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pEg7, a new Xenopus protein required for mitotic chromosome condensation in egg extracts.

Cubizolles F, Legagneux V, Le Guellec R, Chartrain I, Uzbekov R, Ford C, Le Guellec K - J. Cell Biol. (1998)

Bottom Line: This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation.A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts.Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

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Affiliation: Biologie et Génétique du Développement, CNRS UPR 41, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, France.

ABSTRACT
We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

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pEg7 is a component of the 13S condensin complex. (a)  Immunoprecipitation of XCAP-E was performed using preimmune serum (lane 2) or immune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and probed with anti–XCAP-E (top), anti–XCAP-C  (middle), or anti–pEg7 P1 (bottom). An unfertilized egg was used  as an internal standard (lane 1). (b) Immunoprecipitation of  pEg7 was performed using anti–pEg7 P1 immune serum (lane 2)  or preimmune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and  probed with anti–pEg7 P1, anti–XCAP-E, anti–XCAP-C, and  anti–topoisomerase IIα (top to bottom).
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Figure 6: pEg7 is a component of the 13S condensin complex. (a) Immunoprecipitation of XCAP-E was performed using preimmune serum (lane 2) or immune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and probed with anti–XCAP-E (top), anti–XCAP-C (middle), or anti–pEg7 P1 (bottom). An unfertilized egg was used as an internal standard (lane 1). (b) Immunoprecipitation of pEg7 was performed using anti–pEg7 P1 immune serum (lane 2) or preimmune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and probed with anti–pEg7 P1, anti–XCAP-E, anti–XCAP-C, and anti–topoisomerase IIα (top to bottom).

Mentions: To determine if pEg7 is part of the condensin complex, we carried out immunoprecipitation experiments with anti-XCAP-E antibodies (Fig. 6 a). Immunoprecipitates were resolved by SDS-PAGE. After transfer onto nitrocellulose, the blot was probed with anti–XCAP-E, anti– XCAP-C, and anti–pEg7 P1 antibodies. In addition to XCAP-E and XCAP-C, pEg7 was detected in the immunoprecipitates. Relative to the levels found in total unfertilized eggs extracts, the levels of pEg7 in the immunoprecipitates were lower than those of XCAP-E and XCAP-C. This can be accounted for by the existence of an additional 8S complex that contains only XCAP-E and XCAP-C (Hirano et al., 1997). To confirm that pEg7 is physically bound to both XCAP-E and XCAP-C, an immunoprecipitation experiment was performed using the anti–pEg7 P1 antibody (Fig. 6 b). This produced an immunoprecipitate that contained not only pEg7, but also XCAP-E and XCAP-C. Probing the immunoprecipitate with anti–topoisomerase IIα antibodies showed that this protein was absent from the complex. This is in agreement with the previous observation that topoisomerase IIα, although being physically associated with chromatin (Earnshaw et al., 1985), is not a component of the 13S condensin complex (Hirano et al., 1997). Similar results were observed when immunoprecipitations were made using the anti–pEg7 G antibodies (data not shown).


pEg7, a new Xenopus protein required for mitotic chromosome condensation in egg extracts.

Cubizolles F, Legagneux V, Le Guellec R, Chartrain I, Uzbekov R, Ford C, Le Guellec K - J. Cell Biol. (1998)

pEg7 is a component of the 13S condensin complex. (a)  Immunoprecipitation of XCAP-E was performed using preimmune serum (lane 2) or immune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and probed with anti–XCAP-E (top), anti–XCAP-C  (middle), or anti–pEg7 P1 (bottom). An unfertilized egg was used  as an internal standard (lane 1). (b) Immunoprecipitation of  pEg7 was performed using anti–pEg7 P1 immune serum (lane 2)  or preimmune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and  probed with anti–pEg7 P1, anti–XCAP-E, anti–XCAP-C, and  anti–topoisomerase IIα (top to bottom).
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Figure 6: pEg7 is a component of the 13S condensin complex. (a) Immunoprecipitation of XCAP-E was performed using preimmune serum (lane 2) or immune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and probed with anti–XCAP-E (top), anti–XCAP-C (middle), or anti–pEg7 P1 (bottom). An unfertilized egg was used as an internal standard (lane 1). (b) Immunoprecipitation of pEg7 was performed using anti–pEg7 P1 immune serum (lane 2) or preimmune serum (lane 3). Immunoprecipitates were analyzed on SDS-PAGE, transferred to a nitrocellulose filter, and probed with anti–pEg7 P1, anti–XCAP-E, anti–XCAP-C, and anti–topoisomerase IIα (top to bottom).
Mentions: To determine if pEg7 is part of the condensin complex, we carried out immunoprecipitation experiments with anti-XCAP-E antibodies (Fig. 6 a). Immunoprecipitates were resolved by SDS-PAGE. After transfer onto nitrocellulose, the blot was probed with anti–XCAP-E, anti– XCAP-C, and anti–pEg7 P1 antibodies. In addition to XCAP-E and XCAP-C, pEg7 was detected in the immunoprecipitates. Relative to the levels found in total unfertilized eggs extracts, the levels of pEg7 in the immunoprecipitates were lower than those of XCAP-E and XCAP-C. This can be accounted for by the existence of an additional 8S complex that contains only XCAP-E and XCAP-C (Hirano et al., 1997). To confirm that pEg7 is physically bound to both XCAP-E and XCAP-C, an immunoprecipitation experiment was performed using the anti–pEg7 P1 antibody (Fig. 6 b). This produced an immunoprecipitate that contained not only pEg7, but also XCAP-E and XCAP-C. Probing the immunoprecipitate with anti–topoisomerase IIα antibodies showed that this protein was absent from the complex. This is in agreement with the previous observation that topoisomerase IIα, although being physically associated with chromatin (Earnshaw et al., 1985), is not a component of the 13S condensin complex (Hirano et al., 1997). Similar results were observed when immunoprecipitations were made using the anti–pEg7 G antibodies (data not shown).

Bottom Line: This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation.A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts.Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

View Article: PubMed Central - PubMed

Affiliation: Biologie et Génétique du Développement, CNRS UPR 41, Université de Rennes I, Campus de Beaulieu, 35042 Rennes cedex, France.

ABSTRACT
We have isolated a cDNA, Eg7, corresponding to a Xenopus maternal mRNA, which is polyadenylated in mature oocytes and deadenylated in early embryos. This maternal mRNA encodes a protein, pEg7, whose expression is strongly increased during oocyte maturation. The tissue and cell expression pattern of pEg7 indicates that this protein is only readily detected in cultured cells and germ cells. Immunolocalization in Xenopus cultured cells indicates that pEg7 concentrates onto chromosomes during mitosis. A similar localization of pEg7 is observed when sperm chromatin is allowed to form mitotic chromosomes in cytostatic factor-arrested egg extracts. Incubating these extracts with antibodies directed against two distinct parts of pEg7 provokes a strong inhibition of the condensation and resolution of mitotic chromosomes. Biochemical experiments show that pEg7 associates with Xenopus chromosome-associated polypeptides C and E, two components of the 13S condensin.

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Related in: MedlinePlus