mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii

1984 ◽  
Vol 4 (3) ◽  
pp. 424-434
Author(s):  
J A Schloss ◽  
C D Silflow ◽  
J L Rosenbaum
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Control Cell ◽  
Beta Tubulin ◽  
Rna Sequences ◽  
Class V ◽  
Alpha Tubulin ◽  
Flagellar Regeneration ◽  
Flagellar Proteins ◽  
Kinetics Of ◽  
Class Iv

Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas.

scholarly journals mRNA abundance changes during flagellar regeneration in Chlamydomonas reinhardtii.

1984 ◽  
Vol 4 (3) ◽  
pp. 424-434 ◽  
Author(s):  
J A Schloss ◽  
C D Silflow ◽  
J L Rosenbaum
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Control Cell ◽  
Beta Tubulin ◽  
Rna Sequences ◽  
Class V ◽  
Alpha Tubulin ◽  
Flagellar Regeneration ◽  
Flagellar Proteins ◽  
Kinetics Of ◽  
Class Iv

Flagellar amputation in Chlamydomonas reinhardtii induces the accumulation of a specific set of RNAs, many of which encode flagellar proteins. We prepared a cDNA clone bank from RNA isolated from cells undergoing flagellar regeneration. From this bank, we selected clones that contain RNA sequences that display several different patterns of abundance regulation. Based on quantitation of the relative amounts of labeled, cloned cDNAs hybridizing to dots of RNA on nitrocellulose filters, the cloned sequences were divided into five regulatory classes: class I RNAs remain at constant abundance during flagellar regeneration; classes II, III, and IV begin to increase in abundance within a few minutes after deflagellation, reach maximal abundance at successively later times during regeneration, and return to control cell levels within 2 to 3 h; and class V RNA abundance decreases during flagellar regeneration. Alpha- and beta-tubulin mRNAs are included in regulatory class IV. The abundance kinetics of alpha-tubulin mRNAs differ slightly from those of beta-tubulin mRNAs. The availability of these clones makes possible studies on the mechanisms controlling the abundance of a wide variety of different RNA species during flagellar regeneration in Chlamydomonas.

scholarly journals mRNAs for alpha- and beta-tubulin and flagellar calmodulin are among those coordinately regulated when Naegleria gruberi amebae differentiate into flagellates.

1987 ◽  
Vol 105 (3) ◽  
pp. 1303-1309 ◽  
Author(s):  
D K Shea ◽  
C J Walsh
Keyword(s):  
Calcium Binding ◽  
In Vitro Translation ◽  
Class Iii ◽  
Beta Tubulin ◽  
Two Dimensional Gel Electrophoresis ◽  
Alpha Tubulin ◽  
Heat Stable ◽  
Flagellar Proteins ◽  
Naegleria Gruberi

Three of four mRNAs that are specific to the differentiation of Naegleria gruberi amebae into flagellates (Mar, J., J. H. Lee, D. Shea, and C. J. Walsh, 1986, J. Cell Biol., 102:353-361) have been identified as coding for flagellar proteins. The products of these mRNAs, which are coordinately regulated during the differentiation, were identified by in vitro translation of hybrid-selected RNA followed by two-dimensional gel electrophoresis and antibody binding. Six cross-hybridizing clones complementary to a 1.7-kb RNA (class II) all selected mRNA that was translated into two alpha-tubulins. The principal in vitro product, alpha-1, comigrated with a cytoplasmic alpha-tubulin, while the minor product with a more acidic pI, alpha-2, comigrated with flagellar alpha-tubulin. While Naegleria flagellar alpha-tubulin was found to be acetylated based on its reaction with a monoclonal antibody specific to this form, we suggest that alpha-2 is not likely to arise due to acetylation in vitro but probably represents the product of a second alpha-tubulin gene. The class III clone, also complementary to a 1.7-kb RNA, selected beta-tubulin mRNA. In the course of this work it was found, using monoclonal antibodies to the alpha- and beta-subunits of tubulin, that Naegleria alpha-tubulin migrated faster than beta-tubulin on SDS-PAGE. The class IV clone, which hybridizes with a 0.5-kb RNA, selected an mRNA that was translated into a heat stable calcium-binding protein, flagellar calmodulin.

scholarly journals Synthesis, transport, and utilization of specific flagellar proteins during flagellar regeneration in Chlamydomonas.

