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PubMed2005

Mutant analysis reveals a specific requirement for protein P30 in Mycoplasma pneumoniae gliding motility.

The cell-wall-less prokaryote Mycoplasma pneumoniae, long considered among the smallest and simplest cells capable of self-replication, has a distinct cellular polarity characterized by the presence of a differentiated terminal organelle which functions in adherence to human respiratory epithelium, gliding motility, and cell division. Characterization of hemadsorption (HA)-negative mutants has resulted in identification of several terminal organelle proteins, including P30, the loss of which results in developmental defects and decreased adherence to host cells, but their impact on M. pneumoniae gliding has not been investigated. Here we examined the contribution of P30 to gliding motility on the basis of satellite growth and cell gliding velocity and frequency. M. pneumoniae HA mutant II-3 lacking P30 was nonmotile, but HA mutant II-7 producing a truncated P30 was motile, albeit at a velocity 50-fold less than that of the wild type. HA-positive revertant II-3R producing an altered P30 was unexpectedly not fully wild type with respect to gliding. Complementation of mutant II-3 with recombinant wild-type and mutant alleles confirmed the correlation between gliding defect and loss or alteration in P30. Surprisingly, fusion of yellow fluorescent protein to the C terminus of P30 had little impact on cell gliding velocity and significantly enhanced HA. Finally, while quantitative examination of HA revealed clear distinctions among these mutant strains, gliding defects did not correlate strictly with the HA phenotype, and all strains attached to glass at wild-type levels. Taken together, these findings suggest a role for P30 in gliding motility that is distinct from its requirement in adherence.

Adhesins, Bacterial↗
PubMed1999

A MAP kinase encoded by the ubc3 gene of Ustilago maydis is required for filamentous growth and full virulence.

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a budding haploid saprophyte and a filamentous dikaryotic pathogen. We are interested in identifying the genetic determinants of filamentous growth and pathogenicity in U. maydis. To do this, we have taken a forward genetic approach. Previously, we showed that haploid adenylate cyclase (uac1) mutants display a constitutively filamentous phenotype. Mutagenesis of a uac1 disruption strain allowed the isolation of a large number of budding suppressor mutants. These mutants are named ubc, for Ustilago bypass of cyclase, as they no longer require the production of cAMP to grow in the budding morphology. Complementation of one of these suppressor mutants led to the identification of ubc3, which is required for filamentous growth and encodes a MAP kinase most similar to those of the yeast pheromone response pathway. In addition to filamentous growth, the ubc3 gene is required for pheromone response and for full virulence. Mutations in the earlier identified fuz7 MAP kinase kinase also suppress the filamentous phenotype of the uac1 disruption mutant, adding evidence that both ubc3 and fuz7 are members of this same MAP kinase cascade. These results support an important interplay of the cAMP and MAP kinase signal transduction pathways in the control of morphogenesis and pathogenicity in U. maydis.

Amino Acid Sequence↗
PubMed2001

A simple approach to identify the first rice mutants blocked in carotenoid biosynthesis.

Mutations blocking carotenoid biosynthesis, never before described for rice, are valuable for pathway manipulation and study. Similar to defects in ABA biosynthesis, mutations blocking the carotenoid pathway confer vivipary, but in addition also confer an albino seedling phenotype. Pigments extracted from rice mutants exhibiting the double mutant phenotype were analysed by HPLC (high pressure liquid chromatography); these results led to the identification of the first rice mutant accumulating an intermediate of the carotenoid biosynthetic pathway, phytoene, and a second mutant with almost no detectable carotenoids.

Abscisic Acid↗
PubMed2001

Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis.

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.

Amino Acid Sequence↗
PubMed1986

Herpes simplex virus type 2 mutagenesis: characterization of mutants induced at the hprt locus of nonpermissive XC cells.

