Cloning and characterization of the gene that encodes acetyl-coenzyme A carboxylase in the alga Cyclotella cryptica.
The gene that encodes acetyl-coenzyme A carboxylase (ACCase; EC 6.4.1.2) in the eukaryotic alga Cyclotella cryptica has been isolated and cloned, representing the first time that a full-length gene for this enzyme has been isolated from a photosynthetic organism. The gene contains a 447-base pair intron that is located near the putative translation initiation codon and a 73-base pair intron that is located slightly upstream from the region that encodes the biotin binding site of the enzyme. The gene encodes a polypeptide that is predicted to be composed of 2089 amino acids and to have a molecular mass of 230 kDa. The deduced amino acid sequence exhibits strong similarity to the sequences of animal and yeast ACCases in the biotin carboxylase and carboxyltransferase domains. There is less sequence similarity in the biotin carboxyl carrier protein domain, although the highly conserved Met-Lys-Met of the biotin binding site is present. The amino terminus of the predicted ACCase sequence has characteristics of a signal sequence, suggesting that the enzyme is imported into chloroplasts via the endoplasmic reticulum, as has been shown to be the case for certain nuclear-encoded proteins that are transported into the chloroplasts of the diatom Phaeodactylum tricornutum. Southern blot analyses suggest that a single copy of this gene is present in C. cryptica.
Roessler
P G
PG
Applied Biological Sciences Branch, National Renewable Energy Laboratory, Golden, Colorado 80401.
Ohlrogge
J B
JB
D000103:Acetyl-CoA Carboxylase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA; D017377:Diatoms / Q000201:enzymology* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017343:Genes, Plant; D015698:Genomic Library; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D017386:Sequence Homology, Amino Acid
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D004247:DNA; D000103:Acetyl-CoA Carboxylase