Please write down the phenotype and genotype accordingly of cross F, G, and H. Ignore the blank of No. of flies. 

– 71 –

EUKARYOTIC GENE EXPRESSION AND
REGULATION

OBJECTIVES
After completing this exercise, you should be able to:

1. Describe the essential elements of eukaryotic gene expression and regulation

2. Understand and utilize binary systems to express genes in a tissue specific manner

BACKGROUND

In high eukaryotic organisms, sophisticated and coordinated networks of regulatory mechanisms govern
gene activities. An enhancer is a small DNA segment that is a binding site for proteins to enhance
transcription rate. Many enhancers of Drosophila genes were identified by using “enchancer-trap.”
The understanding of basic regulatory mechanisms for the eukaryotic genes has made it possible to
heterologously express human proteins in model organisms. In this laboratory exercise, you will study
gene expression and regulation of a human neuronal toxic protein in Drosophila.

A mutant allele of the human SCA3 gene contains a CAG tri-nucleotide expansion in the coding sequence
(an indel mutation). The allele expresses a mutant protein, SCA-Q78 (or Q78), containing a polypeptide
of 78 glutamines. All expanded polyglutamine proteins are neuronal toxic and cause neuronal diseases.
To express the Q78 protein in Drosophila, a GAL4-UAS binary system was used. Specifically, two
genetically engineered genes were constructed. One of them expresses the yeast Gal4 protein, which is a
DNA-binding protein and a transcription factor. The other expresses the Q78 protein under the control of
the Gal4 protein. Figure 8.1 illustrates the synthesis of Q78.

To express the yeast Gal4 protein, a sequence containing the Gal4 coding region is preceded by
a binding site for a Drosophila transcription factor. Here, the binding site is GMR (glass multiple
repeats) for the glass transcription factor. Thus, this genetically engineered gene is under the control
of the Drosophila glass protein, which expresses primarily in the eye tissues (Figure 8.1a).

In another construct, a yeast upstream activating sequence (UAS) specific for the binding of the
yeast Gal4 protein precedes a partial human gene that contains an expanded (CAG)78 repeat (Figure
8.1b). The human derived gene is expressed when the glass transcription factor becomes available for
binding to GMR, thus expressing Gal4, which in turn binds to the UAS. Therefore expression of Q78
is driven by the expression of the fusion gene, GMR-Gal4.

Figure 8.1 The Gal4-UAS
binary system to express a
human neurotoxic protein
in Drosophila eyes. Two
transgenes were introduced
into the Drosophila germ
line via transposon-mediated
transformation. P indicates a
Drosophila promoter.

– 72 –

MCB 3413: CONCEPTS OF GENETIC ANALYSIS

Taken together, this binary expression system expresses a human protein of interest (Q78) in a neuronal
tissue (the eyes predominantly). The Gal4-UAS system can also be utilized to ectopically over-express a
gene. We will use a genetically engineered transgene containing a UAS that precedes a Drosophila gene,
dikar. Upon expression of Gal4 under the control of the glass-GMR interaction (Figure 8.1a), ectopic
expression of the dikar gene is induced. You will examine the effects of combining the two genetic
backgrounds (an over-expressing dikar gene and an ectopically expressed Q78).

MATERIALS

Peri od I

Dissecting microscope with a light source

Drosophila CO2 work station: CO2 tanks, CO2 pads, and CO2 gun

Fine-bristle brushes

3 vials with fly food (cornmeal/agar-based, semi-solid)
3 vial tops
Index card
Marker to write on the vials
Drosophila stock IDs (full genotypes are listed in Table 8.1):

UAS-dikar virgin females

UAS-Q78

virgin females
GMR-Gal4 males
GMR-Gal4, UAS-Q78 males

Peri od I I

Drosophila CO2 work station: CO2 tanks, CO2 pads, and CO2 gun

Fly morgue (detergent and tap water)

