Mutations affecting adenine phosphoribosyl transferase activity in Chinese hamster cells

Mutants of Chinese hamster ovary cells that were resistant to the adenine analog 2, 6-diaminopurine generally were deficient in the enzyme adenine phosphoribosyl transferase. Such mutants did not occur spontaneously, and were rare even after mutagenesis, presumably because two active genes in this pseudodiploid line must be affected for the recessive drug-resistant phenotype to be expressed. Revertants of one such double mutant were selected on the basis of their ability to utilize adenine as a purine source. These revertants contained reduced levels of enzyme activity and were presumed to be heterozygous for the enzyme structural gene. Forward mutation to diaminopurine resistance, starting with these heterozygous revertants, occurred at a rate 1,000 times greater than that found for the original homozygous cells. The results agree with predictions based on the idea that mutations in the structural gene for the enzyme are responsible for the drug-resistant character of these variants.     

Gene Interactions Affecting Muscle Organization in CAENORHABDITIS ELEGANS

Revertants of unc-15(e73)I, a paralyzed mutant with an altered muscle paramyosin, include six dominant and two recessive intragenic unc-15 revertants, two new alleles of the previously identified suppressor gene, sup-3 V, and a new suppressor designated sup-19(m210)V. The recessive intragenic unc-15 revertants exhibit novel alterations in paramyosin paracrystal structure and distribution, and these alterations are modified by interaction with unc-82(e1220)IV, another mutation that affects paramyosin. A strain containing both unc-15 and a mutation in sup-3 V that restores movement was mutagenized, and paralyzed mutants resembling unc-15 were isolated. Twenty mutations that interfere with suppression were divided into three classes (nonmuscle, sus-1, and mutations within sup-3) based on phenotype, genetic map position and dominance. The nonmuscle mutations include dumpy and uncoordinated types that have no obvious direct effect on muscle organization. Two recessive mutations define a new gene, sus-1 III. These mutations modify the unc-15(e73) phenotype to produce a severely paralyzed, dystrophic double mutant that is not suppressed by sup-3. Five semidominant, intragenic sup-3 antisuppressor mutations, one of which occurred spontaneously, restore the wild-type sup-3 phenotype of nonsuppression. However, reversion of these mutants generated no new suppressor alleles of sup-3, suggesting that the sup-3 antisuppressor alleles are not wild type but may be null alleles.     

Genetic analysis of revertants for the nitrate reductase function of Nicotiana plumbaginifolia

Summary Spontaneous revertants of nitrate reductase (NR)-less mutants were isolated by screening for nitrate utilization in diploid NR^– protoplast cultures of Nicotiana plumbaginifolia. The revertants contained in vivo NR activity in the case of apoenzyme mutants (nia) as well as of a cofactor-deficient (cnx) mutant. Revertants of the NIA type proved to be tetraploid, and genetic analysis showed that only one out of the four NR structural genes had reverted to a functional allele.     

Biochemical Characterization of the ctr Mutants of Escherichia coli

A test procedure based on complementation in mixed extracts is described for the assay of heat-stable protein and enzyme I of the phosphoenolpyruvate-dependent phosphotransferase system. The test was used to assay a collection of pleiotropic carbohydrate mutants of Escherichia coli (ctr mutants) and revertants of these mutants. All mutants were found to lack enzyme I of the phosphoenolpyruvate-dependent transferase system. Revertants of these mutants to complete wild phenotype regained enzyme I-forming ability. Reversion to partial wild type was not accompanied by restoration of enzyme I-forming ability.     

Mutants of Escherichia coli defective in membrane phospholipid synthesis. Properties of wild type and Km defective sn-glycerol-3-phosphate acyltransferase activities.

The sn-glycerol-3-phosphate (glycerol-P) acyltransferase, the first enzyme of membrane phospholipid synthesis in Escherichia coli, was investigated in a wild type and a mutant strain defective in this activity. The mutant strain, selected as a glycerol-P auxotroph, was previously shown to contain a glycerol-P acyltransferase activity with an apparent Km for glycerol-P 10 times higher than that of its parent or revertants. The membranous mutant glycerol-P acyltransferase but did not appear to be thermolabile in vivo. Revertants no longer requiring glycerol-P for growth, showed glycerol-P acyltransferase activity with thermolability properties similar to the wild type. The second phospholipid biosynthetic enzyme, 1-acylglycerol-P acyltransferase, was not thermolabile in membranes containing a thermolabile glycerol-P acyltransferase activity. The pH optimum for the mutant acyltransferase was over 1 pH unit higher than that of the parental activity. Further, the mutant and wild type glycerol-P acyltransferase differed in their response to magnesium chloride and potassium chloride. The palmitoyl-CoA dependence of the wild type and mutant glycerol-P acyltransferase activities were different. The mutant glycerol-P acyltransferase activity was inhibited greater than 90% by Triton X-100 under conditions where the wild type activity was not affected. These experiments provide novel information about the wild type glycerol-P acyltransferase activity of E. coli and provide six additional lines of evidence for the mutant character of the glycerol-P acyltransferase in the mutant strains.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The ery^R and spo^ts phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo⁺), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from ery^R spo^ts strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo⁺ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.     

