Christopher Cronkite

Dr. Peter Wilson

BIOL 280L02
25 Feb. 2013
Analysis of the Lobe, eyeless, and polished Genes of Drosophila melanogaster
Abstract
Prior to the twentieth century, very little was known regarding the inheritance of traits. The rediscovery
of the research performed by Mendel at the start of that century sparked a revolution in genetics.
Mendel's principles, based off of his observations of pea plants, were soon discovered to not
characterize every trait observed in both model systems and humans. New extensions of Mendel's
principles were added as observations and research into genetics continued. A valuable tool for the
study of genetics is the fruit fly Drosophila melanogaster. Due to its short life cycle and visible traits,
D. melanogaster has been used to study the heritability patterns of certain traits. We studied the genetic
basis of heredity for three genes in D. melanogaster concerning eye color and size to determine
whether or not these genes adhered to a Mendelian inheritance pattern. It was concluded that the Lobe
gene does not and rather exhibits an incomplete dominance pattern of heredity whereas the eyeless and
polished genes do follow traditional Mendelian inheritance patterns. This was determined utilizing
monohybrid and dihybrid crosses of the flies.
1. Introduction
Due to the complexity of heredity in humans, it has been necessary for geneticists to utilize
simpler genetic models. There is a vast number of complex traits in humans whose genetic bases are
more complex than a distinguishable phenotype arising from a particular, single genotype. [1] As a
result, geneticists have employed the use of simpler model organisms to study the heritability of
various genes. [2] Through careful study of these model systems, researchers have been able to deduce
various principles regarding the genetics of various traits that allow further insight into how complex

traits function in humans.

The foundation of modern genetics is due primarily to the work of the monk Gregor Johann
Mendel who experimented in the mid-nineteenth century with the garden pea, Pisum sativum. [2] This
plant served as a good model organism for Mendel to study heritability due to its petal physiology. P.
sativum self-fertilizes itself and protects its reproductive organs from the gametes of other organisms
by tightly folding its petals over them. [2] Mendel was able to take these true-breeders and manually
cross them with other pea plants that exhibited different traits. He was then able to analyze the
appearance of certain phenotypes after cross- and later self-fertilization.
As a result of these studies, Mendel concluded with three principles of inheritance, now
understood to be tenets of Mendelian genetics. [1] The first principle is that of dominance. This
principle states that for a given trait with two factors (Mendel's term for allele), there will be one that is
dominant and one that is recessive. [2] Therefore, homozygous dominant and heterozygous individuals
will display the dominant trait while those who are homozygous recessive will exhibit the recessive
phenotype. The second principle, that of segregation, states that during haploid gamete formation, the
two alleles present in a cell will separate from each other. [2] Because of this, the offspring will receive
one allele from one parent and the other allele from the other parent. The third and final principle of
Mendelian genetics is the principle of independent assortment. Utilizing dihybrid crosses of two
different genes, Mendel observed that the alleles from each gene segregated during gamete formation
independently from the alleles of the other genes. [2]
Mendel was able to arrive at these conclusions based off of many years and observing hundreds
of pea plants. [2] In addition to these principles, Mendel also determined statistical probabilities
associated with monohybrid and dihybrid crosses. When performing a monohybrid cross with one
organism homozygous dominant for the trait and the other homozygous recessive, the F1 generation
reveals all dominant phenotypes. However, when the F1 generation self-fertilizes, the F2 generation
yields a combination of dominant and recessive phenotypes at a ratio of 3:1 respectively. [2] Dihybrid