In a previous report, herpes simplex virus type 2 (HSV-2) was shown to increase the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus of nonpermissive rat XC cells (L. Pilon, A. Royal, and Y. Langelier, J. Gen. Virol. 66:259-265, 1985). A series of 17 independent mutants were isolated after viral infection together with 12 spontaneous noninfected mutants to characterize the nature of the mutations induced by the virus at the molecular level. The DNA of the mutants isolated after viral infection was probed with cloned HSV-2 fragments representing the entire genome. In these mutants, no authentic HSV-2 hybridization could be detected. This was indicative of a mechanism of mutagenesis which did not require the permanent integration of viral sequences in the host genome. The structure of the hprt gene was determined by the method of Southern (J. Mol. Biol. 98:503-517, 1975), and the level of hprt mRNA was analyzed by Northern blots. Except for the identification of one deletion mutant in each of the two groups, the HPRT- clones showed no evidence of alteration in their hprt gene. A total of 7 of 12 spontaneous mutants and 11 of 15 mutants isolated from the infected population transcribed an hprt mRNA of the same size and abundance as did the wild-type cells. Thus, the majority of the mutants seemed to have a point mutation in their hprt structural gene. Interestingly, the proportion of the different types of mutations was similar in the two groups of mutants. This analysis revealed that HSV-2 infection did not increase the frequency of rearrangements but rather that it probably induced a general increase of the level of mutations in the cells. This type of response is thought to be compatible with the biology of the virus, and the possible mechanisms by which HSV-2 induces somatic mutations in mammalian cells are discussed.

Animals↗
PubMed1988

Defect in synthesis of deoxyribonucleotides by a bacteriophage T4 nrdB mutant is suppressed on mutation of T4 DNA topoisomerase gene.

Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication. The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB. Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60. We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01. After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures. The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01. However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded. Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression. The accompanying paper (Cook, K. S., Wirak, D. O., Seasholtz, A. F., and Greenberg, G. R. (1988) J. Biol. Chem. 263, 6202-6208) examines the nature of the suppression process at the molecular level.

DNA Topoisomerases, Type I↗
PubMed2006

A secondary metabolite biosynthesis cluster in Trichoderma virens: evidence from analysis of genes underexpressed in a mutant defective in morphogenesis and antibiotic production.

A transcriptional comparison of wild type and a secondary metabolite deficient Trichoderma virens mutant resulted in the identification of six genes similar to those involved in secondary metabolism in other fungi, including four cytochrome P450 genes, one O-methyl transferase and one terpene cylase. Four of the genes (three cytochrome P450s and the cyclase) are located as a cluster. Transcript levels of three of the P450 genes, the O-methyl transferase and the terpene cyclase were measured. These genes are underexpressed in the mutant, which lacks the major secondary metabolites produced by this strain, viridin and viridiol. Expression levels of clones from the differential library with similarity to fungal trehalose synthase and a hydrophobin were also underexpressed in the mutant, while a heat shock protein hsp98 homolog was not. Based on the gene expression pattern and associated secondary metabolite profile, along with similarity to other secondary metabolism pathways in related fungi, we predict that the cluster is associated with the production of a terpene. The terpene could be viridin. This is the first report on cloning of secondary metabolism related genes from T. virens, and of their organization in a cluster, in this biocontrol fungus.

Androstenes↗
PubMed1998

Mutational analysis of the P-glycoprotein first intracellular loop and flanking transmembrane domains.

