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.

Characterization and purification of lupus antigen La, and RNA-binding protein

1985 ◽  
Vol 5 (3) ◽  
pp. 586-590
Author(s):  
A M Francoeur ◽  
E K Chan ◽  
J I Garrels ◽  
M B Mathews
Keyword(s):  
Hela Cell ◽  
Gel Electrophoresis ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
In Vitro Translation ◽  
Cell Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
La Antigen

HeLa cell La antigen, an RNA-binding protein, was characterized by using two-dimensional gel electrophoresis. Eight isoelectric forms (pI 6 to 7) were observed, many containing phosphate. An in vitro translation product similar in size and antigenicity was identified. The HeLa cell protein purified by using an assay based on ribonucleoprotein reconstitution with adenovirus VA RNAI also comprised several isoelectric forms.

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.

Expression of alpha- and beta-tubulin genes during dimorphic-phase transitions of Histoplasma capsulatum

1989 ◽  
Vol 9 (5) ◽  
pp. 2042-2049
Author(s):  
G S Harris ◽  
E J Keath ◽  
J Medoff
Keyword(s):  
Phase Transitions ◽  
Histoplasma Capsulatum ◽  
Blot Hybridization ◽  
Dot Blot ◽  
Dimorphic Fungus ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Two Dimensional Gel Electrophoresis ◽  
Western Blots ◽  
Alpha Tubulin

Recent investigations have confirmed the presence of one alpha-tubulin gene (TUB1) and one beta-tubulin gene (TUB2) in the dimorphic fungus Histoplasma capsulatum. In the present study, Northern blot (RNA blot) analyses revealed multiple alpha-tubulin transcripts and a single beta-tubulin transcript in the yeast and mycelial phases of the high-virulence 217B strain and low-virulence Downs strain. S1 nuclease protection assays demonstrated one initiation start site and two major stop sites for the TUB1 transcripts, suggesting that variations in 3' processing generate the alpha-tubulin messages of 2.5 and 2.0 kilobases. Dot blot hybridization experiments indicated that tubulin gene expression is developmentally regulated during the dimorphic phase transitions. alpha- and beta-tubulin mRNAs increased six- to eightfold during the yeast-to-mycelium conversion and decreased two- to threefold during the reverse transition. These changes in tubulin mRNA content coincided with major morphological events associated with H. capsulatum development. Western blots (immunoblots) of H. capsulatum yeast-specific proteins resolved by two-dimensional gel electrophoresis demonstrated a single alpha- and a single beta-tubulin isoform. Multiple tubulin polypeptides expressed in mycelia are probably products of posttranslational modifications.

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 New poly(A)+RNAs appear coordinately during the differentiation of Naegleria gruberi amebae into flagellates.

1986 ◽  
Vol 102 (2) ◽  
pp. 353-361 ◽  
Author(s):  
J Mar ◽  
J H Lee ◽  
D Shea ◽  
C J Walsh
Keyword(s):  
Blot Analysis ◽  
Rna Synthesis ◽  
In Vitro Translation ◽  
Dot Blot ◽  
Cross Hybridization ◽  
Tubulin Mrna ◽  
Dot Blot Analysis ◽  
Rna Concentration ◽  
Naegleria Gruberi

We have examined the nature of the requirement for RNA synthesis during the differentiation of Naegleria gruberi amebae into flagellates (Fulton, C., and C. Walsh, 1980, J. Cell Biol., 85:346-360) by looking for poly(A)+RNAs that are specific to differentiating cells. A cDNA library prepared from poly(A)+RNA extracted from cells 40 min after initiation of the differentiation (40-min RNA), the time when formation of flagella becomes insensitive to inhibitors of RNA synthesis, was cloned into pBR322. Recombinant clones were screened for sequences that were complementary to 40-min RNA but not to RNA from amebae (0-min RNA). Ten of these differentiation-specific (DS) plasmids were identified. The DS plasmids were found to represent at least four different poly(A)+RNAs based on cross-hybridization, restriction mapping, and Northern blot analysis. Dot blot analysis was used to quantify changes in DS RNA concentration. The four DS RNAs appeared coordinately during the differentiation. They were first detectable at 10-15 min after initiation, reached a peak at 70 min as flagella formed, and then declined to low levels by 120 min when flagella reached full length. The concentration of the DS RNAs was found to be at least 20-fold higher in cells at 70 min than in amebae. The changes in DS RNA concentration closely parallel changes in tubulin mRNA as measured by in vitro translation (Lai, E.Y., C. Walsh, D. Wardell, and C. Fulton, 1979, Cell, 17:867-878).

