Hyung-Soon Yim

One-Step Sequence- and Ligation-Independent Cloning as a Rapid and Versatile Cloning Method for Functional Genomics Studies

ABSTRACT We developed one-step sequence- and ligation-independent cloning (SLIC) as a simple, cost-effective, time-saving, and versatile cloning method. Highly efficient and directional cloning can be achieved by direct bacterial transformation 2.5 min after mixing any linearized vector, an insert(s) prepared by PCR, and T4 DNA polymerase in a tube at room temperature.

Insights into the Evolution of Longevity from the Bowhead Whale Genome

Summary The bowhead whale (Balaena mysticetus) is estimated to live over 200 years and is possibly the longest-living mammal. These animals should possess protective molecular adaptations relevant to age-related diseases, particularly cancer. Here, we report the sequencing and comparative analysis of the bowhead whale genome and two transcriptomes from different populations. Our analysis identifies genes under positive selection and bowhead-specific mutations in genes linked to cancer and aging. In addition, we identify gene gain and loss involving genes associated with DNA repair, cell-cycle regulation, cancer, and aging. Our results expand our understanding of the evolution of mammalian longevity and suggest possible players involved in adaptive genetic changes conferring cancer resistance. We also found potentially relevant changes in genes related to additional processes, including thermoregulation, sensory perception, dietary adaptations, and immune response. Our data are made available online (http://www.bowhead-whale.org) to facilitate research in this long-lived species.

Analysis of the draft genome of the red seaweed Gracilariopsis chorda provides insights into genome size evolution in Rhodophyta

Red algae (Rhodophyta) underwent two phases of large-scale genome reduction during their early evolution. The red seaweeds did not attain genome sizes or gene inventories typical of other multicellular eukaryotes. We generated a high-quality 92.1 Mb draft genome assembly from the red seaweed Gracilariopsis chorda, including methylation and small (s)RNA data. We analyzed these and other Archaeplastida genomes to address three questions: 1) What is the role of repeats and transposable elements (TEs) in explaining Rhodophyta genome size variation, 2) what is the history of genome duplication and gene family expansion/reduction in these taxa, and 3) is there evidence for TE suppression in red algae? We find that the number of predicted genes in red algae is relatively small (4,803-13,125 genes), particularly when compared with land plants, with no evidence of polyploidization. Genome size variation is primarily explained by TE expansion with the red seaweeds having the largest genomes. Long terminal repeat elements and DNA repeats are the major contributors to genome size growth. About 8.3% of the G. chorda genome undergoes cytosine methylation among gene bodies, promoters, and TEs, and 71.5% of TEs contain methylated-DNA with 57% of these regions associated with sRNAs. These latter results suggest a role for TE-associated sRNAs in RNA-dependent DNA methylation to facilitate silencing. We postulate that the evolution of genome size in red algae is the result of the combined action of TE spread and the concomitant emergence of its epigenetic suppression, together with other important factors such as changes in population size.

Analysis of the FGF gene family provides insights into aquatic adaptation in cetaceans

Cetacean body structure and physiology exhibit dramatic adaptations to their aquatic environment. Fibroblast growth factors (FGFs) are a family of essential factors that regulate animal development and physiology; however, their role in cetacean evolution is not clearly understood. Here, we sequenced the fin whale genome and analysed FGFs from 8 cetaceans. FGF22, a hair follicle-enriched gene, exhibited pseudogenization, indicating that the function of this gene is no longer necessary in cetaceans that have lost most of their body hair. An evolutionary analysis revealed signatures of positive selection for FGF3 and FGF11, genes related to ear and tooth development and hypoxia, respectively. We found a D203G substitution in cetacean FGF9, which was predicted to affect FGF9 homodimerization, suggesting that this gene plays a role in the acquisition of rigid flippers for efficient manoeuvring. Cetaceans utilize low bone density as a buoyancy control mechanism, but the underlying genes are not known. We found that the expression of FGF23, a gene associated with reduced bone density, is greatly increased in the cetacean liver under hypoxic conditions, thus implicating FGF23 in low bone density in cetaceans. Altogether, our results provide novel insights into the roles of FGFs in cetacean adaptation to the aquatic environment.

