Suppression of polarity of insertion mutations in the gal operon and N mutations in bacteriophage lambda.

Bacterial mutations (psuA and psu) known for their ability to suppress the polarity on nonsense mutations are shown to suppress the polarity of certain insertion mutations in the gal operon. The short insertion, IS1 (800 nucleotide pairs), is about 15 to 50% suppressed, whereas longer insertions, IS2 (1,400 nucleotide pairs), and IS3 (1,200 nucleotide pairs), are not. Some of the polarity suppressor mutations (psu-1, psu-2, and psu-3) are at least partially permissive for N-gene mutations (N7 and N53) of bacteriophage lambda, suggesting a relationship between natural and mutational polar signals. That this relationship may be complex is indicated by the fact that other suppressor mutations, effective in suppressing nonsense or insertion polarity, fail entirely to permit the growth of lambda N mutants.     

Characterization of strong polar mutations in a region immediately adjacent to the L-arabinose operator in Escherichia coli B-r

Seven l-arabinose-negative mutations are described that map in three genetically distinct regions immediately adjacent to the araO (operator) region of the l-arabinose operon. All seven mutants revert spontaneously, exhibit a cis-dominant, trans-recessive polarity effect upon the expression of l-arabinose isomerase (gene araA), and fail to respond to amber, ochre, or UGA suppressors. Three of these mutants exhibit absolute polarity and are not reverted by the mutangens 2-aminopurine, diethyl sulfate, and ICR-191. These may have arisen as a consequence of an insertion mutation in gene araB or in the initiator region of the l-arabinose operon. The four remaining mutants exhibit strong but not absolute polarity on gene araA and respond to the mutagens diethyl sulfate and ICR-191. Three of these mutants are suppressible by two independently isolated suppressors that fail to suppress known nonsense codons. Partially polar Ara(+) revertants with lesions linked to ara are obtained from three of the same four mutants. These polar mutants, their external suppressors, and their partially polar revertants are discussed in terms of the mechanism of initiation of expression of the l-arabinose operon.     

Dachs: an unconventional myosin that functions downstream of Fat to regulate growth, affinity and gene expression in Drosophila

The dachs gene was first identified almost a century ago based on its requirements for appendage growth, but has been relatively little studied. Here, we describe the phenotypes of strong dachs mutations, report the cloning of the dachs gene, characterize the localization of Dachs protein, and investigate the relationship between Dachs and the Fat pathway. Mutation of dachs reduces, but does not abolish, the growth of legs and wings. dachs encodes an unconventional myosin that preferentially localizes to the membrane of imaginal disc cells. dachs mutations suppress the effects of fat mutations on gene expression, cell affinity and growth in imaginal discs. Dachs protein localization is influenced by Fat, Four-jointed and Dachsous, consistent with its genetic placement downstream of fat. However, dachs mutations have only mild tissue polarity phenotypes, and only partially suppress the tissue polarity defects of fat mutants. Our results implicate Dachs as a crucial downstream component of a Fat signaling pathway that influences growth, affinity and gene expression during development.     

Polarity in gene araB of the l-Arabinose operon in Escherichia coli B/r

A series of mutations are described which map in the araB gene of the l-arabinose operon and exert a polar effect on gene araA, the structural gene for the l-arabinose isomerase. Ten of the 20 araB point mutants examined exhibited absolute polarity and may represent insertions of genetic material into the araB gene. The remaining 10 point mutants exhibit strong polarity (less than 10% of the normal wild-type inducible level of isomerase) and may represent a class of externally suppressible polar mutations other than amber or ochre. Seven of the 12 araB deletion mutants examined, or 58%, exhibit polarity, suggesting that a shift in the reading frame has been generated in the polycistronic message for the l-arabinose operon. The remaining, presumably in-phase, deletion mutants exhibit hyperinducible levels of isomerase, an effect that is eliminated when an araB(+) gene is introduced in the trans position. The hyperinducibility effect is discussed in terms of a model for self-catabolite repression, originally proposed by Katz and Englesberg.     

Suppression of polarity of insertion mutations within the gal operon of E. coli.

Summary Phenotypic revertants of galOP::IS1 and galOP::IS2 mutations have been isolated after mutagenesis with nitrosoguanidine, they are probably caused by mutations in gene suA. The polarity suppressor mutations described in this study and a known mutation in gene suA isolated by D. Morse (Morse and Guertin, 1972) suppress polarity caused by IS1 more effectively than that caused by IS2 or IS4. Furthermore, suppressibility is influenced by the site and orientation of IS integration.The synthesis of the three enzymes in galOP::IS suA double mutants is constitutive and the ratio of the three enzymes is altered in comparison to the wild type. The reasons for constitutive synthesis of the galactose enzymes and for the altered ratio of enzyme synthesis are discussed.     

Characterization of three Agrobacterium tumefaciens avirulent mutants with chromosomal mutations that affect induction of vir genes.

Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes by acetosyringone under conditions in which the parent strain showed vir gene induction. When the mutant bacteria were examined for changes in surface components, it was found that all three of the mutants showed a similar alteration in lipopolysaccharide (LPS). LPS from the mutants was larger in size and more heavily saccharide substituted than LPS from the parent strain. Two of the mutants showed no detectable alteration in outer membrane and periplasmic space proteins. The third mutant, Ivr-225, was missing a 79-kDa surface peptide. The reason(s) for the failure of vir gene induction in these mutants and its relationship, if any, to the observed alteration in LPS are unknown.     

An Escherichia coli gene required for bacteriophage P2-lambda interference.

The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated.     