Peri od II I

Dissecting microscope with a light source
Drosophila CO2 work station: CO2 tanks, CO2 pads, and CO2 gun
Fine-bristle brushes
Fly morgue (detergent and tap water)

Analytic microscope

Digital camera

– 73 –

EUKARYOTIC GENE EXPRESSION AND REGULATION

PROCEDURE

Peri od I
Set up your F0 genetic crosses
1. Refer to pages 4–6 of the lab manual for detailed instructions on fly anesthetization, identifying

sexes and genetic markers, and suggestions on cross set-ups
2. Label three vials “F”, “G”, and “H”; And add your name onto each vial
3. Cross 5-7 healthy UAS-dikar females with 3-5 healthy GMR-Gal4 males in the vial labeled F
4. Cross 5-7 healthy UAS-Q78 females with 3-5 healthy GMR-Gal4 males in the vial labeled G
5. Cross 5-7 healthy UAS-dikar females with 3-5 healthy GMR-Gal4, UAS-Q78 males in the vial

labeled H
6. Record the phenotypes of the parents (F0) used in Crosses F, G, and H in Table 8.1

Peri od II ( This st ep m ay have been performed by your TA)
Remove the F0 generation from the population
1. Refer to pages 4–6 of the lab manual for detailed instructions on fly anesthetization
2. Once the flies have been anesthetized, dispose of them by putting them in the fly morgue as

demonstrated by your TA

Peri od I I I
Record your data
1. Refer to pages 4–6 of the lab manual for detailed instructions on fly anesthetization, identifying

sexes and genetic markers
2. Record the phenotypes of the progeny (F1) from Cross F in Table 8.2

• Increase your magnification enabling you to see the fine eye structure of the flies
• Compare and contrast these eyes with those of their parents. Record MAJOR morphological

differences between the two
3. Record the phenotypes of the progeny (F1) from Cross G in Table 8.2

• Increase your magnification enabling you to see the fine eye structure of the flies
• Compare and contrast these eyes with those of their parents. Record major morphological

differences between the two
4. Record the phenotypes of the progeny (F1) from Cross H in Table 8.2

• Separate Cy from Cy+ flies and record the number of each. You should notice something
strikingly different from the predicted progeny

• Look at the vial for this cross and determine approximately what percentage of pupae has
undergone eclosure (seen as empty pupal cases)

5. Detailed structural examination for the adult eyes with CX 31 analytic microscope
6. Dispose of the flies by putting them in the fly morgue as demonstrated by TA

– 74 –

MCB 3413: CONCEPTS OF GENETIC ANALYSIS

Table 8.1 Genotypes and Phenotypes of F0

Date of cross: / /

Cross ID Male Genotype Female Genotype Male Phenotype* Female Phenotype*

F

eye color:

wing shape:

eye color:
wing shape:

No. of flies: No. of flies:

G

eye color:
wing shape:
eye color:
wing shape:
No. of flies: No. of flies:

H

eye color:
wing shape:
eye color:
wing shape:
No. of flies: No. of flies:

*eyes: red, orange, or “necrotic” looking (lack of uniform color); wing shape: straight or curled

Please Note: CyO is a balancer chromosome for Chromosome 2 with a dominant marker, Cy. GMR-
Gal4, UAS-Q78, and UAS-dikar are transgenes; each is marked with mini-white, a gene that gives rise

to adult eye color ranging from orange to red.

Figure 8.2 Four classes of eye phenotypes including a wild type. The genotypes associated with three
classes of eye defects, as shown in Panel (1), (2), and (3), remain unclear.

X, w GMR-Gal4 + +
Y GMR-Gal4 + +; ; ;

X, w + UAS-dikar +

; ; ;

; ; ; ; ; ;

; ; ; ; ; ;

X, w GMR-Gal4 + +
Y GMR-Gal4 + +

X, w CyO + +
Y GMR-Gal4, + +

X, w + UAS-dikar +

X, w + UAS-Q78 +
X, w + UAS-Q78 +

X, w + UAS-dikar +
X, w + UAS-dikar +

UAS-Q78

– 75 –

LABORATORY REPORT

INTRODUCTION

THEORETICAL QUESTIONS
1. In Cross F, you are creating a fly with ectopic expression of the dikar gene. From this cross, what is

the genotype for a fly that shows this ectopic expression?