crosses led to an F2 phenotypic ratio of 9:3:3:1 for dominant for both traits, dominant for the first trait
and recessive for the other, dominant for the second trait and recessive for the other, and both recessive
phenotypes respectively. [2] These ratios are concurrent with the principles Mendel developed and hold
true for so-called Mendelian traits, those traits that exhibit a distinct phenotypic change resulting from
an altered genotype. [1]
While Mendel's principles and phenotypic ratios hold true for certain traits, there are many more
traits that do not adhere to these generalizations. [1] Mendel assumed that for every trait there was one
specific gene with two possible alleles. [2] He also erroneously concluded that for each gene there was
a dominant and recessive allele, the former of which if present would entirely mask the latter. [2] Due
to research performed in the twentieth century by geneticists like Thomas Hunt Morgan on Drosophila
melanogaster fruit flies, it became evident that Mendelian traits were in the minority to the
overwhelming presence of complex traits, and some understood Mendelian genes were shown to be
actually much more complex. [1] For example, the color of snapdragon flowers do not follow simple
dominance and recessivity principles. [2] Rather, there is a codominance of the alleles, each allele
contributing to the resulting phenotype. Through subsequent esearch into extensions of Mendelian
principles and the relationship of genotypes to phenotypes, geneticists have been able to characterize
more diverse ways that an allele can affect genotypes.
D. melanogaster is an important tool for the study of genetics. Due to a short lifespan and a
large number of progeny for each generation, genetic studies with these fruit flies is both fairly
inexpensive and fast. Also, there are a large number of visible manifestations of mutant genes, or visual
mutations, allowing study of the process of heredity through observation of changed morphology. [2]
As a result, D. melanogaster has become a valuable model organism in determining the complexity of
various traits.
In order to determine whether a certain gene follows principal Mendelian principles or a more
complex system, monohybrid crosses of wild-type (WT) and homozygous recessive D. melanogaster

mutants and dihybrid crosses of fruit fly mutants homozygous recessive for one gene each were
performed. The gene in question for the monohybrid cross was the Lobe gene whose mutation causes
reduction in eye size, and it is expected to be recessive. [3] The genes involved in the dihybrid cross
were eyeless (ey) and polished (pol), the former causing impairment in the growth of the ommatidia [4]
and the latter causing the eye to appear shiny. [5] The resulting F1 and F2 generations were analyzed for
their phenotypes, and, using a Chi-square analysis based off of the expected Mendelian results, the
genes' adherence to Mendelian principles were determined.
2. Methods
2.1. Monohybrid Cross of WT and Lobe Flies
Four male WT D. melanogaster flies and four female Lobe D. melanogaster flies were
introduced into the same fly vial with instant fly food and yeast after being anesthetized by FlyNap.
Flies were left to copulate, and the adult flies were removed after eggs were laid. Larvae were left to
develop into adults who where then observed for phenotypes. The adults were then allowed copulate
and, once the eggs were laid, the adults were removed. The resulting generation of flies were observed
in the same manner. Observations were performed under a light microscope.
2.2. Dihybrid Cross of ey and pol Flies
Four male ey mutant flies were placed in the same fly vial prepared the same way as before as
four female pol mutants. The flies were left to reproduce. Following the laying of eggs, the adults were
removed and the F1 generation of flies was left to grow to adulthood. The flies were anesthetized with
FlyNap and observed for their morphology. They were replaced in the vial and left to reproduce. Once
the eggs were laid, the adults were removed and the eggs were left to develop. Once the F2 generation
reached adulthood, the flies were observed.
3. Results
3.1. Monohybrid Cross of Lobe
The F1 generation following the cross between four male WT flies and four female Lobe flies

yielded 22 WT flies and 82 Lobe flies out of a total of 104 progeny observed. Of the 86 flies from the
F2 generation observed, 11 displayed the WT phenotype and 75 exhibited the Lobe phenotype. (Table
1) There did not appear to be a consistency in either Lobe or WT fly eye size or shape compared to the
P generation.
A Chi-square analysis of the F2 generation was performed using the ratio of 3:1 Lobe:WT. The
Chi-square value is 6.84, and, given one degree of freedom, was compared with the 0.05 critical p
value of 3.84. (Table 2)
Table 1: Monohybrid Cross
F1 Generation
Number of Flies Observed