1982 ◽  
Vol 93 (3) ◽  
pp. 615-631 ◽  
Author(s):  
S P Remillard ◽  
G B Witman
Keyword(s):  
Gel Electrophoresis ◽  
Two Dimensional Gel Electrophoresis ◽  
Whole Cells ◽  
Complex Protein ◽  
Specific Proteins ◽  
Dilution Experiments ◽  
Flagellar Regeneration ◽  
Flagellar Proteins ◽  
Polypeptide Chains ◽  
Kinetics Of

We labeled gametes of Chlamydomonas with 10-min pulses of 35SO4(-2) before and at various times after deflagellation, and isolated whole cells and flagella immediately after the pulse. The labeled proteins were separated by one- or two-dimensional gel electrophoresis, and the amount of isotope incorporated into specific proteins was determined. Individual proteins were identified with particular structures by correlating missing axonemal polypeptides with ultrastructural defects in paralyzed mutants, or by polypeptide analysis of flagellar fractions. Synthesis of most flagellar proteins appeared to be coordinately induced after flagellar amputation. The rate of synthesis for most quantified proteins increased at least 4- to 10-fold after deflagellation. The kinetics of synthesis of proteins contained together within a structure (e.g., the radial spoke proteins [RSP] ) were frequently similar; however, the kinetics of synthesis of proteins contained in different structures (e.g., RSP vs. alpha- and beta-tubulins) were different. Most newly synthesized flagellar proteins were rapidly transported into the flagellum with kinetics reflecting the rate of growth of the organelle; exceptions included a central tubule complex protein (CT1) and an actinlike component, both of which appeared to be supplied almost entirely from pre-existing, unlabeled pools. Isotope dilution experiments showed that, for most quantified axonemal proteins, a minimum of 35-40% of the polypeptide chains used in assembling a new axoneme was synthesized during regeneration; these proteins appeared to have predeflagellation pools of approximately the same size relative to their stoichiometries in the axoneme. In contrast, CT1 and the actinlike protein had comparatively large pools.

scholarly journals Rapid changes in tubulin RNA synthesis and stability induced by deflagellation in Chlamydomonas.

1984 ◽  
Vol 99 (6) ◽  
pp. 2074-2081 ◽  
Author(s):  
E J Baker ◽  
J A Schloss ◽  
J L Rosenbaum
Keyword(s):  
Rna Synthesis ◽  
Synthesis Rate ◽  
Beta Tubulin ◽  
Tubulin Genes ◽  
Induction Signal ◽  
Rapid Changes ◽  
Rapid Accumulation ◽  
Flagellar Regeneration ◽  
Flagellar Proteins ◽  
The Stability

Detachment of the flagella of Chlamydomonas induces a rapid accumulation of mRNAs for tubulin and other flagellar proteins. Measurement of the rate of alpha and beta tubulin RNA synthesis during flagellar regeneration shows that deflagellation elicits a rapid, 4-7-fold burst in tubulin RNA synthesis. The synthesis rate peaks within 10-15 min, then declines back to the predeflagellation rate. Redeflagellation of cells at times before the first flagellar regeneration is completed (and when cells have already accumulated elevated levels of tubulin RNA) induces another burst in tubulin RNA synthesis which is identical to the first in magnitude and duration. This finding indicates that the induction signal may act to simply reprogram the tubulin genes for a transient burst of maximal synthesis. Evidence is presented that the stability of the tubulin RNAs changes during regeneration. Stability changes include both an apparent stabilization during regeneration and accelerated decay following regeneration.

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

1991 ◽  
Vol 113 (3) ◽  
pp. 605-614 ◽  
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.

scholarly journals Kinetics of Interactions between LOV Domains from Chlamydomonas Reinhardtii

2014 ◽  
Vol 106 (2) ◽  
pp. 463a
Author(s):  
Carey K. Johnson ◽  
Kathrin Magerl ◽  
Katee Wyant ◽  
Ashley McDade ◽  
Will Newhart ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Kinetics Of

Tandem arrangement of tubulin genes in the protozoan parasite Leishmania enriettii

