Cloning and Expression Analysis of Alpha-Tubulin Genes in Water Foxtail (Alopecurus aequalis)

Weed Science ◽  
2010 ◽  
Vol 58 (2) ◽  
pp. 89-95 ◽  
Author(s):  
Saima Hashim ◽  
Mayumi Hachinohe ◽  
Hiroshi Matsumoto
Keyword(s):  
Gene Families ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Dependent Manner ◽  
Degenerate Primers ◽  
Specific Expression ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Complete Sequences

Tubulins are encoded by small gene families in plants. Here we report the cloning and characterization of water foxtail α-tubulin genes (AaTUA), an economically important weed. The genome of water foxtail contains TUA family consisting of at least four genes. Using degenerate primers, four partial TUA genes were isolated from the complementary DNA (cDNA) of water foxtail. Using the partial gene sequences, specific primers for each TUA gene were designed and full-length TUA cDNAs were isolated. These genes were designated as AaTUA1 to AaTUA4. The deduced amino acid sequences of AaTUA genes showed significant homology to the TUA genes of barley, corn, and Arabidopsis. The coding sequences of the AaTUA1 and AaTUA3 genes were interrupted by three introns and there were four introns in the coding regions of AaTUA2 and AaTUA4. The organ-specific expression of AaTUA genes showed that AaTUA1 and AaTUA4 were predominantly expressed in all organs examined (root, stem, young leaf, mature leaf, and panicle), whereas AaTUA2 was expressed mainly in roots and AaTUA3 was expressed in stem, root, and panicle. Abscisic acid (ABA) and gibberellic acid (GA) differentially induced the expression of AaTUA1 and AaTUA3. Moreover, trifluralin, propham, and caffeic acid induced the expression of all AaTUA genes in a dose-dependent manner except AaTUA2. This is the first report of complete sequences of AaTUA genes and the first characterization of these genes for any Alopecurus species in the literature.

scholarly journals The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins.

1985 ◽  
Vol 5 (9) ◽  
pp. 2389-2398 ◽  
Author(s):  
C D Silflow ◽  
R L Chisholm ◽  
T W Conner ◽  
L P Ranum
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Gene Products ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Total Complement ◽  
Cloned Gene ◽  
Net Charges

Full-length cDNA clones corresponding to the transcripts of the two alpha-tubulin genes in Chlamydomonas reinhardi were isolated. DNA sequence analysis of the cDNA clones and cloned gene fragments showed that each gene contains 1,356 base pairs of coding sequence, predicting alpha-tubulin products of 451 amino acids. Of the 27 nucleotide differences between the two genes, only two result in predicted amino acid differences between the two gene products. In the more divergent alpha 2 gene, a leucine replaces an arginine at amino acid 308, and a valine replaces a glycine at amino acid 366. The results predicted that two alpha-tubulin proteins with different net charges are produced as primary gene products. The predicted amino acid sequences are 86 and 70% homologous with alpha-tubulins from rat brain and Schizosaccharomyces pombe, respectively. Each gene had two intervening sequences, located at identical positions. Portions of an intervening sequence highly conserved between the two beta-tubulin genes are also found in the second intervening sequence of each of the alpha genes. These results, together with our earlier report of the beta-tubulin sequences in C. reinhardi, present a picture of the total complement of genetic information for tubulin in this organism.

scholarly journals Purification and Characterization of AsES Protein

2013 ◽  
Vol 288 (20) ◽  
pp. 14098-14113 ◽  
Author(s):  
Nadia R. Chalfoun ◽  
Carlos F. Grellet-Bournonville ◽  
Martín G. Martínez-Zamora ◽  
Araceli Díaz-Perales ◽  
Atilio P. Castagnaro ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proteolytic Activity ◽  
Foliar Spray ◽  
Amino Acid Sequences ◽  
Purification And Characterization ◽  
Degenerate Primers ◽  
Acremonium Strictum ◽  
Rapid Amplification

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2̇̄) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins

1986 ◽  
Vol 6 (11) ◽  
pp. 3711-3721
Author(s):  
P J Schatz ◽  
L Pillus ◽  
P Grisafi ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Nucleotide Sequencing ◽  
Cell Fractionation ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Tubulin Genes

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

scholarly journals Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein.

