Structure and expression of an alpha-tubulin gene of Physarum polycephalum.
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 & 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.
Walden
P D
PD
Laboratory of Developmental Biochemistry, National Institute for Medical Research, Mill Hill, London, UK.
Monteiro
M J
MJ
Gull
K
K
Cox
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005455:Fluorescent Antibody Technique; D007438:Introns; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000276:immunology; D010804:Physarum / Q000235:genetics* / Q000276:immunology; D011061:Poly A / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D014404:Tubulin / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D000906:Antibodies; D009842:Oligopeptides; D012333:RNA, Messenger; D014404:Tubulin; D011061:Poly A