Caenorhabditis elegans is a species of microscopic round worm that has been used as a genetic model for over forty years. When in an adverse environment, C. elegans larvae cease reproductive development and enter the stress-resistant dauer stage.
dex-1 mutants of C. elegans are deficient in this protein, resulting in shortened dendrites and a sensitivity to sodium dodecyl sulfate (SDS). SDS will kill any non-dauer C. elegans, but wild type dauers will survive well past the standard concentration of 1% SDS. Thus, treatment with SDS is commonly how labs isolate dauers. By contrast, dex-1 dauers (Fig.3) will die when exposed to 1% SDS, but can potentially survive when exposed to less.
The focus of this lab is to characterize the genetic pathways that facilitate morphological changes that occur during the dauer stage by finding potential interactors of dex-1 during dauer when conducting a suppressor screen

Barnum, Matthew

Flatt, Kristen

Schroeder, Nathan E.

English

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SDS selection
Findings
• After washing off a bit over 100 plates, and exposing 
the worms to SDS, we found 7 independent mutants 
that suppressed their dex-1 phenotype.
Matthew Barnum,1 Kristen Flatt,2 and Nathan Schroeder2,3
Triton College, River Grove, IL1
Neuroscience program, University of Illinois at Urbana-Champaign2
Department of Crop Sciences, College of Agriculture, Consumer and Environmental Sciences University of Illinois at Urbana-Champaign3
Finding dex-1 Phenotype Suppressing Components
Acknowledgments
Financial support was provided by the National Science 
Foundation under grant #NSF REU 1559908/1559929, as part of 
the Phenotypic Plasticity Research Experience for Community 
College Students, through the University of Illinois at Urbana-
Champaign Institute for Genomic Biology and Parkland College. 
http://precs.igb.illinois.edu/
I’d like to thank the project directors, Dr. Nathan Schroeder and 
Dr. C. Britt Carlson. Thank you as well to my mentor Kristen Flatt 
and the whole Schroeder lab for all the encouragement.
References
1) Cassada, R. C., & Russell, R. L. (1975)
2)  Heiman, M. G., & Shaham, S. (2009).
3)  Jorgensen, E. M., & Mango, S. E. (2002)
4)  Sin, O., Michels, H., & Nollen, E. (2014)
5)  Hodgkin, J. Genetic suppression (2005)
Project Outline
Our project set out to find potential interactors of dex-1
during dauer by conducting a suppressor screen
Suppressor Screening
• We started by finding dex-1 mutants that’ve 
suppressed their SDS sensitivity, and we did this 
through mutagenesis and SDS selection. 
• This involved mutating dex-1 mutants with EMS, and 
exposing their F2 progeny to SDS while in dauer. We 
then moved on to mapping the survivors mutation. 
Gene Mapping
• Once we isolate suppressor mutants, we will perform 
several crosses between these mutants and 
mapping strains. 
• Based on the crosses progeny's phenotypic ratios, 
these crosses will tell us what chromosome the 
mutated gene lies on, and further crosses will narrow 
down and eventually determine the map position. 
• After finding that position, we will track the new gene’s 
resulting protein and its effect. 
Mutagenesis
Background
C. elegans:
• Caenorhabditis elegans is a species of microscopic round 
worm that has been used as a genetic model for over forty 
years. 
• When in an adverse environment, C. elegans larvae cease 
reproductive development and enter the stress-resistant 
dauer stage1 (Fig.2).
