Regulation of Sperm-Specific Proteins by IFE-1, a Germline-Specific Homolog of eIF4E, in C. elegans

가와사키이찌로,Myung-Hwan Jeong,심용희 한국분자세포생물학회 2011 Molecules and cells Vol.31 No.2

IFE-1 is one of the five C. elegans homologs of eIF4E, which is the mRNA 5′ cap-binding component of the translation initiation complex eIF4F. Depletion of IFE-1 causes defects in sperm, suggesting that IFE-1 regulates a subset of genes required for sperm functions. To further understand the molecular function of IFE-1, proteomic analysis was performed to search for sperm proteins that are downregulated in ife-1(ok1978); fem-3(q20) mutants relative to the fem-3(q20) control. The fem-3(q20) mutant background was used because it only produces sperm at restrictive temperature. Total worm proteins were subjected to 2DDIGE, and differentially expressed protein spots were further identified by MALDI-TOF mass spectrometry. Among the identified proteins, GSP-3 and Major Sperm Proteins (MSPs) were found to be significantly down-regulated in the ife-1(ok1978) mutant. Moreover, RNAi of gsp-3 caused an ife-1-like phenotype. These results suggest that IFE-1 is required for efficient expression of some sperm-specific proteins, and the fertilization defect of ife-1 mutant is caused mainly by a reduced level of GSP-3.

Cholesterol-Responsive Metabolic Proteins Are Required for Larval Development in Caenorhabditis elegans

가와사키이찌로,심용희,정명환,윤유준,신윤경 한국분자세포생물학회 2013 Molecules and cells Vol.36 No.5

Caenorhabditis elegans, a cholesterol auxotroph, showed defects in larval development upon cholesterol starvation (CS) in a previous study. To identify cholesterol-respon-sive proteins likely responsible for the larval arrest upon CS, a comparative proteomic analysis was performed between C. elegans grown in normal medium supplemented with cholesterol (CN) and those grown in medium not supplemented with cholesterol (cholesterol starvation, CS). Our analysis revealed significant change (more than 2.2-fold, p < 0.05) in nine proteins upon CS. Six proteins were down-regulated [CE01270 (EEF-1A.1), CE08852 (SAMS-1), CE11068 (PMT-2), CE09015 (ACDH-1), CE12564 (R07H5.8), and CE09655 (RLA-0)], and three proteins were up-regu-lated [CE29645 (LEC-1), CE16576 (LEC-5), and CE01431 (NEX-1)]. RNAi phenotypes of two of the down-regulated genes, R07H5.8 (adenosine kinase) and rla-0 (ribosomal protein), in CN were similar to that of larval arrest in CS, and RNAi of a down-regulated gene, R07H5.8, in CS further enhanced the effects of CS, suggesting that down-regulation of these genes is likely responsible for the larval arrest in CS. All three up-regulated genes con-tain putative DAF-16 binding sites and mRNA levels of these three genes were all decreased in daf-16 mutants in CN, suggesting that DAF-16 activates expression of these genes.

Inhibition of Overexpressed CDC-25.1 Phosphatase Activity by Flavone in Caenorhabditis elegans

김구슬,가와사키이찌로,정유훈,심용희 한국분자세포생물학회 2009 Molecules and cells Vol.27 No.3

We previously reported that flavone induces embryonic lethality in Caenorhabditis elegans, which appeared to be the result of cell cycle arrest during early embryogenesis. To test this possibility, here we examined whether flavone inhibits the activity of a key cell cycle regulator, CDC-25.1 in C. elegans. A gain-of-function cdc-25.1 mutant, rr31, which exhibits extra cell divisions in intestinal cells, was used to test the inhibitory effects of flavone on CDC-25 activity. Flavone inhibited the extra cell divisions of intestinal cells in rr31, and modifications of flavone reduced the inhibitory effects. The inhibitory effects of flavone on CDC-25.1 were partly, if not completely, due to transcriptional repression.

Caffeine Induces High Expression of cyp-35A Family Genes and Inhibits the Early Larval Development in Caenorhabditis elegans

민혜민,심용희,가와사키이찌로,강주미 한국분자세포생물학회 2015 Molecules and cells Vol.38 No.3

Intake of caffeine during pregnancy can cause retardation of fetal development. Although the significant influence of caffeine on animal development is widely recognized, much remains unknown about its mode of action because of its pleiotropic effects on living organisms. In the present study, by using Caenorhabditis elegans as a model organism, the effects of caffeine on development were examined. Brood size, embryonic lethality, and percent larval development were investigated, and caffeine was found to inhibit the development of C. elegans at most of the stages in a dosage-dependent fashion. Upon treatment with 30 mM caffeine, the majority (86.1 3.4%) of the L1 larvae were irreversibly arrested without further development. In contrast, many of the late-stage larvae survived and grew to adults when exposed to the same 30 mM caffeine. These results suggest that early-stage larvae are more susceptible to caffeine than later-stage larvae. To understand the metabolic responses to caffeine treatment, the levels of expression of cytochrome P450 (cyp) genes were examined with or without caffeine treatment using comparative microarray, and it was found that the expression of 24 cyp genes was increased by more than 2-fold (p < 0.05). Among them, induction of the cyp-35A gene family was the most prominent. Interestingly, depletion of the cyp-35A family genes one-by-one or in combination through RNA interference resulted in partial rescue from early larval developmental arrest caused by caffeine treatment, suggesting that the high-level induction of cyp-35A family genes can be fatal to the development of early-stage larvae.

