DROSOPHILcross over

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 mediated 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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