Showing papers on "Wild type published in 1998"

Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development

Abstract: The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.

Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1.

Abstract: Analysis of transgenic mice expressing familial amyotrophic lateral sclerosis (ALS)-linked mutations in the enzyme superoxide dismutase (SOD1) have shown that motor neuron death arises from a mutant-mediated toxic property or properties. In testing the disease mechanism, both elimination and elevation of wild-type SOD1 were found to have no effect on mutant-mediated disease, which demonstrates that the use of SOD mimetics is unlikely to be an effective therapy and raises the question of whether toxicity arises from superoxide-mediated oxidative stress. Aggregates containing SOD1 were common to disease caused by different mutants, implying that coaggregation of an unidentified essential component or components or aberrant catalysis by misfolded mutants underlies a portion of mutant-mediated toxicity.

Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2

Abstract: The INK4a-ARF locus encodes two proteins, p16INK4a and p19ARF, that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19ARF can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21Cip1, and to arrest cell proliferation. Binding of p19ARF to p53 requires the ARF N-terminal domain (amino acids 1–62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19ARF in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to ≈75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19ARF restores p53’s ability to induce p21Cip1 and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53-dependent function through an additional mechanism.

Binding, gating, affinity and efficacy: The interpretation of structure-activity relationships for agonists and of the effects of mutating receptors

Abstract: The nature of the problem is illustrated by the curves in Figure 1. A mutation in a receptor is seen to produce 100 fold increase in the EC50 for an agonist (Figure 1a). A ligand binding experiment with the same agonist, on the same mutant receptor (Figure 1b) shows that the measured anity for the binding of the agonist has also been reduced by about 100 fold. Obviously the mutation has a€ected the agonist-binding site, and the mutated amino acid is likely to be part of that site? No! It is not in the least obvious. The example in Figure 1 was calculated on the basis that the anity for the binding step of the reaction was totally una€ected by the mutation (the equilibrium constant for this step was 100 mM for both wild type and mutant). The only di€erence between wild type and mutant receptor in this example is the ability of the receptor, once the agonist has bound, to change conformation to its active state. There is no reason at all why the amino acids that a€ect the ability to change conformation should be anywhere near the agonist binding site. . Binding experiments do not measure affinity (in any sense that is useful for learning about the binding site), for any ligand that causes a conformation change. . The term `apparent affinity' is often used to describe EC50 for the response but it is meaningless (unless you define what you mean by `apparent'). Making this distinction between e€ects on binding and e€ects on conformation change is arguably the fundamental problem of modern molecular studies of receptors. It is not an easy distinction to make, but unless it can be solved, the interpretation of structure-function studies is quite likely to be nonsense. It is not just a theoretical problem; this is how ion channels actually behave. Nevertheless, the very existence of the problem has not always been recognized. For example, statements like the following are not at all uncommon*. (a) `Simplistically, the ecacy of a full agonist can be set equal to 1, that of an antagonist to 0, and that of a partial agonist to a value between 0 and 1' (Ross, 1996, in Goodman & Gilman, 9th Edition). This statement obscures the point that is crucial, both for the interpretation of structureactivity relations and of mutant studies, that ecacy has no upper limit in principle, and that when it is large, changes in it are indistinguishable from changes in anity.

Stabilization of wild-type p53 by hypoxia-inducible factor 1α

Abstract: Although hypoxia (lack of oxygen in body tissues) is perhaps the most physiological inducer of the wild-type p53 gene, the mechanism of this induction is unknown. Cells may detect low oxygen levels through a haem-containing sensor protein. The hypoxic state can be mimicked by using cobalt chloride and the iron chelator desferrioxamine: like hypoxia, cobalt chloride and desferrioxamine activate hypoxia-inducible factor 1alpha (HIF-1alpha), which stimulates the transcription of several genes that are associated with hypoxia. Here we show that these treatments induce accumulation of wild-type p53 through HIF-1alpha-dependent stabilization of p53 protein. Induction of p53 does not occur in either a mutant hepatoma cell line that is unable to induce HIF-1alpha or embryonic stem cells derived from mice lacking HIF-1beta. HIF-1alpha is found in p53 immunoprecipitates from MCF7 cells that express wild-type p53 and are either hypoxic or have been exposed to desferrioxamine. Similarly, anti-haemagglutinin immunoprecipitates from lysates of normoxic PC3M cells that had been co-transfected with haemagglutinin-tagged HIF-1alpha and wild-type p53 also contain p53. Transfection of normoxic MCF7 cells with HIF-1alpha stimulates a co-transfected p53-dependent reporter plasmid and increases the amount of endogenous p53. Our results suggest that hypoxic induction of transcriptionally active wild-type p53 is achieved as a result of the stabilization of p53 by its association with HIF-1alpha.

