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

Tpnl家族是一个与En/ spm相关的转座因子，是日本牵牛花的主要诱变因子。该Tpnl家族的结构特征如下：每一端有28bp的末端倒置重复序列（terminal inverted repeat， TIR）和一个亚末端区域，包括内部高度重复的序列。在这些末端序列之间，有来自宿主基因的群体特异性序列。例如，它们是肌凝蛋白，-半乳糖苷酶和AP2B。通过对Tpn插入突变的分子和表型分析，认为这些非自主的Tpns是由一个已识别的自主元件编码转座酶调动的。为了分离出自主Tpn元件，利用其他En/ spm相关转座元件的保守基序设计了一些引物对。利用这些引物和变异菌株的mRNA进行RT-PCR扩增出部分同源片段。由于自主Tpn元件被认为具有更大的尺寸，因此构建了一个基因组DNA库，并使用RT-PCR产物作为探针进行筛选。虽然部分克隆携带En/ spm相关序列，但没有克隆含有指定Tpnl家族的亚末端序列。有一些Tpn群携带较长的orf，与其他已知基因无同源性。因此，这些元素中的一组被认为是自治元素的删除类型。我现在正在使用这些Tpns的内部探针寻找更长的Tpn元素。另一种策略也被用于比较Ipomoea物种之间的tppn相关序列，因为自主元件被认为存在于所有其他兄弟物种中，但正常的Tpn捕获宿主基因可能在相对较短的时间内重复产生和消除循环。

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.