Genetic dissection of the central pair microtubules of the flagella of Chlamydomonas reinhardtii.

Mutations at two loci, which cause an altered mobility of the flagella, affected the central pair microtubule complex of Chlamydomonas reinhardtii flagella. The mutations at both loci primarily affected the C1 microtubule of the complex. Three alleles at the PF16 locus affected the stability of the C1 microtubule in isolated axonemes. This phenotype has allowed us to determine that at least ten polypeptides of the central pair complex are unique to the C1 microtubule. The motility defect was correlated with the failure to assemble three of these ten polypeptides in vivo. The structural gene product of the PF16 locus was a polypeptide with molecular weight 57,000 as shown by analysis of five intragenic revertants and by analysis of axonemes from dikaryon rescue experiments. Three alleles at the PF6 locus affected the assembly of one of the two projections of the C1 microtubule and this projection was formed by at least three polypeptide components, which are a subset of polypeptides missing in isolated pf16 axonemes. No structural gene product has been identified for the PF6 locus. The gene product is probably not one of the identified projection constituents as shown by analysis of dikaryon rescue experiments. Chemical extraction of isolated wild-type axonemes suggests that at least seven polypeptide components are unique to the C2 microtubule.

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The colR4 and colR15 beta-tubulin mutations in Chlamydomonas reinhardtii confer altered sensitivities to microtubule inhibitors and herbicides by enhancing microtubule stability.

The Journal of Cell Biology ◽

10.1083/jcb.113.3.605 ◽

1991 ◽

Vol 113 (3) ◽

pp. 605-614 ◽

Cited By ~ 54

Author(s):

M J Schibler ◽

B Huang

Keyword(s):

Chlamydomonas Reinhardtii ◽

Missense Mutations ◽

Wild Type ◽

Beta Tubulin ◽

Alpha Tubulin ◽

Microtubule Inhibitors ◽

Microtubule Stability ◽

The Stability ◽

Tubulin Mutations

The colR4 and colR15 beta 2-tubulin missense mutations for lysine-350 in Chlamydomonas reinhardtii (Lee and Huang, 1990) were originally isolated by selection for resistance to the growth inhibitory effects of colchicine. The colR4 and colR15 mutants have been found to be cross resistant to vinblastine and several classes of antimitotic herbicides, including the dinitroanilines (oryzalin, trifluralin, profluralin, and ethafluralin); the phosphoric amide amiprophos methyl; and the dimethyl propynl benzamide pronamide. Like colchicine and vinblastine, the antimitotic effects of these plant-specific herbicides have been associated with the depolymerization of microtubules. In contrast to their resistance to microtubule-depolymerizing drugs, the mutants have an increased sensitivity to taxol, a drug which enhances the polymerization and stability of microtubules. This pattern of altered sensitivity to different microtubule inhibitors was found to cosegregate and corevert with the beta-tubulin mutations providing the first genetic evidence that the in vivo herbicidal effects of the dinitroanilines, amiprophos methyl, and pronamide are related to microtubule function. Although wild-type like in their growth characteristics, the colR4 and colR15 mutants were found to have an altered pattern of microtubules containing acetylated alpha-tubulin, a posttranslational modification that has been associated with stable subsets of microtubules found in a variety of cells. Microtubules in the interphase cytoplasm and those of the intranuclear spindle of mitotic cells, which in wild-type Chlamydomonas cells do not contain acetylated alpha-tubulin, were found to be acetylated in the mutants. These data taken together suggest that the colR4 and colR15 missense mutations increase the stability of the microtubules into which the mutant beta-tubulins are incorporated and that the altered drug sensitivities of the mutants are a consequence of this enhanced microtubule stability.

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Abstract 165: The 18 kDa FGF-2 Prevents the Doxorubicin-induced Cardiac Damage and Amp-activated Kinase Activation, in vitro and in vivo

Circulation Research ◽

10.1161/res.117.suppl_1.165 ◽

2015 ◽

Vol 117 (suppl_1) ◽

Author(s):

Navid Koleini ◽

Jon Jon Santiago ◽

Barbara E Nickel ◽

Robert Fandrich ◽

Davinder S Jassal ◽

...

