Lecture 34, 12/1; Chapter 18, Cell Cycle Regulation and Cancer

Chap 18
Cell Cycle Regulation and cancer.

-Cancer is the 2nd leading cause of death in western world.  1 in 3 people will be diagnosed with cancer at one point in their life.
-1,000,000 new cases of cancer in the U.S. each year.
-500,000 deaths attributed to cancer

Cancer is a genetic disease. 
-The genomic alteration associated with cancer range from single nucleotide changes to large deletions.

• Vast majority of these mutations arise in somatic cells.
• only 1 % of cancers are linked to germline mutations which predispose an individual to cancer.

Most cancers are a result of multiple mutations (6-10 avg) as opposed to a single mutation.

(9:20) Common traits of cancer.

• cell proliferation - abnormal unchecked growth
• Metastisis - cells have the ability to spread to other parts of the body

typically two broad categories of tumors

• benign - tumors that are localized to one region and are not metastisis.
• malignent - tumor which has the ability to spread to other areas of the body

all tumors arise from a single cell.

• begins to accumulate mutations and eventually starts to grow uncontrolably.

(15:25) The idea that "all tumors arise from a single cell" is supported by x inactivation data. 

• cells in a single tumor all share a common inactivated X chromosome.
• Because X inactivation is random the likelihood that all cells in a tumor share an inactivated X chromosome by chance is extremely low.

(18:05) Broadly: Cancer is a multi-step process.

• This idea that "cancer is a multi-step process" is supported by the following data.
• 1.) There is a definite increase in the incidence of cancer as age increases.
• 2.) Also when looking at victims of the atomic bombing in WWII we see that 5-8 years after the bombing there was an increase in the rate of cancer for survivors of the initial blast.

(21:30) Example of the multi-step process: Cervical Cancer.

• In the normal cervix a number of cells, after time, become quiescent cells (a cell that has entered Go phase and stopped dividing). 
  
• However, a number of basal cells exist in the cervix (not quiescent) which are actively dividing or have that ability.
• Occasionally basal cells form mutations.  When these mutations happen they can form a dysplasia (tumor area).  This is early cervical cancer and easily treatable.  Untreated the cells in the dysplasia will develop additional mutations over time.  With time the dysplasia becomes carcinoma which is much more difficult to treat.

(32:15) Genetic causes of cancer
Some cancer cells contain genetic defects affecting DNA repair and genomic stability. 
-On average, cancer cells have higher rates of mutations than normal cells. This suggests problems with DNA repair.
-Mutator phenotype - a cell more prone to mutation.

(34:45) Two types of cancer caused by mutations in the DNA repair system (inheritable forms of cancer)
-Xerodermo pigmentosum - very susceptable to the effects of UV light.  These indivuduals have a mutation in one of the 7 genes involved in nucleotide excision repair. 
-Hereditary nonpolyposis Colon cancer - this is an autosomal dominant allele (will run through families very strongly).  Mutation is involved in genes involved in mismatch repair system.

(39:05) Cancer is often caused by mutations that affect the cell cycle.
One of the hallmarks of cancer is that cells grow and divide at a greatly increased rate.
The cell cycle control system helps to prevent the uncontrolled growth of cells.

• During G1 the cell makes a decision about whether or not to continue to divide. A cell that stops actively dividing enters the Go phase.  In the Go phase cells are metobolically active . . . but not dividing.  A cell in the Go phase is referred to as a quiescent cell.
• Another one of the hallmarks of a cancer cell is that it skips Go phase.  If the cell does enter Go phase it passes through very quickly.
• (45:30) Normal cells will exit the Go phase in response to environmental signals --> A signal transduction pathway accomplishes this. 
  In a cancer cell, the genes in that signal transduction pathway are mutated so that the signal pathway is always on (tells it to grow more). 
  
• The cell cycle has checkpoints to help prevent this from happening: G1s checkpoint, G2 M checkpoint and the M checkpoint.
• BUT mutations in the genes for these checkpoints have an association with cancer.
  

