Mutational analysis of the active site of indoleglycerol phosphate synthase from Escherichia coli.

Indoleglycerol phosphate synthase catalyzes the ring closure of 1-(2-carboxyphenylamino)-1-deoxyribulose 5''-phosphate to indoleglycerol phosphate, the fifth step in the pathway of tryptophan biosynthesis from chorismate. Because chemical synthesis of indole derivatives from arylamino ketones requires drastic solvent conditions, it is interesting by what mechanism the enzyme catalyzes the same condensation reaction. Seven invariant polar residues in the active site of the enzyme from Escherichia coli have been mutated directly or randomly, to identify the catalytically essential ones. A strain of E. coli suitable for selecting and classifying active mutants by functional complementation was constructed by precise deletion of the trpC gene from the genome. Judged by growth rates of transformants on selective media, mutants with either S58 or S60 replaced by alanine were indistinguishable from the wild-type, but R186 replaced by alanine was still partially active. Saturation random mutagenesis of individual codons showed that E53 was partially replaceable by aspartate and cysteine, whereas K114, E163, and N184 could not be replaced by any other residue. Partially active mutant proteins were purified and their steady-state kinetic and inhibitor binding constants determined. Their relative catalytic efficiencies paralleled their relative complementation efficiencies. These results are compatible with the location of the essential residues in the active site of the enzyme and support a chemically plausible catalytic mechanism. It involves two enzyme-bound intermediates and general acid-base catalysis by K114 and E163 with the support of E53 and N184.     

Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation.

Twelve temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1), representing seven complementation groups, were isolated subsequent to 5-bromodeoxyuridine mutagenesis. These mutants were identified by their inability to replicate in a line of monkey (CV-1) cells at 39 C. Seven of these mutants, representing six complementation groups, induced thymidine kinase (tk) and transformed Ltk- cells, a line of mouse L cells lacking tk, to a tk+ phenotype at both the permissive (34 C) and nonpermissive (39 C) temperatures. Thus, the defective cistrons in these six complementation groups, although necessary for lysis, have no essential function in this transformation system. Transformation by these 12 mutants was dependent on prior UV irradiation. Infection of cells with unirradiated virus under conditions which did not permit virus replication was not sufficient to allow cell transformation. Five mutants, representing two complementation groups, were tk- and were incapable of causing the tk--to-tk+ transformation at either 34 C of 39 C. The tk defects in these mutants are probably unrelated to the ts defects, since one of these complementation groups contains a tk+ member. Therefore, transformation of Ltk- cells to a tk+ phenotype by HSV-1 requires an active viral tk gene. One complementation group was represented by a single tk- member. The role of this cistron in transformation remains undetermined since the primary block to transformation is presumed to be the tk- phenotype. Mutants representing the seven complementation groups were unable to replicate at 39 C in two lines of HSV-1-transformed cells, indicating that the activities of resident wild-type copies of the defective cistrons, if present, could not be detected by complementation.     

The α-Glycerophosphate in DROSOPHILA MELANOGASTER II. Genetic Aspects

Seven alleles of the alpha-Glycerophosphate dehydrogenase-1 (alphaGpdh-1) locus of Drosophila melanogaster have been described. These include two naturally occurring electrophoretic variants, one EMS-induced electrophoretic variant, and four EMS-induced "null" or "zero" mutants. With the electrophoretic variants, the locus was mapped to II-20.5 +/- 2.5. A complementation matrix was prepared utilizing the null mutants. Three of the four mutants and a deletion of the locus (Grell 1967) exhibit dosage dependency. The dosage independent mutant exhibits complementation with two of the other null alleles. Flies genetically deficient in alpha-glycerophosphate dehydrogenase are fertile, but their relative viability is severely diminished. Such flies also lose the ability to sustain flight, an observation consistent with the enzyme's function in energy production. The levels of mitochondrial alpha-glycerophosphate oxidase, measured in flies genetically deficient in the cytoplasmic enzyme, were normal.     

