Hall, Julie (2021) International Worm Meeting "Creating choice in molecular genetics lab through the use of toxicology"

Hall, Julie

[International Worm Meeting, 2021]

Some pedagogy techniques focus on giving students choice - what assignments to do, what topic path to study, or what topics within the course an individual wants to learn. The basis behind this is that by giving students choice, they have ownership in their learning and will be more invested in their coursework. For many science courses, however, the choice of what topic to study is not a viable option; students cannot choose to learn about translation and not transcription or leave out entire units of key concepts in a discipline. Regardless, creating choice and ownership is still important in student learning and can be done in a more structured way. In my 300-level molecular genetics lab, students choose a toxicant to investigate for two lab experiments using C. elegans. One lab experiment (Lab 1) focuses on utilizing reverse transcription and PCR to analyze gene expression. The second lab experiment (Lab 2) is a toxicology measure where students pick an outcome (movement, growth, reproduction, or death) to observe after exposure to their toxicant of choice. Students work in pairs to pick a toxicant to study. They perform a literature search to determine two concentrations to test for the experiments and an outcome to test in Lab 2 based on their readings. Lab 1 allows students to learn RNA isolation, PCR, and gel electrophoresis while testing a hypothesis related to gene expression in response to their toxicant. Lab 2 provides more opportunities for students to have choice in which they explore an effect of the toxicant that interests them. Students present their research through the writing of an introduction, results, and discussion sections in the form a journal style paper. Student choice with these two labs provides them with the opportunity to have ownership of the experiments through structured lessons. Students also practice skills of developing research statements and hypothesize through literature research and investigation. These labs are reported as being a favorite within the course and students become invested in conducting the experiments because they got to choose what was being tested (toxicant) and what they wanted to test (outcome).

Hall, Julie et al. (2009) International Worm Meeting "Understanding linkage and recombination using C. elegans RNAi mutants."

Hall, Julie, Zucchero, Theresa

[International Worm Meeting, 2009]

Although students often grasp the concepts of genetic inheritance and DNA structure easily, they sometimes struggle with understanding linkage and recombination frequencies. We set out to design a guided investigative project for undergraduate genetic students to help them better understand the concepts of linkage and recombination frequencies and how these concepts are used in research to genetically map an unknown gene. The goal of this laboratory exercise is to help students understand how a genetic model organism is used in the study of genetics. More specifically, students will be able to determine linkage using basic genetic crosses with marker strains, apply concepts of mapping, and calculate recombination frequencies to map a gene. For this five week project, students work in pairs to map the location of an "unknown" mutant which is defective for a gene involved in RNA interference (RNAi). When mutated, these genes result in RNAi resistance, an easily testable phenotype. Using linkage analysis, students map their "unknown" mutant to one of three chromosomes. Once they have determined on which chromosome their mutant lies, students pick strains from a provided list of mutants with visible phenotypes to use in a three-factor cross. Using the data collected from their three-factor cross, students determine recombination frequencies and an approximate map position. Finally, with the help of the professor, students navigate Wormbase.org to determine the identity of their "unknown" gene. The data the students collected did lead them to map locations that allowed them to correctly predict the identity of their "unknown" gene. Background information was introduced in the form of review articles and primary literature. Worksheets requiring the students to fill in the predicted results were provided to help them better understand the crosses being performed. Additionally, at the end of the project, students were asked to submit a journal-style paper based on their results. Although students were guided through the project, they were required to think critically about their results and the next step of the experiment. This project allowed students to apply knowledge they had learned in the genetics course to an experiment that mimicked a procedure often performed in research.

Hall, Julie et al. (2011) International Worm Meeting "Identification of a pathway involved in the cadmium-responsive transcriptional regulation of metallothionein expression."

Freedman, Jonathan, Hall, Julie

[International Worm Meeting, 2011]

