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1. ABSTRACT
The purpose of this lab experiment was to analyze genes that mediate biological
processes using Caenorhabditis elegans as an ideal genetic model system. The forward
genetic screen technique was performed to identify the genes with associated
phenotypic mutant. This technique was firstly performed by father of C. elegans and
Genetics, Sydney Brenner. We performed the experiment under the supervision of the
teaching assistant. Laboratory procedures were carefully followed, and the data results
were recorded.
Two research questions were raised that (1) whether or not the genetic mutations
of dpy-11 in C.elegans was dominant or recessive (2) how such mutations would alter
the body form which was phenotypically observed. A hypothesis suggested that a
recessive allele in C. elegans would result a dumpy phenotype (dpy). Worms with such
phenotypes would possess short and fat body regardless of the same developing
processes. The study of dumpy mutation in C. elegans played a role in exploring the
mutation mechanisms and associated phenotype in orthologues of human genes.

2. INTRODUCTION
The purpose of this project was to use C. elegans as a genetic model system to
explore the biological processes which were relevant to biological application research.
Forward genetic screen allowed to study specific genes that associated with certain
phenotypes. Such phenotypes of interest were initially observed, and the associated
genes were analyzed.

C. elegans is known as a soil nematode that possess a body length about 1 mm.
the animals live in soil and survive by bacteria. Sydney Brenner was famous for
choosing C.elegans as a model for his studies of genetics mechanisms in the
development of nervous system.

Such animals are considered as a genetic model system due to its simple
anatomical characteristics. C. elegans possesses a transparent body that organs of
interest can be seen under a dissecting microscope. The animals can be cultivated
easily and has a short life cycle of 3 days. There are two sexual classifications in C.

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elegans: males and hermaphrodites. Male worms only have testes and hermaphrodites
have both testes and ovaries. Worms can undergo self-fertilization or cross-fertilization.
In nature, the number of male worms are less than that of hermaphrodite worms. The
adult hermaphrodite worms have about 959 cells while the males have 1031 somatic
cells. Worms’ life span is about 2 to 3 weeks and it requires about 14 hours for the
embryo to develop. Post-embryogenesis involves four stages, as follow: L1, L2, L3, and
L4 (Figure 1). As the food is absent after the L2 stage, an alternative stage called dauer
is formed. This stage can last for many months and the larvae becomes stress
resistance.

Figure 1. Lifecycle of C. elegans

In this project, forward genetic screen technique was used to study the genes
that affected a specific phenotype. C.elegans with mutant genes would possess
different phenotypic features compared to a wild type C. elegans. This mutagenized C.
elegans might reproduce offpring also had such phenotypes. This indicated that the
mutant was inherited and the phenotype was observable in offspring. Such approach to

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screen mutagenized population of animals for phenotypes of interest was called forward
genetic screen. This technique allowed to isolate C.elegans with a specific phenotypic
feature and the associated genes were studied to identify DNA mutation.

Scientists use forward genetic genes to study biological pathway at molecular

level. Mutant genes that resulted an observable phenotype could be analyzed. This
helped broaden understanding of genes, RNA, proteins, and associated biological
processes that were important for tissue and organ function. This scientific approach
was extensively studied to understand many other important biological processes. A
study of mutation of specific genes on nervous system was conducted to understand
the gene function and the behavior resulted from such genes (Brenner S).

In this project, mutagenized C.elegans were isolated to study genes that were
responsible for an expression of dumpy body phenotype. This method was applied to
medical research to study those associated genes with genetic diseases in human. For
example, dpy-2 and dpy-10 were determined their ability to encode collagens (Levy et.
al. 1) Collagen is a structural component in the extracellular matrix in animals. This
protein also plays a role in body morphology. When the genes encoded for such protein
were mutated, the protein function would be altered, therefore, resulted changes in body
morphology. The substitutions of glycine base within gly-X-Y collagen portion could
result in dumpy and left roller (DLRol) phenotypes. These mutants were resulted from
the reduced function of dpy-10 collagen gene. Molecular defects of this gene could
severely affect the morphology of C. elegans.

The approach of this laboratory project was to screen the F2 generation of

animal for dumpy phenotype. Worms with dumpy phenotype possessed shorter body
compared to that of the wildtype. Forward genetic screen technique required the animal
to be exposed under ethyl methanesulfonate (EMS), a mutagen that could cause DNA
mutation. Under the effect of EMS, guanine base would pair with thymine during DNA
synthesis. The lab result was expected to the observation of worms with dumpy
phenotype. Also, genes associated with such phenotype was analyzed for another
potential research project.

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1. METHOD
a. Research design
We were assigned to perform the experiment in a group of four students. This
experiment was designed to perform forward genetic screen to examine F2 generation
of animal with dumpy phenotype. The genes associated with phenotype of interest was
analyzed.

