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

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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.