Isolation and characterization of CHO cell mutants with altered asparagine synthetase.

Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.     

Physiological evidence for an interaction between helix XI and helices I, II, and V in the melibiose carrier of Escherichia coli

In a previous study 23 residues in helix XI of the cysteine-less melibiose carrier were changed individually to cysteine. Several of these cysteine mutants (K377C, A383C, F385C, L391C, G395C) had low transport activity and they were white on melibiose MacConkey fermentation plates. After several days of incubation of these white clones on melibiose MacConkey plates a rare red mutant appeared. The plasmid DNA was then isolated and sequenced. The two second site revertants from K377C were I22S and D59A. This change of aspartic acid to a neutral residue suggests that physiologically there is an interaction between K377 and D59 (possibly a salt bridge). The revertants from A383C were in positions 20 (F20L) and 22 (I22S and I22N). Revertants of F385C were intrahelical changes (I387M and A388G) and a change in C-terminal loop (R441C). Revertants of L391C were in helix I (I22N, I22T and D19E) and helix V (A152S). Revertants of G395C were in helix I (D19E and I22N). We suggest that there is an interaction between helix XI and helices I, II, and V and proximity between these helices.     

Phenotypic reversion in some early blocked sporulation mutants of Bacillus subtilis: isolation and phenotype identification of partial revertants

Single-step mutants of Bacillus subtilis blocked at stage zero of sporulation (spoO mutants) are pleiotropic. They are impaired in several traits, including increased sensitivity to polymyxin B, when compared to the wild type. Revertants for one or another of the traits associated in this pleiotropy have been isolated and classified according to their phenotypes. Attention was centered on partial revertants (spoO still), of which at least three classes could be recognized. Class I partial revertants recovered a high level of resistance to polymyxin and, in a constant order, a variable number of the other traits; some kind of hierarchy is thus revealed to exist among the traits affected in the original pleiotropy. Partial revertants in the other two classes are still hypersensitive to polymyxin. In class II revertants, ability to excrete exoenzymes and antibiotic(s) is recovered to various extents. Only inducibility of nitrate reductase by nitrate is recovered in class III mutants. The possible significance of these observations is discussed.     

Thermoresistant revertants of an Escherichia coli strain carrying tif-1 and ruv mutations: non-suppressibility of ruv by sfi.

Spontaneous thermoresistant revertants were isolated from Tif1 Ruv- and Tif1 Ruv+ strains of Escherichia coli K-12. They were divided into five groups; backmutants to tif+ and recA structural gene mutants accounted for at least two of these groups. Mutations with an unconditional RecA- phenyotype were detected at a higher frequency in the Tif1 Ruv- strains (65%) than in the Tif1 Ruv+ strains (25%). A third group consisted of revertants exhibiting a RecA- phenotype at low temperature. Revertants with normal recombination ability and UV resistance, but with a thermosensitive defect in propagating lambda bio11 phage, were also isolated (group 4). The alleles responsible for this property were cotransducible with the srl gene, suggesting that they are located at the recA locus. Other revertants, which might carry lex, LEXB, or zab mutations, were UV sensitive and were able to propagate lambda bio11 phage (group 5). The sfi mutation, which suppresses filamentation in the Tif1 and UV-sensitive Lon- strains, does not restore UV resistance of the Ruv- mutant.     