• The focus of our lab is characterizing the genetic 
pathways that facilitate morphological changes that occur 
during the dauer stage.
dex-1 mutants
dex-1 mutants of C. elegans:
• DEX-1 is an extracellular matrix protein in C. elegans 
responsible for proper dendrite morphology in the early 
stages of the worms growth.2
• dex-1 mutants are deficient in this protein, resulting in 
shortened dendrites and a sensitivity to sodium dodecyl 
sulfate (SDS).
• SDS will kill any non-dauer C. elegans, but wild type 
dauers will survive well past the standard concentration of 
1% SDS. Thus, treatment with SDS is commonly how labs 
isolate dauers. 
• In contrast, dex-1 dauers (Fig.3) will die when exposed to 
1% SDS, but can potentially survive in less (Fig.1).
Potential types of suppression mutation
Intragenic mutation
• Same site replacement: a form of suppression wherein the 
originally mutated gene is simply mutated back to N2.
• Compensatory second site mutation: a mutation 
elsewhere on the protein that affects it in such a way that 
the original mutation’s phenotype is reduced. Rare.
Extragenic
• Bypass suppression: a mutation that activates an alternate 
pathway, “bypassing” the defective components. 
o For example, a mutation in a non-coding region that 
results in a change in the level of dex-1 expression.
• Suppression by interacting components: a mutation that 
restores an abnormal protein to proper functionality by 
changing an interacting protein.5
o For example, the CUT protein is an interactor with the 
DEX-1 protein, so a change in it could potentially cause a 
suppression by interacting components.
Further Research
Mapping a Gene:
• With the exact mutation site found, we can track the 
gene’s resulting protein’s pathway and determine what 
kind of suppression it is.
Embryo
L1
(adverse conditions)
L2                                      L2d
(Recovery)
L3                                     Dauer
(Recovery)
L4
Adult
General mapping strain   x   Suppressed mutant
(Male)                           (Hermaphrodite)
Chromosome specific               Suppressed 
mapping strain         x              mutant
(Male)                           (Hermaphrodite)
Mapping strain with                 Suppressed
flanking markers        x             mutant
(Male)                          (Hermaphrodite)
Fosmid rescue
Single gene rescue
Fig.5 Protocol for EMS mutagenesis3
Mutating the worms:
• We synced worms up at 
the L4 stage (Fig.4)
• Mutated synced L4’s with 
Ethyl Methanosulfonate
o We were sure to use 
proper safety equipment, 
as EMS is toxic
• Checking every day and 
separating individuals 
when needed, we 
allowed the mutated P0 
generation to lay the F1 
generation, then we let 
those F1 lay the F2 
(which are able to have 
recessive phenotypes).3
• For this reason, these F2 
are the worms we 
screened with SDS.
Fig.4 dex-1 L4 worm. Closeup of vulva.
Screening the worms:
• Got F2 gen to become 
dauers
o We did this by placing 
adults older than L1 at 
25oC. This causes their 
offspring to reach dauer
thanks to a secondary 
mutation in our worms 
known as daf-7 
• Exposed the dauer F2 to 
SDS
• Picked survivors onto 
their own plates
• From here we can move 
onto gene mapping
Fig.6 Protocol for a suppressor screen.4
Fig.1 SDS sensitivity scale.
AGA CCC CGA CCA             (N2 protein)
AGA CCC TGA CCA            (dex-1 protein)2
AGA CCC CGA CCA      (Suppressor’s protein)
Fig.2 N2 dauer
Fig.3 dex-1 dauer


