No abstract

Balancer chromosomes are critical tools for genetic research. In C. elegans, reciprocal translocations that lead to aneuploidy have been widely used to maintain lethal and sterile mutations in stable stocks. Here, we generated a set of aneuploidy-free and structurally defined crossover suppressors that contain two overlapping inversions using the CRISPR/Cas9 system. The toolkit includes 13 crossover suppressors and covers approximately 63% of all C. elegans coding genes. Together with the classical intrachromosomal crossover suppressors, the system now covers 89% of the coding genes. We also labeled the created balancers with fluorescent and phenotypic markers. We show that the crossover suppressors are better for embryonic analysis compared with translocational balancers. Additionally, we demonstrate an efficient method to generate lethal alleles by targeting essential genes on a chromosome balanced with a crossover suppressor. The toolkit will allow more efficient experiments in which lethal and sterile mutants can be analyzed.

Parkinson's disease is neuropathologically characterized by the presence of Lewy bodies, whose major component is alpha-synuclein. We had previously generated transgenic mice that expressed human alpha-synuclein carrying an Ala53Thr point mutation (halpha-syn140m) under the control of the rat tyrosine hydroxylase promoter and found that halpha-syn140m was localized not only in the cytoplasm but also in the nuclei of mesencephalic dopaminergic neurons. In the present study, we carried out immunohistochemical analysis of the brain of Tg mice using anti-PSer129, an antibody that specifically recognizes alpha-synuclein phosphorylated at Ser129. The antibody detected only phosphorylated halpha-syn140m, whereas phosphorylation of endogenous alpha-synuclein, if any, was below the detection limit of the method employed. The analysis showed that approximately one-third of the halpha-syn140m-positive neurons in the midbrain of heterozygous Tg mice were concomitantly reactive to anti-PSer129. The ratio almost doubled in homozygotes, indicating that the phosphorylation level depends directly on the amount of substrate. In addition, the ratio did not change at least up to 48 weeks of age. These data strongly suggest that halpha-syn140m underwent constitutive phosphorylation and that the phosphorylation level was maintained to a certain level until the aged stages. Remarkably, halpha-syn140m localized in the nuclei seemed to be preferentially phosphorylated compared with that in the cytoplasm. Among kinases that have been reported to be involved in the phosphorylation of alpha-synuclein, the beta subunit of casein kinase-2 was detected in the nuclei by immunohistochemistry. These data imply that at least casein kinase-2 is involved in the phosphorylation of halpha-syn140m in the Tg mice.

2-(3,5,6-Trifluoro-2-hydroxy-4-methoxyphenyl)benzoxazole (3) and benzothiazole analogue (4) are prepared by the two-step procedures from the corresponding 2-(pentafluorophenyl)benzazoles. Benzoxazole 3 is applicable to a fluorescent probe sensing magnesium cation, and 4 is suitable for sensing zinc cation. Both fluorophores 3 and 4 are sensitive to the pH change at pH 7-8, resulting in large fluorescence enhancement under basic conditions. Their high sensitivity to pH and selectivity in metal cations are ascribed to the high acidity of the fluorophenol moiety.

Balancer chromosomes are convenient tools used to maintain lethal mutations in heterozygotes. We established a method for engineering new balancers in C. elegans by using the CRISPR/Cas9 system in a non-homologous end-joining mutant. Our studies will make it easier for researchers to maintain lethal mutations and should provide a path for the development of a system that generates rearrangements at specific sites of interest to model and analyse the mechanisms of action of genes.