Molecular cloning and analysis of mutable genes in Japanese Morning Glory.

许多高等植物有表型变异。遗传易变性包括在这类表型中。日本科学家对日本牵牛花和四点钟花进行了广泛的研究。在这项研究中，作者旨在分离和分析日本牵牛花可能的可变基因。第一步是从植物中纯化高分子量的DNA。采用两种方法分离基因；一个是研究牵牛花菌株之间的遗传多态性。二是检测携带茎环结构的DNA片段。通过南方杂交，我发现了不同园艺品系的DNA样本中存在许多多态性。然而，在变异和多态模式之间没有发现相关性。单次插入事件很难推断出一个多态性模式。接下来，我试图通过检测转座子特有的茎和环结构来分离转座子样DNA片段。牵牛花突变体的DNA被DNA酶I消化并变性。单链DNA在分子内形成双链的条件下再退火。单链DNA片段由单链特异性核酸酶消化。将得到的双链DNA克隆到dG-tailed质粒pUC9中。一个由携带克隆转座子样DNA的嵌合质粒组成的池。在检查插入片段的长度和拷贝数后，对具有代表性的5个克隆进行测序。检测到一个倒置的重复序列和片段，显示出菌株间的多态性，与Tam1 （snap dragon的转座子）的茎有60%的同源性。因此，后一种方法被证明是有效的。我将从两个方面进一步研究；多态性和可移动性。

Many higher plants have phenotypic variegations. The genetic mutability is included in this class of phenotypes. Japanese scientists have studied extensively with Japanese Morning Glory and the four o'clock plant. In this study, the author is aiming to isolate and analyse possible mutable genes from Japanese Morning Glory. The first step was to purify higher molecular weight DNA from the plant. Two methods were emplyed for isolation of the genes; one is to examine genetic polymorphisms among strains of Morning Glory. The other is to detect DNA fragments carrying the structure of stem and loop. I found many polymorphisms among DNA samples of various horticultural strains by Southern hybridization. However, no correlation was detected between the mutability and the polymorphic pattern. one polymorphic pattern was difficult to be concluded by single insertional event.Next, I tried to isolate transposon-like DNA fragments by the detection of stem and loop structures which are characteristic of transposons. The DNA of Morning Glory mutants was digested by DNase I and was denatured. The single stranded DNA was reannealed under the condition that makes intramolecular double strands. The fraction of single stranded DNA was digested by single strand specific nuclease. The resulted double stranded DNA was cloned into dG-tailed plasmid pUC9. A pool consisted of chimeric plasmids carrying cloned trasposon-like DNA. After the examination of the length and copy number of the inserts, representative five clones were sequenced. I detected an inverted repetitive sequence and the fragments that showed polymorphism among strains and that had about 60% homology to the stem of Tam1 a transposon of snap dragon).Accordingly, the latter method was shown to be effective. I will further this study from two points; polymorphism and movability.