ISOLATION OF CHROMOSOME-ASSOCIATED PROTEINS FROM DROSOPHILA-MELANOGASTER THAT BIND A HUMAN CENTROMERIC DNA-SEQUENCE

• Type: Article in International Scientific Journal
• Date: 1994

Authors

AVIDES, MC (Author) Other; Sunkel, CE (Author) ICBAS

Journal

Title: Journal of Cell Biology

Vol. 127

Pages: 1159-1171

ISSN: 0021-9525

Publisher: Rockefeller University Press

Indexing

ISI Web of Knowledge - 9 Citations

Scopus - 10 Citations

Other information

Authenticus ID: P-001-J8B

DOI: 10.1083/jcb.127.5.1159

Abstract (EN): The molecular mechanism involved in packaging centromeric heterochromatin is still poorly understood. CENP-B, a centromeric protein present in human cells, is thought to be involved in this process. This is a DNA-binding protein that localizes to the central domain of the centromere of human and mouse chromosomes due to its association with the 17-bp CENP-B box sequence. We have designed a biochemical approach to search for functional homologues of CENP-B in Drosophila melanogaster. This strategy relies upon the use of DNA fragments containing the CENP-B box to identify proteins that specifically bind this sequence. Three polypeptides were isolated by nuclear protein extraction, followed by sequential ion exchange columns and DNA affinity chromatography. All three proteins are present in the complex formed after gel retardation with the human alphoid satellite DNA that contains the CENP-B box. Footprinting analysis reveals that the complex occupies both strands of the CENP-B box, although it is still unclear which of the polypeptides actually makes contact with the DNA. Localization of fluorescein-labeled proteins after microinjection into early Drosophila embryos shows that they associate with condensed chromosomes. Immunostaining of embryos with a polyclonal serum made against all three polypeptides also shows chromosomal localization throughout mitosis. During metaphase and anaphase the antigens appear to localize preferentially to centromeric heterochromatin. Immunostaining of neuroblasts chromosome spreads confirmed these results, though some staining of chromosomal arms is also observed. The data strongly suggests that the polypeptides we have identified are chromosomal binding proteins that accumulate mainly at the centromeric heterochromatin. Furthermore, DNA binding assays clearly indicate that they have a high specific affinity for the human CENP-B box. This would suggest that at least one of the three proteins isolated might be a functional homologue of the human CENP-B.

Language: English

Type (Professor's evaluation): Scientific

No. of pages: 13