1986 ◽  
Vol 6 (3) ◽  
pp. 906-913 ◽  
Author(s):  
E M Elliott ◽  
G Henderson ◽  
F Sarangi ◽  
V Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mammalian Species ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Parental Line ◽  
Alpha Tubulin ◽  
Tubulin Genes

The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.

Complete sequence of three alpha-tubulin cDNAs in Chinese hamster ovary cells: each encodes a distinct alpha-tubulin isoprotein

1986 ◽  
Vol 6 (3) ◽  
pp. 906-913
Author(s):  
E M Elliott ◽  
G Henderson ◽  
F Sarangi ◽  
V Ling
Keyword(s):  
Chinese Hamster Ovary ◽  
Cho Cells ◽  
Chinese Hamster Ovary Cells ◽  
Mammalian Species ◽  
Chinese Hamster ◽  
Amino Acid Sequences ◽  
Parental Line ◽  
Alpha Tubulin ◽  
Tubulin Genes

The genome of Chinese hamster ovary (CHO) cells contains a complex family of approximately 16 alpha-tubulin genes, many of which may be pseudogenes. We present here the complete cDNA sequences of three expressed alpha-tubulin genes; one of these genes has been identified only in CHO cells. The noncoding regions of these three CHO alpha-tubulin genes differed significantly, but their coding regions were highly conserved. Nevertheless, we observed differences in the predicted amino acid sequences for the three genes. A comparison of the CHO alpha-tubulin sequences with all of the sequences available for mammals allowed assignment of the alpha-tubulin genes to three classes. The proteins encoded by the members of two of these classes showed no class-specific amino acids among the mammalian species examined. The gene belonging to the third class encoded an isoprotein which was clearly distinct, and members of this class may play a unique role in vivo. Sequencing of the three alpha-tubulin genes was also undertaken in CMR795, a colcemid-resistant clonal CHO cell line which has previously been shown to have structural and functional alterations in its tubulin proteins. We found differences in the tubulin nucleotide sequence compared with the parental line; however, no differences in the alpha-tubulin proteins encoded in the two cell lines were observed.

The two alpha-tubulin genes of Chlamydomonas reinhardi code for slightly different proteins

1985 ◽  
Vol 5 (9) ◽  
pp. 2389-2398
Author(s):  
C D Silflow ◽  
R L Chisholm ◽  
T W Conner ◽  
L P Ranum
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Cdna Clones ◽  
Gene Products ◽  
Beta Tubulin ◽  
Alpha Tubulin ◽  
Tubulin Genes ◽  
Total Complement ◽  
Cloned Gene ◽  
Net Charges

Full-length cDNA clones corresponding to the transcripts of the two alpha-tubulin genes in Chlamydomonas reinhardi were isolated. DNA sequence analysis of the cDNA clones and cloned gene fragments showed that each gene contains 1,356 base pairs of coding sequence, predicting alpha-tubulin products of 451 amino acids. Of the 27 nucleotide differences between the two genes, only two result in predicted amino acid differences between the two gene products. In the more divergent alpha 2 gene, a leucine replaces an arginine at amino acid 308, and a valine replaces a glycine at amino acid 366. The results predicted that two alpha-tubulin proteins with different net charges are produced as primary gene products. The predicted amino acid sequences are 86 and 70% homologous with alpha-tubulins from rat brain and Schizosaccharomyces pombe, respectively. Each gene had two intervening sequences, located at identical positions. Portions of an intervening sequence highly conserved between the two beta-tubulin genes are also found in the second intervening sequence of each of the alpha genes. These results, together with our earlier report of the beta-tubulin sequences in C. reinhardi, present a picture of the total complement of genetic information for tubulin in this organism.

