The genetics and biochemistry of urease in Ustilago violacea

Two complementing loci in different linkage groups of the basidiomycete Ustilago violacea are involved in urease activity: a structural one (ure-1) and a second inferred to involve a permease (ure-2) locus. Two types of complementing mutations occur in the structural locus: null activity (ure-1a) and obviously reduced activity (ure-1b). The ure-2 mutants lacked urease activity in vivo on the phenol red-urea test medium, but gave extracts with wild-type activity. Extracts from wild-type strains gave one site of urease activity after polyacrylamide gel electrophoresis. A number of ure-1b mutants and active revertants from ure-1a mutants yielded electrophoretically variant urease sites. The results are discussed in terms of enzyme polymorphism in haploid eukaryotes by one (missense) or two (null, then missense) mutations.     

Enzymatic,immunological and phylogenetic characterization of <Emphasis Type="Italic">Brucella suis</Emphasis> urease

Background  

The sequenced genomes of the Brucella spp. have two urease operons, ure-1 and ure-2, but there is evidence that only one is responsible for encoding an active urease. The present work describes the purification and the enzymatic and phylogenomic characterization of urease from Brucella suis strain 1330. Additionally, the urease reactivity of sera from patients diagnosed with brucellosis was examined.     

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.     

Design and Validation of ureC-based Primers for Groundwater Detection of Urea-Hydrolyzing Bacteria

Polymerase chain reaction primers based on the ureC gene are described for use in detecting diverse groundwater urea-hydrolyzing bacteria. Six degenerate primers were designed and evaluated for their ability to detect the gene encoding the large catalytic subunit of urease, ureC. Five combinations of these primers were tested pair-wise and displayed an overlapping detection range for bacterial isolates. Pair L2F/L2R exhibited the greatest detection range for described bacterial species and for bacterial isolates from groundwater samples belonging to the bacterial divisions Firmicutes, Actinobacteria, and the α , β , and γ subdivisions of Proteobacteria. Primers L2F/L2R exhibited a greater detection range than previously described ureC-specific primers, and amplified novel ureC sequences from groundwater isolates in the genera Hydrogenophaga, Acidovorax, Janthinobacterium, and Arthrobacter. A comparative phylogenetic analysis of ureC and 16S rRNA genes was performed to determine the utility of groundwater ureC sequence information as a phylogenetic marker for ureolytic species. Our results were consistent with previous analyses of urease genes which demonstrated that the ureC gene has undergone lateral transfer and is not a robust phylogenetic marker. However, the ureC-specific primers, L2F/L2R, demonstrate a broad detection range for ureolytic species, and can serve to enhance functional diversity analyses of ureolytic bacteria.     

<Emphasis Type="Italic">Brucella suis</Emphasis> urease encoded by <Emphasis Type="Italic">ure</Emphasis> 1 but not <Emphasis Type="Italic">ure</Emphasis> 2 is necessary for intestinal infection of BALB/c mice

Background  

In prokaryotes, the ureases are multi-subunit, nickel-containing enzymes that catalyze the hydrolysis of urea to carbon dioxide and ammonia. The Brucella genomes contain two urease operons designated as ure1 and ure2. We investigated the role of the two Brucella suis urease operons on the infection, intracellular persistence, growth, and resistance to low-pH killing.     

Evidence against mutational "hot-spots" at aggregation loci in Dictyostelium discoideum

Summary Complementation tests were performed on 10 strains of Dictyostelium discoideum which carry developmental mutations representing aggregation loci identified previously in two independent studies. When the 5 aggregation-deficient strains representing loci CG1-5 were fused with the 5 strains carrying mutations at loci agoA-E, all 25 crosses produced aggregation-competent diploids. Complicating factors, such as negative gene interactions and possible interallelic complementation are discussed. The results of this experiment suggest that the 10 aggregation loci identified in the two studies are different and that aggregation loci in D. discoideum are probably not associated with significant mutational hot-spots.     

Resistance to 2-deoxyglucose in yeast: A direct selection of mutants lacking glucose-phosphorylating enzymes

Summary When strains of Saccharomyces cerevisiae carrying a single glucose-phosphorylating enzyme such as hexokinase P1 or hexokinase P2 or glucokinase, are subjected to the selection pressure against the toxic sugar 2-deoxyglucose, the majority of survivors are mutants lacking the respective enzymes. All the 2-deoxyglucose-resistant segregants recovered from backcrosses of these mutants to a wild type strain are glucose-negative and all the sensitive ones are glucose-positive. The hexokinase mutations are located in the same complementation groups as defined by the structural genes of hexokinase P1 and hexokinase P2. No interallelic complementation has been observed either in hexokinase P1 or in hexokinase P2 amongst a total of 4×64, and 5×60 different combinations of independent mutants at the hxk1 and hxk2 loci respectively. There appears to be neither a common genetic regulator controlling two or more of these glucose-phosphorylating enzymes nor a sugar carrier that can be dispensed with.     