WT
22

Lobe
82

F2 Generation
Number of Flies Observed
Chi-Square Value

WT
11

Lobe
75
6.84

Critical Value at p=0.05

3.84

3.2. Dihybrid Cross of ey and pol

Of the 70 fruit flies observed from the F1 generation following the cross of the four ey males
and four pol females, all were WT. The F2 generation revealed a combination of WT, pol, ey, and ey/pol
flies, specifically 42, 15, 10, and 5 observed respectively out of a total of 82. (Table 2)
A Chi-square analysis was performed using the expected Mendelian ratio of 9:3:3:1 of the
WT:ey:pol:ey/pol. The value calculated was 1.192 and was compared to the correlating 0.05 critical p
value considering three degrees of freedom of 7.82. (Table 2)
Table 2: Dihybrid Cross
F1 Generation
Number of Flies
Observed

WT
70

ey
0

pol
0

ey/pol
0

F2 Generation
Number of Flies

WT
42

ey
15

pol
10

ey/pol
5

Observed
Chi-Square Value

1.19

Critical Value at p=0.05

7.82

4. Discussion
While Mendel's three principles correlated with his study of various heritable phenotypes of P.
sativum, there are more exceptions to the rule than adherents. [1] More often than not, phenotypes are
due to a combination of genes functioning together to produce a specific trait. There are also many
genes that have more than just two alleles possible. [2] Furthermore, one allele is not always dominant
over the other, and instead the gene products of two different alleles may function together, creating a
blending of the phenotypes. [2] Nevertheless, understanding Mendelian genetics with the knowledge of
the extensions that may apply are important in understanding complex traits in species like humans.
The results of the monohybrid cross between WT and Lobe flies leads us to believe that Lobe
does not follow a traditional Mendelian inheritance pattern. Assuming that the P generation Lobe flies
were homozygous for the mutant Lobe gene, the F1 generation should have revealed either all WT flies
if the WT was dominant or all Lobe flies if the mutant gene was dominant. However, nearly 79% of the
flies observed exhibited the Lobe phenotype while the rest were WT. It is important to note as well that
there were very few flies observed that displayed the complete size of a WT eye. Instead, there
appeared a spectrum of eye sizes. Analysis of the F1 generation disproves Lobe as adhering to
Mendelian principles as, had it been, there would have been all of either Lobe or WT but none of the
other.
Furthermore, the status of the Lobe gene as Mendelian was disproved by analysis of the F2
generation. If the Lobe gene was Mendelian, there would have been observed a 3:1 ratio of the
dominant phenotype to the recessive phenotype. The actual ratio observed was 6.8:1 Lobe/WT. The
Chi-square value of 6.84 exceeded the value correlating with a p value of 0.05 by 3.00, proving our
hypothesis as invalid. It could already have been deduced from the F1 generation that the Lobe gene

does not follow traditional Mendelian genetics, but the analysis of the F2 generation confirms it.
Since it has been deduced that the Lobe gene does not follow Mendelian principles, it must be
the result of the phenotype being a complex trait It is important to note that, similar to the F1
generation, the eyes of the F2 flies exhibited a varying degree of size and shape, and some of flies
deemed WT had eyes smaller than would be expected from WT flies. This would lead one to believe
that there is a spectrum of eye size being observed from near-WT to Lobe. Studies on the gene product
of Lobe, a PRAS40 ortholog, have revealed its involvement in eye development through regulation of
the Jak/STAT pathway. [3] It has also been shown that Lobe mutants show decreased eye size due to
apoptosis of ommatidia-precursor cells. [3] It is understandable then that heterozygous Lobe flies
would display a fusion phenotype of WT and Lobe. The Lobe gene alleles must display incomplete
dominance. One copy of the WT gene still allows significant eye development, but both alleles must be
present in order for the WT phenotype to appear. Conversely, the mutant Lobe gene mutation must be a
loss of function mutation, but since heterozygotes will retain one functional copy, some eye
development will occur. Based on this conclusion, while the Lobe gene does not follow traditional
Mendelian genetics, it does adhere to the well-charactertized extension of incomplete dominance.
The dihybrid cross between the ey and pol flies revealed more typical Mendelian result.
Analysis of the F1 generation revealed a lack of any mutant as all the flies displayed WT eyes. This is
concurrent with Mendelian principles. Even if one of the F1 flies received a mutated gene from each
parent, it would still have a WT copy of each. The gene product of the WT copy must then be sufficient
enough to produce a WT phenotype, thereby masking the mutant. By preliminary analysis, it appears as
though mutant ey and pol are recessive alleles adhering to the principles of dominance, segregation,
and independent assortment.
Analysis of the F2 generation of the dihybrid cross confirmed preliminary consclusions. The
Chi-square value obtained of 1.19 was significantly less than the maximum critical value associated
with a p value of 0.05. While there was not a perfect 9:3:3:1 ratio of phenotypes, we observed an

