Title: Rapid Integration of Multi-copy Transgenes Using Optogenetic Mutagenesis in Caenorhabditis elegans Noma K, Jin Y Ref: G3 (Bethesda), 8:2091, 2018 : PubMed
Stably transmitted transgenes are indispensable for labeling cellular components and manipulating cellular functions. In Caenorhabditis elegans, transgenes are generally generated as inheritable multi-copy extrachromosomal arrays, which can be stabilized in the genome through a mutagenesis-mediated integration process. Standard methods to integrate extrachromosomal arrays primarily use protocols involving ultraviolet light plus trimethylpsoralen or gamma- or X-ray irradiation, which are laborious and time-consuming. Here, we describe a one-step integration method, following germline-mutagenesis induced by mini Singlet Oxygen Generator (miniSOG). Upon blue light treatment, miniSOG tagged to histone (Histone-miniSOG) generates reactive oxygen species (ROS) and induces heritable mutations, including DNA double-stranded breaks. We demonstrate that we can bypass the need to first establish extrachromosomal transgenic lines by coupling microinjection of desired plasmids with blue light illumination on Histone-miniSOG worms to obtain integrants in the F3 progeny. We consistently obtained more than one integrant from 12 injected animals in two weeks. This optogenetic approach significantly reduces the amount of time and labor for transgene integration. Moreover, it enables to generate stably expressed transgenes that cause toxicity in animal growth.
Subcellular localization of ribosomes defines the location and capacity for protein synthesis. Methods for in vivo visualizing ribosomes in multicellular organisms are desirable in mechanistic investigations of the cell biology of ribosome dynamics. Here, we developed an approach using split GFP for tissue-specific visualization of ribosomes in Caenorhabditis elegans. Labeled ribosomes are detected as fluorescent puncta in the axons and synaptic terminals of specific neuron types, correlating with ribosome distribution at the ultrastructural level. We found that axonal ribosomes change localization during neuronal development and after axonal injury. By examining mutants affecting axonal trafficking and performing a forward genetic screen, we showed that the microtubule cytoskeleton and the JIP3 protein UNC-16 exert distinct effects on localization of axonal and somatic ribosomes. Our data demonstrate the utility of tissue-specific visualization of ribosomes in vivo, and provide insight into the mechanisms of active regulation of ribosome localization in neurons.
Inhibition of Rho-associated coiled-coil forming kinases (ROCKs) reduces allergic airway responses in mice. The purpose of this study was to determine the roles of the two ROCK isoforms, ROCK1 and ROCK2, in these responses. Wildtype (WT) mice and heterozygous ROCK1 and ROCK2 knockout mice (ROCK1(+/-) and ROCK2(+/-), respectively) were sensitised and challenged with ovalbumin. ROCK expression and activation were assessed by western blotting. Airway responsiveness was measured by forced oscillation. Bronchoalveolar lavage was performed and the lungs were fixed for histological assessment. Compared with WT mice, ROCK1 and ROCK2 expression were 50% lower in lungs of ROCK1(+/-) and ROCK2(+/-) mice, respectively, without changes in the other isoform. In WT lungs, ROCK activation increased after ovalbumin challenge and was sustained for several hours. This activation was reduced in ROCK1(+/-) and ROCK2(+/-) lungs. Airway responsiveness was comparable in WT, ROCK1(+/-), and ROCK2(+/-) mice challenged with PBS. Ovalbumin challenge caused airway hyperresponsiveness in WT, but not ROCK1(+/-) or ROCK2(+/-) mice. Lavage eosinophils and goblet cell hyperplasia were significantly reduced in ovalbumin-challenged ROCK1(+/-) and ROCK2(+/-) versus WT mice. Ovalbumin-induced changes in lavage interleukin-13, interleukin-5 and lymphocytes were also reduced in ROCK1(+/-) mice. In conclusion, both ROCK1 and ROCK2 are important in regulating allergic airway responses.
Transcriptional gene silencing (TGS) is the mechanism generally thought by which heterochromatin effects silencing. However, recent discovery in fission yeast of a cis-acting posttranscriptional gene-silencing (cis-PTGS) pathway operated by the RNAi machinery at heterochromatin challenges the role of TGS in heterochromatic silencing. Here, we describe a multienzyme effector complex (termed SHREC) that mediates heterochromatic TGS in fission yeast. SHREC consists of a core quartet of proteins - Clr1, Clr2, Clr3, and Mit1 - which distribute throughout all major heterochromatin domains to effect TGS via distinct activities associated with the histone deacetylase Clr3 and the SNF2 chromatin-remodeling factor homolog Mit1. SHREC is also recruited to the telomeres by multiple independent mechanisms involving telomere binding protein Ccq1 cooperating with Taz1 and the RNAi machinery, and to euchromatic sites, via mechanism(s) distinct from its heterochromatin localization aided by Swi6/HP1. Our analyses suggest that SHREC regulates nucleosome positioning to assemble higher-order chromatin structures critical for heterochromatin functions.
Hepatic lipase (HL) is synthesized in the liver and hydrolyses triglyceride and phospholipids. C-514T polymorphism in HL gene promoter was reported to associate with hepatic lipase activity and plasma lipid levels. We examined whether C-514T polymorphism affects glucose metabolism beyond its effect on plasma lipid levels in nondiabetic Japanese subjects. Gene frequencies of C/C homozygote, C/T heterozygote and T/T homozygote were 18, 51 and 31%, respectively. The allelic frequencies of C and T were 44 and 56%, respectively. T allele frequency was much higher than in Caucasian subjects. Moreover, -514T allele carriers had higher levels of triglyceride (P=0.027), fasting insulin (P=0.016) and HOMA-IR (P=0.033) than non-carriers. In contrast to some former studies, -514T allele affected triglyceride levels and insulin sensitivity. Taken together, HL gene might be one of the important susceptibility genes of type 2 diabetes and the high incidence of type 2 diabetes could be explained by high frequency of -514T allele in the Japanese population. Moreover, since HL and adiponectin showed an additive effect on insulin sensitivity, these genetic variations can be independently associated with insulin sensitivity.
