Cloning and analysis of beta-tubulin gene from a protoctist.
We have isolated and characterized by restriction endonuclease mapping, transcription pattern, and DNA sequencing a beta-tubulin gene from the coenocytic freshwater protoctist, Achlya klebsiana. The gene is intronless and has a single open reading frame that encodes a 444-amino acid residue polypeptide of Mr 49,856. The protein shows a high degree of homology to other beta-tubulins, 85% identity to human beta-tubulin and 89% identity to beta-tubulin of the sporozoan (also a protoctist) Plasmodium falciparum. Fungal beta-tubulins are among the least identical to A. klebsiana beta-tubulin. Through Southern blot hybridization analysis, we determined that there is just one form of beta-tubulin gene in A. klebsiana. Transcription of the gene was studied during sporogenesis. Following induction of sporogenesis, the level of the mRNA increased markedly at 2 h and declined in the next 2 h when mitosis, cytokinesis, and spore development occurred. At the same time, beta-tubulin content increased about 6-fold in the cells. Sporulation in A. klebsiana is not inhibited by antimitotic drugs such as benomyl, colcemid, and colchicine. Benomyl resistance in Neurospora crassa and Aspergillus nidulans has been genetically and molecularly linked to single amino acid substitutions at positions 167 and 165, respectively. The change from phenylalanine to tyrosine conferring benomyl resistance to N. crassa is seen in A. klebsiana, but the valine substitution for alanine in A. nidulans is marked by cysteine replacement in A. klebsiana. The amino acid found at position 165 is not conserved in various beta-tubulins, but phenylalanine at position 167 is extremely conserved.
Department of Microbiology, University of Manitoba, Winnipeg, Canada.
L E
LE
Cameron
J A
JA
Hutsul
L
L
Thorlacius
H B
HB
LéJohn
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D056890:Eukaryota / Q000235:genetics*; D005796:Genes; D006801:Humans; D008969:Molecular Sequence Data; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014404:Tubulin / Q000235:genetics*
D003062:Codon; D014404:Tubulin; D004247:DNA