Helitrons, a novel type of mysterious DNA transposons discovered computationally prior to bench work confirmation, are components ubiquitous in most sequenced genomes of various eukaryotes, including plants, animals, and fungi. There is a paucity of empirical evidence to elucidate the mechanism of Helitrons transposition in plants. Here, by constructing several artificial defective Helitron (dHel) reporter systems, we aim to identify the autonomous Helitrons (aHel) in maize genetically and to demonstrate the transposition and repair mechanisms of Helitrons upon the dHel-GFP excision in maize. When crossing with various inbred lines, several transgenic lines produced progeny of segregated, purple-blotched kernels, resulting from a leaky expression of the C1 gene driven by the dHel-interrupted promoter. Transcription analysis indicated that the insertion of different dHels into the C1 promoter or exon would lead to multiple distinct mRNA transcripts corresponding to transgenes in the host genome. Simple excision products and circular intermediates of dHel-GFP transposition have been detected from the leaf tissue of the seedlings in F₁ hybrids of transgenic lines with corresponding c1 tester, although they failed to be detected in all primary transgenic lines. These results revealed the transposition and repair mechanism of Helitrons in maize. It is strongly suggested that this reporter system can detect the genetic activity of autonomic Helitron at the molecular level. Sequence features of dHel itself, together with the flanking regions, impact the excision activity of dHel and the regulation of the dHel on the transcription level of the host gene.

MuDR, the founder member of the Mutator superfamily and its MURA transcripts, has been identified as toxic sequences to Escherichia coli (E. coli), which heavily hindered the elucidation of the biochemical features of MURA transposase and confined the broader application of the Mutator system in other organisms. To harness less constrained systems as alternatives, we attempted to clone TED and Jittery, two recently isolated autonomous Mutator-like elements (MULEs) from maize, respectively. Their full-length transcripts and genomic copies are successfully cloned when the incubation time for bacteria to recover from heat shock is extended appropriately prior to plating. However, during their proliferation in E. coli, TED transformed plasmids are unstable, as evidenced by derivatives from which frameshift, deletion mutations, or IS transposon insertions are readily detected. Our results suggest that neither leaky expression of the transposase nor the presence of terminal inverse repeats (TIRs) are responsible for the cloning barriers, which were once ascribed to the presence of the Shine–Dalgarno-like sequence. Instead, the internal sequence of TED (from 1250 to 2845 bp), especially the exons in this region, was the most likely causer. The findings provide novel insights into the property and function of the Mutator superfamily and shed light on the dissection of toxic effects on cloning from MULEs.