The nature of mutants induced by ionizing radiation in cultured hamster cells. II. Antigenic response and reverse mutation of HPRT-deficient mutants induced by gamma-rays or ethyl methanesulphonate

A large series of independent mutants deficient in HPRT enzyme activity, isolated from V79-4 hamster cells (Brown and Thacker, 1984), were assessed for properties which reflect the nature of the genetic changes induced. A total of 88 mutants were screened, 43 isolated from gamma-ray-treated cultures and 45 induced by ethyl methanesulphonate (EMS). Firstly, each mutant was assayed for the presence of protein with the antigenic response of HPRT (cross-reacting material, CRM), using an antibody raised against partially purified V79-4 HPRT enzyme. In a competitive inhibition assay, 31% of EMS-induced mutants were CRM-positive compared to 7% of the gamma-ray series. Secondly, each mutant was tested for ability to revert to HPRT proficiency, either spontaneously or after treatment with the powerful mutagen ethyl nitrosourea (ENU). All except 2 of the EMS-induced mutants reverted with ENU, and many reverted spontaneously, under the given conditions. However reversion was not detected in about 80% of gamma-ray-induced mutants, suggesting that the types of forward mutation caused by ionizing radiation differ qualitatively from those caused by EMS. The EMS-induced mutations are likely to be mostly point mutations, with at least 40% of the missense type, while gamma-ray-induced mutations may arise mostly through larger genetic changes.     

The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns. All guaA strains, including three frameshift mutants, produced derepressed levels of IMP dehydrogenase when cultured with a growth-limiting concentration of guanine. The guaB strains were of two types: (i) those producing derepressed levels of XMP aminase, and (ii) those producing basal levels of XMP aminase when grown under conditions of guanine starvation. In the guaB strains of the second type, the expression of the adjacent guaA gene is reduced. It is proposed that this pleiotropic effect of some guaB mutations is a result of polarity. The orientation of polarity suggests the gene order "operator"-guaB-guaA. Gel diffusion studies with IMP dehydrogenase antiserum showed that strains carrying polar guaB mutations do not produce cross-reacting material (CRM). The remaining guaB mutants were either CRM(+) or CRM(-). Mapping the mutations by three-factor crosses showed that polar and nonpolar guaB sites are clustered in a small genetic region cotransducible with guaA. The relative positions of the guaB mutational sites established that the polar mutations lie within the structural gene for IMP dehydrogenase.     

Correlation between mutation type and the production of cross-reacting material in mutants of the A gene of the histidine operon in Salmonella typhimurium

This study concerns the correlation between the type of mutation in a bacterial gene and the ability of the mutant organism to produce immunologically cross-reacting material (CRM). Ninety-five mutants of the A gene of the histidine operon in Salmonella typhimurium have been separated into three classes, by means of mutagenesis and suppression tests, according to the classification scheme devised by H. J. Whitfield, Jr., et al.: (i) missense mutants (in which there is a single amino acid substitution in the A protein); (ii) nonsense mutants (in which various portions of the carboxyl-terminal segment of the A protein are missing); and (iii) deletion and frameshift mutants. Extracts of all mutants were also examined for the presence of CRM. At least 70% of the missense mutants produced CRM. The genetic loci affected in these mutants were randomly distributed throughout the A gene. None of the nonsense, deletion, or frameshift mutants produced CRM. The absence of CRM in the nonsense mutants is in keeping with the finding that removal of the carboxyl-terminal valine residue from the A protein by carboxypeptidase A eliminates antigenicity.     

Galactokinase Mutants of Chinese Hamster Somatic Cells Resistant to 2-Deoxygalactose

The growth of Chinese hamster somatic cells was inhibited by 0.2 mg/cc of 2-deoxygalactose. Mutants partially or fully resistant to 2-deoxygalactose were isolated in a single-step or two-step selection. Some of them did not grow as well as the wild type; one of them which lacked galactokinase(EC.2.7.1.6) activity did not grow at all in galactose medium. The galactokinase kinetic properties (Vmax & kmax of the other mutants and of the wild type were different. Therefore resistance resulted either from the possible absence of galactokinase synthesis or from a structural mutation, possible a missence mutation, in the galactokinase gene.- A simple diagnostic test for juvenile cataract is proposed.     