Interallelic complementation at the pyrF locus and the homodimeric nature of orotate phosphoribosyltransferase (OPRTase) in Mucor circinelloides

Using 5-fluoroorotic acid (5-FOA) as a positive selection system we isolated mutants of Mucor circinelloides altered in the pyrimidine biosynthetic pathway. These mutants were found to be deficient either in orotidine-5′-monophosphate decarboxylase (OMPdecase), or in orotate phosphoribosyltransferase (OPRTase) activity. Complementation tests among mutants lacking OPRTase activity classified them into three groups, thus suggesting the possibility of interallelic complementation. To investigate this hypothesis a cDNA clone corresponding to the OPRTase-encoding gene of M. circinelloides was isolated by direct complementation of E. coli. The genomic copy transformed to prototrophy one member of each of the three classes of OPRTase-deficient mutants. We therefore concluded that they were all altered at the same locus, the pyrF locus. The corresponding alleles were cloned and sequenced. Comparisons of the amino acid sequence of M. circinelloides OPRTase with those of E. coli and S. typhimurium revealed a high degree of similarity in secondary and tertiary structure. As the two bacterial enzymes exist as dimers, a homodimeric quaternary structure of the M. circinelloides mature protein can be assumed. This would also explain the interallelic complementation between some pyrF mutants. The mutations found could affect either the active site or the structure of the dimer interface of the OPRTase. Received: 22 May 1998 / Accepted: 13 August 1998     

Assembly of the mitochondrial membrane system. Genetic complementation of mit- mutations in mitochondrial DNA of Saccharomyces cerevisiae.

A method has been devised to test intergenic complementation of mutations in the mitochondrial DNA of Saccharomyces cerevisiae. The test is based on the observation that diploids issued from pairwise crosses of certain mit- mutants with deficiencies in cytochrome oxidase, or coenzyme QH2-cytochrome c reductase, acquire high levels of respiratory activity shortly after zygote formation. Under our experimental conditions neither biochemical complementation, interallelic complementation, nor recombination has been found to contribute to any significant extent toward the respiration measured in the diploids at early times. The test has been used to study the number of complementation groups represented by a large number of mit- mutants. Results of pairwise crosses of mutants in the oxi 1, oxi 2, oxi 3, cob 1, and cob 2 loci indicate that complementation occurs between the oxi and cob loci between different oxi loci but not between the two cob loci. The five loci have, therefore, been assigned to four different complementation groups.     

The nature of spontaneous mutations during vegetative growth in Schizosaccharomyc pombe

Summary Fifty five purple spontaneous mutants of the haploid fission yeast Schizosaccharomyces pombe have been isolated and distributed, by genetic analyses, between two loci (ad-6 and ad-7) which control two independent steps in the purine biosynthesis.The molecular nature of these mutants has been determined by both their physiological behaviour (leakiness, temperature-sensitivity, pH-sensitivity) and their interallelic complementation ability.It is concluded that during the mitotic cycle the large majority of spontaneous mutations are base-pair substitutions.Publication n. 6 from the Laboratorio di Mutagenesi e Differenziamento del Consiglio Nazionale delle Ricerche (C.N.R.), Pisa, Italy.     

The regulation of urease activity in Aspergillus nidulans

Aspergillus nidulans can utilize urea as a sole source of nitrogen but not as a carbon source. Urea is degraded by a urease. Mutation at any one of three genes, ureB, ureC, and ureD, may result in deficient urease activity. The ureB gene is closely linked to ureA, the structural gene for the urea transport protein. The heat lability of a ureB ^– revertant strain, intragenic complementation tests, and the linkage of ureB to ureA suggest that ureB is the urease structural gene. The ureD gene is probably involved in the synthesis or incorporation of a nickel cofactor essential for urease activity. The function of the ureC gene is not known. Urease is not induced but is subject to nitrogen regulation. The urease activities of ammonium-derepressed mutants show that the effector of nitrogen regulation is more likely to be glutamine than ammonium. When glutamine is present in the medium, urease appears to be inactivated by some means which does not involve a newly synthesized protease or a direct interaction between glutamine and urease.     

Clustering of mutations in methylmalonyl CoA mutase associated with mut- methylmalonic acidemia.

