Chapter 19 - Eukaryotic Genomes Gene Regulation (controlling gene expression – turning genes on/off) Gene expression = Transcription and Translation of

Chapter 19 - Eukaryotic Genomes

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

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

AIM: What is the effect of differentiated gene expression?


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 (proteins) 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 / specialize) into a specific cell type like a neuron or muscle cell. All of their genes have the potential to be turned on/off.


What’s a stem cell?


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

Stem Cells

Stem Cell

More Stem Cells

Differentiated Cells



Where do you hypothesize you would find stem cells?

Stem Cells

Stem Cell

Stem Cells

Differentiated Cells



Where do you hypothesize you would find stem cells?

Stem Cells


1. Embryonic stem cells

- ex. (figure to right) hematopoietic stem cells (HSC) found in bone marrow of femur, hips, sternum, ribs and other bones (see next slide) and can only become either red or white blood cells.

- Found throughout body after embryonic development- Multipotent – means they can become ONLY a limited number of cell types depending on the type of stem cell.

- Found in embryos

- Totipotent – means they can become any cell type (placenta, muscle, neural, epithelial, etc…)- Pluripotent – means they can become any cell type except the placenta (muscle, neural, epithelial, etc…)

2. Adult stem 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 no reason for muscle to ever make insulin.

Active genes

Inactive genes



Active genes

Inactive genes


Is is possible for a differentiated cell like a neuron or muscle cell to dedifferentiate back to a stem cell? What would this require?



It would require genes that have been “permanently” turned off, typically by histone packing (more to come shortly), to be turned back on.


Active genes

Inactive genes

Is this possible? How can we test this?


Let’s try a little experiment:1. Let’s take an ovum (which is a stem cell of course) from some multicellular organism like a sheep and remove the nucleus.

AIM: What is the effect of differentiated gene expression?Is is possible for a differentiated cell like a neuron or muscle cell to dedifferentiate back to a stem cell? What would this require?


Let’s try a little experiment:2. Then let’s take the nucleus from a differentiated cell (let’s say a muscle 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 cannot turn back on the silenced (off) 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.

Embryo = time between conception (fertilization) until eight weeks old


What would you try next?



Let’s try a little experiment:4. We can try to implant the early embryo (blastocyst) 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.

Is this lamb, a clone, genetically identical to the ovum donor, surrogate mother or the nucleus donor?The nucleus donor as the nucleus contained the DNA



This process is called REPRODUCTIVE CLONING.

This indicates that genes in a differentiated nucleus have the “potential” to reactivate and therefore differentiated cells IN THEORY can dedifferentiate.

Does this answer the above question?



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

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 (pharm 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?

Cloned cats…


At least 20 ranging from camels, cats, dogs, a horse all the way to fish, frogs and fruit flies.


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 (their 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.The nucleus would obviously be one of your nuclei and the ovum would come from a donor….



Ethics

Should we be allowed to generate embryos for the sake of using the embryonic stem cells for research/medicine?



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



Bone Marrow Transplant is Stem Cell Treatment

Ex. Patient with Leukemia (white blood cell [leukocyte] cancer).

Destroy all white blood cells of patient using radiation/chemotherapy.

Take bone marrow from matching donor and infuse patient with hematopeotic stem cells.


AIM: Do differentiated cells retain their genetic potential?

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

Bone Marrow Transplant Cures HIV (aside):


AIM: Do differentiated cells retain their genetic potential?Using Stem Cells with Gene Therapy

Gene Therapy involves replacing a mutated gene within an already developed organism with a functional gene (somatic gene therapy) or replacing a gene in a germ line cell (sperm of egg) resulting in a heritable change (germ line gene therapy).

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



Regeneration- Regrowth of a lost of damaged body part

Can differentiated cells dedifferentiate into stem cells in plants?



Fig. 11.3A





Review

- Embryonic vs. adult stem cells

- Therapeutic vs. Reproductive cloning

1. Stem Cells

(toti-/pluripotent)

(multipotent)



Not all genes are going to be silenced for the life of the cell/organism, many will be turned on/off as needed…

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 eukaryotes.


