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PubMed1998

Determination of HPRT mutant frequency and molecular analysis of T-lymphocyte mutants derived from coke-oven workers.

We measured the frequency of mutant (MF) lymphocytes at the hprt locus in a population of 43 coke-oven workers exposed to PAH and in a group of 26 non-exposed workers. A non-significant increase in MF in the exposed group (19.0 +/- 16.3) compared to the non-exposed group (15.8 +/- 14.6) was observed. Moreover, when we considered smoking habits for the overall population, the MF values were higher, although not significantly, in smokers than in non-smokers. For some T-cell mutant clone structural alterations, splicing and coding errors were detected by PCR-based methods. We analysed 161 HPRT- clones, derived from exposed and non-exposed workers by multiplex-PCR and 56 HPRT- clones by reverse transcriptase-PCR. Overall, the percentages of the different types of gene alterations were similar in exposed and non-exposed subjects. Only the frequency of splice mutations in mutant clones derived from coke-oven workers was higher (22%) than in non-exposed donors (11%).

Adult↗
PubMed2003

A shaE deletion mutant showed lower Na+ sensitivity compound to other deletion mutants in the Bacillus subtilis sodium/hydrogen antiporter (Sha) system.

The sodium/hydrogen antiporter (Sha, identical to multiple resistance and pH adaptation: Mrp) encoded by shaABCDEFG is considered to be the major Na+ excretion system in Bacillus subtilis. We constructed deletion mutants of each sha (=mrp) gene and found that a shaE mutant showed lower Na+ sensitivity compared to the other mutants with respect to the growth, sporulation and Na+ efflux activity. ShaE may have a dissimilar role to other Sha proteins in transport functions.

Journal Article↗
PubMed2002

Pathway-specific tumor suppression. Reduction of p27 accelerates gastrointestinal tumorigenesis in Apc mutant mice, but not in Smad3 mutant mice.

Expression of the cyclin-dependent kinase inhibitor p27(Kip1) (p27) is frequently reduced in human colorectal cancer, and this correlates with poor patient prognosis. To clarify the role of p27 in gastrointestinal (GI) cancer, we measured p27 expression, as well as the effect of germline deletion of p27, in 3 different mouse models of GI neoplasia. p27 expression was frequently reduced in GI tumors arising in 1,2-dimethylhydrazine (DMH) treated mice, and in Apc mutant Min/+ mice, but not in GI tumors arising in Smad3 mutant mice. Germline deletion of p27 resulted in accelerated tumor development and increased tumor cell proliferation in both DMH treated and Min/+ mice, but not in Smad3 mutant mice. p27 deficiency also led to increased adenoma to adenocarcinoma progression. These results indicate that reduction of p27 cooperates with mutations in Apc but not in Smad3 during GI tumorigenesis. Thus, tumor suppression by p27 is contingent on the specific oncogenic pathway that drives tumor development.

1,2-Dimethylhydrazine↗
PubMed1992

Construction and characterization of a spectral probe mutant of troponin C: application to analyses of mutants with increased Ca2+ affinity.

A spectral probe mutant (F29W) of chicken skeletal muscle troponin C (TnC) has been prepared in which Phe-29 has been substituted by Trp. Residue 29 is at the COOH-terminal end of the A helix immediately adjacent to the Ca2+ binding loop of site I (residues 30-41) of the regulatory N domain. Since this protein is naturally devoid of Tyr and Trp, spectral features can be assigned unambiguously to the single Trp. The fluorescent quantum yield at 336 nm is increased almost 3-fold in going from the Ca(2+)-free state to the 4Ca2+ state with no change in the wavelength of maximum emission. Comparisons of the Ca2+ titration curves of the change in far-UV CD and fluorescence emission indicated that the latter was associated only with the binding of 2Ca2+ to the regulatory sites I and II. No change in fluorescence was detected by titration with Mg2+. The Ca(2+)-induced transitions of both the N and C domains were highly cooperative. Addition of Ca2+ also produced a red shift in the UV absorbance spectrum and a reduction in positive ellipticity as monitored by near-UV CD measurements. The fluorescent properties of F29W were applied to an investigation of five double mutants: F29W/V45T, F29W/M46Q, F29W/M48A, F29W/L49T, and F29W/M82Q. Ca2+ titration of their fluorescent emissions indicated in each case an increased Ca2+ affinity of their N domains. The magnitude of these changes and the decreased cooperativity observed between Ca2+ binding sites I and II for some of the mutants are discussed in terms of the environment of the mutated residues in the 2Ca2+ and modeled 4Ca2+ states.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

19F NMR of trifluoroacetyl-labeled cysteine mutants of myoglobin: structural probes of nitric oxide bound to the H93G cavity mutant.

