Mutations In Structural Genes Research Articles

Regulation of the degradative pathway enzymes coded for by the TOL plasmid (pWWO) from Pseudomonas putida mt-2.

Pseudomonas putida mt-2 carries a plasmid (TOL, pWWO) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. Characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m-toluate, whereas the inducers of the enzymes responsible for oxidation of m-xylene to m-toluate include m-xylene and m-methylbenzyl alcohol but not m-toluate. A regulatory mutant is described in which m-xylene and m-methylbenzyl alcohol no longer induce any of the pathway enzymes, but m-toluate is still able to induce the enzymes responsible for its own degradation. Among revertants of this mutant are some strains in which all the enzymes are expressed constitutively and are not further induced by m-xylene. A model is proposed for the regulation of the pathway in which the enzymes are in two regulatory blocks, which are under the control of two regulator gene products. The model is essentially the same as proposed earlier for the regulation of the isofunctional pathway on the TOL20 plasmid from P. putida MT20.

Acquisition of ability to utilize Xylitol: disadvantages of a constitutive catabolic pathway in Escherichia coli

Ribitol+ strains of Escherichia coli acquire the ability to utilize xylitol by mutating to constitutive production of the coordinately controlled ribitol catabolic enzymes ribitol dehydrogenase (RDH) and D-ribulokinase (DRK). Such strains concomitantly acquire toxicity to galacitol and L-arabitol, and to D-arabitol if they are unable to utilize it for growth. Strains selected for resistance to these polyols have DRK structural gene mutations or other mutations that eliminate the constitutive production of DRK, consistent with the view that DRK phosphorylates those polyols to toxic substances. Ribitol+ strains selected for growth on 8 mM xylitol fail to grow on 30 mM xylitol. A product of ribitol and xylitol catabolism represses synthesis of RDH, an enzyme required for growth on xylitol. At 30 mM xylitol, greater than 99% of RDH synthesis is repressed. Strains that grow on 8 mM xylitol can mutate to grow on 30 mM xylitol. Such mutants, relieved of this repression, overproduce RDH, resulting in good growth on the poor substrate, xylitol, but poor growth on the normal substrate, ribitol.

510 GALACTOCEREBROSIDASE(GC) CROSS REACTING MATERIAL(CRM) 5X0 IN GLOBOID CELL LEUKODYSTROPHY(GLD)

The primary genetic defect in GLD is a deficiency of GC activity. In order to clarify the nature of the mutation in this disease, we have purified GC from placenta and used antibodies prepared against this normal enzyme to look for CRM in tissues of patients with GLD. Antigenic identity was observed between GC of normal brain, liver and skin fibroblasts. The antibodies were monospecific to this enzyme, precipitated only β-galactosidase activities toward galactosylceramide and lactosylceramide (assay I)and did not react with either GM1 or neutral β-galactosidases. Preparations of 1 brain, 2 livers and 4 fibroblast lines from GLD patients were examined by double gel immunodiffusion against the anti-GC antibodies and found to be CRM positive. The patients' CRM was identical to the normal enzyme and demonstrated a similar electrophpretic mobility on immunoelectrophoresis. However, activity towards naphthol-AS-BI-β-galactoside could be demonstrated in the immune precipitates of normal controls and other sphingo-lipidoses but not in those of the GLD patients. A single radial immunodiffusion assay was developed for quantitation of the CRM in the various tissues independent of its enzymic activity. Quantities of CRM within the normal range were determined in the brain, liver, and skin fibroblasts of the GLD patients. The existence of normal amounts of CRM in the patient's tissues indicates a structural gene mutation producing a protein which has lost its enzymic activity but retained its antigenicity.

Physiological characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein

Studies using isogenic transductant strains mlpA+ and mlpA as well as reversion analysis suggested that the physiological consequences of a structural gene mutation in murein lipoprotein include (i) increased sensitivity toward chelating agents ethylenediaminetetraacetic acid and ethyleneglycol-bis (beta-aminoethyl ether)-N,N-tetraacetic acid, (ii) leakage of periplasmic enzyme ribonuclease, (iii) weakened association between the outer membrane and the rigid layer accentuated by Mg2+ starvation, resulting in the formation of outer membrane blebs, and (iv) decreased growth rate in media of low ionic strength or low osmolarity. It is suggested that the bound form of lipoprotein plays an important role in the maintenance of the structural integrity of the outer membrane of the Escherichia coli cell envelope. Other outer membrane components may also contribute to the anchorage of outer membrane to the rigid layer, probably through ionic interactions with divalent cations. Using the phenotype of ribonuclease leakage as an unselected marker in a three-factor cross with P1 transduction, we were able to establish the gene order of man mlpA aroD pps on the E. coli chromosome.