Demonstration of several independent loci involved in the synthesis of iso-2-cytochrome c in yeast

Summary This study concerns the chromosomal genes controlling the synthesis of cytochrome c in yeast. In the wild type there are two molecular species of cytochrome c : iso-1 (major from) and iso-2 (minor form) which differ in many positions of their amino-acid sequence. A mutation, CY1cy1-1, in the structural gene for iso-1, leads to iso-1 deficiency, while retaining a normal albeit small amount of iso-2-cytochrome c.The cyI-1 mutant does not grow on DL-lactate as sole carbon source, while the wild type does. This property was used for selecting cytochrome c rich revertants (CYT) from cytochrome c deficient strains cy1-1; ca 200 revertants were isolated after extensive nitrous acid mutagenesis from a haploid cy1-1 strain or from a diploid cy1-1/cy1-1 strain and ca 30 of them were analyzed genetically and biochemically. The cytochrome c of seven (CYT) revertants was extracted and characterized; none of them contained iso-1-cytochrome c, but all contained large amount of iso-2-cytochrome csufficient to compensate for the deficiency. It was concluded that none of the revertants resulted from back mutation of cy1-1 and that the cy1-1 mutation is a deletion or some other irreversible aberration. These conclusions were corroborated by genetic analysis. It was shown that every reversion is due to a chromosomal mutation segregating as a single gene. Five unlinked gene loci, CY2A, CY2B, CY2C, CY2D, CY2E, were uncovered in this way. None of them were linked to the CY1 locus. Revertants selected in the diploid strain were dominant or semi-dominant while those selected in the haploid strain were recessive. To the first class belong alleles at loci CY2A, CY2B, CY2C, while to the latter belong alleles at loci CY2D and CY2E.Five unlinked loci are implicated in iso-2-cytochrome c synthesis. Mutations selected at these loci act as suppressors of cytochrome c deficiency caused by a deletion of the CY1 locus. In fact the muations do not restore the synthesis of the deficient protein (iso-1-cytochrome c), but increase the synthesis of an another protein, structurally alike (iso-2-cytochrome c), and having very similar if not identical physiological activity. We propose the term of compensator genes to define this type of mutations. We discuss some possible mechanisms to explain the rarity of compensator mutations and the hypothesis that the locus CY2A could correspond not only to the regulatory gene for iso-2-cytochrome c but also to the structural one.     

Structure and function of the mitochondrial bc1 complex. Properties of the complex in temperature-sensitive cor1 mutants

The properties of the ubiquinol-cytochrome c reductase complex (bc1 complex) have been studied in respiratory defective mutants of Saccharomyces cerevisiae bearing lesions in the core 1 subunit. All the cor1 mutants examined have greatly reduced concentrations of mitochondrial cytochrome b and display succinate-cytochrome c reductase activities near the limits of detection. Two mutants (E576 and C7), however, had 5% of wild type activity when the cells were grown at 23 degrees C, but not at 37 degrees C. The temperature-sensitive phenotype was determined to result from substitution of either Arg or Glu for Gly68 of the core 1 subunit. The respiratory competent revertants E576/R8 and C7/R4 derived from E576 and C7 retain the temperature sensitivity of the original mutants. Both revertants are temperature sensitive in vivo, but only mitochondria isolated from E576/R8 are temperature sensitive in vitro. The bc1 complex of mitochondria isolated from this revertant displays a normal value of the ratio Kcat/Km for cytochrome c and four times higher than the wild type for duroquinol. The succinate-cytochrome c reductase activity of E576/R8 is almost completely abolished after incubation at 37 degrees C for 90 min. It is inferred that the quaternary structure of ubiquinol-cytochrome c reductase complex is more labile at the nonpermissive temperature in the mutant and undergoes an alteration such that cytochrome b is no longer able to receive electrons through either the "o" or the "i" site pathway. The temperature lability and kinetic properties of the mutant enzyme point to a requirement of the core 1 not only for assembly but also for the catalytic activity of the complex.     

Revertants of a trans-dominant S49 mouse lymphoma mutant that affects expression of cAMP-dependent protein kinase

Phenotypic revertants were isolated from an S49 mouse lymphoma tissue culture cell mutant that lacks cAMP-dependent protein kinase (cA-PK) activity (kin-). The mutant phenotype is trans-dominant and results from a lesion that probably lies outside the cA-PK subunit structural genes. The nature of the event that produces the kin- phenotype is unknown. However, the mechanism that is responsible for its behavior is genetically encoded because: spontaneous revertants arise at low frequency; reversion frequency is increased by mutagen treatment; mutagen-specific classes of revertant phenotypes are induced; and some revertants are temperature-sensitive for expression of cA-PK subunit polypeptides. Additional evidence is provided that argues against structural lesions in cA-PK catalytic (C) subunits as explanatory of the kin- phenotype. Kin- cells do not express an immunologically detectable C polypeptide, whereas C expression is restored in revertant cells. Revertants in which phenotype and cA-PK activity levels are only partially restored to that of wild-type cells contain a commensurately reduced amount of C polypeptide. Finally, the structure of C polypeptide in partial revertants is unaltered from that of wild-type C. The evidence supports the hypothesis that the kin- lesion defines a regulatory gene responsible for setting intracellular levels of cA-PK C subunit expression.     