Mutants of satellite bacteriophage P4 that are defective in the suppression of transcriptional polarity

The satellite bacteriophage P4 relies on a helper such as P2 to supply the gene products necessary for virion assembly and cell lysis (Six, 1975). P4 has the unique capacity to activate the late genes of P2 by a mechanism that differs from the one normally used by P2 itself. This process has been termed transactivation (Calendar et al., 1977). In addition, P4 is able to suppress the strong polarity associated with certain P2 amber mutations. The isolation of P4 mutants solely defective in polarity suppression (psu^?) demonstrates that the ability of P4 to suppress polarity is non-essential for P4 growth. In particular, polarity suppression plays no essential role in either transactivation or head size determination. The product of the P4 psu gene has been identified as a 19,900 M_r P4 late protein.     

The rcbA gene product reduces spontaneous and induced chromosome breaks in Escherichia coli

Elevated levels of DnaA cause excessive initiation, which leads to an increased level of double-strand breaks that are proposed to arise when newly formed replication forks collide from behind with stalled or collapsed forks. These double-strand breaks are toxic in mutants that are unable to repair them. Using a multicopy suppressor assay to identify genes that suppress this toxicity, we isolated a plasmid carrying a gene whose function had been unknown. This gene, carried by the cryptic rac prophage, has been named rcbA for its ability to reduce the frequency of chromosome breaks. Our study shows that the colony formation of strains bearing mutations in rep, recG, and rcbA, like recA and recB mutants, is inhibited by an oversupply of DnaA and that a multicopy plasmid carrying rcbA neutralizes this inhibition. These and other results suggest that rcbA helps to maintain the integrity of the bacterial chromosome by lowering the steady-state level of double-strand breaks.     

Evidence for unique DNA repair activity encoded by a cloned Serratia marcescens gene: suppression of Escherichia coli mutations that reduce repair of alkylated DNA.

A recombinant plasmid containing a Serratia marcescens DNA repair gene has been analyzed biochemically and genetically in Escherichia coli mutants deficient for repair of alkylated DNA. The cloned gene suppressed sensitivity to methyl methanesulfonate of an E. coli strain deficient in 3-methyladenine DNA glycosylases I and II (i.e., E. coli tag alkA) and two different E. coli recA mutants. Attempts to suppress the methyl methanesulfonate sensitivity of the E. coli recA mutant by using the cloned E. coli tag and alkA genes were not successful. Southern blot analysis did not reveal any homology between the S. marcescens gene and various known E. coli DNA repair genes. Biochemical analysis with the S. marcescens gene showed that the encoded DNA repair protein liberated 3-methyladenine from alkylated DNA, indicating that the DNA repair molecular is an S. marcescens 3-methyladenine DNA glycosylase. The ability to suppress both types of E. coli DNA repair mutations, however, suggests that the S. marcescens gene is a unique bacterial DNA repair gene.     

Strong-polar mutations in the transferase gene of the galactose operon in E.coli

Summary A class of mutations in the transferase gene of the galactose operon in E. coli is described, which is strongly polar for the synthesis of kinase. The latter enzyme is made only to the extent of about 0.1% of the amount made in the induced wildtype. This amount is not dependent on the map position of the mutations and the residual synthesis is non-inducible. The mutants thus resemble 0° mutants in the same operon.Epimerase, which is coded for by the gene proximal to the transferase gene with respect to the operator, is made in normal amounts and its synthesis is normally inducible.The mutants do not seem to belong either to the nonsense or to the frameshift class on the basis of reversion pattern, suppressibility, and degree of polarity. The possible nature of the mutations is discussed.     

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.     

Daf-2, Daf-16 and Daf-23: Genetically Interacting Genes Controlling Dauer Formation in Caenorhabditis Elegans

Under conditions of high population density and low food, Caenorhabditis elegans forms an alternative third larval stage, called the dauer stage, which is resistant to desiccation and harsh environments. Genetic analysis of some dauer constitutive (Daf-c) and dauer defective (Daf-d) mutants has revealed a complex pathway that is likely to function in particular neurons and/or responding tissues. Here we analyze the genetic interactions between three genes which comprise a branch of the dauer formation pathway that acts in parallel to or downstream of the other branches of the pathway, the Daf-c genes daf-2 and daf-23 and the Daf-d gene daf-16. Unlike mutations in other Daf-c genes, mutations in both daf-2 and daf-23 cause non-conditional arrest at the dauer stage. Our epistasis analysis suggests that daf-2 and daf-23 are functioning at a similar point in the dauer pathway. First, mutations in daf-2 and daf-23 are epistatic to mutations in the same set of Daf-d genes. Second, daf-2 and daf-23 mutants are suppressed by mutations in daf-16. Mutations in daf-16 do not suppress any of the other Daf-c mutants as efficiently as they suppress daf-2 and daf-23 mutants. Third, double mutants between either daf-2 or daf-23 and several other daf-d mutants exhibit an unusual interaction. Based on these results, we present a model for the function of daf-2, daf-23 and daf-16 in dauer formation.     

Polarity of amber mutations and suppressed amber mutations in the galactose operon of E. coli

Summary Amber mutants in the t gene of the galactose operon have been examined for polarity in the presence and absence of the suppressors su _I and su _yMel .In the absence of suppressors there is a gradient of polarity with the more polar mutations nearer the epimerase gene. This polarity is cis-dominant. Amber t mutants have raised epimerase levels but this effect is recessive. The operon is normally inducible in the presence of amber mutations. A double amber mutant had the polarity of the mutation nearest the epimerase end of the gene.In the presence of suppressors there is practically no gradient of polarity. This is in disagreement with the model proposed by Martin et al. (1966) and Yanofsky and Ito (1966). Modifications of this model to fit the present data are proposed.     

Polarity Effects in the Hisg Gene of Salmonella Require a Site within the Coding Sequence

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.