Inhibition of Developmental Processes by Flavone in Caenorhabditis elegans and Its Application to the Pinewood Nematode, Bursaphelenchus xylophilus

심용희,이용욱,가와사키이찌로,임융호,오완석,백융기 한국분자세포생물학회 2008 Molecules and cells Vol.26 No.2

Flavone (2-phenyl chromone) is a well-known plant flavonoid, but its bioactivity has been little explored. Treatment of Caenorhabditis elegans or C. brissage with flavones induced embryonic and larval lethality that was pronounced in early larval stages. This anti-nematodal effect was also observed in the pinewood nematode, B. xylophilus. LD50 values were approximately 100 M for both B. xylophilus and C. elegans. Our results indicate that flavone is an active nematicidal compound that should be further investigated with the aim of developing a potent drug against B. xylophilus.

Identification of cdc25 Gene in Pinewood Nematode, Bursaphelenchus xylophilus, and Its Function in Reproduction

Ye-Na Choi,오봉경,가와사키이찌로,Wan-Suk Oh,이이,백융기,심용희 한국분자세포생물학회 2010 Molecules and cells Vol.29 No.2

The cdc25 gene, which is highly conserved in many eu-karyotes, encodes a phosphatase that plays essential roles in cell cycle regulation. We identified a cdc25 ortholog in the pinewood nematode, Bursaphelenchus xylophilus. The B. xylophilus ortholog (Bx-cdc25) was found to be highly similar to Caenorhabditis elegans cdc-25.2 in sequence as well as in gene structure, both having long intron 1. The Bx-cdc25 gene was determined to be composed of seven exons and six introns in a 2,580 bp region, and was shown to encode 360 amino acids of a protein containing a highly-conserved phosphatase do-main. Bx-cdc25 mRNA was hardly detectable throughout the juvenile stages but was highly expressed in eggs and in both female and male adults. Functional conservation during germline development between C. elegans cdc25 and Bx-cdc25 was revealed by Bx-cdc25 RNA interference in C. elegans.

A Mutation of cdc-25.1 Causes Defects in Germ Cells But Not in Somatic Tissues in C. elegans

Jiyoung Kim,Ah-Reum Lee,가와사키이찌로,Susan Strome,심용희 한국분자세포생물학회 2009 Molecules and cells Vol.28 No.1

By screening C. elegans mutants for severe defects in germline proliferation, we isolated a new loss-of-function allele of cdc-25.1, bn115. bn115 and another previously identified loss-of-function allele nr2036 do not exhibit no-ticeable cell division defects in the somatic tissues but have reduced numbers of germ cells and are sterile, indi-cating that cdc-25.1 functions predominantly in the germ line during postembryonic development, and that cdc-25.1 activity is probably not required in somatic lineages during larval development. We analyzed cell division of germ cells and somatic tissues in bn115 homozygotes with germline-specific anti-PGL-1 immunofluorescence and GFP trans-genes that express in intestinal cells, in distal tip cells, and in gonadal sheath cells, respectively. We also analyzed the expression pattern of cdc-25.1 with conventional and quantitative RT-PCR. In the presence of three other family members of cdc-25 in C. elegans, defects are observed only in the germ line but not in the somatic tissues in cdc-25.1 single mutants, and cdc-25.1 is expressed predomi-nantly, if not exclusively, in the germ line during postem-bryonic stages. Our findings indicate that the function of cdc-25.1 is unique in the germ line but likely redundant with other members in the soma.

Two Mutations in pab-1 Encoding Poly(A)-Binding Protein Show Similar Defects in Germline Stem Cell Proliferation but Different Longevity in C. elegans

고선희,Jae-Hyung Park,Ah-Reum Lee,Eugene Kim,Jiyoung-Kim,가와사키이찌로,심용희 한국분자세포생물학회 2010 Molecules and cells Vol.30 No.2

Four new alleles, bn116, bn117, bn118, and bn119, on LG I were isolated in C. elegans with defects in germline stem cell proliferation. Using genetic mapping and snip-SNP mapping, bn116, bn117, bn118, and bn119 were located 5.0 cM, 1.3 cM, 2.3 cM, and 5.0 cM, respectively, to the right of dpy-5 on LG I. Further, bn116 and bn119 were grouped into the same complementation group by a complementation test. They are loss-of-function recessive alleles that produce homozygous sterile worms whose germ cells do not prolif-erate during larval development. However, the worms con-tained normal somatic gonadal structures including distal tip cells and gonadal sheath cells, suggesting that the defect in germline proliferation was not caused by the absence of somatic signaling. Although DAF-16 was localized to the nucleus in all four mutants, the life span was extended only in the three mutants except bn116. These results suggest that the defect in germline stem cell proliferation, the pres-ence of normal somatic gonadal tissues, and DAF-16 nu-clear translocation were sufficient for extending the lifespan of the bn117, bn118, and bn119 mutants, but not the bn116 mutant. Intriguingly, bn116 and bn119 were identified as two different mutations on the same gene, pab-1, which encodes a poly(A)-binding protein. Therefore, although the bn116 and bn119 mutations cause similar defects in germ cell prolifera-tion, their effects on life span are different.