Keyword(s):

Molecular Weight ◽

Cell Death ◽

Left Ventricular ◽

Left Ventricular Ejection ◽

Wild Type ◽

Rat Cardiomyocytes ◽

Ventricular Ejection Fraction ◽

Compound C

Introduction: Protection of the heart from chemotherapeutic (Doxorubicin, DOX) drug-induced toxicity is a desirable goal, to limit side effects of cancer treatments. DOX toxicity has been linked to the activation (phosphorylation) of the AMP-activated kinase, AMPK. The 18 kDa low molecular weight isoform of fibroblast growth factor 2 (Lo-FGF-2) is a known cardioprotective and cytoprotective agent. In this study we have tested the ability of Lo-FGF-2 to protect from DOX-induced damage in rat cardiomyocytes in vitro, and in transgenic mouse models in vivo, in relation to AMPK activation. Methods: Rat neonatal cardiomyocytes in culture were exposed to DOX (0.5 μM) in the presence or absence of pre-treatment Lo-FGF-2 (10 ng/ml). Compound C was used to block phosphorylation (activity) of AMPK. Levels of cell viability/death (using Calcein-AM/Propidium iodide assay), phospho -and total AMPK, and apoptotic markers such as active caspase 3 were analyzed. In addition, transgenic mice expressing only Lo-FGF2, and wild type mice, expressing both high molecular weight (Hi-FGF2) as well as Lo-FGF2 were subjected to DOX injection (20 mg/kg, intraperitoneal); echocardiography was used to examine cardiac function at baseline and at 10 days post-DOX. Results: DOX-induced cell death of cardiomyocytes in culture was maximal at 24 hours post-DOX coinciding with significantly increased in activated (phosphorylated) AMPK. Compound C attenuated DOX-induced cardiomyocyte loss. Pre-incubation with Lo-FGF-2 decreased DOX induced cell death, and also attenuated the phosphorylation of AMPK post-DOX. Relative levels of phospho-AMPK were lower in the hearts of Lo-FGF2-expressing male mice compared to wild type. DOX-induced loss of contractile function (left ventricular ejection fraction and endocardial velocity) was negligible in Lo-FGF2-expressing mice but significant in wild type mice. Conclusion: Lo-FGF-2 protects the heart from DOX-induced damage in vitro and in vivo, by a mechanism likely involving an attenuation of AMPK activity.

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Tissue transglutaminase-dependent posttranslational modification of the retinoblastoma gene product in promonocytic cells undergoing apoptosis.

Molecular and Cellular Biology ◽

10.1128/mcb.17.10.6040 ◽

1997 ◽

Vol 17 (10) ◽

pp. 6040-6048 ◽

Cited By ~ 92

Author(s):

S Oliverio ◽

A Amendola ◽

F Di Sano ◽

M G Farrace ◽

L Fesus ◽

...

Keyword(s):

Molecular Weight ◽

Gene Product ◽

Posttranslational Modification ◽

Enzyme Inhibitor ◽

Tissue Transglutaminase ◽

U937 Cells ◽

Retinoblastoma Gene ◽

Cell Progression ◽

Retinoblastoma Gene Product

The retinoblastoma gene product (pRB) plays an important role in controlling both cell release from the G1 phase and apoptosis. We show here that in the early phases of apoptosis, pRB is posttranslationally modified by a tissue transglutaminase (tTG)-catalyzed reaction. In fact, by employing a novel haptenized lysis synthetic substrate which allows the isolation of glutaminyl-tTG substrates in vivo, we identified pRB as a potential tTG substrate in U937 cells undergoing apoptosis. In keeping with this finding, we showed that apoptosis of U937 cells is characterized by the rapid disappearance of the 105,000- to 110,000-molecular-weight pRB forms concomitantly with the appearance of a smear of immunoreactive products with a molecular weight of greater than 250,000. The shift in pRB molecular weight was reproduced by adding exogenous purified tTG to extracts obtained from viable U937 cells and was prevented by dansylcadaverine, a potent enzyme inhibitor. The effect of the pRB posttranslational modification during apoptosis was investigated by determining the E2F-1 levels and by isolating and characterizing pRB-null clones from U937 cells. Notably, the lack of pRB in these U937-derived clones renders these p53-null cells highly resistant to apoptosis induced by serum withdrawal, calphostin C, and ceramide. Taken together, these data suggest that tTG, acting on the pRB protein, might play an important role in the cell progression through the death program.

