Isolation of suppressors of temperature-sensitive folding mutations.

Mutations in the tailspike gene (gene 9) of Salmonella typhimurium phage P22 have been used to identify amino acid interactions during the folding of a polypeptide chain. Since temperature-sensitive folding (tsf) mutations cause folding defects in the P22 tailspike polypeptide chain, it is likely that mutants derived from these and correcting the original tsf defects (second-site intragenic suppressors) identify interactions during the folding pathway. We report the isolation and identification of second-site revertants to tsf mutants.     

Isolation and characterization of amber mutations in gene dnaA of escherichia coli K-12.

Amber mutants with defects in the dnaA gene of Escherichia coli K-12 were isolated after localized mutagenesis of the tna-dnaA region of the chromosome. We isolated 36 mutants defective in the initiation of deoxyribonucleic acid replication as determined by their dependence upon integrative suppression by a P2 sig5 prophage. Three of the 36 mutants were shown to contain amber mutations through the use of a temperature-sensitive amber suppressor. These mutations, which mapped between gyrB and tna, were characterized genetically and biochemically as amber mutations in dnaA.     

Identification of new cell division genes in Escherichia coli by using extragenic suppressors.

To facilitate the analysis of the cell division control apparatus in Escherichia coli, we studied extragenic suppressor mutations of a previously characterized temperature-sensitive division mutation, ftsM1. Cells of strain GD40 which harbor this mutation were spread on agar plates and incubated at 42 degrees C, and the surviving cells were analyzed for the presence of a suppressor mutation. One group of suppressed mutants had acquired a new mutation which, by conjugation, was found to be located in the 30- to 40-min region of the E. coli genetic map. The other group comprised revertants carrying a suppressor which appeared to map between thr and leu. This suppressor gene, called sftA, was cloned with a mini-Mu-derived in vivo cloning system by selection for suppression of temperature sensitivity in GD40 cells. Subsequent subcloning of a fragment of the chromosomal DNA from the mini-Mu plasmid into pBR325 resulted in the delineation of the suppressor gene on a 1.8-kilobase XhoI-PvuI fragment. A strain, CV514, which does not express the temperature sensitivity phenotype of the ftsM1 mutation, was found to harbor a natural suppressor of this mutation. UV sensitivity, another known phenotype of the ftsM1 mutation, was also corrected by the presence of the sftA suppressor in the cell. Thus, the characterization of extragenic suppressors may allow the identification of new genes involved in the control of cell division.     

Isolation and characterization of Escherichia coli dnaA amber mutants.

Specialized transducing phage lambda (formula, see text) dnaA-2 was mutagenized, and two derivatives designated lambda (formula) dnaA17(Am) and lambda (formula) dnaA452(Am) were obtained. They did not transduce such mutations as dnaA46, dnaA167, and dnaA5 when an amber suppressor was absent, but they did so in the presence of an amber suppressor. By contrast, they transduced the dna-806 and tna-2 mutations in the absence of an active amber suppressor. The dna-806 and tna-2 mutations are known to be located very close to the dnaA gene, but in separate cistrons. When ultraviolet light-irradiated uvrB cells were infected with the derivative phages and proteins specified by them were analyzed by gel electrophoresis, a 50,000-dalton protein was found to be specifically missing if an amber suppressor was absent. This protein was synthesized when an amber suppressor was present. The dnaA17(Am) mutation on the transducing phage genome was then transferred by genetic recombination onto the chromosome of an Escherichia coli strain carrying a temperature-sensitive amber suppressor supF6(Ts), yielding a strain which was temperature sensitive for growth and deoxyribonucleic acid replication. The temperature-sensitive trait was suppressed by supD, supE, or supF. We conclude that, most likely, the derivative phages acquired amber mutations in the dnaA gene whose product is a 50,000-dalton protein as identified by gel electrophoretic analysis.     

Isolation and characterization of extragenic suppressor strains of Corynebacterium diphtheriae.

