摘要
用质粒ｐＢＲ３２２的ＤＮＡ酶切片段，长度分别为７５０、３７５和１８６ｂｐ作为外源ＤＮＡ在非洲爪蟾 卵提取物中实现了非细胞体系的核装配（核重建）。将分离纯化得到的 ＰＢＲ３２２ ＤＮＡ酶切后经低熔 点琼脂糖回收ＤＮＡ片段，长度为７５０、３７５和１８６ｂｐ，分别加入到爪蟾卵提取物再生系统中温育， 经ＤＡＰＩ染色、孚尔根染色及电子显微镜观察发现能装配大量的重建核。显微分光光度法研究表 明，ＤＮＡ片段在诱导形成重建核的过程中发生了明显的凝集－会凝集变化。α－３２Ｐ－ｄＣＴＰ掺入重建核 的液闪计数结果表明，ＤＮＡ酶切小分子片段诱导形成的重建核具有较高的ＤＮＡ复制活性，而且复 制活性在一定范围内与加入的ＤＮＡ片段长度呈正相关。实验结果表明，非细胞体系中诱导的核重 建不仅与外源ＤＮＡ的种类无关，与外源ＤＮＡ的长度也没有关系。
Cell-free extracts prepared from activated eggs of Xenopus laevis were able to be used in assembling nuclei around different DNA fragments, of which the length is 750bp. 375bp and 186bp respectively. pBR322 was purified and digested with mixed restriction endonucleases. The digested DNA fragments with different lengths (750bp. 375bp and 186bp) were isolat- ed from low temperature agarose and added to the nuclear reconstitution extracts respective- ly. DAPI stain. Feulgen reaction and electronic microscope showed that even the 186bp DNA fragments could assembly many typical nuclei in cell-free extracts. the scanning results of assembled nuclei by microspectrophotometer demonstrated that DNA fragments under- went obvious change from condensation to decondensation during nuclear reconstitution. In- corporation of α-32P-dCTP into DNA suggested that the reconstituted nuclei were capable of DNA replication and the activity is positively related to the length of DNA fragments in a given extent. It seemed that the nuclear reconstitution in Xenopus cell-free extracts is inde- pendent of not only the resources of exogenous DNA, but also the length of DNA.
出处
《动物学报》
SCIE
CAS
CSCD
1996年第4期428-435,共8页
ACTA ZOOLOGICA SINICA
基金
国家自然科学基金资助项目
关键词
非洲爪蟾
卵提取物
非细胞体系
核装配
DNA
DNA fragments, Xenopus egg extracts, Cell-free system, Nuclear recon- stitution, DNA replication.