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NEUROSPORA TREHALASE AND ITS STRUCTURAL GENE

Genetics ◽

10.1093/genetics/110.2.217 ◽

1985 ◽

Vol 110 (2) ◽

pp. 217-227

Author(s):

Christopher White ◽

Deborah B Lee ◽

Stephen J Free

Keyword(s):

Molecular Weight ◽

Linkage Group ◽

Structural Gene ◽

Gene Mutations ◽

Free Medium ◽

Wild Type ◽

Group I ◽

Normal Wild Type

ABSTRACT We have isolated Neurospora trehalaseless mutants and mapped the trehalase structural gene to linkage group I. The structural gene mutations not only affect thermostability and other characteristics of the enzyme but also affect the production of an inhibitor of the wild-type trehalase. The inhibitor appears to be the mutant trehalase. We suggest that the mutant subunits act as inhibitors by entering into the multimeric forms of the enzyme and altering the ability of the normal wild-type subunits to catalyze the cleavage of trehalose.—Wild type trehalase has been purified to near homogeneity, and its characteristics have been studied. It was purified as a tetramer, with each subunit having a molecular weight of 88,000.—We have studied the regulation of trehalase and found the production of trehalase to be glucose repressible. Cells begin to produce trehalase 60 min after being transferred to glucose-free medium.

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Liberation of Zinc-Containing L31 (RpmE) from Ribosomes by Its Paralogous Gene Product, YtiA, in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.188.7.2715-2720.2006 ◽

2006 ◽

Vol 188 (7) ◽

pp. 2715-2720 ◽

Cited By ~ 57

Author(s):

Genki Akanuma ◽

Hideaki Nanamiya ◽

Yousuke Natori ◽

Naofumi Nomura ◽

Fujio Kawamura

Keyword(s):

Bacillus Subtilis ◽

Ribosomal Protein ◽

Gene Product ◽

Binding Affinity ◽

Regulatory Mechanism ◽

Intracellular Concentration ◽

Paralogous Gene ◽

The Stability

ABSTRACT We have found that alternative localization of two types of L31 ribosomal protein, RpmE and YtiA, is controlled by the intracellular concentration of zinc in Bacillus subtilis. The detailed mechanisms for the alternation of L31 proteins under zinc-deficient conditions were previously unknown. To obtain further information about this regulatory mechanism, we have studied the stability of RpmE in vivo and the binding affinity of these proteins to ribosomes in vitro, and we have found that liberation of RpmE from ribosomes is triggered by the expression of ytiA, which is induced by the derepression of Zur under zinc-deficient conditions.

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Poliovirus Polymerase Residue 5 Plays a Critical Role in Elongation Complex Stability

Journal of Virology ◽

10.1128/jvi.02147-09 ◽

2010 ◽

Vol 84 (16) ◽

pp. 8072-8084 ◽

Cited By ~ 20

Author(s):

Sarah E. Hobdey ◽

Brian J. Kempf ◽

Benjamin P. Steil ◽

David J. Barton ◽

Olve B. Peersen

Keyword(s):

Amino Acid ◽

Rna Binding ◽

Critical Role ◽

Polymerase Activity ◽

Structural Basis ◽

Wild Type ◽

Elongation Complex ◽

Virus Growth ◽

The Stability

ABSTRACT The structures of polio-, coxsackie-, and rhinovirus polymerases have revealed a conserved yet unusual protein conformation surrounding their buried N termini where a β-strand distortion results in a solvent-exposed hydrophobic amino acid at residue 5. In a previous study, we found that coxsackievirus polymerase activity increased or decreased depending on the size of the amino acid at residue 5 and proposed that this residue becomes buried during the catalytic cycle. In this work, we extend our studies to show that poliovirus polymerase activity is also dependent on the nature of residue 5 and further elucidate which aspects of polymerase function are affected. Poliovirus polymerases with mutations of tryptophan 5 retain wild-type elongation rates, RNA binding affinities, and elongation complex formation rates but form unstable elongation complexes. A large hydrophobic residue is required to maintain the polymerase in an elongation-competent conformation, and smaller hydrophobic residues at position 5 progressively decrease the stability of elongation complexes and their processivity on genome-length templates. Consistent with this, the mutations also reduced viral RNA production in a cell-free replication system. In vivo, viruses containing residue 5 mutants produce viable virus, and an aromatic phenylalanine was maintained with only a slightly decreased virus growth rate. However, nonaromatic amino acids resulted in slow-growing viruses that reverted to wild type. The structural basis for this polymerase phenotype is yet to be determined, and we speculate that amino acid residue 5 interacts directly with template RNA or is involved in a protein structural interaction that stabilizes the elongation complex.

