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PubMed2003

Identification and characterization of a diamide sensitive mutant of Mycobacterium smegmatis.

A mutant, T7, highly sensitive to oxidative stress as caused by diamide was isolated from a Mycobacterium smegmatis mc(2)155 transposon mutant library. While wild-type M. smegmatis is able to grow well on solid media supplemented with 10 mM diamide, T7 is only able to grow on solid media containing up to 1 mM diamide. This mutant is also sensitive to other thiol modifying agents such as iodoacetamide and chlorodinitrobenzene. By sequencing the genomic DNA flanking the transposon, T7 was found to be mutated in the region upstream of the homolog of M. tuberculosis Rv0274 open reading frame. Sequence analysis revealed that Rv0274 is a member of a superfamily of metalloenzymes comprising enzymes such as extradiol dioxygenases, glyoxalases, and fosfomycin resistant glutathione transferases. Cloning and epichromosomal expression of M. tuberculosis Rv0274 in the mutant resulted in complementation of the sensitivity to diamide.

Amino Acid Sequence↗
PubMed2003

Synthesis of solution-phase combinatorial library of 4,6-diamino-1,2-dihydro-1,3,5-triazine and identification of new leads against A16V+S108T mutant dihydrofolate reductase of Plasmodium falciparum.

An efficient method to synthesize solution-phase combinatorial library of 1-aryl-4,6-diamino-1,2-dihydro-1,3,5-triazine was developed. The strategy involved an acid-catalyzed cyclocondensation between arylbiguanide hydrochlorides and carbonyl compounds in the presence of triethyl orthoacetate as water scavenger. A 96-membered combinatorial library was constructed from 6 aryl biguanides and 16 carbonyl compounds. Screening of the library by iterative deconvolution method revealed two candidate leads which are equally active against wild-type Plasmodium falciparum dihydrofolate reductase, but are about 100-fold more effective against the A16V+S108T mutant enzyme as compared to cycloguanil.

Amino Acid Substitution↗
PubMed2000

Identification of a tumor-derived p53 mutant with novel transactivating selectivity.

MDM2 is a p53-responsive molecule that when overexpressed, can alter growth control pathways via p53-dependent and independent mechanisms. We have identified a mutant p53 containing line that expresses high levels of transcripts that are regulated by the p53-responsive promoter of the MDM2 gene. Analysis of cloned product obtained from these tumor cells revealed that they harbor a mutant p53 protein (possessing an Arg to Gln substitution at codon 213) that is a potent transactivator of MDM2 expression. Consistent with this activity, the R213Q mutant was found to have the ability to interact with DNA sequences located within the MDM2 promoter. In contrast to previously described tumor-derived p53 mutants which retain MDM2 transactivation function and possess partial growth suppressive activity, the R213Q mutant is severely compromised in its ability to induce p53-regulated transcripts that encode for proteins involved in cell-cycle arrest and apoptosis. The R213Q mutant can also be expressed at high levels in stably transfected cells and cells that harbor this mutant possess elevated levels of MDM2 protein. The R213Q mutant was also found to be able to up-regulate MDM2 during a genotoxic stress response. R213Q is the first described tumor-derived p53 mutant that is deficient at up-regulating both cell cycle arrest and apoptotic factors, but is highly proficient at inducing the growth-promoting molecule MDM2.

Apoptosis↗
PubMed2000

Identification of genomic DNA sequences bound by mutant p53 protein (Gly245-->Ser) in vivo.

Mutant p53 proteins were shown to exert complex DNA-interactions in vitro, like binding to MAR-DNA, but so far it is unknown whether such interactions also occur in vivo. Therefore we analysed the binding of mutant (mut) p53 (Gly245-->Ser) in Onda 11 glioma cells to cellular DNA in vivo, using p53-specific chromatin immunoprecipitation (CHIP) after in vivo cross-linking of mut p53 to genomic DNA with cisplatin. We identified genomic DNA fragments to which mut p53 (Gly245-->Ser) could be cross-linked in vivo. Purified recombinant mut p53 (Gly245-->Ser) was able to bind specifically to such elements in PCR-EMSA in vitro, supporting the idea that this mut p53 protein interacts with genomic DNA in vivo. The genomic DNA fragments identified are vastly different in sequence, but display as a common feature a high likelihood to adopt a non B-DNA conformation. Therefore we propose that structural determinants within these DNA elements are important for their interaction with mut p53 (Gly245-->Ser) in vivo. Oncogene (2000) 19, 4178 - 4183

Amino Acid Substitution↗
PubMed1990

Rapid and sensitive techniques for identification and analysis of 'reactive-centre' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas.

Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.

Angioedema↗
PubMed2000

Type II protein C deficiency: identification and molecular modelling of two natural mutants with low anticoagulant and normal amidolytic activity.