Regulation of the neutral phosphatase in Chlamydomonas reinhardi: An immunogenetic study of wild-type and mutant strains

In Chlamydomonas reinhardi, the activity of the neutral phosphatase considerably increases when the cells are grown in the absence of inorganic phosphate (Pi). A comparative immunological study of cells grown on media containing Pi or not indicated that the neutral phosphatase was synthesized de novo. Ten mutants lacking the neutral phosphatase and distributed among three genetic loci (PD2, PD3, PD24) were investigated for their ability to produce cross-reacting material (CRM) antigenically related to the wild enzyme. All mutants were shown to form much less CRM than the wild-type strain. It is proposed that the three genes are involved in the regulation of neutral phosphatase synthesis.     

Aldehyde oxidase cross-reacting material in the Aldox n,cin, mal,and lxd mutants of Drosophila melanogaster

Rocket immunoelectrophoresis was used to estimate aldehyde oxidase cross-reacting material (AO-CRM) in larval hemolymph and adult fly extracts in mutants with reduced AO enzymatic activity. Hemolymph of larvae homozygous for Aldox ⁿ, which is a mutation of the presumed structural gene for AO, contains 30% of the wild-type CRM. The demonstration of AO-CRM in Aldox ⁿ larval hemolymph is surprising since this genotype has been reported to lack CRM. By contrast, adult Aldox ⁿ flies lack detectable CRM. The other AO-deficient mutants that were examined are cin, mal, and lxd; each has appreciable levels of CRM in both larval hemolymph and adult extracts. Detection of CRM in these mutants helps to clarify conflicting reports in the literature.This research was supported by a grant from the Natural Sciences and Engineering Research Council of Canada to L.W.B.     

Regulation of Phosphate Metabolism in NEUROSPORA CRASSA : Identification of the Structural Gene for Repressible Alkaline Phosphatase

Five additional mutants of Neurospora crassa have been isolated that lack the repressible alkaline phosphatase. The mutations in these strains map at a previously assigned locus on Linkage Group V designated pho-2 (GLEASON and METZENBERG 1974). The five new mutants, as well as three previously isolated by GLEASON and METZENBERG (1974), were examined for the presence of cross-reacting material to antibody prepared against purified wild-type enzyme. Two of the mutants produced high levels of cross-reacting material, thus providing evidence that the pho-2 locus includes the structural gene for the repressible alkaline phosphatase. Two revertants were obtained from one of the mutants that contained cross-reacting material. Neither revertant produced an enzyme that could be distinguished physicochemically from that of wild type. A method for measuring very low levels of repressible alkaline phosphatase in crude extracts is also described.     

Mitochondrial binding of a protein affected in mutants resistant to the microtubule inhibitor podophyllotoxin

Specific antibodies to a protein P1 Mr approximately equal to 63,000) from Chinese hamster ovary cells, which is affected in mutants resistant to the microtubule inhibitor, podophyllotoxin, and behaves like a microtubule-related protein by certain criteria [14], have been raised. The antibody reacts specifically with the P1 protein in one- and two-dimensional immunoblots, and a cross-reacting protein of similar molecular mass and electrophoretic mobility is also found in cells from various vertebrate and invertebrate species. The observed similarity in the peptide maps of the cross-reacting protein from human, mouse, Chinese hamster and chicken cells indicates that the structure of this protein should be highly conserved. However, no P1-antibody cross-reacting protein was observed in plants (corn, mung), fungus (Neurospora crassa), yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli and Salmonella typhimurium). Immunofluorescence studies with the P1-antibody show that, in interphase cells of various cross-reacting species, it bound specifically to mitochondria which were associated and distributed on and along the length of microtubules. Similar association and codistribution of mitochondria and microtubules were not observed in mitotic cells. Some implications of the mitochondrial localization of the protein P1 and the observed association between microtubules and mitochondria are discussed.     