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The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5268 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5268-5277 ◽

Cited By ~ 59

Author(s):

W Zerges ◽

J D Rochaix

Keyword(s):

Chlamydomonas Reinhardtii ◽

Rna Binding ◽

Mrna Translation ◽

Binding Activity ◽

Leader Sequence ◽

Sequence Motif ◽

Wild Type ◽

Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

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RNA silencing of hydrogenase(-like) genes and investigation of their physiological roles in the green alga Chlamydomonas reinhardtii

Biochemical Journal ◽

10.1042/bj20100932 ◽

2010 ◽

Vol 431 (3) ◽

pp. 345-352 ◽

Cited By ~ 30

Author(s):

James E. Godman ◽

Attila Molnár ◽

David C. Baulcombe ◽

Janneke Balk

Keyword(s):

Chlamydomonas Reinhardtii ◽

Green Alga ◽

Rna Silencing ◽

Aldehyde Oxidase ◽

Eukaryotic Cell ◽

Unique Combination ◽

Wild Type ◽

Anaerobic Conditions ◽

Production Cell

The genome of the green alga Chlamydomonas reinhardtii encodes two [FeFe]-hydrogenases, HydA1 and HydA2, and the hydrogenase-like protein HYD3. The unique combination of these proteins in one eukaryotic cell allows for direct comparison of their in vivo functions, which have not been established for HydA2 and HYD3. Using an artificial microRNA silencing method developed recently, the expression of HydA1, HydA2 and HYD3 was specifically down-regulated. Silencing of HydA1 resulted in 4-fold lower hydrogenase protein and activity under anaerobic conditions. In contrast, silencing of HydA2 or HYD3 did not affect hydrogen production. Cell lines with strongly (>90%) decreased HYD3 transcript levels grew more slowly than wild-type. The activity of aldehyde oxidase, a cytosolic Fe-S enzyme, was decreased in HYD3-knockdown lines, whereas Fe-S dependent activities in the chloroplast and mitochondria were unaffected. In addition, the HYD3-knockdown lines grew poorly on hypoxanthine, indicating impaired function of xanthine dehydrogenase, another cytosolic Fe-S enzyme. The expression levels of selected genes in response to hypoxia were unaltered upon HYD3 silencing. Together, our results clearly distinguish the cellular roles of HydA1 and HYD3, and indicate that HYD3, like its yeast and human homologues, has an evolutionary conserved role in the biogenesis or maintenance of cytosolic Fe-S proteins.

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Stability and Tick Transmission Phenotype ofgfp-TransformedAnaplasma marginalethrough a CompleteIn VivoInfection Cycle

Applied and Environmental Microbiology ◽

10.1128/aem.02096-10 ◽

2010 ◽

Vol 77 (1) ◽

pp. 330-334 ◽

Cited By ~ 11

Author(s):

Susan M. Noh ◽

Massaro W. Ueti ◽

Guy H. Palmer ◽

Ulrike G. Munderloh ◽

Roderick F. Felsheim ◽

...

Keyword(s):

Reservoir Host ◽

Pathogen Transmission ◽

Infection Cycle ◽

Wild Type ◽

Infection Rates ◽

Tick Infection ◽

Tick Transmission ◽

The Stability ◽

Established Infection

ABSTRACTWe tested the stability and tick transmission phenotype of transformedAnaplasma marginalethrough a completein vivoinfection cycle. Similar to the wild type, thegfp-transformedA. marginalestrain established infection in cattle, a natural reservoir host, and persisted in immune competent animals. The tick infection rates for the transformedA. marginaleand the wild type were the same. However, there were significantly lower levels of the transformedA. marginalethan of the wild type in the tick. Despite the lower levels of replication, ticks transmitted the transformant. Transformants can serve as valuable tools to dissect the molecular requirements of tick colonization and pathogen transmission.

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