2. Draw a diagram illustrating the defect(s) caused by expressing a transgene (UAS-dikar) in the
Drosophila eye tissue. Hint: The defect is shown in one of the panels of Figure 8.2.

RESULTS

Table 8.2 Genotypes and Phenotypes of F1 (crosses F and G)

Cross ID Male Genotype Female Genotype Male Phenotype* Female Phenotype*
F

eyes:

wing shape:
eyes:
wing shape:
No. of flies: No. of flies:
eyes:
wing shape:
eyes:
wing shape:
No. of flies: No. of flies:
G
eyes:
wing shape:
eyes:
wing shape:
No. of flies: No. of flies:
eyes:
wing shape:
eyes:
wing shape:
No. of flies: No. of flies:

*eyes: red, orange, “hairy” looking, or “necrotic” looking (lack of uniform color); wing shape: straight or
curled

Name:_______________________________

Date:_________________ Section:_______

E
U

K
A

R
Y

O
T

IC
G

E
N

E
E

X
P

R
E

S
S

IO
N

A
N

D
R

E
G

U
L

A
T

IO
N

– 76 –

E
U
K
A
R
Y
O
T
IC
G
E
N
E
E
X
P
R
E
S
S
IO
N
A
N
D
R
E
G
U
L
A
T
IO
N

NOTES

DISCUSSION AND CONCLUSIONS
Please refer to HUSKYCT (your scheduled laboratory section webpage) for questions related to this
section. Your assignment must be submitted via HUSKYCT prior to your scheduled laboratory section
meeting time. Data sheets are submitted to your TA in the laboratory classroom. You must submit your
data sheets for each exercise to receive full credit. Failure to submit data sheets for any exercise will
cause you to lose up to 50%.

Table 8.3 Genotypes and Phenotypes of F1 (Cross H)

Cross ID Male Genotype Female Genotype Male Phenotype* Female Phenotype*
H
eyes:
wing shape:

pupae casing:

eyes:
wing shape:
pupae casing:
No. of flies: No. of flies:
eyes:
wing shape:
pupae casing:
eyes:
wing shape:
pupae casing:
No. of flies: No. of flies:

*eyes: red or orange, “hairy” looking, or “necrotic” looking (lack of uniform color); wing shape: straight
or curled; pupae casing: open (clear, hatched) or closed (black, dead)

INTRODUCTION

– 3 –

Laboratory Exercise Guidelines
Please Note: You must type your lab report and submit it on HUSKYCT via “SafeAssign.” This program is
designed to scan your document for plagiarism against your peers (past and current), journal articles,
and other online sources. Please use size 12 font, double-space formatting, and limit it to no more than 4
pages. Hardcopies will not be accepted under any circumstances.

The majority of the lab reports, which are collected and graded in this course, are graded as
follows:

Introduction (10%)
Relevant background information
The major aim(s) of the experiment

Theoretical Questions (30%)
Answer any provided questions
Offer expected results (hypothesis)

Results (30%)
Answer any provided questions
Provide clear and detailed data
Summary of results
Any figures, tables, and graph axes are properly labeled

Discussion/Conclusion (30%)
Answer any provided questions
Summarize experiment
Do your data support the hypothesis?
Describe errors and possible sources of errors
Concluding remarks (significance of results)

– 4 –

MCB 3413: CONCEPTS OF GENETIC ANALYSIS

Fly Pushing
A number of laboratory exercises in this course require the use of Drosophila melanogaster (commonly
referred to as fruit flies) as a genetic model. The following section offers some guidelines for working
with the flies.

