A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts.
Proc Natl Acad Sci U S A
; 94(23): 12378-83, 1997 Nov 11.
Article
em En
| MEDLINE
| ID: mdl-9356457
ABSTRACT
Kinetochores are complex macromolecular structures that link mitotic chromosomes to spindle microtubules. Although a small number of kinetochore components have been identified, including the kinesins CENP-E and XKCM1 as well as cytoplasmic dynein, neither how these and other proteins are organized to produce a kinetochore nor their exact functions within this structure are understood. For this reason, we have developed an assay that allows kinetochore components to assemble onto discrete foci on in vitro-condensed chromosomes. The source of the kinetochore components is a clarified cell extract from Xenopus eggs that can be fractionated or immunodepleted of individual proteins. Kinetochore assembly in these clarified extracts requires preincubating the substrate sperm nuclei in an extract under low ATP conditions. Immunodepletion of XKCM1 from the extracts prevents the localization of kinetochore-associated XKCM1 without affecting the targeting of CENP-E and cytoplasmic dynein or the binding of monomeric tubulin to the kinetochore. Extension of this assay for the analysis of other components should help to dissect the protein-protein interactions involved in kinetochore assembly and function.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Oócitos
/
Cromossomos
/
Cinetocoros
Limite:
Animals
Idioma:
En
Revista:
Proc Natl Acad Sci U S A
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos