ABSTRACT
Pacific saury (Cololabis saira) is a commercially important small pelagic fish species in Asia. In this study, we conducted the first-ever whole genome sequencing of this species, with single molecule, real-time (SMRT) sequencing technology. The obtained high-fidelity (HiFi) long-read sequence data, which amount to ~30-folds of its haploid genome size that was measured with quantitative PCR (1.17 Gb), were assembled into contigs. Scaffolding with Hi-C reads yielded a whole genome assembly containing 24 chromosome-scale sequences, with a scaffold N50 length of 47.7 Mb. Screening of repetitive elements including telomeric repeats was performed to characterize possible factors that need to be resolved towards 'telomere-to-telomere' sequencing. The larger genome size than in medaka, a close relative in Beloniformes, is at least partly explained by larger repetitive element quantity, which is reflected in more abundant tRNAs, in the Pacific saury genome. Protein-coding regions were predicted using transcriptome data, which resulted in 22,274 components. Retrieval of Pacific saury homologs of aquaporin (AQP) genes known from other teleost fishes validated high completeness and continuity of the genome assembly. These resources are available at https://treethinkers.nig.ac.jp/saira/ and will assist various molecular-level studies in fishery science and comparative biology.
Subject(s)
Beloniformes , Fisheries , Animals , Base Sequence , Chromosomes , Fishes/genetics , Biology , Beloniformes/genetics , PhylogenyABSTRACT
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.
Subject(s)
Proteolysis/drug effects , Proteomics/methods , Recombinant Fusion Proteins/metabolism , Animals , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Female , HCT116 Cells , Hippocampus/cytology , Humans , Indoleacetic Acids/pharmacology , Male , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Minichromosome Maintenance Proteins/genetics , Minichromosome Maintenance Proteins/metabolism , Mutation , Neurons/drug effects , Neurons/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Oryza/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Recombinant Fusion Proteins/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
Hepatocytes are an important tool for in vitro toxicology testing. In addition to primary cultures, a limited number of immortalized cell lines have been developed. We here describe a new cell line, designated as HepaMN, which has been established from a liver associated with biliary atresia. Hepatocytes were isolated from a liver of 4-year-old girl with biliary atresia and immortalized by inoculation with CSII-CMV-TERT, CSII-CMV-Tet-Off, CSII-TRE-Tight-cyclin D1 and CSII-TRE-Tight-CDK4R24C (mutant CDK4: an INK4a-resistant form of CDK4) lentiviruses at the multiplicity of infection of 3 to 10. HepaMN cells exhibited morphological homogeneity, displaying hepatocyte-like phenotypes. Phenotypic studies in vivo and in vitro revealed that HepaMN cells showed polarized and functional hepatocyte features along with a canalicular cell phenotype under defined conditions, and constitutively expressed albumin and carbamoyl phosphate synthetase I in addition to epithelial markers. Since HepaMN cells are immortal and subcloned, kinetics and expression profiles were independent of population doublings. HepaMN cells showed increased CYP3A4 expression after exposure to rifampicin, implying that their close resemblance to normal human hepatocytes makes them suitable for research applications including drug metabolism studies.