The isolation and characterization of two different nonsense suppressor strains of Corynebacterium diphtheriae C7 sup+(-)tox- are described. Appropriate lysogens of these strains with corynephage beta, carrying known class II tox premature polypeptide chain termination mutations [C7sup-1(betatox-30) and C7sup-2(betatox-45)], each produce a 62,000-dalton polypeptide with nicotinamide adenine dinucleotide: elongation factor-2 adenosine diphosphate ribosyltransferase activity in addition to a chain-terminated polypeptide of 30,000 or 45,000 daltons, respectively. In addition, purified protein of 62,000 daltons, resulting from the suppression of the nonsense mutations tox-30 and tox-45, will react with antisera purified against the terminal 17,000 daltons of the toxin molecule and are immunologically identical to toxin by radial immunodiffusion. The suppression pattern of lysogenic derivatives of C7sup-1(-)tox- and C7sup-2(-)tox- with other class II and III mutants of corynephage beta was determined.     

Yeast lacking superoxide dismutase. Isolation of genetic suppressors.

Null mutants of superoxide dismutase (SOD) in Saccharomyces cerevisiae are associated with a number of biochemical defects. In addition to being hypersensitive to oxygen toxicity, strains containing deletions in both the SOD1 (encoding Cu/Zn-SOD) and SOD2 (encoding Mn-SOD) genes are defective in sporulation, are associated with a high mutation rate, and are unable to biosynthesize lysine and methionine. The sod-linked defect in lysine metabolism was explored in detail and was found to occur at an early step in lysine biosynthesis, evidently at the level of the alpha-amino adipate transaminase. To better understand the role of SOD in cell metabolism, our laboratory has isolated yeast suppressors that have bypassed the SOD defect ("bsd" strains), that is, S. cerevisiae cells lacking SOD, yet resistant to oxygen toxicity. Two nuclear bsd complementation groups have been identified, and both suppress a variety of biological defects associated with sod1 and sod2 null mutants. These results demonstrate that a single gene mutation can alleviate the requirement for SOD in cell growth. Both bsd complementation groups are unable to utilize many non-fermentable carbon sources, suggesting a possible suppressor-linked defect in electron transport.     

Genetic analysis of flagellar length control in Chlamydomonas reinhardtii: a new long-flagella locus and extragenic suppressor mutations.

Flagellar length in the biflagellate alga Chlamydomonas reinhardtii is under constant and tight regulation. A number of mutants with defects in flagellar length control have been previously identified. Mutations in the three long-flagella (lf) loci result in flagella that are up to three times longer than wild-type length. In this article, we describe the isolation of long-flagellar mutants caused by mutations in a new LF locus, LF4. lf4 mutations were shown to be epistatic to lf1, while lf2 was found to be epistatic to lf4 with regard to the flagellar regeneration defect. Mutations in lf4 were able to suppress the synthetic flagella-less phenotype of the lf1, lf2 double mutant. In addition, we have isolated four extragenic suppressor mutations that suppress the long-flagella phenotype of lf1, lf2, or lf3 double mutants.     

Selection and analysis of rare second-site suppressors of Drosophila RNA polymerase II mutations

We used a mutagenesis and selection procedure in Drosophila melanogaster to recover rare allele-specific suppressor mutations. More than 11 million flies mutant for one of five recessive-lethal mutations in the two largest subunits of RNA polymerase II were selected for additional mutations that restored viability. Forty-one suppressor mutations were recovered. At least 16 are extragenic, identifying a minimum of three loci, two of which do not map near genes known to encode subunits of RNA polymerase II. At most, 25 are intragenic, 4 reverting the initial altered nucleotide back to wild type. Sequence analysis of interacting mutations in the two largest subunits identified a discrete domain in each subunit. These domains might be contact points for the subunits. Finally, our selections were large enough to allow recovery of multiple independent changes in the same nucleotides yet mutations in other equally likely targets were not recovered. The mutations recovered are not random and might provide insights into possible mechanisms for mutagenesis in eukaryotes.     

