Biochemistry/Biophysics Program, Washington State University, 99164-4660, Pullman, WA, USA.
Photosynth Res. 1988 Jan;19(1-2):23-37. doi: 10.1007/BF00114567.
Discoveries of the uptake and expression of various Escherichia coli plasmids by the cyanobacterium Anacystis nidulans and isolated cumber etioplasts are reviewed. In particular, the binding and uptake of nick-translated (32)P-labeled plasmids and the expression of genes in the native plasmids are considered.Permeaplasts of A. nidulans 6301 and isolated EDTA-washed cucumber etioplasts exhibit binding and uptake of DNA that is unaffected by uncouplers of photophosphorylation or by dissipators of transmembrane proton graident. ATP inhibits both binding and udptake by permeaplasts or EDTA-washed etioplasts but the analog AMP-PNP (non-hydrolzable) is noninhibitory. With permeaplasts there is no effect of 20 mM Mg(2+) (in the light) upon intake, whereas with EDTA-washed etioplasts, Mg(2+) at the same concentration inhibits uptake as does 20 mM Ca(2+).The transformation of A. nidulans 6301 to ampicillin-resistance by the plasmid pBR322 is much enhanced in permeaplasts. Indeed extracts of transformed cells catalyze the hydrolosis of the β-lactam nitrocefin. Transfromation of A. nidulans to antibiotic resistance may also be achieved with the plasmids pHUB4 and pCH1. The effect of light on transformation of A. nidulans 6301 differs with different plasmids. In pBR322 transformants the expression of ribulose bisphosphate carboxylase-oxygenase (RuBisCO) is markedly elevated. In these transformants, the foreign plasmid replicates by a pathway involving chromosomal integration and dissociation.The plasmid pCS75, a derivative of pUC9 (and therefore of pBR322) containing a Pst1 insert carrying genes for the large and small (S) subunits of RuBisCO from A. nidulans, is taken up and expressed in EDTA-washed cucumber cotyledon etioplasts. Expression is evidenced by the hydrolysis of nitrocefin and immunoprecipitation of labeled S subunits of RuBisCO (utilizing etioplasts which have been labeled with (35)S-methionine after incubation with pCS75). The plasmid pUC9-CM carrying a cat gene is also expressed in cucumber etioplasts in a manner that demonstrates dependence both on the duration of etioplast washing by EDTA and plasmid concentration. Translation (as measured by (35)S-methionine incorporation) by EDTA-washed etioplasts increases with cotyledon greening. However the enhancement of translation by prior incubation of EDTA-washed plastids with pCS75 decreases to zero during 24hr of cotyledon greening. Results suggest that the expression of foreign DNA in plastids may depend critically upon their developmental state.
本文回顾了蓝藻鱼腥藻和分离的黄化质体对各种大肠杆菌质粒的摄取和表达的研究进展。特别地，考虑了用（32）P 标记的 Nick 翻译质粒的结合和摄取以及天然质粒中基因的表达。鱼腥藻 6301 的渗透质体和分离的 EDTA 洗涤的黄化质体表现出对 DNA 的结合和摄取，该结合和摄取不受光合磷酸化解偶联剂或跨膜质子梯度耗散剂的影响。ATP 抑制渗透质体或 EDTA 洗涤的黄化质体的结合和 udptake，但类似物 AMP-PNP（不可水解）是非抑制性的。对于渗透质体，20 mM Mg2+（在光下）对摄取没有影响，而对于 EDTA 洗涤的质体，相同浓度的 Mg2+ 会抑制摄取，Ca2+ 也会抑制摄取。用质粒 pBR322 将鱼腥藻 6301 转化为氨苄青霉素抗性的效率在渗透质体中大大提高。事实上，转化细胞的提取物催化β-内酰胺硝基头孢菌素的水解。通过质粒 pHUB4 和 pCH1 也可以实现鱼腥藻对抗生素的抗性转化。不同质粒对鱼腥藻 6301 转化的光效应不同。在 pBR322 转化体中，核酮糖二磷酸羧化酶-加氧酶（RuBisCO）的表达显著升高。在这些转化体中，外源质粒通过涉及染色体整合和解离的途径复制。质粒 pCS75 是 pUC9（因此是 pBR322）的衍生物，包含一个 Pst1 插入物，该插入物携带来自鱼腥藻的 RuBisCO 的大亚基和小亚基（S）亚基的基因，被摄取并在 EDTA 洗涤的黄化黄瓜子叶质体中表达。表达通过硝基头孢菌素的水解和 RuBisCO 的标记 S 亚基的免疫沉淀来证明（利用在用 pCS75 孵育后用（35）S-甲硫氨酸标记的质体）。携带 cat 基因的质粒 pUC9-CM 也以依赖于 EDTA 洗涤质体的持续时间和质粒浓度的方式在黄瓜质体中表达。EDTA 洗涤的质体的翻译（通过（35）S-甲硫氨酸掺入来测量）随着子叶的变绿而增加。然而，在用 pCS75 预先孵育 EDTA 洗涤的质体期间，翻译的增强在 24 小时的子叶变绿过程中降低到零。结果表明，质体中外源 DNA 的表达可能严重依赖于其发育状态。
