Harksun Lee et al. (2008) "Molecular genetics of nictation"

Hyesung Kim, Seunghee Nam, Junho Lee, Harksun Lee, Myungkyu Choi

[2008]

"When the food is limited or the population is too crowded, C. elegans enters an alternative stage called the dauer. The dauer larvae usually do not move and pharyngeal pumping is almost arrested. The dauer larvae show an unexpected behaviour, nictation. The dauer larvae climb up onto any projections and while standing by their posterior tip, wave in three-dimensional spirals and loops.(Croll, N.A., and Matthews, B. E., 1977, Biology of nematodes) This behaviour is not shown by animals at any other developmental stage. Although C. elegans is known as the free-living organism in the soil, many Caenorhapditis species associate with other species such as snails and insects. Nictation may be critical for transmission of nematodes to a new niche, and thus for their survival. It is known that there are specific neural circuits for specific behaviours such as forward and backward movements, thermotaxis and chemotaxis. The neural circuit involved in nictation is not studied. We want to elucidate the neural circuit involved in the nictation behaviour. We established the behaviour assay of nictation. We will present the identification of genes required for nictation."

Jim-Ming Lee et al. (2008) ""The 3,UTR of fib-1 is the target of NCL-1 suppression""

Szecheng J. Lo, Jim-Ming Lee, Keng-Poo Tan, Tian Hsiang Ma

[2008]

"The 3,UTR of fib-1 is the target of NCL-1 suppressionJim-Ming Lee, Keng-Poo Tan, Tian Hsiang Ma and Szecheng J. LoDepartment of life Science and Graduate Institute of Microbiology, Chang Gung University, TaoYuan, TaiwanThe nucleolus of eukaryotic cells is known to be the site for ribosome assembly and the numbers and sizes of nucleoli reflect the cellular activity of ribosome synthesis. However, how organisms control the sizes and numbers of nucleoli in various types of cells is poorly understood. Caenorhabditis elegans provides a good system to answer this question. In the wild type of C. elegans, no visible nucleolus is detected in the early embryos till the 100-cell stage and the size of nucleolus is smaller in neurons and hypodermis cells than in intestine and pharynx cells. The ncl-1 mutant exhibits nucleolus starting in the two-cell stage and a larger size of nucleolus in neurons and hypodermis cells of adult worms. Since the brat gene of Drosophila, an ortholog of ncl-1, has been demonstrated as a translational suppressor, we searched for the target gene which is transitionally suppressed by NCL-1 in C. elegans. We identified that fibrillarin (FIB-1), one of the major nucleolar proteins with a high conservation across all eukaryotes, was a possible target of NCL-1. The results of Western blots indicated that FIB-1 was present in the embryos of ncl-1 background worms but little or none in the wild type embryos while the results of real-time RT-PCR revealed that the level of mRNA of FIB-1 was similar in the early embryos from two different genetic background worms. We then used a GFP reporter system which contains the 3,UTR of fib-1 fused to the end of GFP to create transgenic worms in the wild type and ncl-1 background. GFP signals were observed in the most of cells except those in the head and tail regions of wild type worms. In contrast, GFP appeared in the entire worms in the ncl-1 background worms. These results indicated that the 3,UTR of fib-1 is the target of NCL-1 suppression. Whether the NCL-1 binds to the 3,-UTR of fib-1 directly or indirectly remains to be elusive. "

Min-Ho Lee et al. (2003) International Worm Meeting "GLD-1 and Translational Regulation"

Tim Schedl, Min-Ho Lee

[International Worm Meeting, 2003]

