Chapter 11 - The Control of Gene Expression AIM: What is the effect of differentiated gene...

Chapter 11 - The Control of Gene Expression

AIM: What is the effect of differentiated gene expression?

Now that we understand how genes are transcribed into RNA and how that RNA, if it is mRNA, is translated into a polypeptide, it is time to understand how gene transcription and translation are controlled in the cell.


Gene Regulation(controlling gene expression – turning genes on/off)

Gene expression = Transcription and Translation of a gene; the cell expresses the gene



Multicellular organisms are composed of many different types of cells…



What makes these cells different from each other?The same thing that makes a school

different from a bank or a police station different from a fire house…the workers are different!!

(Different cells have different genes turned on/off)

Differential gene expression



Stem Cells

Stem Cell

- cells that have the ability to differentiate (to turn into) a specific cell type like a neuron or muscle cell. All of their genes have the potential to be turned on/off.



Stem Cells can divide to make more stem cells or they can differentiate.

Stem Cells

Stem Cell

Stem Cells

Differentiated Cells



Some genes are turned off for the life of the cell:In differentiated cells, certain genes

are “permanently” shut down by histone packing like the insulin gene in muscle cells. There is not reason for muscle to make insulin.

Active genes

Inactive genes



Can differentiated cells turn back into stem cells (dedifferentiate)?This is not the norm, but is it possible?

Active genes

Inactive genes



Is is possible for a differentiated cell to dedifferentiate back to a stem cell?

Let’s try a little experiment:1. Let’s take an ovum from some multicellular organism like a sheep and remove the nucleus.




Let’s try a little experiment:2. Then let’s take the nucleus from a differentiated cell and put it into the ovum (this is a diploid nucleus of course).

(somatic/differentiated cell’s nucleus)




Let’s try a little experiment:A. What do you predict should happen if differentiated cells can never access the silenced genes? B. What if the genes can be turned back on?

(somatic/differentiated cell’s nucleus)




Let’s try a little experiment:3. It turns out that the genes can be reactivated (they are not permanently turned off) and the “zygote” divides to become an embryo.

The cells of the embryo are called embryonic stem cells. What type of cells can embryonic stem cells differentiate into?These will become the organism so ALL CELL TYPES!

Embryo = conception (fertilization) to eight weeks old




Let’s try a little experiment:3. It turns out that the genes can be reactivated (they are not permanently turned off) and the “zygote” divides to become an embryo. What would you try next?




Let’s try a little experiment:4. We can try to implant the embryo into the uterus of a surrogate mother (a black face ewe in this case) and see what happens…




Let’s try a little experiment:5. Amazingly, the embryo develops and the lamb is born. This lamb is a clone (genetically identical) to the ovum donor or the nucleus donor?The nucleus donor as the nucleus contained the

DNA



Is it possible for a differentiated cell to dedifferentiate back to a stem cell?

This process is called REPRODUCTIVE CLONING.

This indicates that genes in a differentiated nucleus have the “potential” to reactivate and therefore differentiated cells will likely be able to dedifferentiate.

Does this answer the above question?




REPRODUCTIVE CLONING

Dolly (left) and her surrogate mother. A black face sheep cannot give birth to a white face sheep naturally.



What could reproductive cloning be used for?1. Repopulating endangered species…is

there a problem?

2. Clone drug-producing animals

3. Clone genetically-unique animals, etc…Should we do this with humans?

What if you had a reproductive clone. One day you fell ill and needed part of a liver or a kidney or bone marrow?...

There are arguments on both sides…

They are all genetically identical and therefore equally susceptible to the same environmental changes…



How many different animals have been cloned thus far?At least 20 ranging from camels, cats, dogs, a horse all the way to fish, frogs and fruit flies.

Cloned cats…



How many different animals have been cloned thus far?At least 20 ranging from camels, cats, dogs, a horse all the way to fish, frogs and fruit flies.

Cloned cats…that have been genetically modified (next chapter) to glow red.



