Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tubulin binds guanine nucleotides with high affinity and specificity. GTP, an allosteric effector of microtubule assembly, requires Mg2+ for its interaction with beta-tubulin and binds as the MgGTP complex. In contrast, GDP binding does not require Mg2+. The structural basis for this difference is not understood but may be of fundamental importance for microtubule assembly. We investigated the interaction of beta-tubulin with guanine nucleotides using site-directed mutagenesis. Acidic amino acid residues have been shown to interact with nucleotide in numerous nucleotide-binding proteins. In this study, we mutated seven highly conserved aspartic acid residues and one highly conserved glutamic acid residue in the putative GTP-binding domain of beta-tubulin (N-terminal 300 amino acids) to asparagine and glutamine, respectively. The mutants were synthesized in vitro using rabbit reticulocyte lysates, and their affinities for nucleotide determined by an h.p.l.c.-based assay. Our results indicate that the mutations can be placed in six separate categories on the basis of their effects on nucleotide binding. These categories range from having no effect on nucleotide binding to a mutation that apparently abolishes nucleotide binding. One mutation at Asp224 reduced the affinity of beta-tubulin for GTP in the presence but not in the absence of Mg2+. The specific effect of this mutation on nucleotide binding is consistent with an interaction of this amino acid with the Mg2+ moiety of MgGTP. This residue is in a region sharing sequence homology with the putative Mg2+ site in myosin and other ATP-binding proteins. As a result, tubulin belongs to a distinct class of GTP-binding proteins which may be evolutionarily related to the ATP-binding proteins.
J Mol Biol 1992 Sep 05
PMID:Site-directed mutagenesis of the GTP-binding domain of beta-tubulin. 152 95

Two-dimensional gel/western blot analysis was used to characterize alpha- and beta-tubulin isotype expression along the developmental axis of the maize (Zea mays) seedling primary root. We identified four distinct alpha-tubulin isotypes and a minimum of six beta-tubulin isotypes. This analysis showed differences between the alpha- and beta-tubulin isotypes expressed in rapidly dividing tissue at the root tip and differentiated root tissues proximal to the tip. The alpha 1 and alpha 4 isotypes predominated in samples from immature rapidly dividing tissues such as root tips, whereas in mature tissues such as differentiated root and pollen, alpha 2, alpha 3 and alpha 4 isotypes predominated. The beta 1 and beta 2 isotypes were more abundant in protein samples from root cortex than in samples from the root tip or vascular cylinder. In contrast, the beta 4 and beta 5 isotypes appeared to be more abundant in root tip and vascular cylinder samples than in root cortex samples. Hybridization probes from the 3' non-coding region of six alpha-tubulin cDNA clones were used to quantify the levels of corresponding tubulin transcripts in selected tissues, from embryonic to mature and from largely undifferentiated to highly differentiated. The results from these hybridization experiments showed that all of the alpha-tubulin genes were expressed in all tissues examined, although each gene showed a unique pattern of differential transcript accumulation. A transcript produced from cDNA clone representing the tua5 alpha-tubulin gene was translated in vitro and produced an alpha-tubulin that comigrated with the alpha 2 isotype.
J Mol Biol 1992 Sep 05
PMID:Tubulin gene expression in maize (Zea mays L.). Change in isotype expression along the developmental axis of seedling root. 152 5

The cys-3+ gene of Neurospora crassa encodes a bZIP (basic region-leucine zipper) regulatory protein that is essential for sulfur structural gene expression (e.g., ars-1+). Nuclear transcription assays confirmed that cys-3+ was under sulfur-regulated transcriptional control and that cys-3+ transcription was constitutive in sulfur controller (scon)-negative regulator mutants. Given these results, I have tested whether expression of cys-3+ under high-sulfur (repressing) conditions was sufficient to induce sulfur gene expression. The N. crassa beta-tubulin (tub) promoter was fused to the cys-3+ coding segment and used to transform a cys-3 deletion mutant. Function of the tub::cys-3 fusion in homokaryotic transformants grown under high-sulfur conditions was confirmed by Northern (RNA) and Western immunoblot analysis. The tub::cys-3 transformants showed arylsulfatase gene expression under normally repressing high-sulfur conditions. A tub::cys-3ts fusion encoding a temperature-sensitive CYS3 protein was used to confirm that the induced structural gene expression was due to CYS3 protein function. Constitutive CYS3 production did not induce scon-2+ expression under repressing conditions. In addition, a cys-3 promoter fusion to lacZ showed that CYS3 production was sufficient to induce its own expression and provides in vivo evidence for autoregulation. Finally, an apparent inhibitory effect observed with a strain carrying a point mutation at the cys-3 locus was examined by in vitro heterodimerization studies. These results support an interpretation of CYS3 as a transcriptional activator whose regulation is a crucial control point in the signal response pathway triggered by sulfur limitation.
Mol Cell Biol 1992 Apr
PMID:Production of the CYS3 regulator, a bZIP DNA-binding protein, is sufficient to induce sulfur gene expression in Neurospora crassa. 153 30

