The occurrence of hypertetraploid and other unusual polyploid loaches Misgurnus anguillicaudatus among market specimens in Japan

Exotic animals may cause a genetic contamination of indigenous species if they escape and reproduce in wild populations. Loach Misgurnus anguillicaudatus and its related species have been imported to Japan for commercial uses. We collected live loach specimens from central wholesale market in Tokyo. Among 451 specimens, ploidy status was examined by DNA content flow cytometry and polyploid loaches with triploid, tetraploid and other higher DNA content ranges were detected. Hyper-triploid and hyper-tetraploid individuals could be easily detected by flow cytometry using a standard eudiploid, eutriploid and eutetraploid controls and then reproductive capacity of these hyper-polyploid males was examined. Sperm of hyper-triploid males did not exhibit active progressive motility and major populations of spermatozoa or spermatozoon-like cells were detected in triploid and hexaploid ranges. Motile haploid spermatozoa were very few in sperm from hyper-triploid males. Therefore, hyper-triploid males were sterile, whereas hyper-tetraploid males produced fertile hyper-diploid spermatozoa with active progressive motility after contact with ambient water. Viable progeny occurred in the cross between normal wild-type diploid female and hyper-tetraploid males, but androgenotes induced by fertilization of UV-irradiated eggs with sperm of hyper-tetraploid males were inviable hyper-diploid. Cytogenetic analyses in such androgenotes indicated that hyper-tetraploid males should produce hyper-diploid spermatozoa with 2n=54, i.e. presence of four supernumerary micro-chromosomes

Lantibiotic Transporter Requires Cooperative Functioning of the Peptidase Domain and the ATP Binding Domain*

Lantibiotics are ribosomally synthesized and post-translationally modified peptide antibiotics that contain unusual amino acids such as dehydro and lanthionine residues. Nukacin ISK-1 is a class II lantibiotic, whose precursor peptide (NukA) is modified by NukM to form modified NukA. ATP-binding cassette (ABC) transporter NukT is predicted to cleave off the N-terminal leader peptide of modified NukA and secrete the mature peptide. Multiple sequence alignments revealed that NukT has an N-terminal peptidase domain (PEP) and a C-terminal ATP binding domain (ABD). Previously, in vitro reconstitution of NukT has revealed that NukT peptidase activity depends on ATP hydrolysis. Here, we constructed a series of NukT mutants and investigated their transport activity in vivo and peptidase activity in vitro. Most of the mutations of the conserved residues of PEP or ABD resulted in failure of nukacin ISK-1 production and accumulation of modified NukA inside the cells. NukT(N106D) was found to be the only mutant capable of producing nukacin ISK-1. Asn106 is conserved as Asp in other related ABC transporters. Additionally, an in vitro peptidase assay of NukT mutants demonstrated that PEP is on the cytosolic side and all of the ABD mutants as well as PEP (with the exception of NukT(N106D)) did not have peptidase activity in vitro. Taken together, these observations suggest that the leader peptide is cleaved off inside the cells before peptide secretion; both PEP and ABD are important for NukT peptidase activity, and cooperation between these two domains inside the cells is indispensable for proper functioning of NukT