Identification of multicopy suppressors of the pcnB plasmid copy number defect in Escherichia coli

Plasmids containing a ColE1 origin of replication are widely used for cloning purposes in Escherichia coli. Among the host factors that affect the copy number of ColE1 plasmids is the E. coli protein poly(A) polymerase I (PAP I), which regulates the intracellular level of RNA I, a ColE1-encoded negative regulator of plasmid replication. In strains that lack PAP I, RNA I levels are elevated, resulting in reduced levels of ColE1 plasmids in the cell. PAP I is encoded by the gene pcnB. We devised a genetic approach, based on the identification of multicopy suppressor clones, to identify trans-acting factors that can help offset the ColE1 plasmid copy number defect in a pcnB (-) genetic background. Using this strategy, we identified suppressors that mapped to two regions of the E. coli chromosome. The suppressor activity of one of the chromosomal regions was localized to the rssB gene, a response regulator gene known to be involved in the turnover of the stationary-phase sigma factor, RpoS. The second suppressor maps to min 55.4 of the E. coli chromosome, and the factor responsible for the suppressor activity appears to be a novel RNA or protein.     

Isolation and analysis of dominant secA mutations in Escherichia coli.

The secA gene product is an autoregulated, membrane-associated ATPase which catalyzes protein export across the Escherichia coli plasma membrane. Previous genetic selective strategies have yielded secA mutations at a limited number of sites. In order to define additional regions of the SecA protein that are important in its biological function, we mutagenized a plasmid-encoded copy of the secA gene to create small internal deletions or duplications marked by an oligonucleotide linker. The mutagenized plasmids were screened in an E. coli strain that allowed the ready detection of dominant secA mutations by their ability to derepress a secA-lacZ protein fusion when protein export is compromised. Twelve new secA mutations were found to cluster into four regions corresponding to amino acid residues 196 to 252, 352 to 367, 626 to 653, and 783 to 808. Analysis of these alleles in wild-type and secA mutant strains indicated that three of them still maintained the essential functions of SecA, albeit at a reduced level, while the remainder abolished SecA translocation activity and caused dominant protein export defects accompanied by secA depression. Three secA alleles caused dominant, conditional-lethal, cold-sensitive phenotypes and resulted in some of the strongest defects in protein export characterized to date. The abundance of dominant secA mutations strongly favors certain biochemical models defining the function of SecA in protein translocation. These new dominant secA mutants should be useful in biochemical studies designed to elucidate SecA protein's functional sites and its precise role in catalyzing protein export across the plasma membrane.     

Fine structure genetic map and complementation analysis of mutations in the dnaA gene of Escherichia coli

Summary A fine structure genetic map of several mutations in the dnaA gene of Escherichia coli was constructed by the use of recombinant and M13 phages. The dnaA508 mutation was found to be the mutation most proximal to the promoter, while the dnaA203 mutation was found to be the most distal one. The order of mutations established in this analysis was: dnaA508, dnaA167, (dnaA5, dnaA46, dnaA211), dnaA205, dnaA204, dnaA203. The mutations dnaA601, dnaA602, dnaA603, dnaA604 and dnaA606 were found to map very close to each other and close to dnaA205 in the middle third of the dnaA gene. In analysing the dominance relationship all 13 dnaA mutations were found to be recessive to the wild type. Characteristic phenotypes of the dnaA(Ts) mutants, like reversibility of the temperature inactivation of the dnaA protein, cold sensivity of haploid or of merodiploid strains and suppressibility by rpoB mutations, are found to correlate with clusters of mutations within the gene.     

Isolation and characterization of the Escherichia coli msbB gene, a multicopy suppressor of null mutations in the high-temperature requirement gene htrB.