Figure 0.1 Fly workstation example (laboratory classroom set may differ)

I. Fly Anesth etization
1. Unclamp the tube, which connects to the etherizer bottle
2. Bump the bottle gently on the bench top to knock the flies to the bottom of the bottle
3. Quickly remove the bottle top and invert the bottle onto the etherizer bottle
4. Tap the flies out (take care not to tap too hard—fly food may enter the etherizer)
5. Upon removal of the flies from the bottle, replace the bottle top and return the bottle to the tray
6. Unclamp the tube, which connects to the CO2 pad that is located on the dissecting scope
7. Gently empty the flies from the etherizer onto the pad; flies are now anesthetized and ready for

manipulation
*when finished, be sure to re-clamp the tubes that supply CO2 to the etherizer and pad*

– 5 –

INTRODUCTION

II. Identif yi n g S exes an d Gen et ic Markers

S e x :

Using a fine-bristle brush, manipulate the flies in a manner that will enable you to view the posteriors
of the flies. Familiarize yourself with sexing the flies in order to set up your fly crosses. Virgin
females will be used in your cross set-ups. Your TA will explain the significance of using virgins to
set up a genetic cross.

Figure 0.2 Characteristics of adult Drosophila males and females. Males have a dark posterior region
(more obvious in older males) and a brownish ring surrounding the genital plate (Panel a). Females lack
the genital plate ring and are slightly larger than males. Males also contain sex combs, which are bristles
located on the forelimbs (Panel b).

G e n e t i c M a r k e r s :

Table 1 summarizes the recessive and dominant mutations that are used as markers for analysis of
meiotic chromosome segregation. The figure on the following page shows different genetic markers
and the associated phenotype. Manipulate the flies in a manner that will enable you to view their
different anatomical features. Familiarize yourself with these phenotypes.

Table 0.1

Mutation Location Type of Mutation Phenotype
w (white) X recessive white eyes
y (yellow) X recessive yellow body
Cy (Curly) 2 dominant curled wings

Sco (Scutoid) 2 dominant loss of bristles, scutellars
Sb (Stubble) 3 dominant short, thin bristles

– 6 –

MCB 3413: CONCEPTS OF GENETIC ANALYSIS

Figure 0.3 Mutations used as genetic markers to monitor chromosome segregation in meiosis. For further
details, see Table 0.1.

III. Cross S et-u p s

All crosses should consist of 4–5 female virgins and 4–5 males. Record genotypes (if known) and
phenotypes of flies used. Use a fresh food vial for each cross and label it with your name, section number,
and cross identification. Leave vial on its side until all flies have woken up.

Suggestion: In your notebook, record the date on which the cross was set-up.

– 7 –

USE OF MICROPIPETTORS
Micropipettors are designed to deliver from 1 to 1000µl (0.001-1.0ml) with extreme
accuracy within the specified volume range. These pipettors must always be used with
a disposable tip. It is very important to remember that different types of tips are not
always interchangeable between different sizes or brands of pipettors.

Micropipettors are sensitive and expensive lab tools! Please review these
instructions before each use.

1. Check the range of the pipettor. It is displayed at the top of the
pipettor. You will be using pipettors having ranges of 1-10µl,
10-100µl, and 100-1000µl. Do not force the pipettor beyond its
designated range – you will damage the pipettor.

2. Set the desired volume by unlocking and then turning the volume
adjustment knob until the correct volume appears on the digital
indicator. Then re-lock the knob prior to usage. For best results always approach the desired
volume by dialing down from a larger volume setting.

3. Attach a new disposable tip to the shaft of the pipettor. Press the tip on firmly, using a slight
twisting motion, to ensure a positive, airtight seal. Use WHITE tips for the 1-10µl pipettor,
YELLOW tips for the 10-100µl pipettor, and BLUE tips for the 100-1000µl pipettor.

4. Depress the plunger to the FIRST positive stop. This is the volume displayed on the digital
indicator.

5. Holding the pipettor vertically, immerse the lower third of the tip into the sample liquid, and
allow the plunger to return SLOWLY to the UP position. Do not allow it to snap up!