Cloning and expression of a renal Na-Pi cotransport system from flounder

1994 ◽  
Vol 267 (2) ◽  
pp. F311-F317 ◽  
Author(s):  
A. Werner ◽  
H. Murer ◽  
R. K. Kinne
Keyword(s):  
Function Analysis ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Xenopus Laevis Oocytes ◽  
Dependent Manner ◽  
Pseudopleuronectes Americanus ◽  
Cross Hybridization ◽  
Hydropathy Plot ◽  
Cotransport System

Starting with the recently published sequence of the rat renal Na-Pi cotransport system, we have cloned a corresponding cDNA from the kidney of winter flounder (Pseudopleuronectes americanus), designated flounder NaPi-II. Expression of the cognate in vitro transcribed RNA in Xenopus laevis oocytes stimulated Na-dependent Pi transport specifically and in a time- and dose-dependent manner. Apparent affinities of Na and Pi, as well as the pH dependency, were very similar to those found for the mammalian systems. The flounder NaPi-II cDNA is 2,424 base pairs long and encodes a protein of 637 amino acids. The hydropathy plot predicts eight transmembrane spanning domains. In these regions the flounder NaPi-II-deduced protein shows high homology (approximately 80%, identity, approximately 92% similarity) with the amino acid sequences reported for mammalian NaPi-II proteins. However, in the hydrophilic parts of flounder NaPi-II protein, only minimal similarity could be found between fish and mammalian systems (30% homology, 45% similarity). Northern blot analysis with flounder NaPi-II cDNA as a probe confirmed this finding: even under nonstringent washing conditions, no cross-hybridization with mRNA from rat renal cortex was observed. Interestingly, flounder intestine was found to contain high levels of mRNA corresponding to NaPi-II. Supplementary bands of 1.9 and 4.2 kb were observed on Northern blots of renal and intestinal tissue. The close functional relationship of the flounder NaPi-II protein with the previously described Na-Pi cotransport systems and the pronounced differences on the level of their primary structures provide the tools for detailed structure-function analysis of Na-Pi cotransport.

Cloning and expression of the beta- and gamma-subunits of the human epithelial sodium channel

1995 ◽  
Vol 268 (5) ◽  
pp. C1157-C1163 ◽  
Author(s):  
F. J. McDonald ◽  
M. P. Price ◽  
P. M. Snyder ◽  
M. J. Welsh
Keyword(s):  
Human Kidney ◽  
High Sensitivity ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Rat Colon ◽  
Na Channel ◽  
Na Channels ◽  
Gamma Subunits ◽  
Epithelial Na Channel

Amiloride-sensitive Na+ channels are an important component of the Na+ reabsorption pathway in a number of epithelia. Here we report the cloning and characterization of cDNAs encoding two subunits of the human kidney epithelial Na+ channel (beta- and gamma-hENaC). Their predicted amino acid sequences were highly homologous (83-85% identical) to the corresponding subunits reported from rat colon (beta- and gamma-rENaC). Both beta- and gamma-hENaC mapped to human chromosome 16. Northern blot analysis showed high expression of beta- and gamma-hENaC in kidney and lung and differential expression of the three subunits in other tissues. Coexpression of beta- and gamma-hENaC with alpha-hENaC in Xenopus oocytes produced Na+ channels with high selectivity for Na+ and high sensitivity to amiloride. In addition, human subunits were able to substitute for the corresponding rat subunits in forming functional Na+ channels, suggesting conservation of function and suggesting that differences in sequence do not disrupt interactions between subunits. These results suggest that human alpha-, beta-, and gamma-ENaC together form Na+ channels with properties that are similar to those observed in epithelia, and will allow further investigation into the role that these channels may play in human disease.

scholarly journals Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

1986 ◽  
Vol 6 (11) ◽  
pp. 3711-3721 ◽  
Author(s):  
P J Schatz ◽  
L Pillus ◽  
P Grisafi ◽  
F Solomon ◽  
D Botstein
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Budding Yeast ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Nucleotide Sequencing ◽  
Cell Fractionation ◽  
Gene Products ◽  
Yeast Saccharomyces Cerevisiae ◽  
Alpha Tubulin ◽  
Tubulin Genes

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.

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