Two Pleiotropic Classes of daf-2 Mutation Affect Larval Arrest, Adult Behavior, Reproduction and Longevity in Caenorhabditis elegans

Abstract: The nematode Caenorhabditis elegans responds to overcrowding and scarcity of food by arresting development as a dauer larva, a nonfeeding, long-lived, stress-resistant, alternative third-larval stage. Previous work has shown that mutations in the genes daf-2 (encoding a member of the insulin receptor family) and age-1 (encoding a PI 3-kinase) result in constitutive formation of dauer larvae (Daf-c), increased adult longevity (Age), and increased intrinsic thermotolerance (Itt). Some daf-2 mutants have additional developmental, behavioral, and reproductive defects. We have characterized in detail 15 temperature-sensitive and 1 nonconditional daf-2 allele to investigate the extent of daf-2 mutant defects and to examine whether specific mutant traits correlate with each other. The greatest longevity seen in daf-2 mutant adults was approximately three times that of wild type. The temperature-sensitive daf-2 mutants fell into two overlapping classes, including eight class 1 mutants, which are Daf-c, Age, and Itt, and exhibit low levels of L1 arrest at 25.5 degrees. Seven class 2 mutants also exhibit the class 1 defects as well as some or all of the following: reduced adult motility, abnormal adult body and gonad morphology, high levels of embryonic and L1 arrest, production of progeny late in life, and reduced brood size. The strengths of the Daf-c, Age, and Itt phenotypes largely correlated with each other but not with the strength of class 2-specific defects. This suggests that the DAF-2 receptor is bifunctional. Examination of the null phenotype revealed a maternally rescued egg, L1 lethal component, and a nonconditional Daf-c component. With respect to the Daf-c phenotype, the dauer-defective (Daf-d) mutation daf-12(m20) was epistatic to daf-2 class 1 alleles but not the severe class 2 alleles tested. All daf-2 mutant defects were suppressed by the daf-d mutation daf-16(m26). Our findings suggest a new model for daf-2, age-1, daf-12, and daf-16 interactions.

A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes

Abstract: Much attention has focused on the aetiology of oxidative damagein cellular and organismal ageing1,2,3,4. Especially toxic arethe reactive oxygen byproducts of respiration and other biological processes5. A mev-1 (kn1 ) mutant of Caenorhabditis elegans has been found to be hypersensitive to raised oxygen concentrations6,7. Unlike the wild type, its lifespan decreases dramatically as oxygen concentrations are increased from 1 to 60% (ref. 7). Strains bearing this mutation accumulate markers of ageing (such as fluorescent materials and protein carbonyls) faster than the wild type8,9. We show here that mev-1 encodes a subunit of the enzyme succinate dehydrogenase cytochrome b , which is a component of complex II of the mitochondrial electron transport chain. We found that the ability of complex II to catalyse electron transport from succinate to ubiquinone is compromised in mev-1 animals. This may cause an indirect increase in superoxide levels, which in turn leads to oxygen hypersensitivity and premature ageing. Our results indicate that mev-1 governs the rate of ageing by modulating the cellular response to oxidative stress.

Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers

Abstract: Mutations of DNA mismatch repair genes, including the hMLH1 gene, have been linked to human colon and other cancers in which defective DNA repair is evidenced by the associated instability of DNA microsatellite sequences (MSI). Germ-line hMLH1 mutations are causally associated with inherited MSI colon cancer, and somatic mutations are causally associated with sporadic MSI colon cancer. Previ- ously however, we demonstrated that in many sporadic MSI colon cancers hMLH1 and all other DNA mismatch repair genes are wild type. To investigate this class of tumors further, we examined a group of MSI cancer cell lines, most of which were documented as established from antecedent MSI- positive malignant tumors. In five of six such cases we found that hMLH1 protein was absent, even though hMLH1-coding sequences were wild type. In each such case, absence of hMLH1 protein was associated with the methylation of the hMLH1 gene promoter. Furthermore, in each case, treatment with the demethylating agent 5-azacytidine induced expres- sion of the absent hMLH1 protein. Moreover, in single cell clones, hMLH1 expression could be turned on, off, and on again by 5-azacytidine exposure, washout, and reexposure. This epigenetic inactivation of hMLH1 additionally accounted for the silencing of both maternal and paternal tumor hMLH1 alleles, both of which could be reactivated by 5-azacytidine. In summary, substantial numbers of human MSI cancers appear to arise by hMLH1 silencing via an epigenetic mechanism that can inactivate both of the hMLH1 alleles. Promoter methyl- ation is intimately associated with this epigenetic silencing mechanism.