Characterization of cetacean Numt and its application into cetacean phylogeny

The translocations of mitochondrial DNA into chromosomal DNA (nuclear mitochondrial DNA, Numt) are ubiquitous in eukaryotes including yeasts, plants, and animals. The features of Numt and the recent sequencing technology can facilitate an expanded application of Numt into a valuable phylogenetic marker for unresolved taxa. To date, the phylogeny of extant cetaceans has been studied by a variety of morphological and molecular data and still has long attracted attention. Here, the Numts of cattle, two baleen whale and four toothed whales were detected by BLAST-search of the mitochondrial sequences of each species against its corresponding nuclear genome and we investigated the characteristics of cetacean Numt and revisited the phylogeny and evolution of cetartiodactyl using Numts. The content and distribution of Numt length showed similar patterns among six cetacean genomes. Under-representation of D-loop region-derived Numts and different abundance of Numt across D-loop sub-domains were observed in cetacean Numts except sperm whale. Examination of Numt location in cetacean nuclear genomes showed that some of orthologous Numts were integrated into exons, introns, and pseudogenes, suggesting that cetacean Numts may contribute to cetacean biology and evolution. Our phylogenetic study with cetacean Numt based on the maximum likelihood method corresponded to the study from other phylogenetic markers.

Optimizing T4 DNA polymerase conditions enhances the efficiency of one-step sequence- and ligation-independent cloning

Previously, we developed a one-step sequence- and ligation-independent cloning (SLIC) method that is simple, fast, and cost-effective. However, although one-step SLIC generally works well, its cloning efficiency is occasionally poor, potentially due to formation of stable secondary structures within the single-stranded DNA (ssDNA) region generated by T4 DNA polymerase during the 2.5 min treatment at room temperature. To overcome this problem, we developed a modified thermo-regulated one-step SLIC approach by testing shorter T4 DNA polymerase treatment durations (5 s-2.5 min) over a wide range of temperatures (25-75°C). The highest cloning efficiency resulted when inserts with homology lengths <20 bases were treated with T4 DNA polymerase for 30 s at 50°C. This briefer T4 polymerase treatment at a higher temperature helps increase cloning efficiency for inserts with strong secondary structures at their ends, increasing the utility of one-step SLIC for the cloning of short fragments.

The jellyfish genome sheds light on the early evolution of active predation

Background Unique among cnidarians, jellyfish have remarkable morphological and biochemical innovations that allow them to actively hunt in the water column. One of the first animals to become free-swimming, jellyfish employ pulsed jet propulsion and venomous tentacles to capture prey. Results To understand these key innovations, we sequenced the genome of the giant Nomura’s jellyfish (Nemopilema nomurai), the transcriptomes of its bell and tentacles, and transcriptomes across tissues and developmental stages of the Sanderia malayensis jellyfish. Analyses of Nemopilema and other cnidarian genomes revealed adaptations associated with swimming, marked by codon bias in muscle contraction and expansion of neurotransmitter genes, along with expanded Myosin type II family and venom domains; possibly contributing to jellyfish mobility and active predation. We also identified gene family expansions of Wnt and posterior Hox genes, and discovered the important role of retinoic acid signaling in this ancient lineage of metazoans, which together may be related to the unique jellyfish body plan (medusa formation). Conclusions Taken together, the jellyfish genome and transcriptomes genetically confirm their unique morphological and physiological traits that have combined to make these animals one of the world’s earliest and most successful multi-cellular predators.

FGF11 induced by hypoxia interacts with HIF‐1α and enhances its stability

Fibroblast growth factor 11 (FGF11) is an intracellular FGF. Although induction of FGF11 by hypoxia has been observed in several cell types, the molecular function of FGF11 is not clearly understood yet. Here, we investigated the role of FGF11 under hypoxia. We identified hypoxia‐inducible factor‐1α (HIF‐1α) as an interacting protein of FGF11 using immunoprecipitation and mass spectrometry. FGF11 knockdown decreased HIF‐1α protein, while FGF11 overexpression increased it, without affecting HIF‐1α mRNA. Protein stability test and ubiquitination assay showed that FGF11 increased HIF‐1α stability by acting upstream of proteasomal degradation. Altogether, these results suggest a cross‐regulation between HIF‐1α and FGF11, through which hypoxia‐induced FGF11 reinforces hypoxia responses by enhancing the stability of HIF‐1α.