Structure and Expression of the Mitochondrial Citrate Synthase Gene in Carrot Cells Utilizing Al-Phosphate

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We characterized the mitochondrial citrate synthase (mtCS) in selected carrot cells designated as IPG cells (Koyama et al. 1992), which showed a mtCS activity about 1.5 times higher than that of the wild-type (WT) cells. Since the CS in both cell types was completely inhibited by the addition of polyclonal antibodies raised against carrot mtCS, we assumed that the higher CS activity in the IPG cells was caused by the increase of the mtCS activity. Using a partial DNA sequence of mtCS cDNA isolated from cDNA libraries of carrot, both 3' and 5' regions were obtained by rapid amplification of cDNA ends (RACE). Thereafter, full-size cDNA encoding mtCS was isolated from both IPG and WT cells. There was no difference between the DNA sequences of the full-size cDNA from the two cell types. The transcript level determined by quantitative PCR analysis for mRNA was about two times higher in the IPG cells than in the WT cells. Southern hybridization analysis indicated that both cell types contained a single copy of the mtCS gene. It was concluded that the selected IPG cells showed an alteration in mtCS mRNA levels, and displayed a higher CS activity.