The role of individual intracellular (IC) loops linking transmembrane (TM) domains in P-glycoprotein (P-gp) function remains largely unknown. The high degree of sequence conservation of these regions in the P-gp family and other ABC transporters suggests an important role in a common mechanism of action of these proteins. To gain insight into this problem, we have randomly mutagenized a portion of TM2, the entire IC1 loop, TM3, the entire extracellular loop (EC2), and part of TM4, and analyzed the effect of such mutations on P-gp function. Random mutagenesis was carried out using Taq DNA polymerase and dITP under conditions of low polymerase fidelity, and the mutagenized segments were reintroduced in the full length mdr3 cDNA by homologous recombination in the yeast Saccharomyces cerevisiae strain JPY201. The biological activity of mutant P-gp variants was analyzed in yeast by their ability to confer cellular resistance to the antifungal drug FK506 and the peptide ionophore valinomycin, and by their ability to complement the yeast Ste6 gene and restore mating in a yeast strain bearing a null mutation [Raymond, M., et al. (1992) Science 256, 232-4] at this locus. The analysis of 782 independent yeast transformants allowed the identification of 49 independent mutants bearing single amino acid substitutions in the mutagenized segment resulting in an altered P-gp function. The mutants could be phenotypically classified into two major groups, those that resulted in partial or complete overall loss of function and those that seemed to affect substrate specificity. Several of the mutants affecting overall activity mapped in IC1; in particular we identified a segment of four consecutive mutation sensitive residues (TRLT, positions 169-172) with such a phenotype. On the other hand, we identified a cluster of mutants affecting substrate specificity within the short EC2 segment and in the adjacent portion of the neighboring TM4 domain. Expression and partial purification of a representative subset of these mutants showed that in all but two cases, loss of function was associated with loss of drug-induced ATPase activity of P-gp. Therefore, it appears that TM domains, IC and EC loops, are structurally and functionally tightly coupled in the process of drug stimulatable ATPase characteristic of P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Caenorhabditis elegans levamisole resistance genes lev-1, unc-29, and unc-38 encode functional nicotinic acetylcholine receptor subunits.

We show that three of the eleven genes of the nematode Caenorhabditis elegans that mediate resistance to the nematocide levamisole and to other cholinergic agonists encode nicotinic acetylcholine receptor (nAChR) subunits. unc-38 encodes an alpha subunit while lev-1 and unc-29 encode non-alpha subunits. The nematode nAChR subunits show conservation of many mammalian nAChR sequence features, implying an ancient evolutionary origin of nAChR proteins. Expression in Xenopus oocytes of combinations of these subunits that include the unc-38 alpha subunit results in levamisole-induced currents that are suppressed by the nAChR antagonists mecamylamine, neosurugatoxin, and d-tubocurarine but not alpha-bungarotoxin. The mutant phenotypes reveal that unc-38 and unc-29 subunits are necessary for nAChR function, whereas the lev-1 subunit is not. An UNC-29-GFP fusion shows that UNC-29 is expressed in body and head muscles. Two dominant mutations of lev-1 result in a single amino acid substitution or addition in or near transmembrane domain 2, a region important to ion channel conductance and desensitization. The identification of viable nAChR mutants in C. elegans provides an advantageous system in which receptor expression and synaptic targeting can be manipulated and studied in vivo.

Amino Acid Sequence↗
PubMed2002

Identification and expression of human cytomegalovirus transcription units coding for two distinct Fcgamma receptor homologs.

Cellular receptors for the Fc domain of immunoglobulin G (IgG) (FcgammaRs) comprise a family of surface receptors on immune cells connecting humoral and cellular immune responses. Several herpesviruses induce FcgammaR activities in infected cells. Here we identify two distinct human cytomegalovirus (HCMV)-encoded vFcgammaR glycoproteins of 34 and 68 kDa. A panel of HCMV strains exhibited a slight molecular microheterogeneity between Fcgamma-binding proteins, suggesting their viral origin. To locate the responsible genes within the HCMV genome, a large set of targeted HCMV deletion mutants was constructed. The mutant analysis allowed the identification of a spliced UL119-UL118 mRNA to encode vFcgammaR gp68 and TRL11/IRL11 to encode vFcgammaR gp34. Both vFcgammaRs are surface resident type I transmembrane glycoproteins. Significant relatedness of sequences in the extracellular chain of gpUL119-118 and gpTRL11 with particular immunoglobulin supergene family domains present in FcgammaR I and FcgammaRs II/III, respectively, indicates a different ancestry and function of gpUL119-118 and gpTRL11. The HCMV-encoded vFcgammaRs highlight an impressive diversification and redundancy of FcgammaR structures.

