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Molecular cloning of delta 9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation.

ABSTRACT: 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.

SUBMITTER: Nakashima S

PROVIDER: S-EPMC1217476 | biostudies-other | 1996 Jul

REPOSITORIES: biostudies-other

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Project description:BACKGROUND: The ciliated protozoan Tetrahymena thermophila is a well-studied single-celled eukaryote model organism for cellular and molecular biology. However, the lack of extensive T. thermophila cDNA libraries or a large expressed sequence tag (EST) database limited the quality of the original genome annotation. METHODOLOGY/PRINCIPAL FINDINGS: This RNA-seq study describes the first deep sequencing analysis of the T. thermophila transcriptome during the three major stages of the life cycle: growth, starvation and conjugation. Uniquely mapped reads covered more than 96% of the 24,725 predicted gene models in the somatic genome. More than 1,000 new transcribed regions were identified. The great dynamic range of RNA-seq allowed detection of a nearly six order-of-magnitude range of measurable gene expression orchestrated by this cell. RNA-seq also allowed the first prediction of transcript untranslated regions (UTRs) and an updated (larger) size estimate of the T. thermophila transcriptome: 57 Mb, or about 55% of the somatic genome. Our study identified nearly 1,500 alternative splicing (AS) events distributed over 5.2% of T. thermophila genes. This percentage represents a two order-of-magnitude increase over previous EST-based estimates in Tetrahymena. Evidence of stage-specific regulation of alternative splicing was also obtained. Finally, our study allowed us to completely confirm about 26.8% of the genes originally predicted by the gene finder, to correct coding sequence boundaries and intron-exon junctions for about a third, and to reassign microarray probes and correct earlier microarray data. CONCLUSIONS/SIGNIFICANCE: RNA-seq data significantly improve the genome annotation and provide a fully comprehensive view of the global transcriptome of T. thermophila. To our knowledge, 5.2% of T. thermophila genes with AS is the highest percentage of genes showing AS reported in a unicellular eukaryote. Tetrahymena thus becomes an excellent unicellular model eukaryote in which to investigate mechanisms of alternative splicing.

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Molecular cloning of delta 9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation.

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