Previous work established that the htrB gene of Escherichia coli is required for growth in rich media at temperatures above 32.5 degrees C but not at lower temperatures. In an effort to determine the functional role of the htrB gene product, we have isolated a multicopy suppressor of htrB, called msbB. The msbB gene has been mapped to 40.5 min on the E. coli genetic map, in a 12- to 15-kb gap of the genomic library made by Kohara et al. (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987). Mapping data show that the order of genes in the region is eda-edd-zwf-pykA-msbB. The msbB gene codes for a protein of 37,410 Da whose amino acid sequence is similar to that of HtrB and, like HtrB, the protein is very basic in nature. The similarity of the HtrB and MsbB proteins could indicate that they play functionally similar roles. Mutational analysis of msbB shows that the gene is not essential for E. coli growth; however, the htrB msbB double mutant exhibits a unique morphological phenotype at 30 degrees C not seen with either of the single mutants. Analysis of both msbB and htrB mutants shows that these bacteria are resistant to four times more deoxycholate than wild-type bacteria but not to other hydrophobic substances. The addition of quaternary ammonium compounds rescues the temperature-sensitive phenotype of htrB bacteria, and this rescue is abolished by the simultaneous addition of Mg2+ or Ca2+. These results suggest that MsbB and HtrB play an important role in outer membrane structure and/or function.     

Cloning, sequencing, and characterization of multicopy suppressors of a mukB mutation in Escherichia coli

The mukB gene codes for a 177kDa protein, which might be a candidate for a force-generating enzyme in chromosome positioning in Escherichia coli. The mukB106 mutant produces normal-sized, anucleate cells and shows a temperature-sensitive colony formation. To Identify proteins interacting with the MukB protein, we isolated three multicopy suppressors (msmA, msmB, and msmC) to the temperature-sensitive colony formation of the mukB106 mutation. The msmA gene, which could not suppress the production of anucleate cells, was found to be identical to the dksA gene. The msmB and msmC genes suppressed the production of anucleate cells as well as the temperature-sensitive colony formation. However, none of them couid suppress both phenotypes in a mukB null mutation. DNA sequencing revealed that the msmB gene was identicai to the cspC gene and that the msmC gene had not been described before. A homology search revealed that the amino acid sequences of both MsmB and MsmC possessed high similarity to proteins containing the cold-shock domain, such as CspA of E. coliand the Y-box binding proteins of eukaryotes; this suggests that MsmB and MsmC might be DNA-binding proteins that recognize the CCAAT sequence. Hence, the msmB and msmC genes were renamed cspC and cspE, respectively. Possible mechanisms for suppression of the mukB106 mutation are discussed.     

Isolation and characterization of extragenic mutations affecting the expression of the uroporphyrinogen decarboxylase gene (HEM12) in Sacharomyces cerevisiae

Uroporphyrinogen decarboxylase (Uro-d; EC 4.1.1.37), the fifth enzyme in the heme biosynthetic pathway, which catalyzes the sequential decarboxylation of uroporphyrinogen to coproporphyrinogen, is encoded by the HEM12 gene in Saccharomyces cerevisiae. The HEM12 gene is transcribed into a major short mRNA and a minor longer one, approximately 1.35 and 1.55 kb, respectively, in size, and that differ in the 5′ untranslated region. “Uroporphyric” mutants, which have no mutations in the HEM12 gene but accumulate uroporphyrinogen, a phenotype chracteristic of partial Uro-d deficiency, were investigated. Genetic analysis showed that the mutant phenotype depends on the combined action of two unlinked mutations, udt1 and either ipa1, ipa2, or ipa3. ipa1 is tightly linked to HEM12 The mutation udt1 apparently acts specifically on the HEM12 gene, and causes a six to tenfold decrease in the levels of the short HEM12 mRNA, in the β-galactosidase activity of a HEM12-lacZ fusion, in immunodetectable protein and enzyme activity. But heme synthesis is normal and porphyrin accumulation was modest. The mutations ipa1, ipa2, and ipa3 had no phenotype on their own, but they caused an increase in porphyrin accumulation in a udt1 background. This multiplicity of genetic factors leading to uroporphyric yeast cells closely resembles the situation in human porphyria cutanea tarda.     