Finding dex-1 Phenotype Suppressing Components

Scholarship at Parkland

SDS selectionFindings• After washing off a bit over 100 plates, and exposing the worms to SDS, we found 7 independent mutants that suppressed their dex-1 phenotype.Matthew Barnum,1 Kristen Flatt,2 and Nathan Schroeder2,3Triton College, River Grove, IL1Neuroscience program, University of Illinois at Urbana-Champaign2Department of Crop Sciences, College of Agriculture, Consumer and Environmental Sciences University of Illinois at Urbana-Champaign3Finding dex-1 Phenotype Suppressing ComponentsAcknowledgmentsFinancial support was provided by the National Science Foundation under grant #NSF REU 1559908/1559929, as part of the Phenotypic Plasticity Research Experience for Community College Students, through the University of Illinois at Urbana-Champaign Institute for Genomic Biology and Parkland College. http://precs.igb.illinois.edu/I’d like to thank the project directors, Dr. Nathan Schroeder and Dr. C. Britt Carlson. Thank you as well to my mentor Kristen Flatt and the whole Schroeder lab for all the encouragement.References1) Cassada, R. C., & Russell, R. L. (1975)2)  Heiman, M. G., & Shaham, S. (2009).3)  Jorgensen, E. M., & Mango, S. E. (2002)4)  Sin, O., Michels, H., & Nollen, E. (2014)5)  Hodgkin, J. Genetic suppression (2005)Project OutlineOur project set out to find potential interactors of dex-1during dauer by conducting a suppressor screenSuppressor Screening• We started by finding dex-1 mutants that’ve suppressed their SDS sensitivity, and we did this through mutagenesis and SDS selection. • This involved mutating dex-1 mutants with EMS, and exposing their F2 progeny to SDS while in dauer. We then moved on to mapping the survivors mutation. Gene Mapping• Once we isolate suppressor mutants, we will perform several crosses between these mutants and mapping strains. • Based on the crosses progeny's phenotypic ratios, these crosses will tell us what chromosome the mutated gene lies on, and further crosses will narrow down and eventually determine the map position. • After finding that position, we will track the new gene’s resulting protein and its effect. MutagenesisBackgroundC. elegans:• Caenorhabditis elegans is a species of microscopic round worm that has been used as a genetic model for over forty years. • When in an adverse environment, C. elegans larvae cease reproductive development and enter the stress-resistant dauer stage1 (Fig.2).• The focus of our lab is characterizing the genetic pathways that facilitate morphological changes that occur during the dauer stage.dex-1 mutantsdex-1 mutants of C. elegans:• DEX-1 is an extracellular matrix protein in C. elegans responsible for proper dendrite morphology in the early stages of the worms growth.2• dex-1 mutants are deficient in this protein, resulting in shortened dendrites and a sensitivity to sodium dodecyl sulfate (SDS).• SDS will kill any non-dauer C. elegans, but wild type dauers will survive well past the standard concentration of 1% SDS. Thus, treatment with SDS is commonly how labs isolate dauers. • In contrast, dex-1 dauers (Fig.3) will die when exposed to 1% SDS, but can potentially survive in less (Fig.1).Potential types of suppression mutationIntragenic mutation• Same site replacement: a form of suppression wherein the originally mutated gene is simply mutated back to N2.• Compensatory second site mutation: a mutation elsewhere on the protein that affects it in such a way that the original mutation’s phenotype is reduced. Rare.Extragenic• Bypass suppression: a mutation that activates an alternate pathway, “bypassing” the defective components. o For example, a mutation in a non-coding region that results in a change in the level of dex-1 expression.• Suppression by interacting components: a mutation that restores an abnormal protein to proper functionality by changing an interacting protein.5o For example, the CUT protein is an interactor with the DEX-1 protein, so a change in it could potentially cause a suppression by interacting components.Further ResearchMapping a Gene:• With the exact mutation site found, we can track the gene’s resulting protein’s pathway and determine what kind of suppression it is.EmbryoL1(adverse conditions)L2                                      L2d(Recovery)L3                                     Dauer(Recovery)L4AdultGeneral mapping strain   x   Suppressed mutant(Male)                           (Hermaphrodite)Chromosome specific               Suppressed mapping strain         x              mutant(Male)                           (Hermaphrodite)Mapping strain with                 Suppressedflanking markers        x             mutant(Male)                          (Hermaphrodite)Fosmid rescueSingle gene rescueFig.5 Protocol for EMS mutagenesis3Mutating the worms:• We synced worms up at the L4 stage (Fig.4)• Mutated synced L4’s with Ethyl Methanosulfonateo We were sure to use proper safety equipment, as EMS is toxic• Checking every day and separating individuals when needed, we allowed the mutated P0 generation to lay the F1 generation, then we let those F1 lay the F2 (which are able to have recessive phenotypes).3• For this reason, these F2 are the worms we screened with SDS.Fig.4 dex-1 L4 worm. Closeup of vulva.Screening the worms:• Got F2 gen to become dauerso We did this by placing adults older than L1 at 25oC. This causes their offspring to reach dauerthanks to a secondary mutation in our worms known as daf-7 • Exposed the dauer F2 to SDS• Picked survivors onto their own plates• From here we can move onto gene mappingFig.6 Protocol for a suppressor screen.4Fig.1 SDS sensitivity scale.AGA CCC CGA CCA             (N2 protein)AGA CCC TGA CCA            (dex-1 protein)2AGA CCC CGA CCA      (Suppressor’s protein)Fig.2 N2 dauerFig.3 dex-1 dauer

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Finding dex-1 Phenotype Suppressing Components

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