Nitric oxide (NO) binds to the myoglobin (Mb) cavity mutant, H93G, forming either a 5- or 6-coordinate Fe--NO heme complex. The H93G mutation replaces the proximal histidine of Mb with glycine, allowing exogenous ligands to occupy the proximal binding site. In the absence of the covalently attached proximal ligand, NO could bind to H93G from the proximal side of the heme rather than the typical diatomic binding pocket on the distal side when the 5-coordinate complex forms. The question of whether NO binds on the distal or proximal side was addressed by (19)F NMR. Site-directed mutagenesis was used to introduce unique cysteine residues at the protein surface on either the distal (S58C) or proximal (L149C) side, approximately equidistant from and perpendicular to the heme plane of both wild-type and H93G Mb. The cysteine thiols were alkylated with 3-bromo-1,1,1-trifluoroacetone to attach a trifluoroacetyl group at the mutation site. (19)F NMR spectra of 5-coordinate, NO bound S58C/H93G and L149C/H93G double mutants depict peaks with line widths of 100 and 23 Hz, respectively. As fluorine peaks broaden with increasing proximity to paramagnetic centers, such as 5-coordinate Fe--NO, the (19)F NMR data are consistent with NO binding in the distal heme pocket of H93G, even in the absence of a sixth axial ligand. Additionally, (19)F NMR spectra are reported for deoxy, oxy, CO, met CN, and met H(2)O forms of the labeled cysteine mutants. These results demonstrate that the fluorine probes are sensitive to subtle conformational changes in the protein structure due to ligation and oxidation state changes of the heme iron in Mb.

Animals↗
PubMed2002

Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity.

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cerebral cortex, brainstem, and spinal cord. The cytopathological hallmark in the remaining motor neurons of ALS is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. In this paper we report that Dorfin, a RING finger-type E3 ubiquitin ligase, is predominantly localized in the inclusion bodies of familial ALS with a copper/zinc superoxide dismutase (SOD1) mutation as well as sporadic ALS. Dorfin physically bound and ubiquitylated various SOD1 mutants derived from familial ALS patients and enhanced their degradation, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Our results indicate that Dorfin protects neurons by recognizing and then ubiquitylating mutant SOD1 proteins followed by targeting them for proteasomal degradation.

Aged↗
PubMed2004

Two novel mutant human adenylosuccinate lyases (ASLs) associated with autism and characterization of the equivalent mutant Bacillus subtilis ASL.

An Australian patient with autism was found to be heterozygous for two mutations in the gene encoding adenylosuccinate lyase (ASL), resulting in the protein mutations E80D and D87E. The patient's mother carried only the E80D mutation. The equivalent positions are 62 and 69 in Bacillus subtilis ASL. Although both human and B. subtilis enzymes normally have Asp at position 87 (or 69), the B. subtilis ASL has Ile and Asp at 62 and 65, respectively, whereas human ASL has Glu and Arg at the equivalent positions. We have constructed, expressed, and purified the double mutant I62E/D65R as a "humanized" normal B. subtilis enzyme to compare with enzymes with a single mutation at position 62 (I62D/D65R), at position 69 (I62E/D65R/D69E), or at both positions (I62D/D65R/D69E). V(max) for conversion of adenylosuccinate to AMP and fumarate is 0.57 micromol/min/mg for I62E/D65R, 0.064 micromol/min/mg for I62D/D65R, 0.27 micromol/min/mg for I62E/D65R/D69E, and 0.069 micromol/min/mg for I62D/D65R/D69E. The K(m) for adenylosuccinate is elevated in the X62D mutants, and I62D/D65R is the least stable of these ASLs at 37 degrees C. The CD spectra of mutant and wild type enzymes are similar; thus, there are no appreciable structural changes. Clearly the Asp(62) causes the most drastic effect on ASL function, whereas the Glu(69) mutation produces only modest change. These results emphasize the importance of expanding tests for ASL deficiency to individuals with developmental delay of any severity, including individuals with autistic spectrum disorder. This study further demonstrates the usefulness of the B. subtilis ASL as a model to mimic the defective enzyme in ASL deficiency.

Adenosine Monophosphate↗
PubMed1991

Mutant bias in nonlethal selections results from selective recovery of mutants.

