Localization of mitotic factors on metaphase chromosomes.
Localization of mitotic factors on metaphase chromosomes.
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DOI：
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发表时间：
1982-04
影响因子：
4
通讯作者：
R. C. Adlakha;C. Sahasrabuddhe;D. Wright;W. F. Lindsey;P. Rao
中科院分区：
文献类型：
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作者：
R. C. Adlakha;C. Sahasrabuddhe;D. Wright;W. F. Lindsey;P. Rao
本研究的目的是确定是否有丝分裂因子的HeLa细胞，诱导减数分裂成熟，即萌发囊泡破裂（GVBD）和染色体凝聚，当注射到完全成熟的非洲爪蟾卵母细胞，定位在细胞质中或与中期染色体。通过在低盐低渗缓冲液中溶解有丝分裂HeLa细胞并通过离心分离染色体来制备细胞质提取物。用高盐（0. 2 M-NaCl）缓冲液提取与染色体结合的有丝分裂因子。细胞质和染色体蛋白组分进行了评价，其成熟促进活性（MPA）在非洲爪蟾卵母细胞。本研究的结果表明，细胞质和染色体组分在许多方面是相同的，包括它们诱导GVBD的能力，但染色体组分的比活性至少是细胞质组分的三倍。这些数据表明，有丝分裂因子的主要部分是本地化的中期染色体。这种关联似乎不是由于提取过程中有丝分裂蛋白与染色体的偶然结合。此外，当提取物以类似的方式从早期和中期G2期HeLa细胞制备时，只有核提取物具有MPA，并且在细胞质组分中没有发现活性。晚G2期细胞的细胞质和细胞核提取物表现出MPA。这些数据支持这样的结论，即有丝分裂因子一旦合成就优先结合染色质，并且随着细胞在有丝分裂准备中合成更多的这些因子，越来越多的这些因子保留在细胞质中。
The objective of this study was to determine whether the mitotic factors of HeLa cells, which induce meiotic maturation, i.e. germinal vesicle breakdown (GVBD) and chromosome condensation, when injected into fully grown Xenopus laevis oocytes, were localized in the cytoplasm or associated with the metaphase chromosomes. Cytoplasmic extracts were prepared by lysing mitotic HeLa cells in low-salt hypotonic buffer and separating the chromosomes by centrifugation. Th mitotic factors bound to chromosomes were extracted with high-salt (0.2 M-NaCl) buffer. Both the cytoplasmic and chromosomal protein fractions were evaluated for their maturation-promoting activity (MPA) in the Xenopus oocytes. The results of this study indicate that both the cytoplasmic and chromosomal fractions are identical in many respects, including their ability to induce GVBD, but the specific activity of the chromosomal fraction was at least threefold greater than that of the cytoplasmic fraction. These data suggest that a major portion of the mitotic factors is localized on the metaphase chromosomes. This association does not appear to be due to adventitious binding of mitotic proteins to chromosomes during the extraction procedures. Furthermore, when extracts were prepared in a similar way from early- and mid-G2-phase HeLa cells, only the nuclear extracts had MPA and no activity was found in the cytoplasmic fraction. Both the cytoplasmic and nuclear extracts of late-G2 cells exhibited MPA. These data support the conclusion that the mitotic factors become preferentially bound to chromatin as soon as they are synthesized, and as the cell synthesizes more of these factors in preparation for mitosis, increasing amounts of them are retained in the cytoplasm.