What else could we do with this embryo?

?



AIM: How are stem cells generated and used?

We can grow them in a dish (culture them) and then treat the cells with different hormones to get them to differentiate into the cells we want…



What can we use these differentiated cells for?

One could make any cell type they want:

1. Skin cells for burn victims

2. Organs for transplant patients3. Neurons for a person with a spinal cord injury

These transplanted cells will not be rejected (destroyed by the immune system) because they are genetically identical to the patient (your antibodies will not bind to them).

4. Basic scientific research, etc…

What is the advantage of these cells over other neurons or organs in terms of transplants?



This form of cloning is called Therapeutic Cloning.



Ethics

Should we be able to use embryos to get embryonic stem cells?



Recent advances:In 2008, scientists at UCLA figured out how to turn skin cells into embryonic stem cells, alleviating the need for cloning and embryo destruction

Kathrin Plath, UCLA stem cell scientistshttp://www.sciencedaily.com/releases/2008/02/080211172631.htm



Adult stem cells

- Unlike embryonic stem cells, adult stem cells cannot become every cell type…

- Stem cells found within us amongst the differentiated stem cells

Ex. Hematopoietic stem cells- Found in the bone marrow - Divides to make more stem cells, some of which differentiate into all the types of blood cells. - Can be used to treat leukemia or possibly even HIV!

What


AIM: Do differentiated cells retain their genetic potential?

http://www.nature.com/nm/journal/v15/n4/full/nm0409-371.html

Where else do we observe already differentiated cells dedifferentiating and becoming other cells types?



Regeneration- Regrowth of a lost of damaged body part

What about plants? Can differentiated cells dedifferentiate into stem cells?



Fig. 11.3A





Review

- Embryonic vs. adult stem cells- Therapeutic vs. Reproductive cloning

- Stem Cells



Now not all genes are going to be silenced for the life of the cell/organism…

Ex. The genes coding for enzymes that make glycogen in the liver…

If the blood glucose concentration is low, the liver will be releasing glucose, not building glycogen from it. Therefore, the genes should be off. Likewise the genes whose protein products are involved in secreting glucose should be on.

Gene are CONSTANTLY being turned on and off in your cellsLet’s look at how this is accomplished in prokaryotes and then

in eukaryotes.

Fig. 11.1A


NEW AIM: How are genes regulated (controlled) in prokaryotes?

How are genes regulated in prokaryotes?

Lactose (Lac) Operon


AIM: How are genes regulated (controlled) in prokaryotes?In order to begin to understand this process, we will look at a set of three genes involved in lactose metabolism (the hydrolysis of lactose to _______________) called the…

Glucose and galactose

Anatomy of an operon


AIM: How are genes regulated (controlled) in prokaryotes?

Fig. 11.1B

An operon typically contains a:1. Promoter2. Operator

3. A set of genes (3 in this specific case)

The regulatory gene (LacI) is found OUTSIDE of the operon.

A. LacZB. LacYC. LacA

LacZLacY

LacA

4. What critical gene part is missing from this figure?The terminator sequence

The terminator sequence

LacI

The three gene products (can you guess what they might be?):



Fig. 11.1B

1. LacZ codes for β-galactosidase

2. LacY codes for permease

3. LacA codes for transacetylase

LacZLacY

LacA

LacI

- The enzyme that hydrolyzes lactose to glucose and galactose

- A passive lactose transporter protein that sits in the membrane and allow lactose to diffuse into the cell.

- Exact function not yet known…

QUESTION



Fig. 11.1B

If lactose is present around the cell (perhaps it is one of the bacterium in your mouth and you just drank a glass of milk), should these genes be turned on or off?They should be ON since lactose is present and will need to

be hydrolyzed so the glucose can be used to make ATP of for biosynthesis.

Let’s look at how this operon works to control expression of these three genes…

Fig. 11.1B



1. The regulatory gene codes for the repressor protein.A. What does repress mean?

- To preventB. What will this protein do then?