During germ line development, translational control emerges as a heavily utilized mechanism by which gene expression is regulated. The germ line thus provides an excellent model for studying translational regulation. Recently, many RNA binding proteins have been identified as essential factors governing germ line development and early embryogenesis. A comprehensive understanding of how these RNA binding proteins control development remains largely unknown. GLD-1 is a germ line specific, maxi-KH motif containing RNA binding protein that acts as a tumor suppressor and regulates multiple aspects of C. elegans germ cell development, suggesting that it regulates multiple RNAs. The tumorous mutant phenotype, therefore, could result from mis-regulation of mRNA targets during early meiotic prophase. GLD-1 has homologs in Drosohpila (How) and mouse/human (Quaking), therefore conserved throughout evolution. To understand how GLD-1 is regulating mRNA targets to control germ cell development, we have identified multiple in vivo mRNA targets of GLD-1 by their ability to interact with GLD-1 in cytosol extracts. These target mRNAs are preferentially expressed in the germline and as expected, several of them have essential functions in oocyte differentiation and early embryogenesis. We also found that GLD-1 functions as a translational repressor. The expression patterns of four mRNA targets (rme-2, puf-5, oma-1 and oma-2) at the level of protein as well as mRNA are very similar to each other and quite consistent with GLD-1 acting as a translational repressor. We were also able to narrow down GLD-1 binding sites on several targets. They are located at the 5-end, 3-end, or both ends depending on the mRNA target. In a few cases, GLD-1 also can bind to ORF. Currently, we do not know the importance of either the position of GLD-1 binding sites or the number of GLD-1 binding sites. Initial characterization suggests that GLD-1 is likely to repress translation prior to ribosome assembly or soon after ribosome elongation. We found that GLD-1 binds and protects T23G11.2 mRNA from Nonsense Mediated mRNA Decay (NMD), presumably by binding and repressing translation. T23G11.2 mRNA has two small upstream open reading frames in 5-UTR that likely target the mRNA for NMD. Interestingly, when GLD-1 mRNA targets acquire pre-mature stop codons by nonsense mutations, GLD-1 also can protect them from NMD. For NMD to take place, the ribosome has to proceed to the pre-mature stop codon then recruit factors to degrade the mRNA. Analysis of several mRNA targets that contain nonsense mutations suggests that, if bound to ribosome, then the ribosome cannot have elongated to the pre-mature stop codon to trigger NMD during GLD-1 repression.

Lee, Inhwan et al. (2013) International Worm Meeting "Epigentic regulation of L1 longevity."

You, Young-jai, Lee, Inhwan

[International Worm Meeting, 2013]

Animals encounter food infrequently and starvation is a common physiological state in their natural circumstance [1]. Hatching in the absence of food, worms arrest their development at the L1 stage and survive starvation more than two weeks [2]. It was shown that adult longevity is regulated epigenetically by chromatin alterations and the genes that regulate epigenetics [3]. Here, we investigate whether L1 longevity is also regulated epigenetically. When we measured various histone modifications using Western blot, we found histone 3 lysine 4 tri-methylation, known to be a modification to activate gene transcription, is increased in L1 starvation. Moreover, mutants of set-2, which encodes a histone 3 lysine 4 tri-methyl transferase that functions in adult longevity [3], has reduced L1 longevity. There are several genes essential for normal L1 longevity, such as aak-2 and daf-16. Based on our data, we hypothesize that chromatin remodeling through histone 3 lysine 4 tri-methylation regulates transcription of genes involved in L1 longevity. Currently we are testing this hypothesis by measuring expression levels these genes by ChIP-qPCR during L1 starvation in set-2 mutant. References [1] Brian H. Lee, Plus Genetics, 2008 [2] Inhwan Lee, Plus One, 2012 [3] Eric L. Greer, Nature, 2010.

Annie J Lee et al. (2003) International Worm Meeting "Characterization of an sdc-1 Suppressor"

Barbara J Meyer, Annie J Lee

[International Worm Meeting, 2003]

The nematode Caenorhabditis elegans coordinately controls the processes of sex determination and X-chromosome dosage compensation through the sdc genes, sdc-1, sdc-2 and sdc-3. The SDC proteins function together in a complex that associates with the X chromosome to activate dosage compensation and with the her-1 promoter to promote hermaphrodite development. In order to better understand the role of sdc-1 in these processes, a screen for suppressors of sdc-1(null) was performed (1). An interesting sdc-1 suppressor, y63, was identified that differentially suppresses the dosage compensation defect but not the sex determination defect of sdc-1. y63 is a gene-specific, allele-non-specific suppressor, suppressing all six sdc-1 alleles tested, including two amber mutations and a temperature sensitive mutation. Our western blot analysis revealed that SDC-1 protein is not present in embryonic extracts of y63 sdc-1(null) strain suggesting that y63 may function as a bypass suppressor of sdc-1 in dosage compensation. y63 is a semi-dominant suppressor and we are currently studying whether y63 dominance is due to a gain of function mutation or haploinsufficiency. y63 is X-linked but maps to a location distinct from sdc-1 and sdc-2. Using single nucleotide polymorphisms, we have fine mapped y63 to a region of approximately 40 kilobases on the X chromosome, which encompasses 11 open reading frames (ORFs). We are currently performing RNAi and genomic fragment rescue experiments to test the 11 ORFs and preliminary studies have identified one promising candidate. Analysis of this suppressor may lead to the discovery of a factor that contributes to the process of dosage compensation but not sex determination, and may lead to insights into the mechanism by which the SDC proteins function to repress transcription by 2-fold along the entire X chromosome as opposed to by 20-fold at the her-1 locus. 1. Villeneuve, A.M. and B. J. Meyer, (1990) Genetics 124: 91-114

Lee, Hanee (2009) International Worm Meeting "Dissection of the LATS kinase pathway in C. elegans."