The carcinogenic metal cadmium can induce various intracellular stresses. Analysis of transcriptome data from multiple species indicate that cadmium exposure alters the expression of hundreds of genes that are regulated by multiple signal transduction pathways, many of which remain to be defined. In response to cadmium, cells increase the expression of highly conserved, small, cysteine-rich metal-binding proteins known as metallothioneins (MTs), which function in metal detoxification. The nematode C. elegans has two MT genes: mtl-1 and mtl-2. To identify regulatory factors and pathways that control metal-inducible mtl-1 transcription, integrated transgenic strains of C. elegans containing GFP under the control of the 5'-regulatory region of mtl-1 were constructed, pmtl-1::GFP. Transgenic strains constitutively express GFP in the pharynx and following cadmium exposure, express GFP in the intestine. In a reverse genetic screen, genes involved in various stress response pathways were tested for their potential role in controlling mtl-1 expression. Knockout of akt-1 or akt-2 did not affect GFP expression however, the knockout of both genes simultaneously increased expression. AKT-1 is a serine/threonine kinase involved in the insulin signaling pathway and complexes with AKT-2 to regulate transcription of downstream factors. PDK-1 directly interacts with this complex and the knockout of pdk-1 resulted in an increase in expression. Interestingly, mtl-1 transcription was not affected when other insulin signaling pathway genes were knocked out. This suggests that PDK-1 and the AKT-1/2 complex act independently of this pathway to control mtl-1 transcription. To identify other transcriptional regulators, transcription factors involved in various MAPK pathways were tested. Knockout of atf-7 which is involved in the JNK/p38 pathway resulted in an increase in GFP expression. Pathway analysis and RT-PCR data indicate that ATF-7 regulates cadmium-inducible MT transcription downstream of PDK-1 and AKT-1/2. Quantitative RT-PCR also suggests that PMK-1, known regulator of ATF-7; PAX-2, a transcription factor; and TAX-4, a gated ion channel, may also be involved in mtl-1 gene regulation. In a forward genetic screen eleven EMS mutagenized pmtl-1::GFP lines with an increase in GFP after cadmium exposure were isolated. Six of the lines have been mapped to chromosome regions via SNP mapping. Through NextGen sequencing mutations in candidate genes are being identified and further analysis will identify which gene is involved in mtl-1 transcriptional regulation. Through both forward and reverse genetics a pathway responsible for the cadmium-induced transcriptional regulation of mtl-1 is emerging.

Hall, Julie et al. (2009) International Worm Meeting "Identifying cadmium-response transcriptional regulators of the C. elegans metallothionein gene, mtl-1."

Hall, Julie, Freedman, Jonathan

[International Worm Meeting, 2009]

Humans are constantly exposed to the carcinogenic metal cadmium through various environmental routes including diet and cigarette smoke. Cadmium accumulates in cells resulting in a variety of responses including oxidative stress, altered protein activity, inhibition of DNA repair, and the activation of cellular response pathways. In response to cadmium and other metals, cells increase the expression of small cysteine-rich metal-binding proteins that function in metal detoxification and homeostasis known as metallothioneins (MTs). MTs are highly conserved and C. elegans has two MT genes: mtl-1 and mtl-2. Interestingly, C. elegans MT genes lack metal response elements, which are evolutionarily conserved metal regulatory regions found in the promoters of most eukaryotic MT genes. Additionally, a homolog of the cognate transcription factor has not been identified in C. elegans. To identify regulatory factors that control mtl-1 transcription, integrated transgenic strains of C. elegans containing GFP under the control of the 5''-regulatory region of mtl-1 were constructed, pmtl-1::GFP. Similar to what has been reported for other mtl-1 transgenic strains, constitutive GFP expression was observed in the pharynx and following cadmium exposure in the intestine. To identify transcriptional regulators of mtl-1, genes involved in the aging/stress response and oxidative stress pathways were tested to see if they affected GFP expression in pmtl-1::GFP. akt-1(mg144), a gain of function allele, resulted in an increase in GFP expression. AKT-1 is a serine/threonine kinase involved in the insulin signaling pathway. The akt-1 loss of function allele, akt-1(ok525), and other members of the insulin signaling pathway were tested. AKT-1 and AKT-2 act as a complex to regulate the transcription of various genes. akt-1(ok525) or akt-2(ok393) did not affect GFP expression, however, the akt-1(ok525);akt-2(ok393) double-mutant caused an increase in GFP levels. PDK-1 directly interacts with the AKT-1/AKT-2 complex and the pdk-1(sa709) mutation caused an increase in GFP levels in pmtl-1::GFP. Both daf-2, the insulin signaling receptor, and daf-16, the transcription factor that is inhibited by the AKT-1/AKT-2 complex, did not affect GFP expression. Recent evidence has shown that PDK-1 and the AKT-1/AKT-2 complex can regulate the transcription of various genes independent of the insulin signaling pathway. Our data suggests this may be such the case for the transcriptional regulation of mtl-1 in response to cadmium and further pathway analysis and RT-PCR is being conducted to confirm this is the case.

Marinello, Christopher et al. (2017) International Worm Meeting "Investigating the regulatory pathway of C. elegans metallothionein gene mtl-2."