Characterization of two forms of asparaginase in Saccharomyces cerevisiae

Saccharomyces cerevisiae X2180-1A synthesizes two forms of asparaginase: L-asparaginase I, an internal constitutive enzyme, and asparaginase II, an external enzyme which is secreted in response to nitrogen starvation. The two enzymes are biochemically and genetically distinct. The structural gene for asparaginase I (asp 1) is closely linked to the trp 4 gene on chromosome IV. The gene controlling the synthesis of asparaginase II is not linked to either the trp 4 or asp 1 genes. The rate of biosynthesis of asparaginase II is unaltered in yeast strains carrying the structural gene mutation for asparaginase I. Asparaginase II has been purified approximately 300-fold from crude extracts of Saccharomyces by heat and pH treatment, ethanol fractionation, ammonium sulfate fractionation followed by Sephadex G-25 chromatography, and DEAE-cellulose chromatography. Multiple activity peaks were obtained which, upon gas chromatographic analysis, exhibit varying mannose to protein ratios. Asparaginase I has been purified approximately 100-fold from crude extracts of Saccharomyces by protamine sulfate treatment, ammonium sulfate fractionation, gel permeation chromatography, and DEAE-cellulose chromatography. No carbohydrate component was observed upon gas chromatographic analysis. Comparative kinetic and analytic studies show the two enzymes have little in common except their ability to hydrolyze L-asparagine to L-aspartic acid and ammonia.

Restoration of enzyme activity by recessive missense suppressors in the fungus Coprinus.

The acu-1 locus in Coprinus is the structural gene for acetyl-CoA synthetase. Five suppressor gene mutations, which suppress the acu-1,34 missense allele, were induced by mutagen treatment. All five suppressors were shown to have properties expected for tRNA structural gene mutations: they are recessive, they show a gene dosage effect in any doubly heterozygous combination of two sup+ mutations and they are allele specific in action.

Synthesis of tryptophanase in Escherichia coli: Isolation and characterization of a structural-gene mutant and two regulatory mutants

A mutant of E. coli has been isolated that is temperature-sensitive in respect of tryptophanase. When incubated at 60 degrees C, cell-free extracts of the mutant suffer inactivation of enzyme activity much more rapidly than similar extracts of the wild type. After lysogeny with a specialized transducing phage carrying the wild-type tryptophanase gene, the mutant is able to synthesize tryptophanase that is wild-type in its response to treatment at 60 degrees C. It is concluded that the mutation lies in the structural gene for the enzyme. Two further mutants have been isolated that synthesize tryptophanase constitutively. One mutation renders synthesis of the enzyme indifferent to the presence of inducer; the other mutation allows synthesis of the enzyme in the absence of inducer at about 35% of the fully induced wild-type rate. Neither mutation alleviates catabolite repression. Genetic mapping shows that the constitutive mutations lie very close to the structural-gene mutation, on the side of the structural gene distant from bglR.

Recessive dystrophic epidermolysis bullosa: evidence for an altered collagenase in fibroblast cultures.

Collagenase has been implicated in the pathogenesis of blister formation in recessive dystrophic epidermolysis bullosa. In order to examine whether aberrations in this enzyme are important in the disease, fibroblast cultures from two patients were used to compare the properties of the collagenases from the mutant cells with those from control fibroblast lines. Purified procollagenase preparations from the mutant fibroblasts were significantly more thermolabile at low Ca2+ concentration than control enzymes. They also showed a decrease in affinity for Ca2+, a cofactor required both for enzyme activity and thermal stability. In addition, the collagenase from each mutant line displayed diminished specific activity, expressed as activity per unit of immunoreactive protein, with a mean value of 39% of control for one patient's enzyme and 16% for the other. The data support the postulate that, in these two patients, the altered collagenase is the result of a structural gene mutation, a defect in the post-translational modification of the enzyme, or a mutation in a gene regulating the normal degradation of collagenase.

Purine Nucleoside Phosphorylase Deficiency: Altered Kinetic Properties of a Mutant Enzyme

Erythrocyte purine nucleoside phosphorylase from two brothers had 0.5% of normal activity. It differed from the normal enzyme by a tenfold increase in the Michaelis constant for inosine, an inability of inosine to protect against thermal lability, and a more positive net charge. The altered kinetic properties may account for the milder disease in the patients compared to the previously described cases. The data provide evidence for a structural gene mutation and genetic heterogeneity in the new disease of purine nucleoside phosphorylase deficiency and T cell dysfunction.

Evidence for chemically-induced structural gene mutations at the thymidine kinase locus in cultured L5178Y mouse lymphoma cells.