A Role for the AKT1 Potassium Channel in Plant Nutrition

Abstract: In plants, potassium serves an essential role as an osmoticum and charge carrier. Its uptake by roots occurs by poorly defined mechanisms. To determine the role of potassium channels in planta, we performed a reverse genetic screen and identified an Arabidopsis thaliana mutant in which the AKT1 channel gene was disrupted. Roots of this mutant lacked inward-rectifying potassium channels and displayed reduced potassium (rubidium-86) uptake. Compared with wild type, mutant plants grew poorly on media with a potassium concentration of 100 micromolar or less. These results and membrane potential measurements suggest that the AKT1 channel mediates potassium uptake from solutions that contain as little as 10 micromolar potassium.

Retention of wild-type p53 in tumors from p53 heterozygous mice: reduction of p53 dosage can promote cancer formation.

Abstract: Tumor suppressor genes are generally viewed as being recessive at the cellular level, so that mutation or loss of both tumor suppressor alleles is a prerequisite for tumor formation The tumor suppressor gene, p53, is mutated in approximately 50% of human sporadic cancers and in an inherited cancer predisposition (Li-Fraumeni syndrome) We have analyzed the status of the wild-type p53 allele in tumors taken from p53-deficient heterozygous (p53+/-) mice These mice inherit a single null p53 allele and develop tumors much earlier than those mice with two functional copies of wild-type p53 We present evidence that a high proportion of the tumors from the p53+/- mice retain an intact, functional, wild-type p53 allele Unlike p53+/- tumors which lose their wild-type allele, the tumors which retain an intact p53 allele express p53 protein that induces apoptosis following gamma-irradiation, activates p21(WAF1/CIP1) and Mdm2 expression, represses PCNA expression (a negatively regulated target of wild-type p53), shows high levels of binding to oligonucleotides containing a wild-type p53 response element and prevents chromosomal instability as measured by comparative genomic hybridization These results indicate that loss of both p53 alleles is not a prerequisite for tumor formation and that mere reduction in p53 levels may be sufficient to promote tumorigenesis

Multicellular and aggregative behaviour of Salmonella typhimurium strains is controlled by mutations in the agfD promoter

Abstract: A colony morphology type is described in which cells of Salmonella typhimurium form a rigid multicellular network with expression of thin aggregative fimbriae that mediate tight intercellular bonds. Surface translocation of cells on plates and adherence to glass and polystyrene surfaces in biofilm assays are further characteristics of the morphotype. This morphotype (rdar) is normally expressed only at low temperature. However, in two unrelated S. typhimurium strains, spontaneous mutants were found forming rdar colonies independent of temperature. Allelic replacement proved a single point mutation in the promoter region of PagfD in each of the two mutants to be responsible for the constitutive phenotype of a multicellular behaviour. Transcription levels of the two divergently transcribed agf operons required for biogenesis of thin aggregative fimbriae by Northern blot analysis with gene probes for agfA and agfD as well as expression levels of AgfA by Western blotting were compared in the wild type, the constitutive mutants and their respective ompR and rpoS- derivatives. In the wild type the rdar morphotype and expression of thin aggregative fimbriae are restricted to low temperature on plates containing rich medium of low osmolarity, but biogenesis of thin aggregative fimbriae occurs upon iron starvation at 37 degrees C. In the upregulated mutants biogenesis of thin aggregative fimbriae is only abolished at high osmolarity at 37 degrees C and in the exponential phase in broth culture. Control of expression of thin aggregative fimbriae and rdar morphology takes place at the transcriptional level at the agfD promoter. A functional ompR allele is required, however an rpoS mutation abolishes transcription only in the wild type, but has no influence on expression of thin aggregative fimbriae in the constitutive mutants.