Hall, Julie, Henderson, Melissa, Marinello, Christopher

[International Worm Meeting, 2017]

Metallothioneins are small, cysteine-rich, metal-binding proteins that are important in metal detoxification and homeostasis in the cell. MTs are highly conserved and C. elegans has two, mtl-1 and mtl-2. Interestingly, C. elegans metallothionein genes lack MRE's, the well-known transcriptional metal regulatory region found in the promoters of most eukaryotic metallothionein genes. In addition, a homolog of the metal-regulatory transcription factor, MTF-1, has not been identified in the C. elegans genome. Genetic screens have identified a negative regulatory pathway for mtl-1 which includes ATF-7, PMK-1 and members of the insulin signaling pathway, PDK-1 and the AKT-1/-2 complex. As determined by qRT-PCR mtl-2 expression is not affected by these genes found to regulate mtl-1. While it has been identified that ELT-2 is required for metallothionein gut expression this alone does not explain the response to cadmium and other cellular stresses. To identify potential regulators of mtl-2 expression two major strategies are being employed: (1) creation of transgenic strains of C. elegans containing GFP under the control of mtl-2 promoter using the CRIPSR/CAS9 system and (2) forward genetic screen using RNAi knockdown of genes associated with cellular detoxification and response to stress. GFP expression will be measured after exposure to cadmium and other heavy metals along with the knockdown of candidate genes to determine the regulatory pathway of mtl-2.

Sponaugle, Jessica et al. (2017) International Worm Meeting "Understanding the toxicological effects of nutritional and non-nutritional sugars using C. elegans."

Harber, Amanda, Hall, Julie, Sponaugle, Jessica

[International Worm Meeting, 2017]

Sugars are carbohydrates taken into the body through eating or drinking, and are either used to make ATP or stored for a later use in the adipose tissue. The body needs an appropriate amount of sugar in order to function correctly. In fact, the daily recommended amount of sugar is only 32 grams. When the body has more sugar than needed it raises health risk factors including dental caries, obesity, diabetes, and heart diseases. There are two types of sugars: nutritive or nature sugars such as honey and stevia; and nonnutritive which are chemically made and include such things as sucralose, aspartame, and saccharin. Little research has been done on understanding the health risks of nonnutritive sugar. The toxicological effects of two nutritive and two nonnutritive sugars are being investigated in C. elegans to better understand their effects. Nematodes were exposed to the daily recommended amount (32g), two times the recommended amount, three times the recommended amount and the average daily consumption (72g) of sugar and tested for growth and reproduction. In regards to growth there was no change in the rate between any sugars or conditions. Higher concentrations of sugar did appear more sluggish and fatter. Currently reproduction is being investigated and preliminary data suggests that nutritive sugars increase the reproduction rates. These basic toxicological assays as well as aging can help in a better understanding of possible health risks, if any, related to different types of sugars.

Haas, Kathryn L et al. (2011) International Worm Meeting "MTL-1, MTL-2 and CDR-1 are not essential in C. elegans resistance to cadmium."

Hall, Julie, Haas, Kathryn L, Freedman, Jonathan

[International Worm Meeting, 2011]

Consideration of biochemical detoxification of heavy metals, including cadmium, in animals has focused mainly on two classes of metal-binding peptides: the thiol tripeptide, glutathione (GSH), and a class of diverse cysteine-rich low molecular weight peptides, the metallothioneins. The nematode C. elegans contains two metallothionein genes, mtl-1 and mtl-2 that have been presumed to play a dominant role in cadmium detoxification. However knockdown of these genes individually or synchronously by RNA interference and deletion mutations does not result in a cadmium hypersensitive phenotype as compared to wild type. In addition to the metallothioneins as a potential metal detoxification pathway, C. elegans possesses a cadmium specific response gene, cdr-1, which is highly up-regulated upon exposure to cadmium. To test if cdr-1 is a critical gene in metal detoxification in nematodes lacking metallothionein, a triple mutant, mtl-1(tm1770),mtl-2(gk125),cdr-1(tm723), was constructed and tested for hypersensitivity to cadmium by examining the brood size, embryonic lethality, Bagging phenotype and growth. Growth at low cadmium concentrations was the only endpoint in which the triple mutant displayed more sensitivity than the single and double mutants. Although mtl-1, mtl-2 and cdr-1 are highly up-regulated in response to cadmium, these data suggest that they are not the only genes required for the response to cadmium exposure. A likely explanation for the surprising cadmium resistance in mtl-1(tm1770),mtl-2(gk125),cdr-1(tm723) nematodes is a compensatory mechanism that can overcome loss of other metal detoxification peptides. In addition to glutathione alone being a potential pathway in cadmium resistance, a class of metal-binding peptides, the phytochelatins (PCs), has recently been show to impart cadmium resistance in C. elegans. We are now investigating whether the transsulfuration pathway might be a major player in nematode response to cadmium, especially in metallothionein and cdr-1 deficient strains. Experiments using LCMS analysis of various nematode metabolites, including glutathione and phytochelatins in wild type, double mtl-1(tm1770),mtl-2(gk125)and triple mtl-1(tm1770),mtl-2(gk125),cdr-1(tm723) nematodes are currently underway in order to determine relative response of the transsulfuration pathway in response to cadmium exposure in these mutant strains.