1983 ◽  
Vol 3 (6) ◽  
pp. 1070-1076
Author(s):  
S M Landfear ◽  
D McMahon-Pratt ◽  
D F Wirth
Keyword(s):  
Tandem Repeat ◽  
Blot Hybridization ◽  
Protozoan Parasite ◽  
Southern Blot Hybridization ◽  
Beta Tubulin ◽  
Developmentally Regulated ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Southern Blot Hybridization Analysis ◽  
Leishmania Enriettii

The arrangement of developmentally regulated alpha- and beta-tubulin genes has been studied in the parasitic protozoan Leishmania enriettii by using Southern blot hybridization analysis. The alpha-tubulin genes occur in a tandem repeat whose monomeric unit may be represented by a 2-kilobase PstI fragment. Similarly, the beta-tubulin genes probably occur in a separate tandem repeat consisting of approximately 4-kilobase units unlinked to the alpha-tubulin repeats.

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii confers resistance to anti-microtubule herbicides

1993 ◽  
Vol 106 (1) ◽  
pp. 209-218 ◽  
Author(s):  
S.W. James ◽  
C.D. Silflow ◽  
P. Stroom ◽  
P.A. Lefebvre
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Resistance Mutation ◽  
In Vitro Translation ◽  
Predicted Amino Acid Sequence ◽  
Two Dimensional ◽  
Wild Type ◽  
Tubulin Gene ◽  
Alpha Tubulin ◽  
Tubulin Genes

A mutation in the alpha 1-tubulin gene of Chlamydomonas reinhardtii was isolated by using the amiprophos-methyl-resistant mutation apm1-18 as a background to select new mutants that showed increased resistance to the drug. The upA12 mutation caused twofold resistance to amiprophos-methyl and oryzalin, and twofold hypersensitivity to the microtubule-stabilizing drug taxol, suggesting that the mutation enhanced microtubule stability. The resistance mutation was semi-dominant and mapped to the same interval on linkage group III as the alpha 1-tubulin gene. Two-dimensional gel immunoblots of proteins in the mutant cells revealed two electrophoretically altered alpha-tubulin isoforms, one of which was acetylated and incorporated into microtubules in the axoneme. The mutant isoforms co-segregated with the drug-resistance phenotypes when mutant upA12 was backcrossed to wild-type cells. Two-dimensional gel analysis of in vitro translation products showed that the non-acetylated variant alpha-tubulin was a primary gene product. DNA sequence analysis of the alpha 1-tubulin genes from mutant and wild-type cells revealed a single missense mutation, which predicted a change in codon 24 from tyrosine in wild type to histidine in mutant upA12. This alteration in the predicted amino acid sequence corroborated the approximately +1 basic charge shift observed for the variant alpha-tubulins. The mutant allele of the alpha 1-tubulin gene was designated tua1-1.

Dominant effects of tubulin overexpression in Saccharomyces cerevisiae

1989 ◽  
Vol 9 (3) ◽  
pp. 1049-1059
Author(s):  
D Burke ◽  
P Gasdaska ◽  
L Hartwell
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Loss ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Inducible Promoters ◽  
Selective Degradation ◽  
Novel Structure ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Mutant Phenotypes

The consequences of altering the levels of alpha- and beta-tubulin in Saccharomyces cerevisiae were examined by constructing fusions of the structural genes encoding the tubulins to strong galactose-inducible promoters. Overexpression of beta-tubulin (TUB2) was lethal: cells arrested in the G2 stage of the cell cycle exhibited an increased frequency of chromosome loss, were devoid of microtubules, and accumulated beta-tubulin in a novel structure. Overexpression of the major alpha-tubulin gene (TUB1) was not lethal and did not affect chromosome segregation. The rate of alpha-tubulin mRNA and protein synthesis was increased, but the protein did not accumulate. Overexpression of both alpha- and beta-tubulin together resulted in arrested cell division, and cells accumulated excess tubules that contained both alpha- and beta-tubulin. Transient overexpression of both tubulins resulted in a high frequency of chromosome loss. These data suggest that strong selective pressure exists to prevent excess accumulation of microtubules or beta-tubulin and suggest a model by which this goal may be achieved by selective degradation of unassembled alpha-tubulin. Furthermore, the phenotype of beta-tubulin overexpression is similar to the phenotype of a beta-tubulin deficiency. These results add to a number of recent studies demonstrating that mutant phenotypes generated by overexpression can be informative about the function of the gene product.

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