Name: ________________________________________________
INVESTIGATION: REGULATORY SWITCHES OF THE PITX1 GENE IN STICKLEBACK FISH
(modified from: HHMI Biointerac ve: h p://biol.co/2oBSbUK)
PART 1: INTRODUCTION
The types and amounts of proteins produced by a given cell in
the body are very important and carefully regulated.
Transcribing DNA to messenger RNA and transla ng that RNA
to protein is o en referred to as gene expression. Regula ng
that expression simply means turning on or off or increasing
or decreasing the produc on of a given protein. Interes ngly,
protein produc on can be regulated at the transla onal and
transcrip onal steps, as well as a er a protein is produced. In this ac vity, we will talk about how gene expression is
regulated through transcrip on.
The basic elements of transcrip on regula on in eukaryotes are similar to the very well-studied lac and trp operon
systems found in bacterial cells. In both eukaryo c and bacterial systems a protein, either an ac vator or repressor, binds
to a region of the DNA called an “operator” in prokaryotes and a “regulatory switch” or “enhancer” in eukaryotes. The
ac vator or repressor protein acts like the hand that flips the switch, but it can only turn the switch on or off if it can bind
to the specific DNA sequence.
Gene regula on is essen al for the cell to perform the func ons needed to live. In mul cellular eukaryotes, gene
regula on is also important in building bodies. During development, different sets of genes need to be turned on and off
in the right places, at the right mes, and in the right sequence for bodies to be built correctly. In this ac vity, you will
learn about one par cular gene, Pitx1, and how its expression is regulated in different ssues.
Ques ons:
1. Sketch either the trp or lac operon and iden fy the main components of its regulatory system.
2. What is transcrip on? How is it related to gene expression?
3. Describe the difference between an operator and an enhancer.
PART 2: REVIEWING THE REGULATION OF EUKARYOTIC GENE TRANSCRIPTION
Watch the short film, The Making of the Fi est: Evolving Switches, Evolving Bodies. Pay close a en on to how the
switches regulate the expression of the Pitx1 gene in s ckleback embryos.
Use the informa on from the film and your knowledge of eukaryo c gene transcrip on to answer ques ons 5-9.
4. Figure 1 is a diagram, similar to the one shown in the film (8:00-8:34), showing key components of gene transcrip on.
Label the boxes in Figure 1 with the le ers a-e, which correspond to the terms listed below. For example, write le er “a”
in the box poin ng at the protein-coding region.
a. Protein-coding region b. Regulatory switches (or enhancers) c. Promoter d. mRNA e. RNA polymerase
5. Gene transcrip on is a complex process that involves interac ons of proteins and regulatory regions of DNA.
Transcrip on factors specifically bind to the promoter to ac vate transcrip on. Other proteins play a role in these
regulatory regions. Explain the role of each of the following, using the image to help you.
ac vators
mediators
RNA polymerase
PART 3: GENE REGULATION IN DIFFERENT TISSUES
As you saw in the film, the presence or absence of pelvic spines in the s ckleback fish is controlled by whether the Pitx1 gene is
expressed in the pelvic ssue. However, the Pitx1 protein is actually important in building other body parts and is therefore
expressed in mul ple ssues at specific mes.
How is Pitx1 expressed in different ssues? The Pitx1 gene has mul ple regulatory switches that control the expression of the gene in
different ssues: the pituitary, jaw, and pelvic ssues. Having mul ple switches enables Pitx1 to be used many mes in different
contexts and expands the versa lity of that gene. Ac vators present in a par cular ssue bind to a specific sequence on the DNA
and turn Pitx1 on in the appropriate ssues. For example, in the cells that develop into the pelvis there is a specific ac vator
(ac vator 2) that binds to the pelvic switch to transcribe Pitx1 in that ssue. In the jaw, there is a different ac vator (ac vator 1) that
binds to the jaw switch to turn on Pitx1 in the jaw ssue.
Figure 2 illustrates how Pitx1 transcrip on is regulated in different ssues. The center image is that of a s ckleback embryo. The drawings in the surrounding boxes
show the Pitx1 gene region and ac vator proteins present in the jaw, pelvis, eye, or pituitary ssues. Note that for simplicity, we are only showing one ac vator
molecule present in a par cular ssue. In reality, many ac vators are present in a par cular ssue at any one me. Ac vator molecules with specific shading can bind
to switches with the same shading.
6. List all the ssues shown in Figure 2 that express the Pitx1 gene:
7. Consider what was revealed in the film. How can a spined s ckleback fish have the exact same PitX1 sequence as a spineless
s ckleback?
8. Examine the table, if a muta on occurred in the indicated region, which areas would you expect to express the Pitx1 gene. Check
all that apply.
Muta on Jaw Eye Pelvis Pituitary
Ac vator 1
Ac vator 2
Ac vator 3
Ac vator 4
Pitx1 Coding
9. The Pitx1 protein has many important func ons in various ssues during s ckleback development. The complete
absence of Pitx1 protein from all ssues is lethal to the organism. Circle the fish below that lacks Pitx1 expression in the
pelvis and EXPLAIN your choice.
Synthesis Ques ons
10. Explain the role that regulatory switches play in determining whether s ckleback embryos will develop pelvic spines.
11. According to the film, what is the selec ve pressure that led to freshwater s ckleback fish losing their pelvic spines?