scholarly journals Tubulin is phosphorylated at tyrosine by pp60c-src in nerve growth cone membranes.

1990 ◽  
Vol 111 (5) ◽  
pp. 1959-1970 ◽  
Author(s):  
W T Matten ◽  
M Aubry ◽  
J West ◽  
P F Maness
Keyword(s):  
Growth Cone ◽  
Cortical Neurons ◽  
Specific Protein ◽  
Beta Tubulin ◽  
Nerve Growth ◽  
Alpha Tubulin ◽  
Nerve Growth Cone ◽  
Phosphopeptide Mapping

We show here that tubulin is the major in vivo substrate of the tyrosine-specific protein kinase pp60c-src in nerve growth cone membranes. Phosphotyrosine antibodies were used to demonstrate phosphotyrosyl residues in a subpopulation of alpha- and beta-tubulin that was highly enriched in a subcellular fraction of growth cone membranes from fetal rat brain. The presence of phosphotyrosine-modified isoforms of alpha- and beta-tubulin in vivo was confirmed by 32p labeling of rat cortical neurons in culture. Tubulin in growth cone membranes was phosphorylated at tyrosine in endogenous membrane phosphorylation reactions (0.068 mol phosphotyrosine/mol alpha-tubulin and 0.045 mol phosphotyrosine/mol beta-tubulin), and phosphorylation was specifically inhibited by antibodies directed against pp60c-src, which is localized in the growth cone membranes. pp60c-src was capable of directly phosphorylating tubulin as shown in immune complex kinase assays with purified brain tubulin. Phosphopeptide mapping revealed a limited number of sites of tyrosine phosphorylation in alpha- and beta-tubulin, with similar phosphopeptides observed in vivo and in vitro. These results reveal a novel posttranslational modification of tubulin that could regulate microtubule dynamics at the growth cone.

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.

Sequence heterogeneity, multiplicity, and genomic organization of alpha- and beta-tubulin genes in sea urchins

1981 ◽  
Vol 1 (12) ◽  
pp. 1125-1137
Author(s):  
D Alexandraki ◽  
J V Ruderman
Keyword(s):  
Amino Acids ◽  
Ribonucleic Acid ◽  
In Vitro Translation ◽  
Cdna Clones ◽  
Beta Tubulin ◽  
Ribonucleic Acids ◽  
Alpha Tubulin ◽  
Messenger Ribonucleic Acid ◽  
Tubulin Genes ◽  
Terminal Amino

We analyzed the multiplicity, heterogeneity, and organization of the genes encoding the alpha and beta tubulins in the sea urchin Lytechinus pictus by using cloned complementary deoxyribonucleic acid (cDNA) and genomic tubulin sequences. cDNA clones were constructed by using immature spermatogenic testis polyadenylic acid-containing ribonucleic acid as a template. alpha- and beta-tubulin clones were identified by hybrid selection and in vitro translation of the corresponding messenger ribonucleic acids, followed by immunoprecipitation and two-dimensional gel electrophoresis of the translation products. The alpha cDNA clone contains a sequence that encodes the 48 C-terminal amino acids of alpha tubulin and 104 base pairs of the 3' nontranslated portion of the messenger ribonucleic acid. The beta cDNA insertion contains the coding sequence for the 100-C terminal amino acids of beta tubulin and 83 pairs of the 3' noncoding sequence. Hybrid selections performed at different criteria demonstrated the presence of several heterogeneous, closely related tubulin messenger ribonucleic acids, suggesting the existence of heterogeneous alpha- and beta-tubulin genes. Hybridization analyses indicated that there are at least 9 to 13 sequences for each of the two tubulin gene families per haploid genome. Hybridization of the cDNA probes to both total genomic DNA and cloned germline DNA fragments gave no evidence for close physical linkage of alpha-tubulin genes with beta-tubulin genes at the DNA level. In contrast, these experiments indicated that some genes within the same family are clustered.