Mutations have been described in human methylmalonyl CoA mutase (MCM) that exhibit partial defects in enzyme activity, including cobalamin-dependent (i.e., mut-) or interallelic complementation. This work describes mutations in cells from four patients, three of whom exhibit a cobalamin-dependent phenotype and all four of whom exhibit interallelic complementation. Four novel mutations (R694W, G648D, G630E, and G626C) are identified that cluster near the carboxyl terminus of the protein, a region close to another mut- mutation (G717V). Each of these mutations was shown to express a phenotype congruent with that of the parental cell line, after transfection into mut0 fibroblasts, and each exhibits interallelic complementation in cotransfection assays with clones bearing a R93H mutation. The activity of mutant enzymes expressed in Saccharomyces cerevisiae parallels the residual activity of the parental cell lines and exhibits novel sensitivities to pH and salt. The clustering of these mutations identifies a region of MCM that most likely represents the cobalamin-binding domain. The location of this domain, as well as the pattern of sequence preservation between the homologous human and Probiono-bacterium shermanii enzymes, suggests a mechanism for interallelic complementation in which the cobalamin-binding defect is complemented in trans from the heterologous subunits of the dimer.     

Biochemical characterization of mutant propionyl CoA carboxylases from two minor genetic complementation groups

We have biochemically characterized several parameters of propionyl CoA carboxylase (PCC) activity in fibroblast extracts from PCC-deficient patients belonging to the two minor genetic complementation groups, pcc B and pcc BC. Comparison of PCCs from these groups with those of the two major complementation groups, pcc A and pcc C, has demonstrated that PCCs from both the pcc B and pcc BC groups closely resemble each other as well as PCC from the pcc C group. These results further support the hypothesis that the pcc B and pcc BC lines are interallelic with respect to pcc C and consequently that the structural mutations in the PCCs from these groups involve the same subunit.     

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.     

Super-suppressor induced interallelic complementation

Summary In yeast the dominant super-suppressorS ₅ has a distinct expression in heterozygotes depending on the particular combination of alleles at thead ₁ orad ₂ loci. If thead ₁ combination is represented by two suppressible alleles, the phenotype of diploid is wild. If thead ₁ combination consists of a suppressible and a non-suppressible allele the phenotype of the diploid is partially mutant. Such a difference in the manifestation of suppressor depending on the combination of alleles is more pronounced in the case ofad ₂ mutations. In the case when bothad ₂ alleles are suppressible, the diploid is prototrophic, but when only one allele is suppressible, the diploid is an adenineless auxotroph as a rule.This type ofS ₅-effect gave us the possibility to study interallelic complementation atad ₂ locus in presence of the super-suppressor. It was shown that some combinations of noncomplementing alleles do complement as a result of suppression.Comparison of the two complementation maps with and without suppressor is made for thead ₂ locus. The mechanisms of the phenomena and of super-suppression are discussed.     

Characterization of Allozyme Null and Low Activity Alleles from Two Natural Populations of DROSOPHILA MELANOGASTER

Null and low enzyme activity alleles recovered from two natural populations were analyzed for a number of genetic and biochemical properties. Analysis of 58 mutations at 14 loci showed that all but one allele were genetically viable and fertile, four alleles were associated with chromosome rearrangements, 28 alleles retained some enzyme activity, 13 alleles formed an active heterodimer with active alleles and five alleles showed partial interallelic complementation. Available evidence indicates that this sample includes mutations resulting from lesions in both coding and regulatory sequences. Certain mutations may be caused by transposable element insertions.     

Genetic analysis of replicating instabilities in yeast

Strains showing ethyl methanesulfonate (EMS)-induced replicating instability were genetically analysed to test whether within a given line, mosaics from different plating generations carry a mutation at the same site within the locus. A forward mutation system involving five loci controlling adenine biosynthesis in Schizosaccharomyces pombe was used. Genetic analysis was carried out by interallelic complementation and intragenic recombination tests. The data showed that EMS-induced instabilities are site-specific in being confined to the same recombination unit. This finding is discussed in relation to the possible mechanism(s) of replicating instabilities after different mutagenic treatments in a variety of biological systems.     

Molecular and biochemical characterization of urease and survival of Yersinia enterocolitica biovar 1A in acidic pH in vitro

Background  

Yersinia enterocolitica, an important food- and water-borne enteric pathogen is represented by six biovars viz. 1A, 1B, 2, 3, 4 and 5. Despite the lack of recognized virulence determinants, some biovar 1A strains have been reported to produce disease symptoms resembling that produced by known pathogenic biovars (1B, 2-5). It is therefore imperative to identify determinants that might contribute to the pathogeniCity of Y. enterocolitica biovar 1A strains. Y. enterocolitica invariably produces urease and the role of this enzyme in the virulence of biovar 1B and biovar 4 strains has been reported recently. The objective of this work was to study genetic organization of the urease (ure) gene complex of Y. enterocolitica biovar 1A, biochemical characterization of the urease, and the survival of these strains under acidic conditions in vitro.     

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.