- It will prevent the expression of the genes (turn them off)- Any guess how it might do this?

Fig. 11.1B



1. The regulatory gene codes for the repressor protein.C. It represses by binding to the Operator

sequence.

Fig. 11.1B



1. The regulatory gene codes for the repressor protein.C. It represses by binding to the Operator

sequence. -When it binds the operator, it will interfere with RNA polymerase binding to the promoter. The genes are off.

Fig. 11.1B



ALL FOR ONE AND ONE FOR ALL

Notice that all three genes are turned on/off together. Eukaryotes do not typically do this. They turn genes on/off individually.

Fig. 11.1B



Q1. How do you suppose these genes will be turned ON when lactose is present?A1. Somehow the repressor needs to fall off.Q2. How can we get it to fall off? (HINT: you are changing its function)A2. You need to change its structure.

Q3. How can we change the structure?A3. Bind something to it…a ligand.

Q4. What should the ligand be?

Fig. 11.1B



The ligand should be lactose itself since in the presence of lactose these genes should be turned ON.



1. Lactose binds the repressor.2. A conformational (shape) change occurs and the repressor

falls off the operator.3. RNA polymerase now binds to the promoter and begin

transcription of all three genes in one long mRNA.

4. Ribosomes translate the mRNA into proteins.

Activating the operon:



Q1. What will happen when β-galactosidase breaks down most of the lactose?A1. Lactose will fall off the repressor and the repressor will

once again bind to the operator and turn the genes off.Q2. Why not just leave these genes on all the time?

A2. This would be a huge waste of resources…ATP, amino acids, ribosomes, nucleotides, RNA polymerases and space.



Lac repressor protein

Repressor bound to the operator sequence

Lac operon – The video

Tryptophan (Trp) operon



- This operon contains fours genes whose protein products are responsible for synthesizing (making) the amino acid tryptophan.

When would you want to turn these genes on?When tryptophan is NOT present,

because that is when you need to make it…




When would you want to turn these genes on?When tryptophan is NOT present,

because that is when you need to make it…

How does this compare to the lac operon?It is the opposite. You

turn the lac genes ON when lactose is present.




Q. Tryptophan binds to the trp repressor just like lactose binds to the lac repressor. How does this work?

What you know:1. Trp binds to repressor2. When Trp is present, trp synthesis genes are offA. The repressor is active when Trp is bound and inactive when it is not, the opposite of the lac operon.




I do not recommend memorizing the difference. Think about is logically:

1. The repressor bind to the operator2. When it is bound the genes are off

3. You need the lactose break down genes when lactose is present.

4. Therefore, when lactose binds to repressor, it should fall off operator

5. Likewise, when trp is present, the trp synthesis genes are unnecessary because you have it already6. Therefore, Trp when Trp binds to the repressor, the repressor should bind the operator and shut the genes off.

Trp operon





The trp repressor (with trp bound) binding to the operator sequence.


NEW AIM: How are genes regulated in eukaryotes?

How are eukaryotic genes regulated?

Fig. 11.11

1. DNA packing2. Transcription initiation3. Splicing4. mRNA degradation5. Translation initiation6. Protein activation7. Protein Breakdown


AIM: How are genes regulated in eukaryotes?

Fig. 11.6

1. DNA Packing



Eukaryotic gene regulation

Histones can pack genes or entire segments of chromosomes tightly such that transcription factors and RNA polymerases cannot access the DNA. These gene are typically turned off for the life of the cell.

Fig. 11.6



Ex. One of the X chromosomes in XX females (humans included) is randomly silenced by histones. Females, like males, only have one active X chromosome. The other is condensed and called a barr body.





Fig. 11.6




Recall Transcription

Fig. 11.8

2. Transcription Initiation



- Transcription factors are required to start transcription. - Some of these proteins will bind at the promoter.- Others will bind at sequences distant from the gene itself called enhancer sequences.

