| Co-Investigator(Kenkyū-buntansha) |
MATSUKI Naoaki The University of Tokyo, Graduate School of Agricultural and Life Sciences, Inst, 大学院・農学生命科学研究科, 助手 (40251417)
DOI Kunio The University of Tokyo, Graduate School of Agricultural and Life Sciences, Prof, 大学院・農学生命科学研究科, 教授 (70155612)
ONO Ken-ichiro The University of Tokyo, Graduate School of Agricultural and Life Sciences, Prof, 大学院・農学生命科学研究科, 教授 (50111480)
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| Research Abstract |
We studied bovine subjects that exhibited a moderate uncompensated anemia with hereditary spherocytosis inherited in an autosomal incompletely dominant mode and retarded growth. Based on the results of SDS-PAGE,immunoblotting, and electron microscopic analysis by the freeze fracture method, we show here that the proband red cells lacked the band 3 protein completely. To elucidate the molecular basis of this aberrant deficiency of the band 3 protein, we cloned red cell band 3 cDNA (-3.5kb) from normal and the affected animal. Sequence analysis of the proband band 3 cDNA and genomic DNA showed a C * T substitution resulting in a nonsense mutation (CGA * TGA ; Arg * Stop) at the position of Arg^<664> that corresponded to codon 646 in human red cell band 3 cDNA.The proband red cells were deficient in spectrin, ankyrin, actin, and protein 4.2, resulting in a distorted and disrupted membrane skeletal network with decreased density. Therefore, the proband red cell membranes were extremely unstable and showed the loss of surface area in several distinct ways such as invagination, vesiculation, and extrusion of microvesicles, leading to the formation of spherocytes. Total dificiency of band 3 also resulted in defective CL^-/HCO_3^- exchange, causing mild acidosis with decreases in the HCO_3^- concentration and total CO_2 in the proband blood. Our results demonstrate that band 3 indeed contributes to red cell membrane stability, CO_2 transport, and acid-base homeostasis, but is not always essential to survival of this mammal.
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