The proliferation-associated nuclear protein Ki-67 in the bovine system: partial characterisation and its application for determination of the proliferation of Theileria-infected bovine cells.
Theileria annulata-infected bovine cells as well as mitogen-stimulated bovine peripheral blood mononuclear cells (PBMC) express a proliferation-associated nuclear protein equivalent to the human Ki-67 protein. In analogy to the human system, the expression of the bovine Ki-67 protein is restricted to proliferating cells only, since (a) Ki-67 expression paralleled [3H]-thymidine incorporation in concanavalin A (Con A)-stimulated bovine PBMC, (b) Ki-67 was not detectable in quiescent bovine cells, and (c) Ki-67 expression in Theileria-infected cells is related to the presence of the parasites within the cytoplasm of the host cells; upon treatment with the theilericidal drug buparvaquone the parasites are destroyed and the cells cease to proliferate and to express the Ki-67 protein. Western-blot analysis of lysates of proliferating bovine cells revealed that the prototype monoclonal antibody Ki-67 and the new equivalent antibody MIB-1 detected one prominent protein band with an apparent molecular weight of 430 kDa. Two cDNA clones (pUC18.B1.Ki-67 and pUC18.B2.Ki-67) were isolated from a lambdagt11 cDNA library of T. annulata-infected bovine cells by immunoscreening with the monoclonal antibody MIB-1. Comparison of these cDNA sequences with those of the human Ki-67 protein revealed 60-70% identity. Within the "Ki-67 motif", identity proved to be 80% at the amino acid level. The remarkable identity between bovine and human Ki-67 proteins suggests that MIB-1 can be used as a marker for cell proliferation in animal research. In this context we could identify proliferating cells in lymph nodes of Theileria-infected animals and, furthermore, we could distinguish between infected and uninfected proliferating cells using MIB-1 and an antiserum against a recombinant parasite protein designated SA288.
Shayan
P
P
Forschungszentrum Borstel, Germany.
Gerlach
C
C
Hügel
F U
FU
Kay
G
G
Campbell
J D
JD
Gerdes
J
J
Ahmed
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002455:Cell Division; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006801:Humans; D007124:Immunoenzyme Techniques; D019394:Ki-67 Antigen / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000469:parasitology; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D017386:Sequence Homology, Amino Acid; D016796:Theileria annulata / Q000502:physiology*
D018076:DNA, Complementary; D019394:Ki-67 Antigen