We have characterized a nonlethal selection for mutations that allow Escherichia coli to grow on large maltodextrins (Dex+) in the absence of the lamB encoded maltoporin LamB. These Dex+ mutations occur before and after imposition of the selection and the selection does not result in a general increase in mutagenesis. The recovered Dex+ mutations are almost exclusively mutations that alter the ompF gene that encodes a major E. coli porin, OmpF even though analogous mutations in the homologous ompC gene, which encodes the OmpC porin, can confer a Dex+ phenotype. We show that the bias for ompF mutations results from a biased recovery and that the genetic background of the starting strain and the selection itself influences the type of mutants that are recovered. When we use a strain carrying an amber mutation in the lamB gene we observe the same preference for ompF mutations as when we start with a lamB deletion strain. In addition, we show that there is no preferential mutagenesis of the lamB gene during the selection which induces transcription of the lamB gene. We present evidence that the biased recovery of mutants observed in this selection does not result from adaptive or directed mutagenesis and that the phenotypic fitness which allows recovery of Dex+ mutants involves more than the increased ability to take up maltodextrins.

Bacterial Outer Membrane Proteins↗
PubMed2005

The rice mutant dwarf bamboo shoot 1: a leaky mutant of the NACK-type kinesin-like gene can initiate organ primordia but not organ development.

That plant dwarfism is caused by hormonal defects related to gibberellin and brassinosteroid has been well documented. Other contributing elements, however, have not been elucidated. Here, we report on one of the most severe dwarf mutants of rice, dwarf bamboo shoot 1 (dbs1). Most mutant plants died within 1 month after sowing, but a few (5.2%) survived and grew. Vacuolation enlarged cells in the leaf primordia and seminal root before abortion, which disrupted the organized cell files in these organs. Relative to the severe defects in shoot and root growth, the overall structure of the dbs1 embryo was almost normal. Similarly, initiation and organogenesis of the leaf primordia at the shoot apical meristem and those of the lateral root primordia at the root elongation zone occurred normally. These observations suggest that DBS1 is involved in the growth and development of organs but not in organ initiation or organogenesis. Positional cloning of DBS1 revealed that it encoded a NACK-type kinesin-like protein (OsNACK), homologous to the essential components of a mitogen-activated protein kinase cascade during plant cytokinesis. A BLAST search indicated that DBS1 was the only gene encoding the OsNACK-type protein in the rice genome, and the dbs1 mutant produced only small amounts of the translatable DBS1 mRNA. Thus, we conclude that the dbs1 mutation causes a severe defect in DBS1 function but does not completely shut it down. We discuss the leaky phenotype of dbs1 under the restricted functioning of OsNACK.

Cell Division↗
PubMed1975

Temperature-sensitive mutants of foot-and-mouth disease virus: the isolation of mutants and observations on their properties and genetic recombination.

A number of temperature-sensitive mutants were isolated from two strains of foot-and-mouth disease virus (FMDV). Various properties of the mutants were examined including comparative growth curves at permissive and restrictive temperatures, cut-off temperatures, thermal lability and pH sensitivity. Recombination was observed between various pairs of mutants of FMDV strain Pacheco. It occurred early in the growth cycle and the proportion of recombinants remained constant thereafter. Maximum recombination was achieved if the input multiplicity of each virus was 6 p.f.u./cell or greater, provided the ratio of the input multiplicities did not vary by more than a factor of two. Day-to-day variations could be substantially reduced by normalizing recombination frequencies in terms of a standard cross. The results suggested that genetic mapping was possible with two-or three-factor crosses.

Animals↗
PubMed1976

Temperature-sensitive mutants of type 12 adenovirus defective in a late function: protein synthesis and evidence for recombination between mutants in complementation group D.

Four temperature sensitive mutants of adenovirus type 12, classified into complementation group D, synthesize most or all of the late virus-specific polypeptides at the restrictive temperature, as shown by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Infections by these mutants under restrictive conditions apparently result in some over production of the virion hexon polypeptide compared to wild type infection. Genetic recombination between the four mutants has been demonstrated. A partial linear genetic map of the D cistron is presented.

Adenoviridae↗
PubMed1989

Temperature-sensitive mutants of bovine herpesvirus type 1: mutants which make unaltered levels of 'early' glycoproteins but fail to synthesize a 'late' glycoprotein.

The major glycoproteins of bovine herpesvirus type 1 showed distinct temporal patterns of expression. The glycoproteins GVP 11 and GVP 6 as well as its cleavage products, GVP 11a and GVP 16, were expressed early in the infectious process, whereas GVP 9 was expressed late. Temperature-sensitive mutants were developed and characterized. Mutants belonging to two complementation groups were unable to synthesize DNA at 40 degrees C, the non-permissive temperature. In cells infected with these mutants the late glycoprotein GVP 9 was not synthesized at 40 degrees C, whereas the synthesis of the early glycoproteins GVP 11 and GVP 6 continued at wild-type levels. These studies suggest that the transition from early to late glycoprotein synthesis is linked to viral DNA synthesis.

