Molecular cloning of delta 9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation.
In response to a decrease in its growth temperature, the protozoan Tetrahymena is known to increase the level of unsaturated fatty acids in its membrane phospholipids so as to maintain the correct physical state (fluidity) of the membranes. In this organism, synthesis of unsaturated fatty acids is initiated by delta 9 acyl-CoA desaturase. Our previous studies have shown that, during cold adaptation, the activity of microsomal palmitoyl- and stearoyl-CoA desaturase increases, reaching a maximal level at 2 h after a temperature down-shift to 15 degrees C. Two hypotheses have been proposed to explain this increase in desaturase activity: (1) self-regulation via a direct effect of reduced membrane fluidity, and (2) induction of desaturase mRNA. However, the precise mechanism is not clearly understood. In order to obtain further insight into the mechanism of regulation of the desaturase, we have isolated a gene that encodes delta 9 fatty acid desaturase from T. thermophila and examined its expression during cold adaptation. The nucleotide sequence indicates that the 1.4 kbp gene encodes a polypeptide of 292 amino acid residues which shows marked sequence similarity to delta 9 acyl-CoA desaturases from other sources, e.g. rat, mouse, Amblyomma americanum and Saccharomyces cerevisiae. This protein has three histidine-cluster motifs (one HXXXXH and two HXXHH), and two hydrophobic regions which are conserved among delta 9 acyl-CoA desaturases. The level of desaturase mRNA was sensitive to decreasing the temperature of the culture media, and was close to maximal immediately after the temperature was shifted down from 35 degrees C to 15 degrees C (0.8 degrees C/min). Thereafter, the amount of mRNA gradually decreased with time, but remained above the control level for at least 5 h. Furthermore, during the course of the cooling process to 15 degrees C, the increased expression of desaturase mRNA became evident at 27 degrees C. Nuclear run-on analysis and actinomycin D chase experiments revealed that the elevation of the mRNA level was due to increases in both transcription and mRNA stability. These results suggest that the enhanced desaturase activity is controlled, at least in part, at the transcriptional level.
Nakashima
S
S
Department of Biochemistry, Gifu University School of Medicine, Japan.
Zhao
Y
Y
Nozawa
Y
Y
D000222:Adaptation, Physiological / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000096:biosynthesis / Q000235:genetics*; D015870:Gene Expression; D008560:Membrane Fluidity / Q000235:genetics*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D012333:RNA, Messenger / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013230:Stearoyl-CoA Desaturase; D013696:Temperature; D016808:Tetrahymena thermophila / Q000201:enzymology / Q000235:genetics*
D018076:DNA, Complementary; D012333:RNA, Messenger; D044943:Fatty Acid Desaturases; D013230:Stearoyl-CoA Desaturase; C029627:delta-9 fatty acid desaturase