The short-period mutant, toc1-1, alters circadian clock regulation of multiple outputs throughout development in Arabidopsis thaliana

Abstract: The coordination of developmental and physiological events with environmental signals is facilitated by the action of the circadian clock. Here we report a new set of circadian clock-controlled phenotypes for Arabidopsis thaliana. We use these markers together with the short-period mutant, toc1-1, and the clock-controlled cab2::luciferase reporter gene to assess the nature of the circadian clock throughout development and to suggest the position of TOC1 within the circadian clock system. In dark-grown seedlings, the toc1-1 lesion conferred a short period to the cycling of cab2::luciferase luminescence, as previously found in light-grown plants, indicating that the circadian clocks in these two divergent developmental states share at least one component. Stomatal conductance rhythms were similarly approximately 3 hours shorter than wild type in toc1-1, suggesting that a cell-autonomous clockwork may be active in guard cells in 5- to 6-week-old leaves. The effect of daylength on flowering time in the C24 ecotype was diminished by toc1-1, and was nearly eliminated in the Landsberg erecta background where the plants flowered equally early in both short and long days. Throughout a 500-fold range of red light intensities, both the wild type and the mutant showed an inverse log-linear relationship of fluence rate to period, with a 2-3 hour shorter period for the mutant at all intensities. These results indicate that TOC1 acts on or within the clock independently of light input. Temperature entrainment appears normal in toc1-1, and the period-shortening effects of the mutant remain unchanged over a 20 degrees C temperature range. Taken together our results are consistent with the likelihood that TOC1 codes for an oscillator component rather than for an element of an input signaling pathway. In addition, the pervasive effect of toc1-1 on a variety of clock-controlled processes throughout development suggests that a single circadian system is primarily responsible for controlling most, if not all, circadian rhythms in the plant.

The Developmentally Regulated alb1 Gene of Aspergillus fumigatus: Its Role in Modulation of Conidial Morphology and Virulence

Abstract: Aspergillus fumigatus, an important opportunistic pathogen which commonly affects neutropenic patients, produces conidia with a bluish-green color We identified a gene, alb1, which is required for conidial pigmentation The alb1 gene encodes a putative polyketide synthase, and disruption of alb1 resulted in an albino conidial phenotype Expression of alb1 is developmentally regulated, and the 7-kb transcript is detected only during the conidiation stage The alb1 mutation was found to block 1,3,6,8-tetrahydroxynaphthalene production, indicating that alb1 is involved in dihydroxynaphthalene-melanin biosynthesis Scanning electron microscopy studies showed that the alb1 disruptant exhibited a smooth conidial surface, whereas complementation of the alb1 deletion restored the echinulate wild-type surface Disruption of alb1 resulted in a significant increase in C3 binding on conidial surfaces, and the conidia of the alb1 disruptant were ingested by human neutrophils at a higher rate than were those of the wild type The alb1-complemented strain producing bluish-green conidia exhibited inefficient C3 binding and neutrophil-mediated phagocytosis quantitatively similar to those of the wild type Importantly, the alb1 disruptant had a statistically significant loss of virulence compared to the wild-type and alb1-complemented strains in a murine model These results suggest that disruption of alb1 causes pleiotropic effects on conidial morphology and fungal virulence

Virulence of antibiotic-resistant Salmonella typhimurium

Abstract: We show that most Salmonella typhimurium mutants resistant to streptomycin, rifampicin, and nalidixic acid are avirulent in mice. Of seven resistant mutants examined, six were avirulent and one was similar to the wild type in competition experiments in mice. The avirulent-resistant mutants rapidly accumulated various types of compensatory mutations that restored virulence without concomitant loss of resistance. Such second-site compensatory mutations were more common then reversion to the sensitive wild type. We infer from these results that a reduction in the use of antibiotics might not result in the disappearance of the resistant bacteria already present in human and environmental reservoirs. Thus, second-site compensatory mutations could increase the fitness of resistant bacteria and allow them to persist and compete successfully with sensitive strains even in an antibiotic-free environment.

