Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells

1987 ◽  
Vol 7 (8) ◽  
pp. 2700-2707
Author(s):  
B Boggs ◽  
F Cabral
Keyword(s):  
Molecular Weight ◽  
Gene Product ◽  
Cho Cells ◽  
Peptide Mapping ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Beta Tubulin Gene

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

scholarly journals Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

1987 ◽  
Vol 7 (8) ◽  
pp. 2700-2707 ◽  
Author(s):  
B Boggs ◽  
F Cabral
Keyword(s):  
Molecular Weight ◽  
Gene Product ◽  
Cho Cells ◽  
Peptide Mapping ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Beta Tubulin Gene

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.

scholarly journals Griseofulvin resistance mutation of Chinese hamster ovary cells that affects the apparent molecular weight of a congruent to 200,000-dalton protein.

1984 ◽  
Vol 4 (9) ◽  
pp. 1761-1768 ◽  
Author(s):  
R S Gupta
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Peptide Mapping ◽  
Chinese Hamster Ovary Cells ◽  
Resistance Mutation ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Resistant Mutant ◽  
Single Step ◽  
Cross Resistance

A single-step griseofulvin-resistant mutant (GrsR-4) of CHO cells which exhibit very specific cross-resistance towards certain microtubule inhibitors showed the absence of a protein of molecular weight congruent to 200,000 (designated P5) and the concomitant presence of a new protein spot, M5, of lower molecular weight (Mr congruent to 180,000) which is not present in other cell lines. Peptide mapping studies showed that proteins P5 and M5 are related to each other and that M5 may be missing a peptide fragment present in P5. In GrsR-4 X GrsS cell hybrids, both P5 and M5 were present in equal amounts, which provided evidence against post-translation mechanisms in the origin of M5 and indicated that the GrsR-4 mutant most likely contains a nonsense mutation in the structural gene for protein P5, which causes its premature termination and leads to the formation of M5. Our studies also showed that in different Chinese hamster cell lines the two alleles of the protein P5 are nonidentical and make protein products which differ from each other in isoelectric points. It is suggested that protein P5 and its isoelectric variant P6 may constitute microtubule-associated proteins.

Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation

1986 ◽  
Vol 6 (5) ◽  
pp. 1422-1429
Author(s):  
C Whitfield ◽  
I Abraham ◽  
D Ascherman ◽  
M M Gottesman
Keyword(s):  
Cho Cells ◽  
Single Gene ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Subunit ◽  
Steady State Levels

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

Griseofulvin resistance mutation of Chinese hamster ovary cells that affects the apparent molecular weight of a congruent to 200,000-dalton protein

1984 ◽  
Vol 4 (9) ◽  
pp. 1761-1768
Author(s):  
R S Gupta
Keyword(s):  
Molecular Weight ◽  
Cell Lines ◽  
Peptide Mapping ◽  
Chinese Hamster Ovary Cells ◽  
Resistance Mutation ◽  
Chinese Hamster ◽  
Apparent Molecular Weight ◽  
Resistant Mutant ◽  
Single Step ◽  
Cross Resistance

A single-step griseofulvin-resistant mutant (GrsR-4) of CHO cells which exhibit very specific cross-resistance towards certain microtubule inhibitors showed the absence of a protein of molecular weight congruent to 200,000 (designated P5) and the concomitant presence of a new protein spot, M5, of lower molecular weight (Mr congruent to 180,000) which is not present in other cell lines. Peptide mapping studies showed that proteins P5 and M5 are related to each other and that M5 may be missing a peptide fragment present in P5. In GrsR-4 X GrsS cell hybrids, both P5 and M5 were present in equal amounts, which provided evidence against post-translation mechanisms in the origin of M5 and indicated that the GrsR-4 mutant most likely contains a nonsense mutation in the structural gene for protein P5, which causes its premature termination and leads to the formation of M5. Our studies also showed that in different Chinese hamster cell lines the two alleles of the protein P5 are nonidentical and make protein products which differ from each other in isoelectric points. It is suggested that protein P5 and its isoelectric variant P6 may constitute microtubule-associated proteins.

scholarly journals Transfer and amplification of a mutant beta-tubulin gene results in colcemid dependence: use of the transformant to demonstrate regulation of beta-tubulin subunit levels by protein degradation.

1986 ◽  
Vol 6 (5) ◽  
pp. 1422-1429 ◽  
Author(s):  
C Whitfield ◽  
I Abraham ◽  
D Ascherman ◽  
M M Gottesman
Keyword(s):  
Cho Cells ◽  
Single Gene ◽  
Chinese Hamster ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Subunit ◽  
Steady State Levels

Total genomic DNA from a temperature-sensitive, colcemid-resistant Chinese hamster ovary (CHO) cell mutant expressing an electrophoretic variant beta-tubulin was used to transform wild-type CHO cells to colcemid-resistant cells at 37 degrees C. Southern blot analysis of the transformant demonstrated the three- to fivefold amplification of one of many beta-tubulin sequences compared with that of the wild type or mutant, thereby identifying a functional tubulin gene in CHO cells. This amplification of one tubulin-coding sequence resulted in a threefold increase in two beta-tubulin mRNA species, suggesting that both species may be encoded by a single gene. Pulse-chase experiments showed that in the transformant, total beta-tubulin was synthesized and degraded faster than in the revertant or wild-type cells, so that the steady-state levels of beta-tubulin and alpha-tubulin were unchanged in the transformant compared with those of wild-type, mutant, or revertant cells. Increased ratios of mutant to wild-type beta-tubulin made the transformant dependent on microtubule-depolymerizing drugs for growth at 37 but not 34 degrees C and supersensitive to the microtubule-stabilizing drug taxol at 34 degrees C.

scholarly journals Nucleotide sequence analysis of beta tubulin gene in a wide range of dermatophytes

2014 ◽  
Vol 52 (7) ◽  
pp. 674-688 ◽  
Author(s):  
Ali Rezaei-Matehkolaei ◽  
Hossein Mirhendi ◽  
Koichi Makimura ◽  
G. Sybren de Hoog ◽  
Kazuo Satoh ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Nucleotide Sequence ◽  
Nucleotide Sequence Analysis ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Wide Range ◽  
Beta Tubulin Gene

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast

1985 ◽  
Vol 5 (5) ◽  
pp. 1093-1099
Author(s):  
R J Schmidt ◽  
N W Gillham ◽  
J E Boynton
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Ribosomal Protein ◽  
Apparent Molecular Weight ◽  
Protein L ◽  
Mature Form ◽  
Chloroplast Protein Synthesis ◽  
Made In

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.

The beta tubulin gene of Eimeria tenella

1996 ◽  
Vol 76 (1-2) ◽  
pp. 315-319 ◽  
Author(s):  
Guan Zhu ◽  
Janet S. Keithly
Keyword(s):  
Eimeria Tenella ◽  
Tubulin Gene ◽  
Beta Tubulin ◽  
Beta Tubulin Gene
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