NEW AIM: How are genes regulated in eukaryotes?

How are eukaryotic genes regulated?

Fig. 19.3

1. DNA/Chromatin packing2. Transcription initiation3. Splicing (RNA processing)4. Transport to Cytoplasm5. mRNA degradation6. Translation initiation7. Protein modification/activation8. Protein Breakdown


AIM: How are genes regulated in eukaryotes?

Fig. 11.6

1. DNA/Chromatin Packing



Eukaryotic gene regulation

Histones (“beads”) can pack genes or entire segments of DNA (“string”) 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





What is a nucleosome’s structure?1. The core is composed of 8 proteins

(H2A, H2B, H3 and H4 – two of each) known as histones. DNA wraps twice around the core. The N-terminal tails of the histones hang out from the nucleosome.2. Another histone (H1), not technically part of the nucleosome, clamps the DNA to the core.





Heterochromatin

Euchromatin-DNA wrapped around nucleosomes. - Nucleosomes not bound to each other- This is the form of an active gene (a gene that can be transcribed if desired by RNA polymerase)

- Nucleosomes binds to each other with help of additional histone called H1 condensing the DNA.- These genes are silenced and cannot be transcribed.

Ex. Gene for insulin in cells other than pancreatic beta cells.





EuchromatinHow does the chromatin stay in this “loose” euchromatin conformation?

Histone Acetylation

The N-terminal tails have the amino acid lysine to which an acetyl group is added preventing the nucleosomes from packing.

= acetyl (memorize it)





Histones RegulationIn addition to acetylation, histones can be modified on their N-termini a number of other ways as shown in this figure.

-CH3 = methyl (memorize it)

For example, methylation appears to promote condensation.





These additional levels of condensing require non-histone proteins and occur only during prophase

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




DNA methylation (an aside)




In addition to histone methylation, most plants and animals use methylation of the DNA itself on the base of cytosine (see below) to regulate gene expression:

DNA methyltransferase

Excessive methylation of a gene appears to be associated with turning a gene off…

DNA methylation as a mode of epigenetic inheritance




What is epigenetics?

Any modification to the genome that results in a change in function, but does not change the DNA sequence

Ex. Environmental chemicals, DNA methylation, histone acetylation. Epigenetic inheritance -

Some of these modifications can be inherited (i.e. DNA methylation patterns) making them significant in terms of diversity and evolution.http://classic.the-scientist.com/blog/display/55342/

Fig. 19.3




Recall Transcription

Fig. 11.8

2. Transcription Initiation



Recall the structure of a eukaryotic gene:


Fig. 11.8




Transcription factors (TF’s) are required to start transcription. A. General transcription factors are required for the transcription of all genes. These are the ones that bind at the promoter and interact with RNA polymerase II.

NO TF’s, NO Transcription

Eukaryotic gene regulationControl elements

General Transcription factors

Enhancer proteins

Don’t memorize this level of detail unless you have nothing else to do. First email me though and I will find you something else to do.



All of the TF’s in this diagram are general TF’s needed by every gene to be transcribed.


Transcription factors (TF’s) are required to start transcription.


Fig. 11.8




Transcription factors (TF’s) are required to start transcription. A. General transcription factors are required for the transcription of all genes. These are the ones that bind at the promoter and interact with RNA polymerase II. B. Specific transcription factors (either activators or repressors) will bind at DNA sequences called control elements (enhancer regions or repressor regions)distant from the gene itself and turn the gene on or off.

Eukaryotic gene regulationControl elements

General Transcription factors

Enhancer proteins

Fig. 11.8




Recap:


Control element = enhancer or repressor regionActivator TF’s bind enhancer

regions and promote gene expressions Repressors TF’s bind to repressor regions and inhibit gene expression

Mediator Proteins

Mediate (bridge) the interaction between activators and general TF’s




Combinatorial Gene Activation


Genes are typically regulated by a number of different enahancer/repressor regions.