Lee, Hanee

[International Worm Meeting, 2009]

The LATS kinase pathway is one major conserved mechanism governing organ size regulation and cancer development. The fact that some components of the LATS kinase pathway are mutated or hypermethylated in cancer suggests that LATS kinase pathway is important in tumorigenesis. Since most components of LATS kinase pathway are conserved well in C.elegans, C.elegans is a good model organism to study about LATS kinase pathway. In our previous study, we found that wts-1 is involved in the cellular polarity of intestinal cell. Loss of wts-1 causes larval arrest phonotype because of the intestinal defects. In other species such as mammal and fly, LATS kinase is known to act through YAP/yki, the transcription coactivator, but it is possible that YAP is not the only substrate. We tried to find the downstream gene of wts-1 by using EMS random mutagenesis in C.elegans. As a result, we obtained four suppressor lines that suppress larval arrest phenotype of wts-1. We are trying to find out which genes are mutated in these lines by SNP mapping. We found that two of them are recessive and the others dominant. One of them is linked at X chromosome. With these data, we will determine which genes act downstream of wts-1 and it will make advanced understanding about the LATS kinase pathway.

Jeong-Eui Lee et al. (2008) "Physiological characterization of Diploscapter sp. strain LKC25"

Young-Ki Paik, David J. Chitwood, Hyoe-Jin Joo, Lynn K. Carta, Pan-Young Jeong, Wan-Suk Oh, Jeong-Eui Lee

[2008]

"Diploscapter sp., a free-living nematode related similar to Caenorhabditis elegans, has been experimentally shown to vector food-borne enterohemorrhagic pathogens such as Escherichia coli O157:H7 and Salmonella enterica. Lack of standard laboratory procedures for the cultivation of Diploscapter has hindered the study of this organism. In this study, we developed optimal culture conditions for Diploscapter coronatus strain LKC25 and studied the basic physiology of this nematode. We found that NGM-G containing 1.5% (w/w) gellan gum supported optimal growth of Diploscapter sp. Progression from eggs to gravid adults occurred over a period of 106 to 110 h (4.4 to 4.6 d) and was associated with four distinct larval stages. Mean life span was 12.2 d at 30&degC, 24.3 d at 25&degC, and 37.1 d at 20&degC (max at 20&degC, 56 d). Brood size was similar at 20&degC (107) and 25&degC (108), but decreased at 30&degC (95), suggesting that growth temperatures above 25&degC adversely affect these worms and confirming that optimal growth occurs at around 20&degC. Crowding of D. coronatus in liquid culture did not induce entry into the dauer state. Unlike C. elegans, this nematode also did not enter the dauer state in response to temperature or starvation. Establishment of D. coronatus culture conditions enabled a preliminary screen of an antifungal chemical library for nematode growth-inhibiting compounds. Using a newly established high-throughput screening method (Oh et al., unpublished data), we identified multiple anthelmintic agents. Their antinematodal activity relative to known drugs (e.g., fosthiazate) is discussed. (Supported by a grant from a Forest Science & Technology Project [No. S110707L0501501] through the Korea Forest Service.)"

Lee DK et al. (1995) International C. elegans Meeting "ACQUIRED THERMOTOLERANCE IN C.ELEGANS"

Han C-T, Lee DK

[International C. elegans Meeting, 1995]