The gene encoding laccase in the chestnut blight fungus, Cryphonectria parasitica, has been cloned and characterized. The predicted C. parasitica laccase amino acid sequence (591 aa) was 57% identical to the Neurospora crassa laccase sequence and contained four potential copper-binding regions that are conserved in a number of copper-binding proteins. Treatment of a virulent C. parasitica strain with 3 microM cycloheximide resulted in a marked increase in laccase mRNA accumulation, whereas identical treatment of an isogenic strain that contained a hypovirulence-associated virus failed to significantly increase laccase mRNA levels. In contrast, the accumulation of mRNAs encoding beta-tubulin, actin, or glyceraldehyde-3-phosphate dehydrogenase was not appreciably altered by either the presence of a hypovirulence-associated virus or treatment with cycloheximide. These results provide evidence that the expression of a specific fungal gene encoding a known protein product is selectively modulated by a hypovirulence-associated virus.
Mol Plant Microbe Interact
PMID:Molecular analysis of the laccase gene from the chestnut blight fungus and selective suppression of its expression in an isogenic hypovirulent strain. 153 23

One gene and two cDNAs encoding three different beta-tubulins (TUB1, TUB2, TUB3) of pea have been cloned and sequenced. The derived amino acid sequences show between 92% and 96% identity relative to one another and to most other beta-tubulins of higher plants and green algae. Two notable extremes are the high similarity of 98% between pea TUB3 and maize beta-tubulin 2 and the relatively low similarity (90%) of the hypocotyl-specific beta-tubulin 1 of soybean to the pea sequences. These similarities do not reflect the molecular phylogeny but rather differences in evolutionary rate of beta-tubulins which are differentially regulated during plant development. Genomic Southern blots reveal a beta-tubulin gene family in pea with at least four separate members including two TUB1 genes, one TUB2 gene and one TUB3 gene. This contradicts an earlier report by Raha et al. (Plant Mol Biol 9: 565-571, 1987) suggesting a tandem repeat organization of tubulin genes in pea. The pea TUB1 gene has two introns in identical positions compared to the beta-tubulin genes from Arabidopsis and soybean. In an attempt to reconstruct the universal ancestor of all present-day tubulin genes the intron positions in 38 different alpha- and beta-tubulin genes from plants, animals, fungi and protozoa were compared. This comparison shows that the primordial gene probably had many introns (more than 20) separating 'protoexons' of 15 to 20 codons in agreement with the 'exon theory of genes'. It also supports the view that, during the course of evolution, introns have shifted and were deleted preferentially in the 3' part of the genes. Similar observations have been made previously for other genes. They can be interpreted in terms of a homologous recombination of genes with their modified (incorrectly spliced) and reverse-transcribed pre-mRNAs.
Plant Mol Biol 1992 Feb
PMID:The beta-tubulin gene family of pea: primary structures, genomic organization and intron-dependent evolution of genes. 155 42

The effect of the vinca alkaloid drugs, vincristine, vinblastine, catharanthine, and vindoline, on the aging process of tubulin has been examined. It was found that addition of vincristine or vinblastine accelerated by a factor of 3-3.5 the transformation of tubulin from the 5.8 S alpha-beta-tubulin dimer to paucidisperse polymers, with an average sedimentation coefficient of 9 S, previously observed in the absence of drugs (V. Prakash and S. N. Timasheff, 1982, J. Mol. Biol. 160, 499-515). This transformation of tubulin from 5.8 S to "9 S" followed pseudo-first-order kinetics whether the starting protein was predominantly dimeric (i.e., at low drug concentration) or self-associated into the reversible linear polymers induced by the vinca alkaloid drugs at high drug concentration (G. C. Na and S. N. Timasheff, 1980, Biochemistry 19, 1355-1365; V. Prakash and S. N. Timasheff, 1985, Biochemistry 24, 5004-5010). Identical kinetics were found in a fluorescence examination of the loss by tubulin of its ability to bind colchicine specifically, indicating that the rate determining step is a protein conformational change that induces a major change in the far uv circular dichroism spectrum of tubulin. The found lack of an effect of dithiothreitol on the aging and aggregation processes is consistent with the irreversible aggregation being due to the intermolecular coalescence of nonpolar patches on the protein. The observations that vincristine binds to aged tubulin and that the aging of tubulin is accompanied by quenching of the tryptophan fluorescence similar to that which occurs on the binding of the vinca drugs has led to the proposal that the vinca alkaloids stabilize the aged conformation of the protein by interacting with nonpolar regions that may be related to the aggregation sites.
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PMID:Aging of tubulin at neutral pH: the destabilizing effect of vinca alkaloids. 157 10