6. Wait 1-2 seconds to ensure that the full sample volume has been drawn into the tip.

7. Withdraw the tip from the solution. Should any liquid remain on the outside of the tip, wipe it off
carefully with a Kimwipe®. (Do not touch the tip opening!)

8. To dispense the sample, place the tip end against the inside wall of the tube, flask, etc. and
depress the plunger to the FIRST stop. Wait several seconds, then depress to the SECOND stop
(the bottom of the stroke) to expel any residual liquid in the tip.

9. Keeping the plunger depressed, withdraw the tip from the tube, and then allow the plunger to
return slowly to the UP position. Discard the tip into the sharps container by pushing the ejector
button.

INTRODUCTION

– 8 –

MCB 2610: FUNDAMENTALS OF MICROBIOLOGY LAB MANUAL

LABORATORY NOTEBOOK SUGGESTIONS
1. THREE-RING BINDER AS A LAB NOTEBOOK
Keep your lab notes in a three-ring binder with extra paper available for recording observations.

2. LABEL EACH NOTEBOOK ENTRY
The lab notebook should be a well organized, easy to use compilation of your procedures,

observations, and results. It is not necessary to repeat the details of procedures already in your
lab manual, but you should always indicate by a suitable label, the conditions under which a
procedure was applied immediately above or adjacent to any list of results.

3. ORGANIZE YOUR NOTEBOOK CAREFULLY
The lab notebook is a scientist’s primary source of laboratory experimental procedures and

data collected. Memory fades quickly, hence results should always be written down at the time
they are observed, along with any other significant factors. You should keep a complete, well
organized record of your observations. You will use this data for diagnosing unknowns later in the
course.

4. ANSWER QUESTIONS ABOUT LAB EXERCISES
In addition to results, most exercises include questions and provide a space for discussion/

conclusions. These should be answered at the end of the write up for that exercise and handed
in when requested by your lab instructor in the format requested. In writing up each lab, answer
the question, “What did this exercise show?” Be sure to explain any unusual, contradictory, or
inconclusive results.

5. KEEP ALL WRITE-UPS FOR EACH EXERCISE IN ONE PLACE
All the results of a given exercise should be written up in one place even if the data are collected

over several lab periods.

2019

Lab Report 7: Gene Expression and Regulation

MCB 3413 Laboratory

Introduction

This lab explores aspects of both prokaryotic and eukaryotic gene expression including glucose

and lactose

m

ediated regulation of the lac operon and eukaryotic gene regulation observed in

genetic crosses of drosophila melanogaster. The lac operon is a region of genes that controls

the metabolism of lactose in response to the levels of glucose and lactose present. It does this

via a negative regulator, the lac repressor, and the catabolite activator protein, a positive

regulatory element (Intrieri & Zhang 2019). Once activated, the lac operon causes production of

the β-galactosidase enzyme, which catalyzes hydrolysis of lactose into glucose and galactose.

Normally, the lac repressor binds to the operator and prevents it from activating the lac operon.

When lactose is introduced, it binds to the lac repressor and prevents it from stopping the

activation of the lac operon. Similarly, the CAP is an element of positive regulation which

activates the lac operon when bound by cAMP. cAMP is a signaling molecule whose

concentration is dependent on the levels of glucose present. High glucose levels result in low

cAMP levels, while low levels cause high levels of cAMP and consequently, activation of the lac

operon and metabolism of lactose (Intrieri & Zhang 2019). Due to these regulation elements,

the optimal conditions for activation of the lac operon include high levels of lactose and low

levels of glucose. This experiment explored these concepts through three tests in which either

glucose, lactose, or a combination of the two was present. ONPG, a dye added before

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incubation, allows the levels of β-galactosidase activity to be observed through the yellow color

that it takes on in response to high levels of activity.

1. A: The cis-elements of the lac operon circuit are the operator, the promoter, and the CAP

binding site

B: The trans-elements of the lac operon circuit are the lac repressor and the CAP.