Chinese hamster cells mutant at the hypoxanthine-guanine phosphoribosyltransferase (GPRT) locus. V. Complementation analysis of ts mutants

Four temperature-sensitive HPRT clones were used for hybridological analysis, which led to increase in complementation rate about 5 times. The probability of complementation, in respect of the HPRT locus proved to be rather high: 14 of 45 hybridization-tested mutants had complementation ability (including 3 ts mutants). Analysis of the complementation rate among mutants revealed clear-cut dependence on the selection conditions: clones grown in a medium with 8-azaguanine showed most frequent complementation. The use of mutants with a new phenotype in hybridization analysis revealed four additional complementation groups, three of which are made of temperature-sensitive clones. Biochemical analysis revealed the presence of hybrid forms of the HPRT enzyme in all hybrids tested. This confirms the intragenic character of complementation. At present, the functional map of the HPRT locus is represented by 9 groups, including a group of mutants with no complementation ability.     

The mechanism of interallelic complementation at the INO1 locus in yeast: Immunological analysis of mutants

Summary The ino1 locus of yeast has been demonstrated to be the structural gene for the repressible enzyme, L-myo-inositol-1-phosphate synthase (Donahue and Henry 1981 a). We have screened a large number of allelic representatives of the ino1 locus for the presence of protein which cross reacts with antibody produced in response to purified wild type inositol-1-phosphate synthase. Approximately 50% of all ino1 representatives screened by immunoprecipitation produce a protein of 62,000 molecular weight, identical in size to the wild type enzyme subunit. These mutants (termed crm⁺) were tested for expression of the 62,000 MW protein under conditions which are repressing for the wild type enzyme (greater than 25 M exogenous inositol). The protein produced by the crm⁺ mutants, like the active enzyme in wild type yeast, is repressed in the presence of high levels of exogenous inositol. In addition, we have reassessed the interallelic complementation pattern observed among mutants at the ino1 locus. The entire pattern of interallelic complementation is temperature sensitive.     

Isolation and Characterization of X-Linked Mutants of DROSOPHILA MELANOGASTER Which Are Sensitive to Mutagens

Thirteen X-linked mutants have been isolated in Drosophila melanogaster which render male and homozygous female larvae sensitive to the mutagen methyl methanesulfonate. Their characterization and preliminary assignment to functional groups is described. Four of these mutants are alleles of mei-41 (Baker and Carpenter 1972). Like previously isolated alleles of this locus, these mutants reduce fertility and increase loss and nondisjunction of the X-chromosome in homozygous females. The remaining mutants have been tentatively assigned to six functional groups (two mutants to the mus(1)101 locus, two to mus(1)102 , two to mus(1)103, and one each to mus(1)104, mus(1)105 , and mus(1)106). Several of the complementation groups can be distinguished on the basis of nondisjunction and cross sensitivity to mutagens. Females homozygous for the mei-41, mus(1)101 and mus(1)102 mutants exhibit elevated levels of nondisjunction. Mutants belonging to complementation groups mei-41, mus(1)101, and mus(1)104 are sensitive to nitrogen mustard (HN2) in addition to their MMS sensitivity. Among these mutants there is currently a direct correlation between sensitivity to HN2, sensitivity to 2-acetylaminofluorene and a deficiency in post-replication repair ( Boyd and Setlow 1976). Only the mei-41 mutants are hypersensitive to UV radiation, although several of the mutants exhibit sensitivity to gamma-rays. Semidominance is observed in female larvae of the mei-41, mus(1)104, and mus(1)103 mutants after exposure to high concentrations of MMS. The properties of the mutants generally conform to a pattern which has been established for related mutants in yeast. Additional properties of these mutants are summarized in Table 9.     

Nature of the Complementation Products Formed by a Complementing Mutant of Neurospora crassa

The mutant am-14 produces no active nicotinamide adenine dinucleotide phosphate-linked glutamate dehydrogenase (GDH) and no protein showing immunological cross-reaction with the enzyme. Nevertheless, it shows complementation with several other am mutants in heterokaryons. Active GDH can be extracted from heterokaryons formed from am-14 and other mutants which, by themselves, produce more or less inactive varieties of the enzyme. The enzyme from am-14 + am-3 heterokaryons can be partially separated from am-3 mutant GDH on a diethylaminoethyl cellulose column. It is characterized by abnormally high thermolability and by a capacity for activation by glutamate. By the same procedure as brings about hybridization between mutant GDH proteins, it has been possible to recover enzyme with the properties of pure am-3 GDH from a partially purified am-14 + am-3 GDH preparation which was initially substantially free of unhybridized am-3 enzyme. This is interpreted as evidence that the active complementation product is a hybrid oligomer containing am-3 monomers and also am-14 monomers, the latter being unable to aggregate by themselves. Heterokaryons formed from am-14 and wild type produce GDH of abnormally high thermolability, presumably due to the formation of am-14 + am(+) hybrids.     

