The Responses of Archezoa and Archaebacteria to The Antibodies Against Mammalian Centromere/Kinetochore Proteins

To understand the structure and function of the centromere/kinetochore thoroughly,we need information on its origin and evolution,since every living organization has its own origin and evolutionary history.We analyzed the protein components of the centromere/kinetochore in one archezoa,three archeabacteria and one eubacterium by western blotting with the human centromere protein B (CENP-B) monoclonal antibody,mACA-2,which recognizes the amino end region of human CENP-B; CENP-B polyclonal antibody ra-ACA-2; and mAb37A5,a monoclonal antibody against a peptide of the kinetochore in CHO cells.Giardia lamblia was used as a representative of archezoa,which possess no mitochondria and has 70S prokaryotic ribosomes and therefore is considered the most primitive eukaryotic cell.Three archeabacteria,Halobacterium dachaidenesis sp.nov.F3,Sulfosphaerellus thermoacidophilum S-5,and Thermoplasma acidophilum were used as the representatives of each existing group of archeabacteria—methanogenous-halophilic group,sulfur-dependent thermophilic group and thermoplasma group respectively.E.coli was used the representative of eubacteria.On western blots using the CENP-B monoclonal antibody,mACA-2,Giardia lamblia reacted an 80 kD band that is similar to one of the three bands tound in other protists such as Euglena gracilis and Oxyrris marina (Wu et al.,1996c).Three archeabacteria and E.coli gave negative response to mACA-2 antibody.The CENP-B polyclonal antibody,ra-ACA-2,recognized similar 60 kD and 30 kD bands in Giardia lamblia and the three archeabacteria.The other protists tested also exhibited the two bands,and an additional 50 kD band (Wu et al.,1995a-c).The reference E.coli gave 60 kD band and two bands about 80 kD which were different from those of either archeabacteria or giardia.Western blots of G.lamblias extracts using the kinetochore protein monoclonal antibody,mAb37A5,Giardia lamblia gave 45 kD,50 kD and 120 kD bands,of which the 45 kD and 120 kD bands were also found in other tested protists (Wu et al.,1994).Two archeabacteria,Halobacterium and Sulfosphaerellus,produced 40 kD,45 kD,and 50 kD bands.Another archeabacterium Thermoplasma gave a 35 kD band besides those three.Eubacterium E.coli did not give any positive response.From the results,we can suggest a evolutionary line of mAb37A5 antigen from archeabacteria to protists:Thermoplasma,35 kD,40 kD,45 kD and 50 kD;Halobacterium and Sulfospherellus,40 kD,45 kD and 50 kD;Giardia,45 kD,50 kD and 120 kD;Euglena and Oxyrris,45 kD and 120 kD (Wu et al.,1994).Our results support the hypothesis that the eukaryotic cell evoloved from an ancient archeabacterium,and the nucleus of Giardia is indeed one of the most primitive of the eukaryotic nucleus.Therefore it is in the intermediate position between archeabacteria and eukaryotic cells.Our results suggest that although the centromere/kinetochore is a characteristic structure peculiar to eukaryotic cells,its protein molecular ancestors originated in the archeabacterial ancestor of dukaryotic cells.The differentiation of CENP-Bs N-terminal structure most have occurred after the archeabacterial stage.Acknowledegements:We would like to thank Prof.Dr.Dennis G.Searcy of Univ.of Massachusetts,USA,for giving Thermoplasma acidophilum;Prof.Dr.William C.Earnshaw of Johns Hopkins Univ.School of Medicine,USA,for giving mACA-2 and ra-ACA-2 antibodies;Prof.Dr.Ronald Hancock of Laval Univ.,Canada,for giving mAb37A5 antibody;and Prof.Dr.Laurence D.Etkin of the Department of Molecular Genetics,UTMDACC,USA,for the language.improvement of English manuscript.