidA expression. Constitutive mutations affecting the control sites of uidA gene probably characterize two distinct attachment sites in the operator locus for each of the repressor molecules.     

Identification of a new inhibitor of essential division gene ftsZ as the kil gene of defective prophage Rac.

A gene function carried by a plasmid, causing arrest of cell division in Escherichia coli, has been identified as the product of a short open reading frame of the prophage Rac, previously designated orfE, expressed only under conditions of prophage induction. Because Rac carries a killing function expressed under conditions of zygotic induction, an orfE-defective Rac+ strain was constructed. This strain had lost the killing function, indicating that orfE is kil. Division inhibition by kil was specifically relieved by overexpression of essential division gene ftsZ. The kil gene product acts independently of the min operon, and its effects are increased in conditions of high cyclic AMP (cAMP) receptor protein-cAMP complex levels in the cell. Furthermore, at high levels of expression, kil product distorts the rod shape of the cells. These features distinguish kil-encoded protein from the inhibitory product of gene dicB, which occupies a similar genetic location in Kim (Qin), another defective prophage of Escherichia coli.     

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.     

A new genetic method for isolating functionally interacting genes: high plo1(+)-dependent mutants and their suppressors define genes in mitotic and septation pathways in fission yeast

We describe a general genetic method to identify genes encoding proteins that functionally interact with and/or are good candidates for downstream targets of a particular gene product. The screen identifies mutants whose growth depends on high levels of expression of that gene. We apply this to the plo1(+) gene that encodes a fission yeast homologue of the polo-like kinases. plo1(+) regulates both spindle formation and septation. We have isolated 17 high plo1(+)-dependent (pld) mutants that show defects in mitosis or septation. Three mutants show a mitotic arrest phenotype. Among the 14 pld mutants with septation defects, 12 mapped to known loci: cdc7, cdc15, cdc11 spg1, and sid2. One of the pld mutants, cdc7-PD1, was selected for suppressor analysis. As multicopy suppressors, we isolated four known genes involved in septation in fission yeast: spg1(+), sce3(+), cdc8(+), and rho1(+), and two previously uncharacterized genes, mpd1(+) and mpd2(+). mpd1(+) exhibits high homology to phosphatidylinositol 4-phosphate 5-kinase, while mpd2(+) resembles Saccharomyces cerevisiae SMY2; both proteins are involved in the regulation of actin-mediated processes. As chromosomal suppressors of cdc7-PD1, we isolated mutations of cdc16 that resulted in multiseptation without nuclear division. cdc16(+), dma1(+), byr3(+), byr4(+) and a truncated form of the cdc7 gene were isolated by complementation of one of these cdc16 mutations. These results demonstrate that screening for high dose-dependent mutants and their suppressors is an effective approach to identify functionally interacting genes.     

Genetic Fine Structure of the BRONZE Locus in Maize

The bronze (bz) locus in maize, located in the short arm of chromosome 9 (9S), is the structural gene for the anthocyanin biosynthetic enzyme UFGT. The gene has been cloned and its physical map has been oriented relative to the centromere of 9S. We report here the genetic fine structure mapping of several biochemically characterized EMS-induced bz-E mutations, derived from the Bz-W22 isoallele, and Ds insertion bz-m mutations, derived from the Bz-McC isoallele. Two UFGT(-), CRM(+ ) mutants (bz-E2 and bz-E5), which genetically identify coding sequences in the gene, and three UFGT(-), CRM(- )bz-E mutants were mapped against the Ds insertion mutants bz-m1 and bz-m2(DI) by selecting Bz intragenic recombinants from heterozygotes of the type bz-E/bz-m . The exclusive occurrence of one recombinant outside marker class allowed the unambiguous placement of the mutants in a genetic fine structure map. Peculiarly, the two CRM(+)bz-E mutants lie upstream of the three CRM(-)bz-E mutants and at a considerable genetic distance. The UFGT allozymes encoded by the progenitor alleles Bz-W22 and Bz-McC differ in two properties, thermal stability and activity. The sites responsible for these properties were mapped as unselected markers among the Bz intragenic recombinants. The thermal stability site, which also identifies a coding region of the gene, mapped very close to the CRM(+)bz-E mutant sites. The site responsible for variation in activity, which probably identifies a region involved in regulation of expression of the bz locus, mapped at the 5' or proximal end of the locus. It was found to be inseparable from the Ds insertion in bz-m1 that lies very close to the 5' end of the transcribed region.-Evidence was obtained that the insertion of Ds within the bz gene has a suppressing effect on intragenic recombination. Additional data are also presented supporting our observation that Ds affects the pattern of intragenic recombination at bz.-Based on the total genetic length of the bz gene and on the physical size of the transcribed region, we estimate that one unit of recombination at bronze corresponds to 14 kb of DNA. This estimate is more than 100 times smaller than the average value for the whole genome and implies that there may be regions, such as bronze, that serve as hotspots for recombination.     