This work describes the isolation and characterization of a full-length cDNA clone encoding beta-tubulin from the pathogen Pneumocystis carinii. P. carinii contains a single gene encoding beta-tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the beta-tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. carinii beta-tubulin and those of fungal beta-tubulins resistant to benomyl, a beta-tubulin-binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.
Mol Microbiol 1992 Apr
PMID:Cloning and sequence of a beta-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy. 158 27

Beet yellows virus (BYV) genome encodes a 65 kDa protein homologous to the HSP70 family of cellular heat-shock proteins (Agranovsky, A.A., Boyko, V.P., Karasev, A.V., Koonin, E.V. and Dolja, V.V. (1991) J. Mol. Biol. 217, 603-610). The respective gene was cloned and expressed in vitro yielding a product of the expected size (p65). This product was found to bind to the purified microtubules with a binding constant of 4 x 10(-7) M. The binding of p65 was stimulated if ATP presented in the translation mixture was hydrolyzed by apyrase. Removal of the short C-terminal domains of alpha- and beta-tubulin by subtilisin digestion abolished the binding, demonstrating its specificity. The possible role of p65 association with microtubules in the movement of virus within and/or between plant cells is proposed.
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PMID:HSP70-related 65 kDa protein of beet yellows closterovirus is a microtubule-binding protein. 161 94

Regeneration of cilia in Tetrahymena cells treated with 1 micrograms/ml of 4',6-diamidino-2-phenylindole (DAPI) takes place about 30 min later then in untreated controls. Analysis of RNA isolated at 50-th min of reciliation, that is when the control cells-but not DAPI-treated ones-have just restored their motility, revealed that; - 1. Two peaks of polyadenylated RNA were present in DAPI-treated cells as well as in the control ones; - 2. The mobility of the polyadenylated RNA was the same in the two compared cell samples; - 3. In wheat germ cell-free translational system these poly (A+)-RNA probably directed the synthesis of alpha- and beta-tubulin; - 4. The amount of polyadenylated RNA in compared cell samples was different, in DAPI-treated Tetrahymena its amount was estimated at 1.43% vs. 1.89% in control ones.
Mol Cell Biochem 1990 Jan 18
PMID:Polyadenylated RNA during DAPI-arrested regeneration of Tetrahymena cilia. 168 6

Angiotensinogen mRNA is found in many extrahepatic tissues, where it may participate in local angiotensin-generating systems. In this study we explore the feasibility of using anti-sense RNA to decrease angiotensinogen production in rat H4IIEC3 hepatoma cells. An amplifiable shuttle vector was modified to allow the production of high levels of stable anti-sense RNA from two regions of the mouse angiotensinogen gene under the control of the inducible sheep metallothionein promoter. Stably transformed, clonal cell lines expressing anti-sense RNA for angiotensinogen were isolated after selection with the aminoglycoside G418. Subsequently, the number of chromosomally integrated copies of the angiotensinogen anti-sense constructs was coamplified by methotrexate selection for dihydrofolate reductase activity carried on the shuttle vector. With a 20- to 30-fold induction of the anti-sense RNAs, the target angiotensinogen mRNA level was reduced to 25-30% of control values. The specificity of this effect was confirmed by showing no decrease in either beta-tubulin or neomycin phosphotransferase mRNA levels. Using tissue-specific promoters, it should be possible to direct these effects to specific organs in transgenic mice. However, in agreement with results from other groups, our findings suggest that it will not be possible to eradicate completely the target gene product using the anti-sense RNA strategy.
J Mol Endocrinol 1990 Apr
PMID:Inducible anti-sense RNA for angiotensinogen in stably transformed hepatoma cell lines. 169 79


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