Therefore, activation requires a number of different activators to be present at the same time:




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


b. Signal transduction occurs (a story you should know well…) and a TF is activated usually via phosphorylation.c. This TF, assuming it to be an activator, will undergo a conformation change resulting in exposure of the nuclear localization signal allowing entrance to the nucleus. It will then bind to specific enhancer control elements to promote expression of these genes.

EXAMPLE:

Fig. 19.3




Fig. 11.9

3. Alternative RNA splicing




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

Fig. 11.9

3. Alternative RNA splicing




Many proteins are involved in regulating which splice variant is formed…Don’t memorize…

Fig. 19.3




5. mRNA degradation




A. Generic degradation

Mediated by enzymatic removale of tail and cap followed by nuclease digestion.

5. mRNA degradation




B. RNA interference (RNAi)

In the case of RNAi, a gene codes for a special type of RNA called a small interfering RNA (siRNA) that is complementary to the target mRNA.In the end, a enzyme called RISC (RNA-induced silencing complex) binds to the siRNA, which anneals to the mRNA. RISC then cuts the mRNA or prevents translation…

5. mRNA degradation




B. RNA interference (RNAi)

Figure from book:

DICER is an enzyme that cuts the initial RNA transcript resulting in smaller pieces that will function as siRNA (or micro RNA, miRNA).

5. mRNA degradation




B. RNA interference (RNAi) as a tool

This means we can shut down any mRNA we want by sending in a complementary RNA that can be recognized by DICER.

http://www.nature.com/nrg/multimedia/rnai/index.html

Fig. 19.3




6. Translation Initiation




NO IF’s, NO Translation

Like transcription, translation also requires other proteins to start called initiation factors (IF’s - prokaryotes) or elongation factors (elF’s – eukaryotes (that is what the “e” is for).

6. Translation Initiation




Don’t memorize, just understand concept

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

Eukaryotes of course are more complicated…

Fig. 19.3




Fig. 11.10

7. 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.

A. Activation by Proteolysis (cutting the protein)

Fig. 11.10

7. Protein activation




(pre-insulin)

Activation/Inactivation through addition of a phosphate, which you should be very familiar with at this point.

B. Phosphorylation

Fig. 19.3




8. 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”.

8. Protein Degradation




What is the signal to degrade a particular protein?

Ubiquitination

The protein to be degraded is tagged. It is marked by the enzymatic addition of ubiquitin (a small protein itself). Not just one, but many in a row shown as green spheres below. Ubiquitin in the mark of death. If a chain of them are attached to you, you will be shredded.

A single ubiquitin protein

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

Fig. 19.3




Clearly more complex than prokaryotes…

Chapter 19 - Eukaryotic GenomesNEW AIM: What is the genetic basis of cancer?

How does one get cancer?

Genetic change falling into one of three categories:

1. Mutation

2. Movement of DNA within the genome3. Amplification of a gene

Let’s look at what this means…

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 internal signal through relay proteins.

Fig. 11.15A



Let’s say the signal molecule (ligand) is a growth factor (GF) and the new proteins being made activates cell division (instructs the cell to produce cyclin and proceed past the G1 checkpoint).

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 the receptor or 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 normal gene that when modified causes cancer is called a proto-oncogene.

OncogeneThe modified form of the gene that causes cancer. Does not NEED to be a mutation…

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

How a proto-oncogene can become an oncogene.