Amino Acid Sequence↗
PubMed2003

Rapid identification and 3D reconstruction of complex cardiac malformations in transgenic mouse embryos using fast gradient echo sequence magnetic resonance imaging.

Developmental malformations of the heart in mouse embryos are commonly studied by histological sectioning. This is slow, labour intensive, and results in the loss of three-dimensional (3D) information. Magnetic resonance studies of embryos typically use spin-echo sequences, using prolonged acquisition times (>36 h) or perfusion with contrast agents to enhance resolution and contrast. This is technically difficult, and requires significant amounts of operator time. We imaged paraformaldehyde fixed embryos using a fast spoiled 3D gradient echo sequence with T(1)-weighting, in unattended overnight runs of less than 9 h. In wild-type embryos, we visualised normal cardiac structures, including cardiac chambers, the ventricular septum, primary and secondary atrial septa, valves, superior and inferior vena cava, aorta, pulmonary artery, and ductus arteriosus. In embryos lacking Cited2 (a transcriptional co-activator required for normal heart development), we identified cardiac malformations including atrial and ventricular septal defects, cono-truncal defects, and aortic arch malformations. We generated 3D reconstructions of normal and mutant hearts using contour identification and surface rendering computer software. The malformations were confirmed by histological sectioning. Our data indicate that fast gradient echo sequence magnetic resonance imaging can be used to rapidly and accurately identify complex cardiovascular malformations in transgenic and mutant mouse embryos.

Animals↗
PubMed2006

Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function.

The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn4451, consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243-261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598-707). In these mutants, removal of residues 598-707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533-583). Analysis of mutations within the aa 243-261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.

Amino Acid Sequence↗
PubMed2003

The LKA gene is a BRASSINOSTEROID INSENSITIVE 1 homolog of pea.

Brassinosteroids (BRs) are growth-promoting plant steroid hormones, and in garden pea (Pisum sativum L.), the lka mutant is defective in BR perception. Here, we show that LKA encodes P. sativum BRI1 (PsBRI1), a homolog of BRI1, which is the Arabidopsis leucine-rich repeat receptor-like kinase/BR receptor. PsBRI1 was isolated by screening a pea cDNA library using Arabidopsis BRI1 cDNA as the probe. PsBRI1 is predicted to encode a 1188-amino-acid protein that has 78% similarity with Arabidopsis BRI1. Sequence analysis of PsBRI1 in the lka mutant led to the identification of a missense mutation that converts the highly conserved aspartic acid residue to asparagine, which is located in the leucine-rich repeat, just before the island domain that may bind BR or a BR-protein complex. The mutation identified in PsBRI1 co-segregated with the semi-erectoide lka phenotype. Transcript analysis of LKA/PsBRI1 indicates that it is ubiquitously expressed in pea and that the expression was downregulated by exogenous BR. The lka mutant was then utilized in further studies to analyze the independent actions of BR and gibberellin (GA) through the characterization of BR response on GA mutants and GA response on BR mutants.

Amino Acid Sequence↗
PubMed2002

Expression and characterization of GSK-3 mutants and their effect on beta-catenin phosphorylation in intact cells.