Dopa decarboxylase from Drosophila melanogaster

Summary Dopa decarboxylase (EC 4.1.1.26) has been purified to near homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 113,000 measured by sucrose gradient sedimentation and 102,000 measured by variable porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions revealed the enzyme consists of two subunits of molecular weight 54,000. The affinity of the enzyme for L-dopa is 30-fold greater than for L-tyrosine. Activity is strongly inhibited by heavy metal ions and the sulfhydryl reagent N-ethylmaleimide. N-acetyl dopamine acts as a competitive inhibitor of the enzyme.Antibodies were elicited against the purified enzyme and measurements of the amount of cross-reacting material (CRM) in two groups of mutants were made. The first group comprised the recessive lethal mutants l(2)amd. Heterozygous mutant stocks are hypersensitive to -methyl dopa, an inhibitor of dopa decarboxylase. These stocks were found to have nearly normal amounts of CRM and enzyme activity.A second group of recessive lethal mutants, characterized by lower levels of dopa decarboxylase, was also analysed. These mutants, designated l(2) Ddc, as heterozygotes exhibited CRM levels between 25 and 75% of normal. Although they are alleles at a single locus, they were classifiable into three distinct groups whose properties readily could be ascribed to a homodimeric structure of the enzyme. This structure would also account for the pattern of intracistronic complementation exhibited by the mutants. Finally, the severity of the mutant defects, as judged by our measurements of CRM and activity, closely parallels that deduced from their complementation pattern. We conclude that these mutations are lesions in the structural gene for dopa decarboxylase.     

Tryptophan synthetase: its charmed history

Tryptophan synthetase was initially selected as a subject for investigation of the relationship between gene structure and protein structure. Early studies with this enzyme first demonstrated the existence in mutants of immunologically cross-reacting material (CRM) and the restoration of a wild-type enzyme by genetic suppression. Fine structure analyses with E. coli tryptophan synthetase missense mutants proved the colinearity of gene structure and catalytic capabilities of this enzyme have been subjects for numerous studies.     

Analysis of HGPRT- CRM+ human lymphoblast mutants.

Three 6-thioguanine-resistant mutants of the human diploid lymphoblast line MGL-8 were studied. The inactivation by heat of both HGPRT activity and antigenicity of the HGPRT immunologically cross-reacting material of the A30 mutant cells were not protected by PRPP, indicating that the HGPRT in A30 cells has an altered PRPP binding site, leading to lack of stabilization and rapid degradation of the enzyme. Two dimensional separations of the immunoprecipitates from extracts of the parental and mutant cell lines showed that the A35 mutant CRM has a more acidic isoelectric pH, while the A30 CRM has a more basic isoelectric pH and that the A30 protein has a faster rate of degradation than the wild-type HGPRT. The A30 CRM also has a smaller molecular size than the wild-type enzyme.     

Ribosomal protein S14 is altered by two-step emetine resistance mutations in Chinese hamster cells.

Four two-dimensional polyacrylamide gel electrophoresis systems were used to identify 78 Chinese hamster cell ribosomal proteins by the uniform nomenclature based on rat liver ribosomal proteins. The 40S ribosomal subunit protein affected by Chinese hamster ovary (CHO) cell one-step emetine resistance mutations is designated S14 in the standard nomenclature. To seek unambiguous genetic evidence for a cause and effect relationship between CHO cell emetine resistance and mutations in the S14 gene, we mutagenized a one-step CHO cell mutant and isolated second-step mutant clones resistant to 10-fold-higher concentrations of emetine. All of the highly resistant, two-step CHO cell mutants obtained displayed additional alterations in ribosomal protein S14. Hybridization complementation tests revealed that the two-step CHO cell emetine resistance mutants were members of the same complementation group defined by one-step CHO cell mutants, EmtB. Two-step mutants obtained from a Chinese hamster lung cell emetine-resistant clone belong to the EmtA complementation group. The two-step and EmtB mutants elaborated 40S ribosomal subunits, which dissociated to 32S and 40S core particles in buffers containing 0.5 M KCl at 4 degrees C. In contrast, 40S ribosomal subunits purified from all EmtA, one-step EmtB EmtC mutants, and wild-type CHO and lung cells were stable at this temperature in buffers containing substantially higher concentrations of salt. Thus, two-step emtB mutations affect the structure of S14 protein directly and the stability of the 40S ribosomal subunit indirectly.     

