Analysis of autonomous element of Tpnl family in the Japanese morning glory

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Tpnl family, an En/Spm-related transposable element is a major mutagen of the Japanese morning glory (1pomoea nil). Structural feature of this Tpnl family is the following : Each end has 28bp of terminal inverted repeat (TIR) and a subterminal region including highly repetitive sequences of the inner side. Between these terminal sequences, there are group-specific sequences derived from host genes. For example, they are myosin, beta-galactosidase, and AP2B. By the molecular and phenotypic analysis of Tpn insertion mutations, it is thought that these non-autonomous Tpns are mobilized by an identified autonomous element coding transposase. In order to isolate autonomous Tpn element, some pairs of primers were designed using conserved motif of other En/Spm-related transposable element. Some homologous fragments were amplified using these primers and mRNA of mutable strains by the RT-PCR experiment. Since autonomous Tpn elements were thought to have more larger size, a cosmid library of genomic DNA was constructed and it was screened using RT-PCR products as probes. Although some clones isolated carried En/Spm-related sequences, no clone contain subterminal sequences which specify Tpnl family. There are some Tpn groups carrying longer ORFs which have no homology with other known genes. Therefore, one group of these elements is thought to be a deleted type from autonomous element. I am now searching for longer Tpn element using internal probe of these Tpns. Another strategy is also employed to compare Tpnl-related sequences among Ipomoea species since autonomous elements are thought to exist in all other sibling species, but normal Tpn capturing host genes might repeated generation-and-elimination cycle in relatively short period.