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Smith, P. A. 2009. Phenotypes to genotypes using C. elegans. Pages 349-356, in Tested
Studies for Laboratory Teaching, Volume 30 (K.L. Clase, Editor). Proceedings of the
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Phenotypes to Genotypes Using C. elegans
Pliny A. Smith
Biology Department
Lake Forest College
555 N. Sheridan Road
Lake Forest, IL 60045
[email protected]
Biography
Pliny A. Smith obtained his B.A. in Biology from Grinnell College in 1992 and his Ph.D. from the
University of Missouri in 2001. He has been an assistant professor in the Biology Department of
Lake Forest College since 2006. Pliny teaches Developmental Biology, Organism Biology, the
Biology of Aging. He uses C. elegans both in the classroom as well as to train undergraduates to
carry out original research.
Introduction
Background: This lab is a variation of the classic microbiology unknown identification labs;
however, it uses the metazoan C. elegans and genetics. Students will be given a series of
unidentified plates containing coded worm strains; known only to the instructor. By examining the
phenotypes and other properties of the worms, the students will be instructed to identify the
genotypes of each unknown from a chart of possible genotypes and phenotypes. Some of the
unknowns cannot be distinguished from each other without additional testing, which the students
should conduct to complete their project. These include tests for conditional alleles, dominance, and
complementation of alleles. Deciding appropriate tests to conduct helps to develop the student’s
ability to use available information and reagents in the design of a hypothesis.
Reasons to use C. elegans for this laboratory. Caenorhabditis. elegans is an excellent organism
for studying developmental genetics. As a free-living soil nematode, C. elegans is safe for
undergraduate students to handle, and teaches skills including use of a microscope and manipulation
of microscopic animals. The worm is simple to culture, requiring only small agar plates inoculated
with non-pathogenic E. coli as food (Brenner, 1974). In addition, thousands of genetically distinct
worm strains are available from the C. elegans Genetics Center at the University of Minnesota. C.
elegans have the advantage over flies in that they remain on Petri dishes; they don’t fly away. C.
elegans can be stored at -80˚C for years; they do not require the isolation of virgins for mating.
Completion of genetic crosses takes only a few days and the crosses are easily performed by students
using a stereo microscope, a wire tip (worm pick), and an alcohol burner. Many phenotypes are
temperature sensitive (conditional alleles), providing another opportunity for students to test
hypotheses. This protocol describes how students can identify genotypes of worms, both behavioral
and anatomical, using phenotypic observations and simple experiments.
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Student Outline
Objectives:
1. Observe Caenorhabditis elegans nematode life stages, sexes, and phenotypes.
2. Identify genotypes of worms using phenotypic observations.
3. Perform a genetic cross of C. elegans and predict the outcomes.
Introduction:
This laboratory investigation connects an organism’s genotype to its phenotype. Using the
information provided in Tables 1 and 2, most populations of worms can be accurately identified
using a microscope and an eyelash to manipulate individuals. However, phenotypes are not always
distinct to one allele of a particular gene; often alleles of different genes may affect the same trait of
an organism. In such cases, a geneticist must devise experiments that determine if the predicted
genotype is present.
The C. elegans worms used in this laboratory are identified by a strain name, i.e., N2, which
refers to the laboratory that created the strain. Genes are identified by a 3-letter code such as unc,
dpy, or bli followed by a number, i.e., unc-32. The genotypes of published C. elegans strains and the
meaning of every C. elegans gene name can be found at http://www.wormbase.org.
Materials
• Worm pick
• Alcohol lamps
• Known and Unknown worm strains on NGM agar plates (see Tables 1 and 2)
• Fresh NGM agar plates (60 x 15 mm) inoculated with E. coli
• Zoom stereo microscopes with at least 30X magnification
• Parafilm and plastic boxes for plate storage
Experimental Procedure.
1. Obtain three plates of mixed-stage known worm strains, N2 (Bristol wildtype), CB4856
(Hawaiian wildtype), and CB164 (dpy-17).
2. Observe each plate using a stereo microscope and record observations concerning size,
movement, and morphology for each type of worm.
