The Detection of Centromere Proteins in Guglena gracilis

In this paper we report our detection of centromere proteins in Euglena gracilis.With Immunofluorescent microscopy stained with Chinese ACA antiserum,in the interphase nucleus of HepⅡ cells (human throat cancer cells) only very minute pots—precentromere could be observed.When Euglena was stained with the same technique,there were only patched within nuclei composed of minute fluorescent spots;no precentromere could be distinguished,although all the nuclei gave positive reaction.Perhaps the distribution of centromere kinetochore proteins within the interphase nucleus of protists was different from that of higher organisms.After sequential selective extractions to prepare nuclerar matrix,the nuclei of Euglena still gave reactions to prepare nuclear matrix,the nuclei of Euglena still gave reaction to ACA serum under immunofluorescent microscope.This fact indicates that the centromere/kinetochore proteins of protists are also tightly combined with nuclear matrix,just as those of higher organisms do.This tight combination with nuclaer matrix was further proved with immnoblotting.The positive band-pattern of Euglena cells was still similar to that of HepⅡ after immunoblotting with Chinese ACA serum,although the band number was slightly fewer.The molecular weights of positive bands indicate that euglenoid has likely possessed the main components of centromere proteins; CENP-A,CENP-B,CENP-C and CENP-D etc.The blotting results with Maca-2 antibody showed that HepⅡ cells gave two bands (80 Kd,stained deely and 120 Kd,stained slightly) Euglena gave three bands 80 Kd,120 Kd,and 150 Kd.The blotting results with ra-ACA-2 antiserum were some what different,although it also recognized human CENP-B protein.All HepⅡ and Euglena gave 50 Kd,60 Kd and 80 Kd band.In the immunoblotting with monoclonal antibody mAb37A5,HepⅡ cells gave two bands very similar to those of CHO cells.Euglena showed two bands,one identical to the tetrahymena band,another being about 55 Kd.