Let’s look at some specific examples…

Amplification (overproduction)

Fig. 19.11

Ras



Ras is a G protein normally activated by an RTK pathway. Ras is commonly mutated in many cancers resulting in a hyperactive version…

Fig. 19.11

Fig. 11.15A



If you get a mutation in one proto-oncogene like Ras, does that mean you get cancer?No, it takes more than one mutation in one

gene to cause cancer…read on about tumor suppressor genes…



They can be:

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

1. TF’s that activate proteins, which prevent cell division or cause apoptosis like p53.

Let’s look at p53 in more detail…

p53 (53 for 53,000-dalton mw) is a TF that is activated in response to excessive DNA damage through a phosphorylation cascade (see above).- p53 turns on genes involved in inhibiting the cell cycle

and if DNA damage is too great, apoptosis genes as well.



p53“The guardian angel of the genome”



They can be:

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

1. TF’s that activate proteins, which prevent cell division or cause apoptosis like p53.

2. DNA repair proteins like BRCA-1 and BRCA-2, which prevent mutations obviously.

OR

Fig. 11.16B

Both BRCA1 and BRCA2 are DNA repair proteins – fix DNA 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.





Based on what you have learned thus far, what genetic changes are necessary to cause cancer?


AIM: What is the genetic basis of cancer?You would need a mutation in BOTH tumor suppressor genes and one oncogene…why both tumor supressor genes?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.


AIM: What is the genetic basis of cancer?Additional genetic changes are typically required like activation of telomerase and genes involved in cell migration.Explain.Telomerase is needed to maintain the length of the ends of chormosomes (telomeres) since they shorten with each division thanks to the lagging strand, and in order to be cancerous the cells need to be able to migrate.

Fig. 11.17A



Fig. 11.16B

Reminder: BRCA1 (BReast CAncer) and BRCA2 are DNA repair proteins – fix DNA breaks.

Breast Cancer is the second most common type of cancer next to Prostate Cancer.

~230,000 new cases a year in females

Inheriting one mutated BRCA1 allele gives you a 60% chance of developing breast cancer before the age of 50 compared to 2% normally.



Predisposition to Cancer

Fig. 11.17A



Conclusion:



1. Multiple mutations are required for cancer to occur

a. A proto-oncogene must be modified by one of the methods discussed to an oncogene promoting cell growthb. Tumor suppressor genes must be rendered inactive so they don’t inhibit division or cause apoptosis.c. Additional genetic changes must occur like activation of telomerase and/or genes involved in motility.



Old chart, prostate now higher than breast…


AIM: Eukaryotic non-coding regions

Eukaryotic genomes consist mostly of non-coding regions in addition to genes called “junk DNA”…is it really junk though??A. 98.5% of our genome

does NOT code for mRNA, tRNA or rRNA!!!B. Most of this DNA is repetitive DNA (DNA sequence of various length that just keep repeating over and over)C. ~44% of human genome consists of transposable elements!



Transposable Elements

Two types in Eukaryotes

1. Transposons

2. Retrotransposons-most transposable elements in eukaryotes are retro



Not all of our genes exist as isolated islands in the genome:…

A. 50% of our genes are arranged in multigene families – collections of similar or identical genes.

B. The genes coding for the three pieces or rRNA (18S, 5.8S and 28S) are grouped and this group is repeated 100’s to 1000’s of times so that ribosomes can be made super quickly and efficientyl. The three genes are made as a single transcript and then cleaved apart.C. The globin genes (code for hemoglobin subunits) are clustered together as well. Hemoglobin is composed of two alpha and two beta subunits. The alpha subunits are clustered on chromosome 16 and the betas on chromosome 11.



Gene duplication followed by mutation is key in evolution

Ex. The hemoglobin subunits

Duplication of an ancestral globin gene freed one up to be mutated resulting in two alleles (alpha and beta). This was followed by transposition as they are on different chromosomes, more duplication and then more mutation…



Exon Shuffling within the genome can lead to the evolution of new genes:

Shown to the right are a series of genes in humans each composed of numerous exons as indicated by colored boxes.

Notice how the many different genes have similar exons (same color box) and many of the exons are repeated in a given gene.

It is clear that exons are being moved around and duplicated through time resulting in the evolution of new genes…

Different exons (colored boxes)

genes

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Chapter 19 - Eukaryotic Genomes Gene Regulation (controlling gene expression – turning genes on/off) Gene expression = Transcription and Translation of