3. An eyelash pick can be used to lightly touch the worm to see if it responds to tactile stimuli.
4. Check for the presence of male worms.
5. Obtain a set of unknown worm strains; a coded number marks each.
6. Observe the plates using a stereo microscope as before, making careful observations.
7. Compare the worms on each plate for:
a. Vulva morphology – Vulva not present, Multi-vulva, Protruding vulva.
b. Coordination – Slow moving, Rolling, and Insensitive to touch.
c. Body Morphology – Small, Dumpy, Round head, Blistered.
8. Compare the phenotypes of each worm to the list of genotypes and phenotypes provided in
the table and enter the unknown strains code in the appropriate box.
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9. In some cases, a clear choice cannot be made initially; an experiment must be performed to
identify the worm strains.
10. Design the experiment, along with appropriate controls.
11. For each experiment, write your predictions of possible outcomes, you may need to create a
Punnett Square.
12. Return at the specified time to follow-up the observations for the experiment and complete
Table 2 using the new data collected.
Table 1. Known Worm Strains
Strain
Genotype
CB4856
Hawaii WT clone
N2
Bristol WT clone
Phenotype
WT, burrows more than N2; males leave a
copulatory plug after mating.
Wildtype – standard strain for C. elegans research
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Table 2. Unknown Worm Strains
Number
Strain
Genotype
BA17
fem-1(hc17) IV
CB1309
CB30
CB3970
CB3991
MT1201
MT3283
MT8189
SM1310
SM139
SU93
M154
CB164
Phenotype
Feminization of animals at 25˚C (TS); no
sperm made at higher temperatures (Nelson et
al., 1978)
lin-2(e1309) X
Vulvaless
sma-1(e30) V
Adults are small with blunt head, larvae have
white blunt heads, recessive (McKeown et al.,
1998)
unc-4(e120) bli-1(e769) Slow moving, with blistered skin
II
sma-8(e2111) V.
Short, blunt head. (Dominant)
egl-9(n571) V
Egg laying defective
him-5(e1490)V,
egl- High incidence of males, hermaphrodites are
15(n484) X
egg laying defective (Egl) (Harfe et al., 1998)
lin-15(n765ts) X
Muv phenotype (TS) grow at 25˚C for good
multivulval phenotype (Herman et al., 1990)
tbx-2(ok529)/
dpy- Dpy/Unc worms, WT, and Tbx-2 worms, all
17(e164), unc-32(e184) have ceh-22::GFP.
ceh-22:GFP (tinman,
III;
ceh-22::GFP nkx2.5 homolog) is expressed in the pharynx,
(integrated)
but it is much lighter than myo-2::GFP (Smith
et al., 2007; Johnstone et al., 1992; Okkema et
al., 1994; Mango, 2007)
dpy-7(e1324ts) X
Dumpy, (TS); more dpy at higher temperatures
(Gilleard et al., 1997)
ajm-1::GFP pRF4 IV ajm-1::GFP – adherens junction GFP, worms
MH27::GFPm roll [jds1] roll if born at > 20˚C, but not at 15˚C (Koppen,
et al., 2001)
sma-1(?);mIs11IV
Short, blunt head (recessive), GFP expression
with myo-2:GFP, pes-10::GFP, and gut::GFP.
Strong GFP expression in pharynx muscle in
adults; embryonic expression in body cells
Dpy-17(e164) III
Short, Dpy worms (Johnstone et al., 1992)
Notes for Instructor
Genetics of C. elegans. C. elegans have five pairs of autosomes and a pair of sex chromosomes;
XX animals are hermaphroditic and self-fertile. XO animals are rarely seen unless they result from a
mating between a male (XO) and a hermaphrodite (XX). Without pre-existing males in a culture,
XO males may infrequently arise from spontaneous chromosomal non-disjunction of the X
chromosome during meiosis. Male sperm are larger than hermaphrodite sperm and always outcompete hermaphrodite sperm; therefore a mating between a hermaphrodite and male results in all
cross-progeny (LaMunyon et al., 1995).