Immunochemical studies on cultured fibroblasts from patients with homocystinuria due to cystathionine beta-synthase deficiency.

Fibroblast extracts from 20 individuals with homocystinuria due to cystathionine beta-synthase deficiency were analyzed for the presence of immunoreactive synthase antigen as cross-reacting material (CRM). CRM was quantitated by competitive and direct immunotitration using rabbit antiserum against homogeneous human liver synthase. The lower limit of sensitivity for detection of CRM was 1.5% of the amount of synthase antigen in control extracts. Each of 14 mutant extracts with detectable synthase activity had detectable CRM ranging from 5% to 100% of the amount found in control extracts. No statistically significant correlation was observed between the percent residual activity and the percent CRM. Of six mutant extracts without measurable catalytic activity, three had no detectable CRM, while three had 13%, 17%, and 26% CRM, respectively. These results extend our information about the biochemical heterogeneity previously found in synthase deficiency, and emphasize that such deficiency is caused by a wide array of mutations affecting the structural locus for cystathionine beta-synthase.     

Mutations of Chinese Hamster Somatic Cells from 2-Deoxygalactose Sensitivity to Resistance

In Chinese hamster somatic cells, the spontaneous change of phenotype from 2-deoxygalactose sensitivity to resistance was studied using fluctuation test experiments à la Luria and Delbrück (1943) for four Chinese hamster cell strains derived from V79. The results are consistent with true mutational events. The mutation rates are in the range of 1 to 3.5 X 10(-5) per cell per generation. The relationship between the 2-deoxyglactose resistance and the galactokinase markers is discussed.     

The molecular basis for the two different clinical presentations of classical pyruvate carboxylase deficiency

Eight cases of isolated human pyruvate carboxylase deficiency were examined from seven families. Although all patients presented with a chronic lacticacidemia, two particular patients presented with the added features of hyperammonemia, citrullinemia, and hyperlysinemia. When cultured skin fibroblasts from these patients were examined for their ability to synthesize [3H]biotin-containing proteins, it was found that the two patients who presented with hyperammonemia, citrullinemia, and hyperlysinemia did not synthesise a protein of the correct subunit molecular weight (Mr = 125 K daltons) corresponding to pyruvate carboxylase. In addition, when skin fibroblast proteins were labeled with [35S]methionine, cross-reacting material (CRM) corresponding to pyruvate carboxylase was immunoprecipitated by antipyruvate carboxylase antiserum in most patients, but again the two patients with the atypical presentation showed no CRM. We propose that the different clinical presentation of human pyruvate carboxylase deficiency is a manifestation of two different mutations in the pyruvate carboxylase gene, one that results in the synthesis of a relatively inactive pyruvate carboxylase protein CRM(+ve) and one that results in the lack of expression of the gene in the form of a recognizable protein CRM(-ve).     

Synthesis and degradation of alcohol dehydrogenase in wild-type and Adh-null activity mutants of Drosophila melanogaster

Both the amount and the size of alcohol dehydrogenase-like cross-reacting material was determined in 14 ethyl methanesulfonate (EMS)-induced alcohol dehydrogenase-null activity mutants. In 11 mutants cross-reacting material of the same apparent molecular weight as alcohol dehydrogenase was detected, while in 3 mutants no cross-reacting material was found. In all cases, the amount of cross-reacting material found in the mutants was lower than that in wild-type flies. High, intermediate, and low cross-reacting material-producing mutants showed similar initial rates of incorporation of labeled amino acid into alcohol dehydrogenase-like protein, presumably reflecting similar rates of synthesis. If the rate of synthesis of cross-reacting material is the same in the mutants as in the wild type, then the different levels of cross-reacting material must be due to different rates of degradation.Supported by NIH Grants GM-18254 and ES-01527 and DOE Contract EY-76-S-2965.     