Reconstitution of succinate dehydrogenase in Bacillus subtilis by protoplast fusion

Bacillus subtilis succinate dehydrogenase (SDH) is composed of two unequal subunits designated Fp (Mr, 65,000) and Ip (Mr. 28,000). The enzyme is structurally and functionally complexed to cytochrome b 558 (Mr, 19,000) in the membrane. A total of 21 B. subtilis SDH-negative mutants were isolated. The mutants fall into five phenotypic classes with respect to the presence and localization of the subunits of the SDH-cytochrome b558 complex. One class contains mutants with an inactive membrane-bound complex. Membrane-bound enzymatically active SDH could be reconstituted in fused protoplasts of selected pairs of SDH-negative mutants. Most likely reconstitution is due to the assembly of preformed subunits in the fused cells. On the basis of the reconstitution data, the mutants tested could be divided into three complementation groups. The combined data of the present and previous work indicate that the complementation groups correspond to the structural genes for the three subunits of the membrane-bound SDH-cytochrome b558 complex. A total of 31 SDH-negative mutants of B. subtilis have now been characterized. The respective mutations all map in the citF locus at 255 degrees on the B. subtilis chromosomal map. In the present paper, we have revised the nomenclature for the genetics of SDH in B. subtilis. All mutations which give an SDH-negative phenotype will be called sdh followed by an isolation number. The designation citF will be omitted, and the citF locus will be divided into three genes: sdhA, sdhB, and sdhC. Mutations in sdhA affect cytochrome b558, mutations in sdhB affect Fp, and mutations in sdhC affect Ip.     

Studies on mutants affecting amidophosphoribosyltransferase activity in Saccharomyces cerevisiae

Mutants at the ade4 locus of yeast were isolated following mutagenesis of ade+ and ade2 with ultraviolet light (UV), ethylmethane sulphonate, and the acridine half mustard ICR-170. Tests for interallelic complementation, osmotic remediality, temperature sensitivity, and mutagen-specific reversion were carried out on 19 mutants. Six mutants showed interallelic complementation and fell into four groups, defining three complons. Three mutants were osmotic remedial and the same three were temperature sensitive. Three mutants induced by ICR-170 gave purine-excreting revertants, designated Pur6 or ade4.RCF, after exposure to UV. Activity of amidophosphoribosyltransferase (PRPPAT) was assayed in the ade4 mutants and other alleles at this locus. The ade4 mutants lacked activity of the enzyme; the alleles su-pur+, su-pur, PUR6, and Pur6, showed different levels of activity. The enzyme was subject to feedback inhibition by AMP and IMP in su-pur+ and PUR6; su-pur was hypersensitive to inhibition by AMP, whereas Pur6 was slightly resistant. Purine synthesis de novo was shown to be repressible in su-pur+ and constitutive in PUR6 and Pur6 by following the accumulation of aminoimidazole ribotide in the presence and absence of cycloheximide. These observations were confirmed by direct assay of enzyme activity.     

Active-site assembly and mode of DNA cleavage by Flp recombinase during full-site recombination.

A combination of half-site substrates and step arrest mutants of Flp, a site-specific recombinase of the integrase family, had earlier revealed the following features of the half-site recombination reaction. (i) The Flp active site is assembled by sharing of catalytic residues from at least two monomers of the protein. (ii) A Flp monomer does not cleave the half site to which it is bound (DNA cleavage in cis); rather, it cleaves a half site bound by a second Flp monomer (DNA cleavage in trans). For the lambda integrase (Int protein), the prototype member of the Int family, catalytic complementation between two active-site mutants has been observed in reactions with a suicide attL substrate. By analogy with Flp, this observation is strongly suggestive of a shared active site and of trans DNA cleavage. However, reactions with linear suicide attB substrates and synthetic Holliday junctions are more compatible with cis than with trans DNA cleavage. These Int results either argue against a common mode of active-site assembly within the Int family or challenge the validity of Flp half sites as mimics of the normal full-site substrates. We devised a strategy to assay catalytic complementation between Flp monomers in full sites. We found that the full-site reaction follows the shared active-site paradigm and the trans mode of DNA cleavage. These results suggest that within the Int family, a unitary chemical mechanism of recombination is achieved by more than one mode of physical interaction among the recombinase monomers.     

Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster

Summary Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPR-Tase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consists of two mutants that each specify severly reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and OD-Case reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and OD-Case in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.     

Genes for the alpha and beta subunits of the phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli.

The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subunit was altered. The method was in vitro complementation; mutant cell extracts were mixed with purified separated alpha or beta subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when alpha was added, but not when beta was added: these are, therefore, assumed to carry lesions in the gene for the alpha subunit. Two other mutants gave the opposite response and are presumably beta mutants. Enzyme activity is also generated when alpha and beta mutant extracts are mixed, but not when two alpha or two beta mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both alpha and beta mutants are capable of generating the native form, although alpha mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the alpha and beta subunits (designated pheS and heT, respectively) were mapped. The gene order, as determined by transduction is aroD-pps-pheT-pheS. The pheS and pheT genes are close together and may be immediately adjacent.     

β-Glucuronidase Mutants of the Nematode CAENORHABDITIS ELEGANS

Using a screening procedure that is based on a histochemical stain for the enzyme beta-glucuronidase, we have isolated several mutants of the nematode Caenorhabditis elegans affected in beta-glucuronidase activity. All of the mutations fall into one complementation group and identify a new gene, gus-1, which has been mapped on the right arm of linkage group I (LG I), 1.1 map units to the left of unc-54. The mutants have no visible phenotype, and their viabilities and fertilities are unaffected. Linked revertants of two of the mutations have been isolated. They restore enzyme activity to almost wild-type levels; the beta-glucuronidase that one of the revertants produces differs from that of the wild type. We propose that gus-1 is the structural locus for beta-glucuronidase.     

Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli

A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

A deletion mutant of the type IC restriction endonuclease EcoR1241 expressing a novel DNA specificity.

We have developed a complementation assay which allows us to distinguish between mutations affecting subunit assembly and mutations affecting DNA binding in the DNA recognition subunit (HsdS) of the multimeric restriction endonuclease EcoR1241. A number of random point mutations were constructed to test the validity of this assay. Two of the mutants produced were found to be truncated polypeptides that were still capable of complementation with the EcoR1241 Hsd subunits to give an active restriction enzyme of novel DNA specificity. The N-terminal variable domain (responsible for recognition of GAA from the EcoR1241 recognition sequence GAAnnnnnnRTCG) and the spacer region (central conserved region) is intact in both of these mutants. One of these mutant genes (hsdS(delta 50) has been cloned as an active Mtase. Purification of the Mtase proved to be difficult because the complex is weak. However, Mtase activity was obtained from a soluble cell extract, and this allowed us to determine the DNA recognition sequence of the Mtase to be GAAnnnnnnnTTC. This recognition sequence is an inverted repeat of 5'-end of the EcoR1241 recognition sequence. This suggests that the mutant Mtase is assembled from two inverted HsdS(D50) subunits, possibly held together by the HsdM subunits.     

Enzyme Alterations in Tyrosine and Phenylalanine Auxotrophs of Salmonella typhimurium

The enzyme activities specified by the tyrA and pheA genes were studied in wildtype strain Salmonella typhimurium and in phenylalanine and tyrosine auxotrophs. As in Aerobacter aerogenes and Escherichia coli, the wild-type enzymes of Salmonella catalyze two consecutive reactions: chorismate --> prephenate --> 4-hydroxy-phenylpyruvate (tyrA), and chorismate --> prephenate --> phenylpyruvate (pheA). A group of tyrA mutants capable of interallelic complementation had altered enzymes which retained chorismate mutase T activity but lacked prephenate dehydrogenase. Similarly, pheA mutants (in which interallelic complementation does not occur) had one group with altered enzymes which retained chorismate mutase P but lacked prephenate dehydratase. Tyrosine and phenylalanine auxotrophs outside of these categories showed loss of both activities of their respective bifunctional enzyme. TyrA mutants which had mutase T were considerably derepressed in this activity by tyrosine starvation and consequently excreted prephenate. A new and specific procedure was developed for assaying prephenate dehydrogenase activity.     