HOS1, a Genetic Locus Involved in Cold-Responsive Gene Expression in Arabidopsis

Abstract: Low-temperature stress induces the expression of a variety of genes in plants. However, the signal transduction pathway(s) that activates gene expression under cold stress is poorly understood. Mutants defective in cold signaling should facilitate molecular analysis of plant responses to low temperature and eventually lead to the identification and cloning of a cold stress receptor(s) and intracellular signaling components. In this study, we characterize a plant mutant affected in its response to low temperatures. The Arabidopsis hos1-1 mutation identified by luciferase imaging causes superinduction of cold-responsive genes, such as RD29A , COR47 , COR15A , KIN1 , and ADH. Although these genes are also induced by abscisic acid, high salt, or polyethylene glycol in addition to cold, the hos1-1 mutation only enhances their expression under cold stress. Genetic analysis revealed that hos1-1 is a single recessive mutation in a nuclear gene. Our studies using the firefly luciferase reporter gene under the control of the cold-responsive RD29A promoter have indicated that cold-responsive genes can be induced by temperatures as high as 19°C in hos1-1 plants. In contrast, wild-type plants do not express the luciferase reporter at 10°C or higher. Compared with the wild type, hos1-1 plants are less cold hardy. Nonetheless, after 2 days of cold acclimation, hos1-1 plants acquired the same degree of freezing tolerance as did the wild type. The hos1-1 plants flowered earlier than did the wild-type plants and appeared constitutively vernalized. Taken together, our findings show that the HOS1 locus is an important negative regulator of cold signal transduction in plant cells and that it plays critical roles in controlling gene expression under cold stress, freezing tolerance, and flowering time.

An ABC-transporter of Arabidopsis thaliana has both glutathione-conjugate and chlorophyll catabolite transport activity.

Abstract: An ABC-transporter of Arabidopsis thaliana exhibiting high sequence similarity to the human (MRP1) and yeast (YCF1) glutathione-conjugate transporters has been analysed and used to complement a cadmium-sensitive yeast mutant (DTY168) that also lacks glutathione-conjugate transport activity. Comparison of the hydrophobicity plots of this A. thaliana MRP-like protein with MRP1 and YCF1 demonstrates that the transmembrane domains are conserved, even at the N-terminus where sequence identity is low. Cadmium resistance is partially restored in the complemented ycf1 mutant, and glutathione-conjugate transport activity can be observed as well. The kinetic properties of the A. thaliana MRP-like protein (AtMRP3) are very similar to those previously described for the vacuolar glutathione-conjugate transporter of barley and mung bean. Furthermore, a hitherto undescribed ATP-dependent transport activity could be correlated with the gene product, i.e. vesicles isolated from the complemented yeast, but not from DTY168 or the wild type, take up the chlorophyll catabolite Bn-NCC-1. The results indicate that the product of the MRP-like gene of A. thaliana is capable of mediating the transport of the two different classes of compounds.

Identification of a polyketide synthase gene ( pksP ) of Aspergillus fumigatus involved in conidial pigment biosynthesis and virulence

Abstract: Aspergillus fumigatus is an important pathogen of the immunocompromised host causing pneumonia and invasive disseminated disease with high mortality. Previously, we identified a mutant strain (white, W) lacking conidial pigmentation and, in addition, the conidia showed a smooth surface morphology, whereas wild-type (WT) conidia are grey-green and have a typical ornamentation. W conidia appeared to be less protected against killing by the host defence, e.g., were more susceptible to oxidants in vitro and more efficiently damaged by human monocytes in vitro than WT conidia. When compared to the WT, the W mutant strain showed reduced virulence in a murine animal model. Genetic analysis suggested that the W mutant carried a single mutation which caused all of the observed phenotypes. Here, we report the construction of a genomic cosmid library of A. fumigatus and its use for complementation of the W mutant. Transformation of the W mutant was facilitated by co-transformation with plasmid pHELP1 carrying the autonomously replicating ama1 sequence of A. nidulans which also increased the transformation efficiency of A. fumigatus by a factor of 10. Using this cosmid library a putative polyketide synthase gene, designated pksP (polyketide synthase involved in pigment biosynthesis) was isolated. The pksP gene has a size of 6660 bp. pksP consists of five exons separated by short (47–73 bp) introns. Its deduced open reading frame is composed of 2146 amino acids. The pksP gene complemented both the white phenotype and the surface morphology of the W mutant conidia to wild type. Whereas W mutant conidia caused a strong reactive oxygen species (ROS) release by polymorphonuclear leukocytes, the ability of pksP-complemented W mutant conidia to stimulate ROS release was significantly reduced and comparable to that of WT conidia. In addition, the complemented strains showed restored virulence in a mouse model.

Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3‐kinase

Abstract: p85/p110 phosphoinositide 3‐kinase (PI3K) is a heterodimer composed of a p85‐regulatory and a p110‐catalytic subunit, which is involved in a variety of cellular responses including cytoskeletal organization, cell survival and proliferation. We describe here the cloning and characterization of p65‐PI3K, a mutant of the regulatory subunit of PI3K, which includes the initial 571 residues of the wild type p85α‐protein linked to a region conserved in the eph tyrosine kinase receptor family. We demonstrate that this mutation, obtained from a transformed cell, unlike previously engineered mutations of the regulatory subunit, induces the constitutive activation of PI3K and contributes to cellular transformation. This report links the PI3K enzyme to mammalian tumor development for the first time.

A Mutant of Arabidopsis Lacking a Chloroplastic Isoamylase Accumulates Both Starch and Phytoglycogen

Abstract: In this study, our goal was to evaluate the role of starch debranching enzymes in the determination of the structure of amylopectin. We screened mutant populations of Arabidopsis for plants with alterations in the structure of leaf starch by using iodine staining. The leaves of two mutant lines stained reddish brown, whereas wild-type leaves stained brownish black, indicating that a more highly branched polyglucan than amylopectin was present. The mutants were allelic, and the mutation mapped to position 18.8 on chromosome 1. One mutant line lacked the transcript for a gene with sequence similarity to higher plant debranching enzymes, and both mutants lacked a chloroplastic starch-hydrolyzing enzyme. This enzyme was identified as a debranching enzyme of the isoamylase type. The loss of this isoamylase resulted in a 90% reduction in the accumulation of starch in this mutant line when compared with the wild type and in the accumulation of the highly branched water-soluble polysaccharide phytoglycogen. Both normal starch and phytoglycogen accumulated simultaneously in the same chloroplasts in the mutant lines, suggesting that isoamylase has an indirect rather than a direct role in determining amylopectin structure.

A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation

Abstract: Previously, a promoter was identified in Lactococcus lactis that is specifically induced by chloride. Here, we describe the nucleotide sequence and functional analysis of two genes transcribed from this promoter, gadC and gadB. GadC is homologous to putative glutamate-gamma-aminobutyrate antiporters of Escherichia coli and Shigella flexneri and contains 12 putative membrane-spanning domains. GadB shows similarity to glutamate decarboxylases. A L. lactis gadB mutant and a strain that is unable to express both gadB and gadC was more sensitive to low pH than the wild type when NaCl and glutamate were present. Expression of gadCB in L. lactis in the presence of chloride was increased when the culture pH was allowed to decrease to low levels by omitting buffer from the medium, while glutamate also stimulated gadCB expression. Apparently, these genes encode a glutamate-dependent acid resistance mechanism of L. lactis that is optimally active under conditions in which it is needed to maintain viability. Immediately upstream of the chloride-dependent gadCB promoter Pgad, a third gene encodes a protein (GadR) that is homologous to the activator Rgg from Streptococcus gordonii. gadR expression is chloride and glutamate independent. A gadR mutant did not produce the 3kb gadCB mRNA that is found in wild-type cells in the presence of NaCl, indicating that GadR is an activator of the gadCB operon.

Divergent cAMP Signaling Pathways Regulate Growth and Pathogenesis in the Rice Blast Fungus Magnaporthe grisea

Abstract: cAMP is involved in signaling appressorium formation in the rice blast fungus Magnaporthe grisea. However, null mutations in a protein kinase A (PKA) catalytic subunit gene, CPKA , do not block appressorium formation, and mutations in the adenylate cyclase gene have pleiotropic effects on growth, conidiation, sexual development, and appressorium formation. Thus, cAMP signaling plays roles in both growth and morphogenesis as well as in appressorium formation. To clarify cAMP signaling in M. grisea , we have identified strains in which a null mutation in the adenylate cyclase gene ( MAC1 ) has an unstable phenotype such that the bypass suppressors of the Mac1 − phenotype ( sum ) could be identified. sum mutations completely restore growth and sexual and asexual morphogenesis and lead to an ability to form appressoria under conditions inhibitory to the wild type. PKA assays and molecular cloning showed that one suppressor mutation ( sum1-99 ) alters a conserved amino acid in cAMP binding domain A of the regulatory subunit gene of PKA ( SUM1 ), whereas other suppressor mutations act independently of PKA activity. PKA assays demonstrated that the catalytic subunit gene, CPKA , encodes the only detectable PKA activity in M. grisea. Because CPKA is dispensable for growth, morphogenesis, and appressorium formation, divergent catalytic subunit genes must play roles in these processes. These results suggest a model in which both saprophytic and pathogenic growth of M. grisea is regulated by adenylate cyclase but different effectors of cAMP mediate downstream effects specific for either cell morphogenesis or pathogenesis.