Two mutations in exons 3 and 9 of the protein C gene were identified by amplification and sequencing from symptomatic probands referred for venous thromboembolism and thrombophilia screening. The phenotype associated with the mutations is a type II protein C deficiency with normal amidolytic activity. In one family, the mutation in exon 3 (G3545-->A), which predicts an R9 to H substitution in the Gla domain, was identified. A mutation in exon 9 (G10899-->A), which predicts an R352 to W substitution in the catalytic site, was identified in the second family and has been reported previously in association with type II deficiency with low amidolytic activity. Western blotting of the purified proteins from the probands' plasma did not show any abnormal migratory pattern. Molecular modelling suggested a possible impairment in the recently described Na+ binding pocket for the R352-->W mutant. No conclusions could be drawn relative to the R9-->H mutant.

Blotting, Western↗
PubMed1999

FtsZ dimerization in vivo.

A hybrid assay, based on the properties of the lambda repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N-terminal end of the lambda cI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional lambda repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N-terminal of about 150 amino acids; the C-terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ.

Bacterial Proteins↗
PubMed2000

Identification and characterization of in vivo attenuated mutants of Brucella melitensis.

Brucella melitensis 16M is a Gram-negative alpha2-proteobacterium responsible for abortion in goats and for Malta fever in humans. This facultative intracellular pathogen invades into and survives within both professional and non-professional phagocytes. Signature-tagged mutagenesis (STM) was used to identify genes required for the in vivo pathogenesis of Brucella. A library of transposon mutants was screened in a murine infection model. Out of 672 mutants screened, 20 were not recovered after a 5 day passage in BALB/c mice. The attenuation of 18 mutants was confirmed using an in vivo competition assay against the wild-type strain. The 18 mutants were characterized further for their ability to replicate in murine macrophages and in HeLa cells. The sequences disrupted by the transposon in the mutants have homology to genes coding for proteins of different functional classes: transport, amino acid and DNA metabolism, transcriptional regulation, peptidoglycan synthesis, a chaperone-like protein and proteins of unknown function. The mutants selected in this study provide new insights into the molecular basis of Brucella virulence.

Animals↗
PubMed1995

Phosphorylation of casein kinase II by p34cdc2. Identification of phosphorylation sites using phosphorylation site mutants in vitro.

The alpha and beta subunits of casein kinase II are dramatically phosphorylated in cells that are arrested in mitosis (Litchfield, D. W., Lüscher, B., Lozeman, F. J., Eisenman, R. N., and Krebs, E.G. (1992) J. Biol. Chem. 267, 13943-13951). Comparative phosphopeptide mapping experiments indicated that the mitotic phosphorylation sites on the alpha subunit of casein kinase II can be phosphorylated in vitro by p34cdc2. In the present study, we have demonstrated that a glutathione S-transferase fusion protein encoding the C-terminal 126 amino acids of the alpha subunit is phosphorylated by p34cdc2 at the same sites as intact casein kinase II, indicating that the mitotic phosphorylation sites are localized within the C-terminal domain of alpha. Four residues within this domain, Thr-344, Thr-360, Ser-362, and Ser-370, conform to the minimal consensus sequence for p34cdc2 phosphorylation. Synthetic peptides corresponding to regions of alpha that contain each of these residues are phosphorylated by p34cdc2 at these sites. Furthermore, alterations in the phosphorylation of the glutathione S-transferase proteins encoding the C-terminal domain of alpha are observed when any of the four residues are mutated to alanine. When all four residues are mutated to alanine, the fusion protein is no longer phosphorylated by p34cdc2 at any of the sites that are phosphorylated in mitotic cells. These results indicate that Thr-344, Thr-360, Ser-362, and Ser-370 are the sites on the alpha subunit of casein kinase II that are phosphorylated in mitotic cells.

Amino Acid Sequence↗
PubMed1987

Identification and characterization of a mouse cell mutant defective in the intracellular transport of glycoproteins.

We have isolated a mutant line of mouse L cells, termed gro29, in which the growth of herpes simplex virus (HSV) and vesicular stomatitis virus (VSV) is defective. The block occurs late in the infectious cycle of both viruses. We demonstrate that HSV and VSV enter gro29 cells normally, negotiate the early stages of infection, yet are impaired at a late stage of virus maturation. During VSV infection of the mutant cell line, intracellular transport of its glycoprotein (G protein) is slowed. Pulse-chase experiments showed that oligosaccharide processing is impeded, and immunofluorescence localization revealed an accumulation of G protein in a juxtanuclear region that contains the Golgi complex. We conclude that export of newly made glycoproteins is defective in gro29 cells, and speculate that this defect may reflect a lesion in the glycoprotein transport apparatus.

Animals↗
PubMed1991

Identification of oda6 as a Chlamydomonas dynein mutant by rescue with the wild-type gene.