8.4:3:2:1 ratio out of 72 flies. Additionally, the appearance of the recessive phenotypes in the F2
generation following their disappearance in the F1 following Mendelian principles. The F1 generation
must have been genotypically heterozygous, the recessive traits becoming visible again as the alleles
being passed down included both dominant WT and recessive mutants. If the F2 fly received two
mutant ey or pol alleles, it would display the mutant phenotype. The fly would only display the double
mutant phenotype if it received all mutant alleles. This is all concurrent with Mendelian genetics.
Therefore, there is significant evidence supporting the hypothesis that the mutant alleles of ey and pol
are both recessive mutations that follow Mendelian principles.
While our conclusions are sound based off of our observations, there are a few limitations with
our experimental design. First of all, the research conducted was on a very small sample size, the
largest batch observed being 104 flies. In order to precisely and accurately compare our observations
with the probabilities associated with Mendelian genetics, a much larger sample size is needed. An
increase in the number of flies studies would decrease error inherent in statistical analyses.
Additionally, there were several limitations concerning the phenotypes studied. The varying degrees of
eye size of the Lobe flies created trouble in determining WT or mutant phenotypes in several of the
flies. It my be possible to find an ortholog of this gene in a separate model organism whose mutant
phenotype is easier to distinguish from the WT one.
While hailed as the Father of Modern Genetics, the principles that Mendel deduced from his
studies on P. savitum do not always pertain to certain genes. According to our study, the ey and pol
genes of D. melanogaster adhere to the principles of Mendelian genetics. There is a distinct dominance
of the WT alleles over the mutant ones, and each gene assorted independently of the other. On the other
hand, the Lobe gene was not observed to follow traditional Mendelian genetics. Instead, the mutant and
WT allele displayed incomplete dominance as the WT gene product of heterozygous flies still
displayed some eye growth and development. While these genes have no direct correlation with human
traits, understanding the genetic basis of inheritance of both simple and complex traits enables

scientists to derive patterns and determine how complex human traits are passed from one generation to
the next.

Bibliography
1.

Zwick, M.E., D.J. Cutler, and A. Chakravarti, Patterns of genetic variation in Mendelian and
complex traits. Annu Rev Genomics Hum Genet, 2000. 1: p. 387-407.

2.

Snustad, D.P.a.M.J.S., Genetics. 6th ed. Genetics. 2012, Hoboken, NJ: John Wiley & Sons. 766.

3.

Wang, Y.H. and M.L. Huang, Reduction of Lobe leads to TORC1 hypoactivation that induces
ectopic Jak/STAT signaling to impair Drosophila eye development. Mech Dev, 2009. 126(10): p.
781-90.

4.

Czerny, T., et al., twin of eyeless, a second Pax-6 gene of Drosophila, acts upstream of eyeless
in the control of eye development. Mol Cell, 1999. 3(3): p. 297-307.

5.

Santiago, E., et al., The distribution of spontaneous mutations on quantitative traits and fitness
in Drosophila melanogaster. Genetics, 1992. 132(3): p. 771-81.