Koga, Scarlett et al. (2017) International Worm Meeting "Cellular effects of copper chloride and nickel sulfate in Caenorhabditis elegans."

Koga, Scarlett, Waldroupe, Jennifer, Campbell, Kaitlyn, Hall, Julie

[International Worm Meeting, 2017]

Copper and nickel are elemental nutrients that are necessary for human biochemical and physiological functions and also used by healthcare professionals to help patients overcome disease and surgeries. In high concentrations, exposure to these metals can cause adverse health consequences damaging organ functionality. These metals are naturally found in food sources such as crab, almonds, cocoa, black pepper, and many more. Exposure to nickel and copper can also occur through contamination of water and soil due to manufacturing of metals, forest fires, and decaying vegetation. To further understand the cellular effects of copper and nickel exposure, apoptosis and cell cycle arrest were investigated in the nematode C. elegans. EC10, EC30, and EC50 concentrations were determined using a growth response assay for copper. Due to extreme phenotypic abnormalities observed at the EC30 and EC50 nickel concentrations, studies with nickel were performed below the calculated EC10. For copper (III) chloride the determined EC10, EC30, and EC50 were 200 M, 300 M, and 400 M, respectively. For nickel (II) sulfate, the EC10 concentration was calculated to 800 M and additional concentrations of 200 M, 400 M, and 600 M were used. Apoptosis was assessed in the germline after 24h exposure to copper or nickel. After exposure to 400 M copper, statistically significant increase in germline apoptosis was observed when compared to the untreated. Exposure to nickel sulfate demonstrated similar trends in apoptosis with statistically significant differences at 400 M, 600 M, and 800 M (2.72, 3.6, and 4, respectively) when compared to the untreated (0.89). Due to induced embryonic apoptosis and phenotypic abnormalities observed during exposure to metals, reproduction assays were performed to investigate the effect of metal toxicity on average brood size and embryonic lethality. Trends demonstrated a drastic decrease in reproduction and an increase in embryonic lethality with increased concentrations of copper and nickel. Cell cycle arrest and chromosome number are being assessed to determine other possible pathways effected by these metals. These results indicate that copper and nickel may effect germ cell development and/or embryonic development from parental exposure through apoptosis and other DNA damage pathways.

Consortium TCeGS (1994) Worm Breeder's Gazette "The C. elegans Genome Sequencing Project: A Progress Report"

Consortium TCeGS

[Worm Breeder's Gazette, 1994]

The C. elegans genome sequencing project: A progress report. The C. elegans Genome Consortium, Genome Sequencing Center, Washington University School of Medicine, St. Louis, Missouri, USA and Sanger Centre, Hinxton Hall, Cambridge, UK.

Nakashima, Aisa et al. (2009) International Worm Meeting "RNAi Spreading Mutants rsd-2 and rsd-6 are Deficient for Germ Cell Immortality."

Ahmed, Shawn, Garwood, Lauren, Nakashima, Aisa, Alvares, Stacy, Hall, Julie, LaRoque, Jan, Zucchero, Theresa

[International Worm Meeting, 2009]

Germ cells represent canonical stem cells that possess the remarkable quality of being able to proliferate from one generation to the next, indefinitely, free of replicative damage. mortal germline (mrt) mutants initially display normal levels of fertility, but become progressively sterile when grown for multiple generations. Of 16 mrt mutants identified in a pilot EMS screen (Ahmed and Hodgkin, 2000), most were temperature-sensitive and only became sterile when grown at 25 deg C. Of the ts mutants, two were resistant to RNAi feeding constructs targeting embryonic lethal genes: 1c and 10i. Genetic mapping experiments, complementation tests for RNAi resistance, and DNA sequencing revealed that 1c and 10i contain mutations in rsd-6 and rsd-2, respectively. Independently isolated alleles of rsd-6 and rsd-2 confer progressive sterility when propagated at 25 deg C, and complementation tests confirmed that these genes are required for germ cell immortality. rsd-6 and rsd-2 have been reported to be deficient for spreading of dsRNA-mediated RNAi from somatic cells to germ cells (Tijsterman et al., 2004). However, our results indicate that RSD-6 may be required in germ cells to promote germ cell immortality, whereas it functions in somatic but not germ cells to promote RNAi spreading. In addition, other RNAi-spreading defective mutants do not exhibit progressive sterility. These results indicate that RSD-6 and RSD-2 are likely to promote germ cell immortality via a mechanism that is distinct from their role in RNAi spreading.