2. A: In a medium of lactose, E. coli would grow somewhat, because without positive regulation by

CAP, activation of the lac operon would be limited to the lactose/lac repressor interaction,

limiting its ability to cleave lactose into glucose for cellular respiration.

B: In a medium of only glucose, E. coli would not grow as it wouldn’t have any lactose to

metabolize into glucose.

C: In a medium of both lactose and glucose, E. coli’s ability to metabolize lactose would still be

limited by the lack of CAP activation by cAMP, so activation of the lac operon would be limited

to the lactose/lac repressor interaction, causing lowered glucose production.

The eukaryotic portion of this lab explored gene regulatory elements of eukaryotes

including the GMR (glass multiple repeat) and UAS (upstream activating seqence) enhancers.

Specifically, these elements were explored through their role in the expression of mutations in

the eye structures. The GAL4-UAS system directs gene expression to a certain part of the body,

in this case the eyes, through a GMR enhancer upstream of Gal4, which is bound only by glass

proteins specific to the eyes. Once Gal4 is synthesized, it binds the UAS enhancer of another

sequence and in turn activates either the Q78 or the dikar gene downstream (Intrieri & Zhang

2019).

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1. In these crosses, the ectopic dikar gene is expressed in the eye tissues of the flies,

resulting in one of three mutant phenotypes of eye defects of varying color.

2.

Cross F Cross G Cross H

+¿
+¿
¿

+¿
UAS dikar

;¿

+¿
GMR Gal 4

;¿

X , w
Y

;¿
+¿
+¿
¿

+¿
UAS Q 78

;¿
+¿
GMR Gal 4
;¿
X , w
Y
;¿
+¿
+¿
¿
+¿
UAS dikar
;¿

+¿
Cyo

;¿
X , w
Y
;¿
+¿
+¿
¿

+¿
UASdikar

;¿

+¿
GMR Gal 4
UAS Q78

;¿
X , w
Y
;¿

M/Orange/Straight M/Orange/Straight M/Orange/Curly M/White/Straight
+¿
+¿
¿

+¿
UAS dikar
;¿
+¿
GMR Gal 4
;¿

X , w
X , w

;¿
+¿
+¿
¿
+¿
UAS Q 78
;¿
+¿
GMR Gal 4
;¿
X , w
X , w
;¿
+¿
+¿
¿
+¿
UAS dikar
;¿
+¿
Cyo
;¿
X , w
X , w
;¿
+¿
+¿
¿
+¿
UASdikar
;¿
+¿
GMR Gal 4
UAS Q78
;¿
X , w
X , w
;¿

F/Orange/Straight F/Orange/Straight F/Orange/Curly F/White/Straight

Results

Table 1 Legend: +++ = strongly yellow ++ = moderately yellow + = weakly yellow O = not yellow

10 Minutes 20 Minutes 30 Minutes

Lactose + ++ +++

Glucose O O O

Lactose and Glucose + + ++

Conclusion

The results of the prokaryotic experiment were mostly consistent with the response predicted

for each level of glucose and lactose. For lactose only, β-galactosidase activity steadily increased

throughout incubation, while it remained at no activity for glucose only. Although it showed less

change than expected, the mixture of glucose and lactose also followed predictions and rose in

activity steadily. The eukaryotic portion also saw the predicted results of the genetic cross, with

both F and G displaying the same phenotype of orange eyes with straight wings. Cross H

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displayed a mixture of phenotypes, both white eyes with straight wings and orange eyes with

curly wings. White eyes were most likely due to the double dose of the Q78 and dikar genes

present in some fly genotypes, while the curly wings of the others were most likely caused by

the presence of the Cyo gene.

References

Intrieri, G., Zhang, P., & University of Connecticut Department of Cell Biology (2019) Concepts of

Genetic Analysis: Laboratory Manual. Plymouth, MI: Macmillan Learning Curriculum Solutions.

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