L5178Y mouse lymphoma cells normally appear to possess two functional thymidine kinase alleles (TK+/+). TK-deficient (TK-/-) clonal lines can be derived from these cells by treatment with EMS or other mutagens. Mezger-Freed [12] has argued that such stable phenotypic variants do not arise as the result of gene mutations but instead represent epigenetic events such as normally occur during differentiation without any permanent gene alteration. If this be so, then rare TK+/- revertants arising in TK-/- cultures should possess TK enzyme identical with one of those present in the original TK+/+ cells, since only depression of the TK gene is involved. Our studies show that this is not the case. Among the mutant TK enzymes analyzed in vitro (those from parental TK+/- lines, each derived in turn from separate TK-/- lines) differences were found in (1) solubility in saline; (2) solubility in 3 M LiCl; (3) Km's; and (4) ATP-Mg2+ requirements. These findings were incompatible with a non-mutational model for the production of these stable variants and, in conjunction with reversion-rate data, they tended to favor either direct structural gene modifications or mutations affecting the expression of adult and fetal enzymes.

Detection of active heteropolymeric beta-glucuronidase in hybrids between mouse cells and human fibroblasts with beta-glucuronidase deficiency.

Heteropolymeric beta-glucuronidases are detected in somatic cell hybrids between mouse cells and human fibroblasts deficient in beta-glucuronidase (beta-D-glucuronide glucuronosohydrolase, EC 3.2.1.31) by electrophoresis and column chromatography. Specific antisera against human beta-glucuronidase prepared in mice recognize these heteropolymeric beta-glucuronidases. Our results demonstrate that synthesis of mutant subunits of variant beta-glucuronidase continues in deficient human fibroblasts; these mutant subunits of human beta-glucuronidase retain the ability to associate with normal subunits of mouse beta-glucuronidase to form the enzymatically and immunologically active tetramer. These studies demonstrate the usefulness of interspecific cell hybrids for study of structural gene mutations of polymeric enzymes, such as those of beta-glucuronidase in mucopolysaccharidosis type VII.

Reduced ferrochelatase activity: a defect common to porphyria variegata and protoporphyria.

Erythroid ferrochelatase activity has been studied in the normoblasts of patients with porphyria variegata and protoporphyria. Two methods were used for the investigation: one using intact cells and the other lysed cells, each measuring the amount of haem synthesized by normoblasts. In patients with porphyria variegata, ferrochelatase activity estimated by both methods was approximately 50% of the normal, and in protoporphyria the ferrochelatase activity was normal in intact normoblasts but was 20% of the normal in sonicated normoblasts (marrow lysates). It is suggested therefore that in porphyria variegata a dominantly inherited structural gene mutation results in an active ferrochelatase whereas in protoporphyria the genetic mutation results in an unstable ferrochelatase. The mechanism of the enzyme instability is not known though a number of postulates are discussed.

Mutants of Chinese hamster ovary cells with altered glucose-6-phosphate dehydrogenase activity.

Two mutant clones of a Chinese hamster ovary cell line deficient in glucose-6-phosphate dehydrogenase (G6PD) activity have been characterized. In each case, there is evidence that a structural gene mutation has taken place. The first mutant produces 11% specific enzyme activity compared to wild-type parental cells, but this residual activity is much more heat sensitive than that of the wild type. The second mutant contains no residual activity, but a revertant was isolated that exhibits a partial restoration of G6PD activity with, again, an increased heat sensitivity. The selection of G6PD+ cells from G6PD- populations can be effected by exploiting the increased sensitivity of the latter to diamide, a compound that depletes the cell of reduced glutathione.

IMMUNOCHEMICAL ANALYSES IN ORNITHINE TRANSCARBAMYLASE DEFICIENCY

Ammonia intoxication is the clinical hallmark of X-linked ornithine transcarbamylase (OTC) deficiency. Hemizygous affected males have less residual OTC activity and more serious clinical consequences than do heterozygous females. We now describe some immunochemical features of mutant OTC in affected males and females. A monospecific antiserum against purified bovine hepatic OTC has been prepared in rabbits. Immunotitration shows that the antiserum can completely precipitate human OTC and that a given amount complexes bovine and human activity comparably. We have employed this antiserum in an Ouchterlony double diffusion system and in a classical immune precipitation inhibition assay. This latter method consistently detects <1% of control cross-reacting material (CRM). Livers from four affected males (OTC activities <0.1-10% of control) and two females (8 and 20%) have been studied with these techniques. Whereas only the female with 20% of residual activity shows a precipitin line by the Ouchterlony technique, both females and the two males with the greatest residual activities (10 and 0.3%) have detectable CRM by immune precipitation inhibition. In these four cases the percent of residual CRM approximates that of enzymatic activity. In the two males with enzymatic activity <0.1%, no CRM has been found. The observed agreement between residual enzymatic activity and CRM may reflect structural gene mutations which alter the rate of degradation and/or synthesis of OTC protein. The findings are equally consistent, however, with regulatory mutations controlling OTC synthesis.

Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells

Two-dimensional polyacrylamide gel electrophoresis is used to visualize the regulatory subunit of cAMP-dependent protein kinase from cultured S49 mouse lymphoma cells and to demonstrate its in vivo phosphorylation. Regulatory subunits from mutant cells with altered kinases exhibit at least two patterns of charge shifts consistent with substitutions of single amino acids. The direct demonstration of structural alteration of this protein provides strong evidence for structural gene mutation in this cultured cell system. While mutant and wild-type gene products co-exist in the mutant cells, there is apparently preferential expression and phosphorylation of mutant subunit in these heterozygotes.

Genetically determined differences in concentrations of isosccepting tRNAs in Escherichia coli

Two examples of genetically determined altered concentrations of isoaccepting tRNAs are presented. The concentrations of isoaccepting tRNAsThr are selectively changed by a mutation causing a fourfold overproduction of the cognate aminoacyl-tRNA-synthetase, the threonyl-tRNA synthetase, whereas the distribution of isoaccepting tRNAs of four control tRNA-species in these E. coli mutants was not affected by that mutation. Secondly evidence is presented for a correlation between mutations in structural genes of aminoacid biosynthetic enzymes and alterations in concentrations of cognate isoaccepting tRNAs in two different E. coli strains, auxotrophic for threonine, isoleucine/valine and leucine, and arginine respectively.

Biosynthesis of bacterial glycogen: genetic and allosteric regulation of glycogen biosynthesis in Salmonella typhimurium LT-2

Structural gene mutants of the glycogen biosynthetic enzymes adenosine diphosphate glucose pyrophosphorylase (glgC) and glycogen synthase (glgA) were isolated and partially characterized. The cotransduction frequencies of these genes with the aspartic semialdehyde dehydrogenase (asd) and glycerol-3-phosphate dehydrogenase (glpD) genes suggested the unambiguous gene order of glpD glgA glgC asd. The results of the three-factor cross glpD- glgA- glgC+ X glpD+ glgA+ glgC- were consistent with the proposed order. A simultaneous and approximately equivalent derepression of the glgC, glgA, and glgB (branching enzyme) gene products was observed in the late logarithmic-early stationary phase of growth on enriched media. These results are consistent with the coordinately regulated synthesis of the three glycogen biosynthetic enzymes in Salmonella typhimurium.

Electrophoretic variation in partial deficiencies of hypoxanthine-guanine phosphoribosyltransferase.

In the study of inherited abnormalities of man the demonstration of variable properties of the assayable gene products have provided evidence for structural gene mutations. Different variants from the normal have suggested heterogeneity of these mutations. Structural alterations of hypoxanthine-guanine phosphoribosyltransferase (HGPRT) have been expressed by abnormalities of enzyme kinetic properties and physical properties in circulating erythrocytes and cultured cells.KeywordsMutant EnzymePartial DeficiencyElectrophoretic VariationAdenine PhosphoribosyltransferaseNormal Specific ActivityThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Dominant and recessive informational suppressors of a missense mutation in Coprinus.

Twenty-one suppressor gene mutations which suppress the met-5.1 missense mutation of Coprinus were separated into six groups (A-F) on the basis of dominance or recessiveness, linkage to the met-5 locus, comlementation in heterozygous cells and growth behaviour. The actual number of suppressor loci could not be determined because crosses between suppressed mutants were inviable. The allele specificity of group A, C, D and F suppressors was confirmed by appropriate crosses. Group B and E suppressors were not tested because of close linkage to the met-5 locus. No evidence for functional suppression of met-5 mutations was obtained thus it is likely that all the suppressors cause translational corelation of met-5.1. Suppressors in four groups (C-F) have properties expected of tRNA structural gene mutations: the group C mutation is dominant, the other mutations are recessive but do not complement in heterozygous cells. The relative efficiencies of the tRNA species involved was assessed by comparing the degree to which the different sup+ mutations depressed the growth rate on methionine supplemented medium. The dominant mutation depressed growth to the greatest extent and is, therefore, the most efficient suppressor. The least efficient suppressors did not depress growth at all. When growth was compared on minimal medium it was found that the more efficient the suppressor the less well it restored growth. The mutations in groups A and B depressed growth more than the tRNA mutations but affect some other component in translation because they are recessive and complement normally. It is suggested that they may act to alter tRNA modifying enzymes.

Molecular analysis of spontaneous somatic mutants

Eukaryotic structural gene mutations occurring spontaneously in a mouse myeloma cell line offer the opportunity to study somatic mutation in animal cells at the molecular level. Studies on the myeloma protein and on mRNA have enabled us to characterise four such mutants representing four different mutation mechanisms. The results may have some bearing on the origin of antibody diversity.