| 摘要: |
| 于1982年12月—1984年5月，以青岛汇泉湾习见的马粪海胆为材料，运用0.5 mol/L KCl或0.5 mol/L LiCl诱导法得到其卵母细胞；差速离心技术提取其线粒体；SDS萃取到线垃体核酸；再以羟基磷庆石柱层析分离出线粒体核酸各组分。结果表明，从所得各种分离产物测不出任何蛋白质。所以，应用上述各种方法和技术，能够有效地分离出海胆卵母细胞线粒体中的DNA，rRNA，tRNA和多核苷酸。 |
| 关键词: 马粪海胆 卵母细胞 线粒体 差速离心 羟基磷灰石柱层析 核酸 |
| DOI： |
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| THE ISOLATION OF MITOCHONDRIAL DNA AND RNAs FROM SEA URCHIN HEMICENTROTUS PULCHERRIMUS OOCYTES |
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Yu Fucai, Ma Li
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Institute of Oceanology, Academia Sinica, Qingdao 266071
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| Abstract: |
| The common sea urchin, Hemicentrotus pulcherrimus from the Qingdao coastal sea was used in the experiment carried out during 1982 - 1984. Oocytes were obtained by using 0.5 mol/L KCl or LiCl stimulation. Oocyte mitochondria was isolated from oocytes with differential centrifugal technique and the nucleic acid was extracted with sodium dodecyl sulfate from mitochondria, and then DNA and RNAs were separated when phosphate buffer of different concentrations were used to elute nucleic acid of mitochondria on the chromatographic column of hydroxyapatite.
Many oocytes were ovulated from sea urchins when they were stimulated with K+ or Li+ while placed aboral surface down on a beaker of 0.5 mol/L KCI or 0.5 mol/L LiC1 solution. The oocytes isolated in this way did not contain the cytoplasmic lags and adherent clumps of cytoplasm. The total nucleic acid was 3 385 μg per gram of oocyte mitochondria in early development oocyte. The fraction eluted with 0.25 mol/L phosphate buffer corresponds to mitochondrial DNA, rRNA with 0.15 mol/L phosphate buffer and eluted peak of tRNA appeared in 0.1 mol/L as a single peak before rRNA. Ultraviolet spectral analysis revealed that DNA UV absorption in ratio of A260/A280 was between 1.96 - 1.98, 2.08 - 2.17 for rRNA, 2.00 - 2.17 for tRNA and 0.48 - 0.50 for DNA, 0.49 - 0.51 for rRNA, 0.50 - 0.52 for tRNA in A230/A260. The biochemical analysis showed that these products were free of proteins. The above findings indicated that the procedures are useful for the separation of DNA and various RAN. |
| Key words: Hemicentrotus pulcherrimus, Oocyte, Mitochondria, Differential centrifugation, Hydroxyapatite chromatography column, Nucleic acids |
