Cloning of a cDNA for rape chloroplast 3-isopropylmalate dehydrogenase by genetic complementation in yeast.
Both insect and mammalian genes have previously been cloned by genetic complementation in yeast. In the present report, we show that the method can be applied also to plants. Thus, we have cloned a rape cDNA for 3-isopropylmalate dehydrogenase (IMDH) by complementation of a yeast leu2 mutation. The cDNA encodes a 52 kDA protein which has a putative chloroplast transit peptide. The in vitro made protein is imported into chloroplasts, concomitantly with a proteolytic cleavage. We conclude that the rape cDNA encodes a chloroplast IMDH. However, Southern analysis revealed that the corresponding gene is nuclear. In a comparison of IMDH sequences from various species, we found that the rape IMDH is more similar to bacterial than to eukaryotic proteins. This suggests that the rape gene could be of chloroplast origin, but has moved to the nucleus during evolution.
Department of Cell Research, Swedish University of Agricultural Sciences, Uppsala.
M
M
Ellerström
L G
LG
Josefsson
L
L
Rask
H
H
Ronne
D050539:3-Isopropylmalate Dehydrogenase; D000429:Alcohol Oxidoreductases / Q000235:genetics*; D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D015139:Blotting, Southern; D001937:Brassica / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D005658:Fungi / Q000235:genetics; D015723:Gene Library; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D010802:Phylogeny; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid
D012313:RNA; D004247:DNA; D000429:Alcohol Oxidoreductases; D050539:3-Isopropylmalate Dehydrogenase