Structure and expression of an alpha-tubulin gene of Physarum polycephalum
| Autor: | Keith Gull, Robert A. Cox, Mervyn J. Monteiro, Paul D. Walden |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
Molecular Sequence Data
Fluorescent Antibody Technique Physarum polycephalum macromolecular substances Molecular cloning Biochemistry Antibodies Physarum Antibody Specificity Tubulin Gene expression Gene cluster Animals Amino Acid Sequence RNA Messenger Cloning Molecular Gene Peptide sequence chemistry.chemical_classification Base Sequence biology Nucleic acid sequence biology.organism_classification Molecular biology Introns Amino acid chemistry Electrophoresis Polyacrylamide Gel Poly A Oligopeptides |
| Popis: | Fragments of Physarum polycephalum DNA generated by partial digestion with Sau3A were cloned into phage-lambda EMBL4. A recombinant (phage-lambda E alpha Tu) containing an alpha-tubulin (E alpha-tubulin) gene was isolated. The E alpha-tubulin gene is part of the alt B locus. The gene was sequenced and was found to contain seven intervening sequences. The alpha-tubulin isotype (E alpha-tubulin) encoded by the gene has a methionine residue at the C-terminus. The E alpha-tubulin gene has much in common with the N alpha-tubulin gene cloned into phage-lambda NM1149 [M. J. Monteiro and R. A. Cox (1987) J. Mol. Biol. 193, 427-438]. However, the gene products E alpha-tubulin and N alpha-tubulin differ in amino acid sequence near to the C-terminus. E-peptide (corresponding to amino acids 440-448 of E alpha-tubulin) and N-peptide (corresponding to amino acids 437-445 of N alpha-tubulin) were synthesised and used to raise antibodies (E-antibodies and N-antibodies). The antibodies were used to show that on two-dimensional gel electrophoresis N alpha-tubulin travels to the alpha 1 position and E alpha-tubulin moves to the alpha 2 position. Gene-specific DNA probes were used to show that transcripts of the E alpha-tubulin gene were present in the plasmodial but not in the amoebal phase of the life cycle. The E- and N-antibodies detected E alpha- and N alpha-tubulins in plasmodia but not in amoebae, confirming that the expression of the E alpha- and N alpha-tubulin genes is regulated during development. E alpha- and N alpha-tubulin were shown to be components of spindle microtubules by indirect immunofluorescence microscopy. |
| Databáze: | OpenAIRE |
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