Eukaryotic cells have evolved a mechanism that delays the onset of anaphase until chromosomes are properly positioned on the spindle. To understand the molecular basis of such surveillance mechanism in human cells, we have cloned a full-length cDNA encoding a putative mitotic checkpoint kinase termed hBub1. Sequence comparison reveals that hBub1 is a structurally conserved protein, sharing 23% amino acid residue identity with BUB1 of budding yeast. In addition, the NH2-terminal portion (161 amino acids) of hBub1 shows a significant homology to yeast MAD3, a protein also known to be involved in the mitotic checkpoint response pathway. Northern blot analyses show that the hBub1 mRNA level is abundantly expressed in tissues or cells with a high mitotic index. When Dami cells undergo terminal differentiation after treatment with phorbol ester, hBub1 expression in this cell line is down-regulated rapidly. The hBub1 protein level is low in G1 and remains relatively constant in S, G2, and M phases. Immunofluorescence analysis shows that hBub1 protein colocalizes with a centromere-kinetochore antigen CREST in interphase, mitotic prophase, and nocodazole-treated cells. Antibody electroporation experiments show that hBub1 is an important component of the spindle checkpoint pathway. Furthermore, fluorescence in situ hybridization analysis maps the hBub1 gene to chromosome 2q12-13. Our studies suggest that hBub1 expression is restricted to proliferating cells and appears to be involved in regulating cell cycle progression. The molecular cloning of hBub1 cDNA will facilitate the study of its role in spindle checkpoint control as well as its potential role in certain genetic disorders.

• PMID: 9790499
• PubMed

Reference Type

Journal Article | Research Support, U.S. Gov't, P.H.S.

Authors

Ouyang B, Lan Z, Meadows J, Pan H, Fukasawa K, Li W, Dai W

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