This lab experiment allowed to practice worm picking methods to transfer animal.
We applied laboratory techniques to cultivate and screen for a phenotype of interest.
Lab safety technique was carefully followed which helped avoiding contamination of
culture. This project required about two weeks to cultivate C. elegans and collect data
result as well. Animals were checked daily.

Methods to pick and cultivate C. elegans should be known clearly. Knowledge of

experimental procedure was acquired to carry out the experiment properly. The
phenotype of interest was identified on F2 generation. Incorrect identification would
cause error in worm cultivation and genetic analysis.
b. Instrument
The model organism for this project was the N2 strains of C. elegans. A stock of
P0 mutagenized worms was obtained. P0 worm population was exposed to mutagen
EMS. An amount of M9 buffer was also obtained for washing step. C.elegans were
cultured on Nematode Growth Media (NGM) which seeded with E. coli OP50 as a food
source for worm development. A pasture pipet with mounting a platinum wire was used
to move from plates to plates.
c. Procedure

An amount of 50 wildtype L4 Larvae to young adult hermaphrodite worms were

collected and washed with M9 buffer. The worms were resuspended in 3 ml of M9
buffer. An amount of 20ul of EMS was added to 0.980 ml of M9 buffer and gently mixed.
An amount of 3 ml of worms was transferred into the 15 ml conical tube of EMS. The
potential of leakage was reduced by covering the tube. The tube was then placed on
rocker at 200C for 4 hours. The final concentration of EMS was 50 mM. The worms were
centrifuged and the supernatant was removed. The worms were washed with M9 buffer.

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A sterile glass pipette was used to transfer worms to the edge of bacterial lawn on NGM
plates. These plates contained P0 population of mutagenized worms that would be used
to obtain the F2 generation. The collected EMS waste was neutralized with a mixture of
an equal volume of 0.1M NAOH 20% and Na2S2O3.

The P0 mutagenized worms were obtained by students. The project was

focused on the observation of dumpy body phenotypic feature and genes that
associated with such phenotype. A pasture pipet with mounting a platinum wire was
used to move from plates to plates. The wire was incinerated often to avoid plate
contamination. Worm picking technique needed to be practiced to avoid poking a hole in
agar plates. Worms might crawl into the holes and died. The plates were labeled and
the generations were kept track. One P0 adult were transferred to each of five POA
plates and these worms were allowed to grow for about 1 to 2 days. The worms was
then transferred to each of five POB plates. After 24 hours, each POB plate was flamed
to kill P0 adults. The worms of POA plates were then transferred to F1A plates. The
same procedure was applied to transfer worms to F1B plates. After 24 hours, the eggs
were laid and the F1A or F1B plates were flamed to kill F1 adults. On F1A and F1B
plates, the eggs were allowed to hatch and F2 mutant was screened. A worm with a
specific phenotype was picked and transfer to F2 small plates. When eggs were laid, F2
generation was flamed and allow for F3 generation to hatch. F3 phenotype was
expected to possess inherited phenotype.

2. RESULT AND DISCUSSION

a. Result
The approach of this project was to conduct
forward genetic screen and the F2 generation worms were
screened for a phenotype of interest. The experimental
result was expected to obtain dumpy body phenotypic
features in offspring generations. The genes associated to
this phenotype was analyzed for further studies. Up to
Figure 2. C. elegans in
today, the worms were cultivated to F1A and F1B plates
F1A plates
(Figure 2). A large number of eggs and adult worms were

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observed in those plates. Mold existed in several plates. This indicated that the plates
were contaminated due to error in laboratory safety. In general, the worms underwent a
normal development process. The presence of eggs was also observed. As mentioned
before, the result was expected that the offspring generation would possess inherited
mutant with dumpy body feature.
b. Discussion

In this project, the phenotype of interest was dumpy body. Worms with such
phenotypes appeared to be short and fat compared to normal wildtype. The changes in
body morphology was caused by recessive mutant alleles at autosomal dpy locus. This
mutation was proposed as a result from the overexpression of X-linked genes.The
forward genetic screen technique was thought to be a powerful method to gain
understanding of genetic pathways for three reasons (Jorgensen and Mango 5). A
group of many genes that associated with a specific phenotype could be identified.
However, the gene that function in more than one genetic pathway might be missed
identified due to its association with more than one phenotype besides the selected
phenotype. Second, the screen allowed to identify mutations that unusually arise. For
example, the mutation of RAS signaling protein pathway led to lethality. The loss of
alleles that associated with such mutations was lined to viable and vulvaless
phenotypes. This played a role in defining the components of RAS signaling pathway
(Jorgensen and Mango 5). Third, function and structure of protein could be analyzed
based on the screen results. The sex determination and dosage compensation
defective (SDC-3) proteins were analyzed for 16 mutations. Two domains existed in
SDC proteins. The genetic expression on the two X chromosomes on hermaphrodite C.
elegans was suppressed on the zinc-finger domain. This downregulation process was to
match the gene dosage in the single X chromosome male C. elegans. The remaining
SDC-3 protein domain shared many characteristics with the ATP-binding domain of
myosin. This domain was required to determine sex in hermaphrodite. With the help of
genetic screen technique, researchers were allowed to identify genes that associated
with certain biological processes at molecular levels.

