Protein knockdown using the auxin-inducible degron (AID) technology is useful to study protein function in living cells because it induces rapid depletion, which makes it possible to observe an immediate phenotype. However, the current AID system has two major drawbacks: leaky degradation and the requirement for a high dose of auxin. These negative features make it difficult to control precisely the expression level of a protein of interest in living cells and to apply this method to mice. Here, we overcome these problems by taking advantage of a bump-and-hole approach to establish the AID version 2 (AID2) system. AID2, which employs an OsTIR1(F74G) mutant and a ligand, 5-Ph-IAA, shows no detectable leaky degradation, requires a 670-times lower ligand concentration, and achieves even quicker degradation than the conventional AID. We demonstrate successful generation of human cell mutants for genes that were previously difficult to deal with, and show that AID2 achieves rapid target depletion not only in yeast and mammalian cells, but also in mice.

A significant feature of the genomes of Lepidoptera, butterflies and moths, is the high conservation of chromosome organization. Recent remarkable progress in genome sequencing of Lepidoptera has revealed that syntenic gene order is extensively conserved across phylogenetically distant species. The ancestral karyotype of Lepidoptera is thought to be n = 31; however, that of the most well-studied moth, Bombyx mori, is n = 28, and diverse studies suggest that three chromosomal fusion events occurred in this lineage. To identify the boundaries between predicted ancient fusions involving B. mori chromosomes 11, 23 and 24, we constructed fluorescence in situ hybridization (FISH)-based chromosome maps of the European corn borer, Ostrinia nubilalis (n = 31). We first determined a 511 Mb genomic sequence of the Asian corn borer, O. furnacalis, a congener of O. nubilalis, and isolated bacterial artificial chromosomes and fosmid clones that were expected to localize in candidate regions for the boundaries using these sequences. Combined with FISH and genetic analysis, we narrowed down the candidate regions to 40 kb-1.5 Mb, in strong agreement with a previous estimate based on the genome of a butterfly, Melitaea cinxia. The significant difference in the lengths of the candidate regions where no functional genes were observed may reflect the evolutionary time after fusion events.

We have identified and characterized a nitrate-inducible ferredoxin (Fd) i n maize (Zea mays L.) roots by structural analysis of the purified protein and by cloning of i t s cDNA and gene. In maize Fd isoproteins are encoded by a small multigene family, and the nitrate-inducible Fd was identified as a novel isoprotein, designated Fd VI, which differed from any Fd I to Fd V identified to date. In the roots of seedlings cultured without nitrate, Fd VI was undetectable.However, during the induction of the capacity for nitrate assimilation, the amount of Fd VI increased markedly within 24 h. Concurrentiy, the levei of transcript for Fd VI increased, but more quickly, reaching a maximal leve1 within 2 h with kinetics similar to those of nitrite reductase and Fd-NADP+ reductase. Fd 111 was constitutively expressed in roots, and no such changes at the protein and mRNA levels were observed during the nitrate induction. I n the 5' flanking region of the gene for Fd VI only, we identified NIT-2 motifs, which are widely found in genes for enzymes related to nitrogen metabolism. These data indicate that Fd V I i s co-induced with the previously characterized enzymes involved i n nitrate assimilation, and they suggest that the novel Fd isoprotein, distinct from the constitutively expressed Fd, might play an important role as an electron carrier from NADPH to nitrite reductase and other Fd-dependent enzymes in root plastids.

Optical investigation and manipulation constitute the core of biological experiments. Here, we introduce a new borosilicate glass material that contains the rare-earth ion terbium(III) (Tb3+), which emits green fluorescence upon blue light excitation, similar to green fluorescent protein (GFP), and thus is widely compatible with conventional biological research environments. Micropipettes made of Tb3+-doped glass allowed us to target GFP-labeled cells for single-cell electroporation, single-cell transcriptome analysis (Patch-seq), and patch-clamp recording under real-time fluorescence microscopic control. The glass also exhibited potent third harmonic generation upon infrared laser excitation and was usable for online optical targeting of fluorescently labeled neurons in the in vivo neocortex. Thus, Tb3+-doped glass simplifies many procedures in biological experiments.