Identification of proteins and mRNAs in isolated yellow crescents of ascidian eggs

Development ◽  
1985 ◽  
Vol 89 (1) ◽  
pp. 275-287
Author(s):  
William R. Jeffery
Keyword(s):  
Gel Electrophoresis ◽  
In Vitro Translation ◽  
Supernatant Fraction ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Mass Fractionation ◽  
Specific Subset ◽  
Mesenchyme Cells ◽  
Tail Muscle

The yellow crescent or myoplasm is a localized cytoplasmic region in eggs of the ascidian Styela that is partitioned to the larval tail muscle and mesenchyme cells during embryonic development. To determine whether the myoplasm contains a specific subset of maternal macromolecules, its abundant proteins and mRNAs were identified and compared to those present in the remainder of the egg. This was accomplished by exploiting a newly developed method for the mass fractionation of yellow crescents which is based on the presence of a unique cytoskeletal domain in the yellow crescent region. The fractionation yields a yellow crescent fraction (YCF) representing the myoplasm and a supernatant fraction representing the nonmyoplasmic regions. The YCF comprises structures which contain the characteristic myoplasmic organelles and about 10% of the protein, 8% of the RNA, and 1–3% of the poly (A) of whole eggs. Two-dimensional gel electrophoresis indicated that the YCF contains 15 polypeptides that are undetectable in the supernatant fraction and 43 polypeptides that are significantly depleted in the latter fraction. The proteins restricted to the YCF are both of cytoskeletal and noncytoskeletal origin. In vitro translation of RNA in a message-dependent lysate and analysis of [35S]methionine-labelled polypeptide products by two-dimensional gel electrophoresis did not reveal qualitative differences between the YCF and the supernatant fraction. Furthermore, the mRNAs coding for two polypeptides that were localized in the myoplasm were not restricted to the YCF. The results suggest that qualitative differences in proteins but not in prevalent mRNAs exist between the yellow crescent and the other cytoplasmic regions of Styela eggs.

In vitro posttranslational modification of lamin B cloned from a human T-cell line

1990 ◽  
Vol 10 (5) ◽  
pp. 2164-2175
Author(s):  
K M Pollard ◽  
E K Chan ◽  
B J Grant ◽  
K F Sullivan ◽  
E M Tan ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Posttranslational Modification ◽  
In Vitro Translation ◽  
Heptad Repeat ◽  
Two Dimensional Gel Electrophoresis ◽  
Lamin B ◽  
Human T Cell ◽  
Human T Cell Line

Autoimmune diseases are characterized by spontaneously occurring autoantibodies which have proven to be useful reagents for the characterization of specific nuclear proteins. Using a monoclonal autoantibody (72B9) derived from a murine lupus strain, we have cloned a cDNA from the human T-cell line MOLT-4, which encodes nuclear lamin B. The identity of the encoded protein as lamin B was established by both biochemical and immunological criteria. Inspection of the deduced amino acid sequence of lamin B revealed the presence in coil 1B of the alpha-helical domain of a leucine heptad repeat region. Analysis of mRNA in HL60 and MOLT-4 cells, which express only lamin B, or HeLa cells, which express all three major lamins (A, B, and C), together with the comigration of in vitro-translated product with isolated HeLa cell lamin B by two-dimensional gel electrophoresis, suggests that a single lamin B is expressed in mammalian somatic cells. In vitro translation with the cDNA clone revealed an EDTA-sensitive posttranslational modification which resulted in an increase in the apparent molecular weight to that equivalent to the native in vivo-synthesized lamin B protein. This in vitro modification included incorporation of a product of mevalonolactone and required an intact carboxy terminus.

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