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Strain Names. A C. elegans strain name consists of one to three CAPITAL letters followed by a
number. This identifies the laboratory that created the strain (a genetically distinct and defined line
of worms). For example, SM1310 denotes the 1310th strain that Susan E. Mango’s lab created.
Gene Names and Alleles. A C. elegans gene is either named after the mutant phenotype (unc-32,
let-60, sma-1), or after the class of protein it encodes (tbx-2, ins-2, ajm-1). Because a gene may have
more than one allele, there is often included an allele designation. This code consists of one to three
lowercase letters followed by a number that identifies the alleles source. For example, dpy-17(e164)
represents the 164 allele isolated by Jonathan Hodgkin’s laboratory (e).
Genotypes. When listed the genotype of a worm strain, the alleles are listed, starting from the left
arm to the right arm of each chromosome, which is denoted by a Roman numeral following the
alleles as in the following example: unc-4(e120) bli-1(e769) II. Heterozygous strains would add a
slash mark describing the alternate alleles on the homologous chromosome.
Proteins. The protein encoded by lin-15 gene is written as LIN-15 (all CAPITALS, non-italic).
Phenotype. Capitalize the first letter of a phenotype description and do not use italics. For
example, Uncoordinated animals such as those with a homozygous unc-32 genotype would display
an Unc-32 phenotype.
Descriptions of Gene Classes. The following is a list of the classes of genes suggested in this
protocol.
dpy "dumpy" (short and fat) body shape. Different dpy mutants have characteristic shapes
from “football” to slightly fat and may be dominant or recessive.
unc Uncoordinated movement. Unc mutants do not crawl the same as wild type, but this
may vary from total paralysis, constant coiling of their tails, a loss of touch response, or the
inability to back up.
sma Small animals. Sma animals do not appear Dpy (short and fat), but are overall smaller
than wild-type worms.
egl Egg-laying defective. Egl worms often end their lives with the Bag (bag of worms)
phenotype because the embryos hatch inside the mother who soon dies.
let Lethals. These may vary from embryonic death to failure to molt into an adult.
bli Blister phenotype. Adult animals have defects in their skin resulting in a bubbled or
blistered appearance.
lin Lineage defective. Cells fail to follow the wild type developmental program resulting in
various phenotypes related to the gain or loss of cell types.
fem Feminization of XX and XO animals. The Fem phenotype results in only female germ
cells being generated by worms, resulting in loss of self-fertility.
him High incidence of males. Chromosomal non-disjunction of the X chromosome occurs
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more frequently than in wild type, resulting in an increased number of spontaneous males.
tbx-2 T Box gene 2 of C. elegans. An evolutionarily conserved gene necessary for foregut
development in C. elegans. Homozygous lethal.
Maintaining worms. Worms are best grown on 30 mm or 60 mm NGM agar plates inoculated with
the E. coli strain OP50 that serves as the worm’s food (Wood, 1988). The plates should be stored in
a plastic box to prevent drying.
Worms are ectotherms, and their development is affected substantially by temperature. C.
elegans are grown at 15ºC, 20ºC, or 25ºC. At 20ºC, it takes approximately 3 days for a freshly laid
embryo to develop into a reproductive adult. At 15ºC it takes about 6 days to reach maturity. Higher
temperatures (20ºC-25ºC) will increase the rate of development, but can affect reproductive success
(Wood, 1988).
Worm populations grow exponentially; each hermaphrodite is self-fertile and will produce up
to 300 offspring in a few days. Single worms can be moved to a new plate with a worm pick or
larger numbers can be transferred using sterile water and a pipette. Care should be taken that the
worms being transferred to new plates display the correct phenotype.
Staging the worms. C. elegans are readily staged and sexed using a dissecting microscope.. The
embryos are oval in shape, with a length of ~100 µm and a width of about 60 µm. The embryos
remain constant in size during development, but the size of the cells within the embryo decreases as
cell divisions take place.