Glycogen synthase kinase-3 (GSK-3) is a serine-threonine kinase that is involved in multiple cellular signaling pathways, including the Wnt signaling cascade where it phosphorylates beta-catenin, thus targeting it for proteasome-mediated degradation. Unlike phosphorylation of glycogen synthase, phosphorylation of beta-catenin by GSK-3 does not require priming in vitro, i.e. it is not dependent on the presence of a phosphoserine, four residues C-terminal to the GSK-3 phosphorylation site. Recently, a means of dissecting GSK-3 activity toward primed and non-primed substrates has been made possible by identification of the R96A mutant of GSK-3beta. This mutant is unable to phosphorylate primed but can still phosphorylate unprimed substrates (Frame, S., Cohen, P., and Biondi R. M. (2001) Mol. Cell 7, 1321-1327). Here we have investigated whether phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin requires priming through prior phosphorylation at Ser(45) in intact cells. We have shown that the Arg(96) mutant does not induce beta-catenin degradation but instead stabilizes beta-catenin, indicating that it is unable to phosphorylate beta-catenin in intact cells. Furthermore, if Ser(45) in beta-catenin is mutated to Ala, beta-catenin is markedly stabilized, and phosphorylation of Ser(33), Ser(37), and Thr(41) in beta-catenin by wild type GSK-3beta is prevented in intact cells. In addition, we have shown that the L128A mutant, which is deficient in phosphorylating Axin in vitro, is still able to phosphorylate beta-catenin in intact cells although it has reduced activity. Mutation of Tyr(216) to Phe markedly reduces the ability of GSK-3beta to phosphorylate and down-regulate beta-catenin. In conclusion, we have found that the Arg(96) mutant has a dominant-negative effect on GSK-3beta-dependent phosphorylation of beta-catenin and that targeting of beta-catenin for degradation requires prior priming through phosphorylation of Ser(45).

Animals↗
PubMed2001

Mutational analysis of the feline immunodeficiency virus matrix protein.

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.

Amino Acid Sequence↗
PubMed2004

Isolation of long-lived mutants in Caenorhabditis elegans using selection for resistance to juglone.

The accumulation of molecular damage from attack by reactive oxygen species is one cause of aging. Therefore, some mutant organisms showing increased resistance to reactive oxygen species should live longer. We show that selection for Caenorhabditis elegans mutants that are resistant to juglone, a reactive oxygen species-generating compound, leads to the identification of long-lived mutants. Indeed, four of six resistant mutants isolated were also long-lived. This study illustrates once more the strong relationship between oxidative stress and the aging processes.

Aging↗
PubMed1993

Characterization of proteins that interact with the cell-cycle regulatory protein Ran/TC4.

The human Ras-related nuclear protein Ran/TC4 (refs 1-4) is the prototype of a well conserved family of GTPases that can regulate both cell-cycle progression and messenger RNA transport. Ran has been proposed to undergo tightly controlled cycles of GTP binding and hydrolysis, to operate as a GTPase switch whose GTP- and GDP-bound forms interact differentially with regulators and effectors. One known regulator, the protein RCC1 (refs 12, 13), interacts with Ran to catalyse guanine nucleotide exchange, and both RCC1 and Ran are components of an intrinsic checkpoint control that prevents the premature initiation of mitosis. To test and extend the GTPase-switch model, we searched for a Ran-specific GTPase-activating protein (GAP), and for putative effectors (proteins that interact specifically with Ran/TC4-GTP). We report here the identification of a Ran GAP and its use to characterize the GTP-hydrolysing properties of mutant Ran proteins, and the identification and cloning of a binding protein specific for Ran/TC4-GTP.

Amino Acid Sequence↗
PubMed1978

Methods for detection of hemoglobin variants and hemoglobinopathies in the routine clinical laboratory.

Many mutant hemoglobins and hemoglobinopathies can be identified with a high degree of specificity in the routine clinical laboratory. The most frequent abnormalities--those involving Hb S or C--are usually easily detectable in small amounts of sample analyzed by two simple methods of electrophoresis: cellulose acetate at pH 8.5 and citrate agar at pH 6. Some rarer mutants, e.g., Hb O, Hope, and Camden, can also be recognized by these two methods. Presumptive identification of other relatively frequent mutants, such as Hb D Los Angeles (Punjab) and Hb G Philadelphia, can be accomplished with additional data obtained from globin electrophoresis on cellulose acetate in acidic and alkaline buffers containing urea and 2-mercaptoethanol (or dithioerythritol). Electrophoretic profiles are presented of about a dozen hemoglobins likely to be encountered in screening programs in the U.S. Methods are also presented for identifying other genetic hemoglobin abnormalities--various types of thalassemia, Hb M, unstable hemoglobins, and those of the newborn.

Electrophoresis, Agar Gel↗