NO TF’s, NO Transcription


2. Transcription Initiation



a. A signal molecule (ligand) like growth factor will bind to a surface receptor.


b. Signal transduction occurs and a TF is activated.c. This TF will enter the nucleus and turn on genes involved in activating cell division.

EXAMPLE:

Fig. 11.9

3. Alternative splicing




- Alternative splicing can control how much mRNA is synthesized of each alternative transcript.

4. mRNA degradation




5. Translation Initiation




Like transcription, translation also requires other proteins to start called initiation factors (IF’s).

NO IF’s, NO Translation

Fig. 11.10

6. Protein activation




(pre-insulin)

Insulin is made as a single polypeptide, which then fold into its inactive form. An enzyme will cut (cleave) the polypeptide forming the active protein form of insulin.

7. Protein Degradation




When a protein is no longer needed (the cell has enough product of a certain enzyme) it can be degraded – broken down into its amino acids, which are then recycled into new polypeptides.

This is accomplished by a large assembly (complex) of proteins called the proteosome.

It is really a “polypeptide shredder”.

AIM: How are genes regulated (controlled) in eukaryotes?

Fig. 11.11

1. DNA packing2. Transcription initiation3. Splicing4. mRNA degradation5. Translation initiation6. Protein activation7. Protein Breakdown



Clearly more complex than prokaryotes…


NEW AIM: What is the genetic basis of cancer?

How does one get cancer?Mutations in the DNA of genes responsible for controlling the cell cycle (cell division).

The products of these genes are typically involved in regulating gene expression…

Fig. 11.15A


AIM: What is the genetic basis of cancer?

Signal transduction pathway

- process by which the cell converts one signal into another

In this case (to the right) an external signal is converted into an multiple internal signal through relay proteins.

Fig. 11.15A



Let’s say the signal molecule (ligand) is growth factor (GF) and the new proteins being made activate cell division.

growth factor (GF)

Activates division

Fig. 11.15A



If there were no growth factor there should be no…

Activates division



If there were no growth factor there should be no……new protein being made and cell division should….

be off.

Q. What if there is a mutation in the gene of one of the relay proteins that changes its shape so that it is always on?

Fig. 11.16A



The transduction pathway will always be on regardless of growth factor…This can lead to uncontrolled cell division…cancer.



Proto-oncogene

A gene that when modified causes cancer is called a proto-oncogene.Oncogene

The mutated form of the gene.

Proto = “before”oncos = “tumor” or cancerGene = gene

Fig. 11.15A

How a proto-oncogene can become an oncogene.



- proto-oncogenes are often signal transduction proteins that promote cell division

Fig. 11.15A



If you get a mutation in a proto-oncogene, does that mean you get cancer?

No, it takes more than one mutation in one gene to cause cancer…read on.

Fig. 11.16B



They are typically TF’s that activate proteins, which prevent cell division or cause apoptosis.

Cells have genes that code for proteins that inhibit cell division called tumor suppressor genes.


AIM: What is the genetic basis of cancer?What would need to happen in order to get cancer in a cell that already has an oncogene?

Fig. 11.16B



You would need a mutation in BOTH tumor suppressor genes…why?Just because you knocked out one, the other can still function and stop the division (two hit hypothesis).Why don’t both proto-oncogenes need to be modified/mutated? These proteins activate and you only need one oncogene to activate the pathway.

Fig. 11.16B


Both BRCA1 and BRCA2 are DNA repair proteins - fixes breaks.

Mutations in the BRCA1 gene increase the risk breast, ovarian, Fallopian tube, prostate and colon cancers.

Over 600 different mutations have been identified

Among breast cancer patients of Jewish ancestry, 10% had mutations in one of these two genes.

Fig. 11.17A



Fig. 11.17B



Conclusion:



1. Multiple mutations are required for cancer to occur

a. A proto-oncogene must be mutated to an oncogene promoting cell growth

b. Tumor suppressor genes must be mutated and rendered inactive so they don’t inhibit division or cause apoptosis.

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