Larval worms are distinguished by size and morphology. L1s are smaller than L2s, which
are smaller than L3s, which in turn are smaller than L4s. Adults are largest and are morphologically
distinct from L4s by containing either male or female external genitalia. The adult hermaphrodite
contains developing embryos inside its uterus as well.
Contamination. Care should be taken to prevent foreign material from contacting the worm plates
or bacterial or fungal contamination can occur. Bacteria other than the normally used OP50 strain
will make it more difficult to see the worms. Fungus will decrease the fertility, survival of the
worms and obstruct microscopic observation as well.
Other Resources.
WormBase: http://wormbase.org
WormBase is a publicly funded, online database of nematode genomics, literature, reagents, and
anything else worm related. WormBase can be used to research the phenotypes of alleles that
students are investigating.
WormBook: http://wormbook.org
Contains chapters concerning C. elegans development, genetics, cell biology, evolution, as well
as worm methods and protocols
Wormatlas: http://wormatlas.org
A free source of diagrams, descriptions, and photographs of worm anatomy.
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C. elegans Genetic Center (CGC): http://www.cbs.umn.edu/CGC/
Source of worm and bacterial strains used in C. elegans labs.
Materials
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•
•
•
•
•
•
•
•
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•
•
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Platinum/iridium wire
Standard Pasteur pipets
Alcohol lamps
A Bunsen burner
Tooth picks
Nail polish
Worm strains (see Table I)
NGM agar plates (60x15mm) inoculated with E. coli
E. coli (the OP50 strain works best)
Zoom stereo microscopes with at least 30X magnification
15˚C incubator
25˚C incubator
Parafilm and plastic boxes for plate storage
Protocols.
Creating eyelash picks
1. Place a small drop of nail polish on the end of a toothpick
2. Pluck an eyelash or eyebrow and insert the follicular (large) end into the nail polish.
3. Let dry and do not sterilize in a flame.
Creating a worm pick
1. Cut a 1.0 cm piece of platinum/iridium wire.
2. Insert 1/3 of the wire into a shorten Pasteur pipet, holding the wire in place with a pair of
forceps.
3. Place the end of the pipette containing the wire into a gas flame and twist the wire with the
forceps to melt the glass and close the end.
4. Let the pick cool and flatten and bend the end of the wire to form a scoop.
Making NGM agar plates
1. Autoclave the following:
2.5 g Bacto Peptone
3.0 g NaCl
20 g Bacto Agar
975 mL distilled H2O
2. After autoclaving, add:
25 ml 1M Sodium Phosphate, pH 6.0
1 mL 1M CaCl2
1 mL 1M MgSO4
1 mL 10 mg/ml Cholesterol in Ethanol
3. Pour ~10 mL of media into each 60 x 15 mm plastic Petri dish, an automatic plate pouring
machine is very useful for making consistent plates
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4. Inoculate NGM plates with 50–100 µL of fresh OP50 culture and allow to grow overnight.
Literature Cited
Brenner, S. 1974. The genetics of Caenorhabditis elegans. Genetics, 77:71-94.
Gilleard, J.S., Barry, J.D., and Johnstone, I.L. 1997. cis regulatory requirements for hypodermal cellspecific expression of the Caenorhabditis elegans cuticle collagen gene dpy-7. Molecular and
Cellular Biology, 17:2301-11.
Harfe, B.D., Vaz Gomes, A., Kenyon, C., Liu, J., Krause, M., and Fire, A. (1998). Analysis of a
Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of
mesodermal patterning. Genes and Development. 12:2623-35.
Herman, R.K., and Hedgecock, E.M. 1990. Limitation of the size of the vulval primordium of
Caenorhabditis elegans by lin-15 expression in surrounding hypodermis. Nature 348, 169171.
Johnstone, I.L., Shafi, Y., and Barry, J.D. 1992. Molecular analysis of mutations in the
Caenorhabditis elegans collagen gene dpy-7. Embo Journal. 11:3857-63.
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LaMunyon, C.W. and Ward, S. 1995 Sperm precedence in a hermaphroditic nematode
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Mango, S.E. 2007. The C. elegans pharynx: a model for organogenesis. WormBook 1-26.
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