We have studied about acquired thermotolerance in wild type C. elegans. Thermotolerance was determined as worms show normal movement and laying eggs 72 hours after severe heat shock for 2.5 hrs at 37 degrees Celsius. More than 90% of severely heat shocked worms died without pre-heat shock, In contrast to that, more than 85% of severely heat shocked worms survived if worms were heat shocked at 33 degrees Celsius, 35 degrees Celsius and 37 degrees Celcius for 1 hour prior to severe heat shock. In other words pre-heat shocked worms gained thermotolerace. The incubation duration giving maximum thermotolerance at 20 degrees Celsius between pre-heat shock and severe heat shock (recovery) was vary by the temperature of pre-heat shock. Worms incubated for 4 hrs and 8 hrs at 20 degrees Celcius after pre-heat shocked at 35 degrees Celsius and 37 degrees Celsius respectively showed maximum thermotolerance. But no recovery incubation was necessary for the pre-heat shocked worms at 33 degrees Celsius to gain thermotolerance, The relationship of thermotolerance and hsp synthesis was investigated showing that accumulation of hsps were required to g ive resistance to heat shock. Especialy, the accumulation of minimum amount of hsp73 was needed for the gaining the thermotolerance. To reveal the mechanism of gaining thermo tolerance in C. elegans, the mechanism of hsp73 expression is under investigation.

Lee, J. et al. (2017) International Worm Meeting "Microtubule glutamylation is dispensable for embryonic development."

Badecker, KE, Chawla, DG, Shah, R, Suri, P, Lee, J., Brewster, M, Peel, N

[International Worm Meeting, 2017]

Glutamylation, the covalent attachment of glutamic acid to tubulin in the polymerized microtubule, is enriched on long-lived microtubules, and is proposed to contribute to centriole stability, cilia motility and axon function. Glutamylation of the microtubules is catalysed by a family of tubulin tyrosine ligase like (TTLL) enzymes. Comprehensive in vivo analyses of the function of tubulin glutamylation have proved challenging because of the existence of a large family of TTLL enzymes in many species. To investigate the function of tubulin glutamylation we have generated a ttll-4(tm3310); ttll-11(tm4059); ttll-15(tm3871) ttll-5(tm3360) ttll-9(tm3889) quint mutant that lacks all five C. elegans glutamylating TTLL enzymes. The quint mutant shows normal embryonic viability and brood size indicating that the centrioles are functional and the microtubules of the spindle are competent for cell division. Recent evidence suggests that in human cells glutamylation modulates microtubule severing by spastin and katanin. To investigating whether a similar regulatory mechanism also exists in worms we have combined our quint mutant with mei-1(GOF) and mei-1(LOF) alleles of katanin (mei-1). We do not, however, observe any suppression of the mei-1-associated lethality when microtubule glutamylation is lost. Our data therefore suggest that, contrary to expectations, microtubule glutamylation is not essential for C. elegans viability. We do however observe a male mating defect indicative of ciliary dysfunction. We are currently undertaking a detailed analysis of microtubule dynamics to determine whether minor perturbations of microtubule function occur in the absence of glutamylation.

Jungsoo Lee et al. (2007) International Worm Meeting "Chronic effects of Ethanol in C. elegans."

Steven L. McIntire, Jungsoo Lee

[International Worm Meeting, 2007]

Ethanol can affect multiple signaling pathways. Some of these pathways have been implicated in acute or chronic behavioral responses to ethanol (Diamond and Gordon, 1997). Ethanol alters the behavioral responses of C. elegans at the same tissue concentrations that cause intoxication in humans (Davies et al., 2003). Similar molecular pathways appear to contribute to the acute neurodepressive effects of ethanol that are observed in C. elegans and mammalian systems. Ethanol exposure during development causes a variety of morphological changes and a retardation of growth. We are interested in identifying the molecular pathways that contribute to the developmental and behavioral defects that are observed after chronic ethanol exposure. We have observed that ethanol inhibits the growth of wild-type animals. To better understand the mechanisms contributing to this effect, we carried out screens for mutants resistant to the chronic effects of ethanol. We have screened 5,000 haploid genomes and have isolated 14 candidates showing resistance to ethanol inhibition of growth. Several mutants are also resistant to acute effects of ethanol on locomotion and egg laying. Some of the mutants have a defect in the acute tolerance that occurs during a continuous ethanol exposure. None of the mutants have an obvious behavioral phenotype in the absence of ethanol. One possibility is that chronic ethanol induces oxidative stress by enhancing reactive oxygen species (ROS) formation or by decreasing oxidative defenses (Sun et al., 2001). To assess the relationship between resistance to ethanol and oxidative stress, we examined sensitivity of the mutants to paraquat. A subset of the mutants is also resistant to paraquat. We have mapped two mutants to the center of chromosome X and are currently in the process of cloning these genes. Reference: Diamond I and Gordon AS (1997). Cellular and molecular neuroscience of alcoholism. Physiol Rev. 77(1):1-20. Review Davies AG et al. (2003). A central role of the BK potassium channel in behavioral responses to ethanol in C. elegans. Cell 115(6):655-66.