Allelspezifische suppressormutationen vonSchizosaccharomyces pombe

By comparing the intragenic distribution of suppressor sensitive mutants in fine structure maps, 13 allele specific suppressor mutations (isolated from revertants in adenine dependent mutants of constitutionad ₇) have been analyzed for their allele specific patterns of action in three different groups of mutants blocked in adenine biosynthesis. The 13 suppressor mutations, which have resulted from mutations at seven different suppressor loci, are characterized by four different suppression patterns. Three of these patterns, which partially overlap, are not locus specific since they include sensitive mutants at each of the three lociad ₇, ad₆ andad ₁ studied. The relative frequency of mutants sensitive to one or the other of the suppressors of this type, the absence of osmotic-remedial strains among the suppressor sensitive mutants, and the polarized complementation behaviour of one suppressiblead ₆ mutant and two suppressiblead ₁ mutants capable of interallelic complementation, suggest that the suppression mechanism involves misreading of a mutant triplet of the nonsense type.     

An analysis of xanthine dehydrogenase negative mutants of the rosy locus in Drosophila melanogaster.

Eighteen alleles of the rosy locus in Drosophila melanogaster were characterized to identify putative nonsense mutants. Seven alleles exhibited no evidence of intragenic complementation, no evidence of immunological complementation, no evidence of immunological cross-reactivity to antibodies elicited by wild type xanthine dehydrogenase (XDH), and of course were completely deficient in measurable XDH activity. It is possible that one or more of these highly negative ry alleles are nonsense mutants. The remaining eleven ry alleles code for XDH molecules that retain some antigenic similarities to the wild type enzyme as assessed by immunoelectrophoresis and six of these eleven were capable of intragenic complementation.     

A role for lipopolysaccharide in turkey tracheal colonization by Bordetella avium as demonstrated in vivo and in vitro

We isolated two insertion mutants of Bordetella avium that exhibited a peculiar clumped-growth phenotype and found them to be attenuated in turkey tracheal colonization. The mutants contained transposon insertions in homologues of the wlbA and wlbL genes of Bordetella pertussis. The wlb genetic locus of B. pertussis has been previously described as containing 12 genes involved in lipopolysaccharide (LPS) biosynthesis. Polyacrylamide gel analysis of LPS from B. avium wlbA and wlbL insertion mutants confirmed an alteration in the LPS profile. Subsequent cloning and complementation of the wlbA and wlbL mutants in trans with a recombinant plasmid containing the homologous wlb locus from B. avium eliminated the clumped-growth phenotype and restored the LPS profile to that of wild-type B. avium. Also, a parental level of tracheal colonization was restored to both mutants by the recombinant plasmid. Interestingly, complementation of the wlbA and wlbL mutants with a recombinant plasmid containing the heterologous wlb locus from B. pertussis, B. bronchiseptica, or Bordetella parapertussis eliminated the clumped-growth phenotype and resulted in a change in the LPS profile, although not to that of wild-type B. avium. The mutants also acquired resistance to a newly identified B. avium-specific bacteriophage, Ba1. Complementation of both wlbA and wlbL mutants with the homologous wlb locus of B. avium, but not the heterologous B. pertussis locus, restored sensitivity to Ba1. Complementation of the wlbL mutant, but not the wlbA mutant, with the heterologous wlb locus of Bordetella bronchiseptica or B. parapertussis restored partial sensitivity to Ba1. Comparisons of the LPS profile and phage sensitivity of the mutants upon complementation by wlb loci from the heterologous species and by B. avium suggested that phage sensitivity required the presence of O-antigen. At the mechanistic level, both mutants showed a dramatic decrease in serum resistance and a decrease in binding to turkey tracheal rings in vitro. In the case of serum resistance, complementation of both mutants with the homologous wlb locus of B. avium restored serum resistance to wild-type levels. However, in the case of epithelial cell binding, only complementation of the wlbA mutant completely restored binding to wild-type levels (binding was only partially restored in the wlbL mutant). This is the first characterization of LPS mutants of B. avium at the genetic level and the first report of virulence changes by both in vivo and in vitro measurements.