Bernier F, Pallotta D, Lemieux G
Biochim Biophys Acta. 1986 Aug 22;867(4):234-43. doi: 10.1016/0167-4781(86)90039-4.
A cDNA library was constructed using the poly(A)+ RNA extracted from spherulating Physarum polycephalum microplasmodia. This library (740 clones) was screened by differential hybridization with 32P-labeled poly(A)+ RNA from growing plasmodia and developing spherules. The results showed that at least 30% of the clones corresponded to mRNAs expressed specifically in spherulating plasmodia. The 35 spherulation-specific cDNA clones giving the strongest hybridization signals were analysed. From this group, four different sequences complementary to very abundant mRNAs were identified. They each accounted for 1.5% of 4.5% of all the clones in the library and probably represented the most abundant spherulation-specific mRNAs. In addition, four less abundant mRNAs were identified from stage-specific clones giving weaker hybridization signals. These sequences represented individually between 0.3% and 0.7% of the clones in the library. Northern blots showed that these eight different sequences were absent from plasmodia and were most abundant 24-36 h after the induction of spherulation. Similar results were also obtained when spherulation was induced by the addition of a sublethal concentration of ferrous iron ions to the growth medium. Hybridization of the spherule-specific clones to Southern blots of genomic DNA suggested the presence of one copy for each gene.
使用从多核变形虫微原质团形成孢子时提取的聚腺苷酸加尾RNA构建了一个cDNA文库。用来自生长中的原质团和发育中的孢子的32P标记的聚腺苷酸加尾RNA通过差异杂交筛选该文库（740个克隆）。结果表明，至少30%的克隆对应于在形成孢子的原质团中特异性表达的mRNA。对给出最强杂交信号的35个孢子形成特异性cDNA克隆进行了分析。从该组中，鉴定出四个与非常丰富的mRNA互补的不同序列。它们各自占文库中所有克隆的4.5%的1.5%，可能代表了最丰富的孢子形成特异性mRNA。此外，从给出较弱杂交信号的阶段特异性克隆中鉴定出四个丰度较低的mRNA。这些序列分别占文库中克隆的0.3%至0.7%。Northern印迹显示，这八个不同的序列在原质团中不存在，并且在孢子形成诱导后24 - 36小时最为丰富。当通过向生长培养基中添加亚致死浓度的亚铁离子诱导孢子形成时，也获得了类似的结果。孢子特异性克隆与基因组DNA的Southern印迹杂交表明每个基因存在一个拷贝。
