Transposition of cyanobacterium insertion element ISY100 in Escherichia coli.
The genome of the cyanobacterium Synechocystis sp. strain PCC6803 has nine kinds of insertion sequence (IS) elements, of which ISY100 in 22 copies is the most abundant. A typical ISY100 member is 947 bp long and has imperfect terminal inverted repeat sequences. It has an open reading frame encoding a 282-amino-acid protein that appears to have partial homology with the transposase encoded by a bacterial IS, IS630, indicating that ISY100 belongs to the IS630 family. To determine whether ISY100 has transposition ability, we constructed a plasmid carrying the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible transposase gene at one site and mini-ISY100 with the chloramphenicol resistance gene, substituted for the transposase gene of ISY100, at another site and introduced the plasmid into an Escherichia coli strain already harboring a target plasmid. Mini-ISY100 transposed to the target plasmid in the presence of IPTG at a very high frequency. Mini-ISY100 was inserted into the TA sequence and duplicated it upon transposition, as do IS630 family elements. Moreover, the mini-ISY100-carrying plasmid produced linear molecules of mini-ISY100 with the exact 3' ends of ISY100 and 5' ends lacking two nucleotides of the ISY100 sequence. No bacterial insertion elements have been shown to generate such molecules, whereas the eukaryotic Tc1/mariner family elements, Tc1 and Tc3, which transpose to the TA sequence, have. These findings suggest that ISY100 transposes to a new site through the formation of linear molecules, such as Tc1 and Tc3, by excision. Some Tc1/mariner family elements leave a footprint with an extra sequence at the site of excision. No footprints, however, were detected in the case of ISY100, suggesting that eukaryotes have a system that repairs a double strand break at the site of excision by an end-joining reaction, in which the gap is filled with a sequence of several base pairs, whereas prokaryotes do not have such a system. ISY100 transposes in E. coli, indicating that it transposes without any host factor other than the transposase encoded by itself. Therefore, it may be able to transpose in other biological systems.
Urasaki
Akihiro
A
Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan.
Sekine
Yasuhiko
Y
Ohtsubo
Eiichi
E
D000595:Amino Acid Sequence; D001483:Base Sequence; D000458:Cyanobacteria / Q000235:genetics* / Q000254:growth & development; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017422:Sequence Analysis, DNA; D019895:Transposases
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D019895:Transposases