New suppressor mutation sur0B of spo0B and spo0F mutations in Bacillus subtilis

Two extragenic suppressor mutations, sur0B20 and sur0F1, which restore the sporulation of spo0B or spo0F mutants of Bacillus subtilis to the wild-type level, were obtained. These suppressor mutations were located in the spo0A gene. Their location is close to that of the sof-1 mutation, which suppresses spo0B, spo0E and spo0F mutations. However, spo0 strains bearing the sur0B20 mutation differed in several phenotypic characteristics from spo0 mutants bearing the sof-1 suppressor. Nucleotide sequence analysis revealed that the sur0B20 and sur0F1 mutations resulted in Glu14 to Val and Asn12 to Lys conversion, respectively, in the spo0A gene. This result indicates that sur0B20 is a new suppressor of spo0b and spo0F mutations, whereas sur0F1 is identical to sof-1.     

Genome-Wide Mutation Avalanches Induced in Diploid Yeast Cells by a Base Analog or an APOBEC Deaminase

Genetic information should be accurately transmitted from cell to cell; conversely, the adaptation in evolution and disease is fueled by mutations. In the case of cancer development, multiple genetic changes happen in somatic diploid cells. Most classic studies of the molecular mechanisms of mutagenesis have been performed in haploids. We demonstrate that the parameters of the mutation process are different in diploid cell populations. The genomes of drug-resistant mutants induced in yeast diploids by base analog 6-hydroxylaminopurine (HAP) or AID/APOBEC cytosine deaminase PmCDA1 from lamprey carried a stunning load of thousands of unselected mutations. Haploid mutants contained almost an order of magnitude fewer mutations. To explain this, we propose that the distribution of induced mutation rates in the cell population is uneven. The mutants in diploids with coincidental mutations in the two copies of the reporter gene arise from a fraction of cells that are transiently hypersensitive to the mutagenic action of a given mutagen. The progeny of such cells were never recovered in haploids due to the lethality caused by the inactivation of single-copy essential genes in cells with too many induced mutations. In diploid cells, the progeny of hypersensitive cells survived, but their genomes were saturated by heterozygous mutations. The reason for the hypermutability of cells could be transient faults of the mutation prevention pathways, like sanitization of nucleotide pools for HAP or an elevated expression of the PmCDA1 gene or the temporary inability of the destruction of the deaminase. The hypothesis on spikes of mutability may explain the sudden acquisition of multiple mutational changes during evolution and carcinogenesis.     

Resistance of Escherichia coli to penicillins: fine-structure mapping and dominance of chromosomal beta-lactamase mutations.

Seven Escherichia coli K-12 mutants with a lowered chromosomal beta-lactamase activity were analyzed genetically. The beta-lactamase-negative mutants isolated from ampA1-carrying strains (resistant to 10 microgram of ampicillin per ml) all carried genetic lesions very close to the ampA1 mutation, which was still present. In an earlier report, two of the mutations mediating a beta-lactamase-negative phenotype (L. G. Burman, T. Park, E. B. Linstr?m, and H. G. Boman, J. Bacteriol. 116:123-130, 1973) were shown to have occurred in the structural gene for beta-lactamase, designated ampC. It is suggested that all beta-lactamase-negative mutants studied here were altered in ampC. The relative order of ampC mutations was (ampC1, ampC8)-ampC9-(ampC12, ampC14)-ampC11, and the gene order was found to be ampC-1mpA-purA. The ampA1 allele was dominant over its wild-type allele but acted only cis and not trans, suggesting that ampA is the promoter or operator region for ampC. A gene dosage effect was found for strains homozygous for ampA+ ampC+ or ampA1 ampC+. Heterozygotes carrying the ampC8 allele on the chromosome showed an apparent derepression of the episomal ampC allele, suggesting a role for beta-lactamase in its own regulation.     

Identification and Characterization of Three Genes That Affect Expression of Adh2 in Saccharomyces Cerevisiae

Using a new selection protocol we have identified and preliminarily characterized three new loci (ADR7, ADR8 and ADR9) which affect ADH2 (alcohol dehydrogenase isozyme II) expression. Mutants were selected which activate ADH2 expression in the presence of an over-expressed, normally inactive ADR1 allele. The mutants had very similar phenotypes with the exception that one was temperature sensitive for growth. In the absence of any ADR1 allele, the mutants allowed ADH2 to partially escape glucose repression. However, unlike wildtype strains deleted for ADR1, the mutants were able to efficiently derepress ADH2. The mutations allowed a small escape from glucose repression for secreted invertase, but had no effect on the glucose repression of isocitrate lyase or malate dehydrogenase. The mutations were shown to be nonallelic to a wide variety of previously characterized mutations, including mutations that affect other glucose-repressed enzymes.