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 The dumpy phenotype was thought to observe in XX C. elegans but not in XO
C. elegans due to the recessive alleles located at dpy-21 locus (Meneely and Wood 1).
Dpy-21 was thought to be resulted from many interactions in several regions of the X
chromosome. This mutation also was thought to regulate the expression of X
chromosome. This meant such mutation also likely to be involved in the interpretation of
X chromosome does to determine sex and to compensate dosage. The experimental
results suggested that dpy-21 were able to perform two functions. First, the ability to
suppress X chromosome expression as a part of dosage compensation mechanism.
Second, the ability to trigger male development pathway (Meneely and Wood 14).

Another research approach to molecular genetic analysis of the C. elegans

stated that dpy-2 and dpy-10 genes involved in collagen encoding. Dumpy phenotype
resulted from the reduction in dpy-10 gene functions. The analysis results suggested
that dpy-10 gene activity played a role in morphology determination of organisms. As
mentioned before, dpy-2 ad dpy-10 were responsible for encoding collagen protein.
Each cuticle collagen gene contained an amino non-Gly-X-Y region, and repetitive Gly-
X-Y regions. These collagen domain organization were similar to vertebrate collagen
type IX and XII (Levy et al 8). Both mutations resulted glycine substitution with another
amino acid within the protein portion. This molecular defect was also observed in
human disease, for example, osteogenesis imperfecta, in which glycine was substituted
in alpha chain 1 of type 1 collagen (Levy et al 11). Also, the mutation was related to
dwarfism and Ehlers – Danlos syndromes (Levy et al11). Such clinical disorders
reflected the abnormal tissue distributions of a particular collagen.

Body morphology of C. elegans was also influenced by other mutants that

had impacts on body length regulation pathways. Long-term starvation in eating-
defective animals induced the autophagy pathways which led to the decrease in fat
deposition, small cells, and small body size in starved worms (Morck and Pilon 1).
Autophagy is a degradation mechanism to destroy cells in the body. The results
suggested that the feeding defective mutants were thinner and shorter than the wildtype
at L4 stage and adult stages. The reduction of body length correlated with the reduction

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of cell sizes (Morck and Pilon 8).This work showed that there was variety of mutants
that caused the reduction in body length via many genetic pathways.

C. elegans was successfully utilized for many diverse biological research

fields. The animal develope on NGM plated seeded with E. coli and somehow infected
by other bacteria and fungi. A research approach to the ability to immune against those
infections on the epidermis tissue was conducted. The research mainly focused on the
signaling pathways that activated by wounding and infections. This mechanism served
to protect the host animal (Taffoni and Pujol 1). C. elegans possessed a flexible external
cuticle layer that secreted by epidermis. The body shape of the worms was determined
by the collagenous extracellular matrix (ECM). These components also played a role in
locomotion via their attachment to the muscles. Cuticles were composed of collagen
proteins and non-structural proteins. The C. elegans cuticles were considered to be
analogous to stratum corneum (SC) skin layer in mammals. Both structures functioned
as external barriers.

Fungi and bacteria infected the nematode via adherence to carbohydrate-rich

cuticle layers. The wounding or infected site induced the expression of nlp genes via G-
protein receptor called DCAR-1 (Taffoni and Pujol 5). DCAR-1 was initially activated by
4- hydroxyphenyl lactic acid (HPLA). A cascade of reactions occurred in response to the
activation. The level of HPLA was elevated after infection and in mutagenized dpy-10
worms. HPLA also seemed to act as damage-associated molecular pattern (DAMP) in
mammals. In fact, high level of DAMP was detected in patients with phenylketonuria
syndrome.
In general, the encoding pathway of collagen proteins was affected by
mutations of associated genes. The mutation in an associated gene could defect the
synthesis protein pathways. The cuticle layer of C. elegans was analogous to dermal
layer in human and other protein tissue components. This suggested a research
approach to human diseases such as acral lentiginous melanoma, basal cell
carcinomas (skin cancers), and any wound healing processes that linked with synthesis
of collagen. At the wound site, collagen accelerated the healing process by initiation the
growth of new tissues. Through studies of the dumpy mutation in C. elegans, the

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mechanism of genetic disorders and cancerous disease were discovered. Those
mechanism could be manipulated by applying RNAi technique to silencing the genes of
interests for the purpose of further researches.

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