Characterization of acid phosphatase-1 null activity mutants of Drosophila melanogaster

A complementation analysis was performed on 15 Acph-1 ⁿ alleles. Only three of these alleles proved to be nonleaky and to exhibit no evidence of complementation. These were then tested immunologically to determine their level of antigenically cross-reacting material (CRM). Their CRM levels were virtually zero, as compared to low, but positive, levels for two other homozygous Acph-1 ⁿ mutants and considerably higher levels for heteroallelic combinations of some of the leaky alleles. Acid phosphatase-1 enzyme subunits, formed by dissociating native enzyme, have close to 100% CRM activity in tests with antibodies elicited by native enzyme.Submitted by John Bell in partial fulfillment of the requirements for a Doctor of Philosophy degree from Cornell University.     

Sequence analysis of mitochondrial chloramphenicol resistance mutations in Chinese hamster cells

A series of mitochondrially inherited chloramphenicol-resistant (CAP-R) mutants were isolated in Chinese hamster cells. To determine whether the Chinese hamster CAP-R mutations were homologous to those isolated in mouse and human cell culture systems, we determined the nucleotide sequence of the region of the mitochondrial 16S rRNA gene spanning the peptidyl transferase-encoding region for eight CAP-R mutant lines in addition to the parental wildtype line. Three main conclusions are drawn from these studies. (1) Although the region of the gene encoding the peptidyl transferase domain is highly conserved relative to that of mice and rats, the contiguous sequences show less conservation. This sequence divergence not only includes the accumulation of single base pair replacements, but also the presence of small insertions or deletions. (2) For six of the CAP-R mutants, heteroplasmic single base pair changes were detected. These mapped to the same sites within the peptidyl transferase domain as the mutations found previously in mouse and human CAP-R mutants. (3) Two Chinese hamster CAP-R mutants, both with an unusual drug resistance phenotype, did not carry any mutations within the CAP-R peptidyl transferase domain. However, both carried a heteroplasmic mutation at the position corresponding to nucleotide 2505 of the mouse 16S rRNA gene, a site predicted to map within a stem/loop structure attached to this key domain of the ribosome. This is the first evidence for mitochondrial CAP-R mutations that map outside the peptidyl transferase region.     

Isolation and characterization of dominant mutations resistant to carbon catabolite repression of galactokinase synthesis in Saccharomyces cerevisiae.

Seven dominant mutations showing greatly enhanced resistance to the glucose repression of galactokinase synthesis have been isolated from GAL81 mutants, which have the constitutive phenotype but are still strongly repressible by glucose for the synthesis of the Leloir enzymes. These glucose-resistant mutants were due to semidominant mutations at either of two loci, GAL82 and GAL83. Both loci are unlinked to the GAL81- gal4, gal80, or gal7 X gal10 X gal1 locus or to each other. The GAL83 locus was mapped on chromosome V at a site between arg9 and cho1. The GAL82 and GAL83 mutations produced partial resistance of galactokinase to glucose repression only when one or both of these mutations were combined with a GAL81 or a gal80 mutation. The GAL82 and GAL83 mutations are probably specific for expression of the Leloir pathway and related enzymes, because they do not affect the synthesis of alpha-D-glucosidase, invertase, or isocitrate lyase.     

Identification and characterization of a third complementation group of emetine-resistant Chinese hamster cell mutants.

We have isolated emetine-resistant cell lines from Chinese hamster peritoneal fibroblasts and have shown that they represent a third distinct class or complementation group of emetine-resistant mutants, as determined by three different criteria. These mutants, like those belonging to the two other complementation groups we have previously defined, which were isolated from Chinese hamster lung and Chinese hamster ovary cells, have alterations that directly affect the protein biosynthetic machinery. So far, there is absolute cell line specificity with respect to the three complementation groups, in that all the emetine-resistant mutants we have isolated from Chinese hamster lung cells belong to one complementation group, all those we have isolated from Chinese hamster ovary cells belong to a second complementation group, and all those isolated from Chinese hamster peritoneal cells belong to a third complementation group. Thus, in cultured Chinese hamster cells, mutations in at least three different loci, designated emtA, emtB, and emtC, encoding for different components of the protein biosynthetic machinery, can give rise to the emetine-resistant phenotype.