小眼虫的着丝粒蛋白检查

THE DETECTION OF CENTROMERE PROTEINS IN Euglena gracilis

为了从起源与进化的角度考察着丝粒蛋白，我们从比酵母更低等的原生生物着手，检查它们的着丝粒蛋白。另文我们已报道了对四膜虫研究的结果，本文报道的是对小眼虫（Ｅｎｇｌｅｎａｇｒａｃｉｌｉｓ）的检查。我们的结果表明：眼虫细胞核里存在的ＡＣＡ抗原的分子量，包括了用ＡＣＡ血清在高等生物中检出来的ＣＥＮＰ－Ａ、ＣＥＮＰ－Ｂ、ＣＥＮＰ－Ｃ和ＣＥＮＰ－Ｄ这几类基本的着丝粒蛋白组分。作免疫荧光染色后，细胞核呈很强的阳性反应，但分辨不出前着丝粒小点。核仁不被荧光染色，除了核仁外似乎整个核都被染色。这可能反映了抗原的分布在核内不是集中成点状的。经过制备核骨架的程序处理后，细胞核仍为阳性（尽管荧光较弱）。如此看来，眼虫至少已有相当大一部分的ＡＣＡ抗原是与核骨架结合着的。对分离的细胞核作相应的抽提处理后再作免疫印迹检查，得到的阳性反应带也证明了这一点。

In this paper we report our detection of centromere proteins in Euglena gracilis. WithImmunofluorescent microscopy stained with Chinese ACA antiserum, in the interphase nucleus of HepⅡ cells (human throat cancer cells) only very minute pots-precentromere could beobserved. When Euglena was stained with the same technique, there were only patched withinnuclei 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 ofEuglena still gave reactions to prepare nuclear matrix, the nuclei of Euglena still gave reactionto ACA serum under immunofluorescent microscope. This fact indicates that thecentromere/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 furtherproved with immnoblotting.The positive band-pattern of Euglena cells was still similar tothat of HepⅡ after immunoblotting with Chinese ACA serum,although the band numberwas slightly fewer.The molecular weights of positive bands indicate that euglenoid has likelypossessed the main components of centromere proteins; CENP-A,CENP-B, CENP-C andCENP-D etc. The blotting results with mACA-2 antibody showed that HepⅡ cells gave twobands (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 80kD band.In the immunoblofting with monoclonal antibody mAb37AS, HepⅡ cells gave twobands very similar to those of CHO cells. Euglena showed two bands,one identical to thetetrahymena band, another being about 55 kD.'