Regulation of polar surface structures in Caulobacter crescentus: Pleiotropic mutations affect the coordinate morphogenesis of flagella, pili and phage receptors

Summary A large number of Caulobacter mutants resistant to DNA or RNA phages were isolated. These phage-resistant mutants exhibited phenotypic variations with respect to cell motility and sensitivity to other phages.The majority of the mutants was resistant to both DNA and RNA phages tested. In addition, these mutants were either motile or non-motile. The analysis of spontaneous revertants from these mutants indicated that a single mutation is involved in these phenotypic variations. Other mutants were resistant to RNA phages and only to a certain DNA phage tested, and were also motile or non-motile.Several temperature-sensitive phage-resistant mutants were also isolated. One of them, CB13 ple-801, exhibited the wild type phenotype when grown at 25°C. However, at a higher temperature (35°C), the mutant cells became non-motile and resistant to both DNA and RNA phages. These phenotypes seem to be attributed to the concommitant loss of flagella, pili and phage receptors. In other respects (cell growth and morphology, and asymmetric stalk formation), CB13 ple-801 was normal at 35°C. The spontaneous revertants from CB13 ple-801 simultaneously regained the wild type phenotypes in all respects.It is suggested that a single mutation pleiotropically affects the formation of flagella, pili and phage receptors.     

Substitutions in the protease (3Cpro) gene of poliovirus can suppress a mutation in the 5' noncoding region.

The poliovirus mutant 5NC-11 has a 4-base insertion at position 70 within the 5' untranslated region and is deficient in RNA synthesis. Revertants from 5NC-11 were isolated, showing a partial recovery of wild-type levels of RNA synthesis. The 5' noncoding region of those revertants contained the mutation intact; mix-and-match experiments with the cDNA from these revertants revealed that a restricted region within the 3C gene was the site of the suppressing mutations in the revertants. The suppressors were point mutations, confirmed by introducing them into the 3C gene by site-directed mutagenesis. Although complementation studies indicated that the suppressors were cis active, we believe that protein changes rather than RNA sequence alterations are responsible for the suppression because RNA changes that did not alter protein sequence had no effect, whereas various protein alterations were suppressive. The results therefore imply that protein 3C interacts with the 5' end of the RNA and may play a role in RNA replication.     

Isolation and physical mapping of temperature-sensitive mutants defective in heat-shock induction of proteins in Escherichia coli

Summary Mutants of Escherichia coli K12 that are partially or totally defective in induction of major heat-shock proteins and cannot grow at high temperature (42° C) were isolated by localized mutagenesis. These mutants carry a single mutation in the gene htpR (formerly hin) located at min 76 on the E. coli genetic map. Some mutants exhibit delayed (partial) induction of heat-shock proteins or require a higher temperature for induction than the wild type, whereas others are not induced under any of these conditions. The maximum temperature that allows growth varies among different mutants and is correlated with the residual induction capacity. Temperature-resistant revertants obtained from each mutant are fully or partially recovered in heat-shock induction. These results indicate that the inability of htpR mutants to grow at high temperature is due to the defect in heat-shock induction. In addition, a couple of mutants was found that produce significantly higher amounts of heat-shock proteins even at 30° C.The htpR gene has been cloned into plasmid pBR322 using the above mutants, and was localized to a DNA segment of 1.6 kilobase pairs. The mutants harboring certain palsmids that carry a part of htpR produce temperature-resistant recombinants at high frequency. This permits further localization of mutations within the htpR gene. Analysis of proteins encoded by each of the recombinant plasmids including the one carrying a previously isolated amber mutation (htpR165) led to the identification of a protein with an apparent molecular weight of about 36,000 daltons as the htpR gene product.     

Revertants of a streptomycin-resistant, oligosporogenous mutant ofBacillus subtilis

Summary Revertants of a streptomycin-resistant (Str^R), oligosporogenous (Spo^-) mutant ofBacillus subtilis were selected for the ability to sporulate. The revertants obtained fell into two phenotypic classes: Str^S Spo⁺ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Str^R Spo⁺, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 in the 50S subunit were detected in Str^R Spo⁺ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the Str^R revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Str^R parental strain. The Str^R parent grew slowly and sporulated at approximately 1% of the wild type level. The Str^S revertants closely resembled the wild type strain with regard to growth and sporulation. The Str^R revertants grew at rates intermediate between those of the Str^R parent and wild type, and sporulated at wild type levels.