Missense and nonsense suppressors can correct frameshift mutations

Missense and nonsense suppressor tRNAs, selected for their ability to read a new triplet codon, were observed to suppress one or more frameshift mutations in trpA of Escherichia coli. Two of the suppressible frameshift mutants, trpA8 and trpA46AspPR3, were cloned, sequenced, and found to be of the +1 type, resulting from the insertion of four nucleotides and one nucleotide, respectively. Twenty-two suppressor tRNAs were examined, 20 derived from one of the 3 glycine isoacceptor species, one from lysT, and one from trpT. The sequences of all but four of the mutant tRNAs are known, and two of those four were converted to suppressor tRNAs that were subsequently sequenced. Consideration of the coding specificities and anticodon sequences of the suppressor tRNAs does not suggest a unitary mechanism of frameshift suppression. Rather, the results indicate that different suppressors may shift frame according to different mechanisms. Examination of the suppression windows of the suppressible frameshift mutations indicates that some of the suppressors may work at cognate codons, either in the 0 frame or in the +1 frame, and others may act at noncognate codons (in either frame) by some as-yet-unspecified mechanism. Whatever the mechanisms, it is clear that some +1 frameshifting can occur at non-monotonous sequences. A striking example of a frameshifting missense suppressor is a mutant lysine tRNA that differs from wild-type lysine tRNA by only a single base in the amino acid acceptor stem, a C to U70 transition that results in a G.U base pair. It is suggested that when this mutant lysine tRNA reads its cognate codon, AAA, the presence of the G.U base pair sometimes leads either to a conformational change in the tRNA or to an altered interaction with some component of the translation machinery involved in translocation, resulting in a shift of reading frame. In general, the results indicate that translocation is not simply a function of anticodon loop size, that different frameshifting mechanisms may operate with different tRNAs, and that conformational features, some far removed from the anticodon region, are involved in maintaining fidelity in translocation.     

pryB mutations as suppressors of arginine auxotrophy in Salmonella typhimurium.

Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation were found to grow more slowly in minimal medium than pyrH+ controls. The addition of arginine or citrulline but not ornithine restored normal growth rates. This requirement for arginine was completely suppressed by pyrB mutations and partially suppressed by pyrC and pyrD mutations. No suppression was observed with mutants at the pyrF locus. Introduction of leaky mutation argI2002 resulted in a more extreme arginine requirement and accentuated suppression by pyrB mutations. Suppression by the pyrC and pyrD mutations was reduced as a result of the incorporation of the leaky argI2002 allele. These results indicate that in pyrH700 strains carbamoyl phosphate is preferentially directed toward the formation of intermediates in the pyrimidine biosynthetic pathway. Arginine auxotrophy results from the reduced availability of carbamoyl phosphate for the biosynthesis of arginine. Suppression of this arginine dependence for growth is used as a convenient positive selection technique for pyrB mutations.     

Intra- and extragenic marker haplotypes of CFTR mutations in cystic fibrosis families

Summary In order to facilitate the screening for the less common mutations in the cystic fibrosis (CF) gene viz., the CF transmembrane conductance regulator gene (CFTR), marker haplotypes were determined for German nonCF (N) and CF chromosomes by polymerase chain reaction analysis of four polymorphisms upstream of the CF gene (XV-2c, KM.19, MP6-D9, J44) and six intragenic polymorphisms (GATT, TUB9, M470V, T854T, TUB18, TUB20) that span the CFTR gene from exon 6 through exon 21. Novel informative sequence variants of CFTR were detected in front of exons 10 (1525-61 A or G), 19 (3601-65 C or A), and 21 (4006-200 A or G). The CF locus exhibits strong long-range marker-marker linkage disequilibrium with breakpoints of recombination between XV-2c and KM.19, and between exons 10 and 19 of CFTR. Marker alleles of GATT-TUB9 and TUB18-TUB20 were found to be in absolute linkage disequilibrium. Four major haplotypes encompass more than 90% of German N and CF chromosomes. Fifteen CFTR mutations detected on 421 out of 500 CF chromosomes were each identified on one of these four predominant 7-marker haplotypes. Whereas all analysed F508 chromosomes carried the same KM.19-D9-J44-GATT-TUB9-M470V-T854T haplotype, another frequent mutation in Germany, R553X, was identified on two different major haplotypes. Hence, a priori haplotyping cannot exclude a particular CF mutation, but in combination with population genetic data, enables mutations to be ranked by decreasing probability.