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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.

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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

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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

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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*

eye color:

wing shape:

eye color:

wing shape:

No. of flies: No. of flies:

eye color:

wing shape:

eye color:

wing shape:

No. of flies: No. of flies:

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

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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*

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:

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

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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*

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)