Animals↗
PubMed2005

Structure of the wild-type TEM-1 beta-lactamase at 1.55 A and the mutant enzyme Ser70Ala at 2.1 A suggest the mode of noncovalent catalysis for the mutant enzyme.

One of the best-studied examples of a class A beta-lactamase is Escherichia coli TEM-1 beta-lactamase. In this class of enzymes, the active-site serine residue takes on the role of a nucleophile and carries out beta-lactam hydrolysis. Here, the structures of the wild-type and the S70G enzyme determined to 1.55 and 2.1 A, respectively, are presented. In contrast to the previously reported 1.8 A structure, the active site of the wild-type enzyme (1.55 A) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X-ray crystal structure of the S70G mutant has an altered Ser130 side-chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild-type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active-site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.

Alanine↗
PubMed1989

Functional analyses of Proteus mirabilis wild-type and mutant RecBCD enzymes in Escherichia coli reveal a new mutant phenotype.

Cloned into Escherichia coli the recB, recC and recD genes of Proteus mirabilis produce a recBCD enzyme (exoV) functional in recombination and DNA repair. The direction of transcription of recB, recC and recD, the sizes of the enzyme subunits, and their composition in the active enzyme are similar to that observed for the E. coli enzyme. In lambda crosses, the P. mirabilis enzyme has only about 40% of the Chi activity of the E. coli enzyme. The recBCD genes were also cloned from an exoV mutant of P. mirabilis which is u.v.-sensitive and partly deficient in exoV. The defect was attributed to the recB gene by complementation studies. In a recBCD deletion strain of E. coli, the enzyme from the mutant produced 40% of conjugational recombinants and had retained about 25% of Chi activity. However, it did not restore normal DNA repair, cell viability or recombination in lambda crosses and P1 transduction. The new mutant phenotype is discussed in the light of the assumption that prokaryotic recBCD enzymes can promote recombination in a Chi-dependent and a Chi-independent manner.

Chromosome Mapping↗
PubMed1995

Mouse protection induced by Pseudomonas aeruginosa PAC1R and its defective mutants, Salmonella minnesota Re-mutant and Escherichia coli O14.

Pseudomonas aeruginosa PAC1R and its defective mutants (acetone-killed bacteria), Salmonella minnesota Re mutant (acetone-killed bacteria and Re-LPS) and Escherichia coli O14 (acetone-killed bacteria and enterobacterial common antigen, ECA) were studied in a mouse active protection test. Immunized mice were challenged with wild-type P. aeruginosa strains. It was established that P. aeruginosa LPS-defective mutants induced cross-immunity against different Fisher immunotypes of P. aeruginosa. S. minnesota Re-LPS and ECA gave mice protection against P. aeruginosa.

Animals↗
PubMed2000

Construction of region-specific partial duplication mutants (merodiploid mutants) to identify the regulatory gene for the glucan-binding protein C gene in vivo in Streptococcus mutans.

The gbpC gene encoding the glucan-binding protein C which is involved in dextran (glucan)-dependent aggregation (ddag) of Streptococcus mutans has been identified by random mutagenesis. We analyzed ddag(-) mutants containing the intact gbpC gene and found that these mutants possessed a large and characteristic duplication of a region of the chromosome which was responsible for the phenotype. Based upon characterization of these duplications, we developed a strategy to introduce a duplication into any specific region of the chromosome of these organisms. The 690-bp gene responsible for the ddag(-) phenotype was identified within a 60-kb region by observing ddag (positive or negative) phenotypes of successively constructed specific duplication mutants.

Bacterial Proteins↗
PubMed1989

Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo.

The ompC, ompD, and ompF genes encode the three major porins of Salmonella typhimurium. ompR encodes a positive regulator required for the expression of ompC and ompF. Transposon-generated mutations in ompC, ompD, ompF, and ompR were introduced into the S. typhimurium mouse virulent strain SL1344 by P22-mediated transduction. Following preliminary characterization in vitro, the strains were used to challenge BALB/c mice by using the oral or intravenous route. Strains harboring ompC or ompF mutations were as virulent as SL1344 after oral challenge. Strains harboring ompD mutations had a slight reduction in virulence. In contrast, ompR mutants failed to kill BALB/c mice after oral challenge and the intravenous 50% lethal dose was reduced by approximately 10(5). The ompR mutants persisted in murine tissues for several weeks following oral or intravenous challenge. Furthermore, mice orally immunized with these ompR mutant strains were well protected against challenge with virulent SL1344.

Administration, Oral↗