We find that two Chlamydomonas outer arm dynein assembly loci, oda6 and oda9, are located on the left arm of linkage group XII, in the vicinity of the previously mapped locus for a 70,000 Mr dynein intermediate chain protein. Restriction fragment length polymorphism mapping indicates that this dynein gene is very closely linked to the oda6 locus. A cDNA clone encoding the 70,000 Mr protein was isolated, sequenced, and used to select genomic clones spanning the corresponding locus from both wild-type and oda6 libraries. When wild-type clones were introduced into cells containing an oda6 allele, the mutant phenotype was rescued, while no rescue was observed after transformation with oda6 clones. Genetic analysis further revealed that newly introduced gene copies were responsible for the rescued phenotype and thus confirms that ODA6 encodes the 70,000 Mr dynein intermediate chain protein. The inability of oda6 mutants to assemble any major outer arm dynein subunits shows that this protein is essential for assembly of stable outer dynein arms. This is the first use of transformation with a wild-type gene to identify the product of a Chlamydomonas mutant.

Amino Acid Sequence↗
PubMed1998

Identification of three highly attenuated Salmonella typhimurium mutants that are more immunogenic and protective in mice than a prototypical aroA mutant.

A panel of Salmonella typhimurium 14028s mutants, which were previously shown to be highly attenuated in the BALB/c mouse model of infection, were analyzed for their potential as live Salmonella oral-vaccine candidates. A prototypical aroA mutant was chosen as a basis of comparison. From the panel of mutants initially chosen for this study, three mutants with comparable levels of attenuation elicited higher Salmonella-specific serum immunoglobulin G (IgG) and/or mucosal secretory-IgA antibody titers than the aroA vaccine strain. The three mutants, CL288, CL401, and CL554, also elicited a better protective immune response than the aroA control strain, after a single oral dose of 1 x 10(9) to 2 x 10(9) bacteria.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2000

Identification of a Mycobacterium bovis BCG auxotrophic mutant that protects guinea pigs against M. bovis and hematogenous spread of Mycobacterium tuberculosis without sensitization to tuberculin.

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Animals↗
PubMed2002

Identification of motility and autoagglutination Campylobacter jejuni mutants by random transposon mutagenesis.

Campylobacter jejuni has been identified as the leading cause of acute bacterial diarrhea in the United States, yet compared with other enteric pathogens, considerably less is understood concerning the virulence factors of this human pathogen. A random in vivo transposon mutagenesis system was recently developed for the purpose of creating a library of C. jejuni transformants. A total of 1,065 C. jejuni transposon mutants were screened for their ability to swarm on motility agar plates and autoagglutinate in liquid cultures; 28 mutants were subsequently identified. The transposon insertion sites were obtained by using random-primed PCR, and the putative genes responsible for these phenotypes were identified. Of these mutants, all 28 were found to have diminished motility (0 to 86% that of the control). Seventeen motility mutants had insertions in genes with strong homology to functionally known motility and chemotaxis genes; however, 11 insertions were in genes of unknown function. Twenty motility mutants were unable to autoagglutinate, suggesting that the expression of flagella is correlated with autoagglutination (AAG). However, four mutants expressed wild-type levels of surface FlaA, as indicated by Western blot analysis, yet were unable to autoagglutinate (Cj1318, Cj1333, Cj1340c, and Cj1062). These results suggest that FlaA is necessary but not sufficient to mediate the AAG phenotype. Furthermore, two of the four AAG mutants (Cj1333 and Cj1062) were unable to invade INT-407 intestinal epithelial cells, as determined by a gentamicin treatment assay. These data identify novel genes important for motility, chemotaxis, and AAG and demonstrate their potential role in virulence.

Agglutination↗
PubMed1984

Cloning genes for proline biosynthesis from Neisseria gonorrhoeae: identification by interspecific complementation of Escherichia coli mutants.

DNA from Neisseria gonorrhoeae KH45 was partially digested with Sau3A and inserted into the BamHI site of the cloning vector pLES2 . After introduction into Escherichia coli JM83 by transformation, two different size classes of plasmids were isolated that could complement the proAB deletion of JM83 . These plasmids ( pLES4 and pLES7 ) were characterized by restriction endonuclease digestion. Southern hybridization demonstrated that the inserts had sequence homology. Various deletions of these plasmids were constructed that had lost the ability to complement the proA lesion of chi 463, the proB lesion of chi 340, or both (plasmids pLES9 , pLES8 , and pLES10 , respectively). These deleted plasmids were introduced into a proline-requiring strain of N. gonorrhoeae, F62, with plasmids pLES4 , pLES7 , and pLES8 possessing the ability to correct the proline requirement of F62. Further analysis indicated that the hybrid plasmids were stably maintained as plasmids in N. gonorrhoeae.

Aldehyde Oxidoreductases↗
PubMed1990

Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.

Amino Acid Sequence↗