Cloning of cDNAs encoding XXXX dehydrogenase-2a and b, enzymatic characterization, and up-regulated expression of the genes in @entity12638 diapause eggs exposed to 5   C.

@entity535 @entity5121 @entity12638 @entity72616 @entity72617 @entity526
We previously cloned a cDNA for @entity5121 dehydrogenase (SDH1) from @entity12638 . In the present study we cloned two additional cDNAs encoding SDHs (designated as SDH2a and @entity72616 ). The @entity535 sequences of SDH2ab were almost the same and had higher similarity to the SDHs of other organisms than to @entity12638 SDH1. The SDH2ab and SDH1genes were located in tandem within about 40 kbp on chromosome 21. SDH2ab mRNAs increased after exposing diapause eggs to 5   C for 40 days, beginning at 2 days post-oviposition, to break diapause. However, they were at very low levels in diapausing eggs incubated at 25   C continuously from oviposition. These changes in expression pattern of SDH2ab mRNA were almost the same as that of SDH1 mRNA. To understand whether SDH1 and SDH2 were responsible for the @entity72617 activity seen in diapause eggs exposed to 5   C for more than 60 days, we expressed a His-tagged SDH2a fusion protein in @entity526 and examined its enzymatic parameters. The maximum activity of SDH2a observed at pH 8.4   9.0, and the Km value for @entity5121 was 12.6 mM, similar to the kinetic properties of other SDHs. Due to the significantly higher similarity between SDH2a and b, they were thought to have similar kinetic properties. Therefore, we purified @entity72617 from @entity12638 diapause-terminated eggs exposed to 5   C for 300 days which showed higher @entity72617 activity using two-step affinity chromatography. The highly purified @entity72617 showed a higher Km value (125 mM) for @entity5121 , being similar to the value (136 mM) determined previously from Eadie-Hofstee plots using egg crude extract as an enzyme source; additionally, the plots showed one slope indicating one Km value. Moreover, in silico analysis indicated that no @entity72617 genes other than SDH1 and 2ab are present in @entity12638 genomic DNA. These results suggest that SDH1 activity may be responsible for the majority of the increased @entity72617 activity seen in diapause eggs after acclimation to 5   C rather than SDH2ab. Further, the relative sequence divergence among these genes is consistent with the idea/hypothesis that the original @entity72617 gene was first duplicated into SDH1 and SDH2, and then SDH2 was duplicated into the SDH2a and @entity72616 genes.