Aluminum Resistance in the Arabidopsis Mutant alr-104 Is Caused by an Aluminum-Induced Increase in Rhizosphere pH

Abstract: A mechanism that confers increased Al resistance in the Arabidopsis thaliana mutant alr -104 was investigated. A modified vibrating microelectrode system was used to measure H + fluxes generated along the surface of small Arabidopsis roots. In the absence of Al, no differences in root H + fluxes between wild type and alr -104 were detected. However, Al exposure induced a 2-fold increase in net H + influx in alr -104 localized to the root tip. The increased flux raised the root surface pH of alr -104 by 0.15 unit. A root growth assay was used to assess the Al resistance of alr -104 and wild type in a strongly pH-buffered nutrient solution. Increasing the nutrient solution pH from 4.4 to 4.5 significantly increased Al resistance in wild type, which is consistent with the idea that the increased net H + influx can account for greater Al resistance in alr -104. Differences in Al resistance between wild type and alr -104 disappeared when roots were grown in pH-buffered medium, suggesting that Al resistance in alr -104 is mediated only by pH changes in the rhizosphere. This mutant provides the first evidence, to our knowledge, for an Al-resistance mechanism based on an Al-induced increase in root surface pH.

Cytoplasmic Regulation of the Movement of E-Cadherin on the Free Cell Surface as Studied by Optical Tweezers and Single Particle Tracking: Corralling and Tethering by the Membrane Skeleton

Abstract: The translational movement of E-cadherin, a calcium-dependent cell–cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and α-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm φ colloidal gold or 210-nm φ latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell–cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 μm by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients ( D micro) of 1.2 × 10−10 and 2.1 × 10−10 cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 μm2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average D micro for Fusion measured by SPT was small (0.2 × 10−10 cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by ( a ) tethering to actin filaments through catenin(s) (like Fusion) and ( b ) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/μm, respectively, indicating a difference in the skeletal structures that produce these two effects.

Complementation of the Arabidopsis pds1 Mutation with the Gene Encoding p-Hydroxyphenylpyruvate Dioxygenase

Abstract: Plastoquinone and tocopherols are the two major quinone compounds in higher plant chloroplasts and are synthesized by a common pathway. In previous studies we characterized two loci in Arabidopsis defining key steps of this biosynthetic pathway. Mutation of the PDS1 locus disrupts the activity of p-hydroxyphenylpyruvate dioxygenase (HPPDase), the first committed step in the synthesis of both plastoquinone and tocopherols in plants. Although plants homozygous for the pds1 mutation could be rescued by growth in the presence of homogentisic acid, the product of HPPDase, we were unable to determine if the mutation directly or indirectly disrupted HPPDase activity. This paper reports the isolation of a cDNA, pHPPD, encoding Arabidopsis HPPDase and its functional characterization by expression in both plants and Escherichia coli. pHPPD encodes a 50-kD polypeptide with homology to previously identified HPPDases, including 37 highly conserved amino acid residues clustered in the carboxyl region of the protein. Expression of pHPPD in E. coli catalyzes the accumulation of homogentisic acid, indicating that it encodes a functional HPPDase enzyme. Mapping of pHPPD and co-segregation analysis of the pds1 mutation and the HPPD gene indicate tight linkage. Constitutive expression of pHPPD in a pds1 mutant background complements this mutation. Finally, comparison of the HPPD genomic sequences from wild type and pds1 identified a 17-bp deletion in the pds1 allele that results in deletion of the carboxyterminal 26 amino acids of the HPPDase protein. Together, these data conclusively demonstrate that pds1 is a mutation in the HPPDase structural gene.