(50:25)Apoptosis - programmed cell death.  A pathway  a cell can activate when its too damaged to continue.  When it is activated the cell self-destructs.

• BUT mutations in the genes for apoptosis are also associated with cancer.
  

Lecture 31, 11/19; Chapter 17, Regulation of Gene Expression in Eukaryotes

Regulation of gene expression in eukaryotes

Gene expression in a multi-cellular organism is very different than in a prokaryote. One such difference is cellular differentiation.

-Cellular differentiation: start out with general undefined cells then turn on different genes to make a different type of cell or cell types. This process is CRITICAL. You need to turn on the correct genes at the correct times otherwise you will get death.

• ex. of cellular differentiation --> you need different things in a muscle cell then in a nerve cell.

(3:35) How the regulation of gene expression differs in Eukaryotes

• Eukaryotic cells are larger and more complex than prokaryotes. Within this, DNA is packed into chromatin with histone proteins. Chromatin remodeling is a key step in the regulation of gene expression in a eukaryote. So if you don't have your DNA in a form that is accessible to RNA POL to copy you can essentially turn off transcription by shuttin down chromatin remodiling.
  
• Eukaryotes typically have their DNA in multiple chromosomes not one chromosome (typically seen in prokaryotic organism)
  
• Because DNA is in the nucleus and the ribosomes are at the endoplasmic reticulum (ER) in the cytosol, transcription and translation are seperated spatially and temporally (occur at different times). In a prokaryote: almost as quickly transcription starts to produce RNA, ribosomes come in, bind that RNA and start to translate that RNA. In eukaryotes these processes are seperated.
• mRNA molecules are modified prior to exiting the nucleus in a eukaryote. That modification includes splicing.
  
• (8:50) Eukaryotic mRNA molecules are more stable than prokaryotic, they have a longer half-life (amount of time they exist). Partly because Prokaryotes need to rapidly respone to changing conditions. Eukarytoes don't experience this as much.
• In eukaryotes regulation can also occur at the level of translation. You have a more stable mRNA molecule but you may alter or regulate the translation of an individual mRNA molecule because it's not being produced and degrated as quickly.
  
• While all eukaryotic cells contain complete copies of their genome (all chromosomes + DNA in all cells) different cells express different subsets of genes.
• Broadly: the process of regulation in a eukaryote is a more complex process then what is seen in a prokaryote.

Differences in regulation of gene expression	Prokayote	Eukaryote
1st difference		larger and more complex
2nd difference	DNA typically in one chromosome	DNA in multiple chromosomes
3rd difference	transcription and translation happen almost simultaneously	transcription and translation are seperated spatially and temporally
4th difference		mRNA molecules are modified prior to exiting the nucleus
5th difference	Prokaryotes need to rapidly respond to changing conditions - less stable.	more stable, longer half-life
6th difference		regulation can also occur at the level of translation
7th difference		contain complete copies of their genome in each cell

(14:45) Chromosome organization in the nucleus influences gene expression

• During interphase, the DNA found in the nucleus is in a relaxed state. However, there is stil orginization to how DNA is organized within the nucleus. This organization plays a key role in the regulation of gene expression.
• Within the nucleus, each unique chromosome exists in a chromosome territory.
• The regions between the chromosome territory's are called interchromosomal domains.
• (19:45) Within the nucleus the arrangement is as follows:
• Chromosomes with small numbers's of genes have thier chromosome territory on the outside of the nuclues.
• Chromosomes with larger numbers of genes exist in chromsome territiores towared the inside of the nucleus.
• It has been proposed that the genes being actively transcribed on a chromosome will be found toward the edge of the chromosome territory. This suggests that RNA POL lives in the interchromsomal domains. The genes need to be brought close to those areas so they can be transcribed.
  
• Once we get a chromosome in position the intition of transcription begins --> two steps.
• Chromatin Remodeling
• We need to recruit a number of factors (typically proteins) that help initiate transcription.

(27:10) Transcription Initiation

• There are three common cis acting elements in Eukaryotic transcription initiation: promoters, enhancers and silencers.
• The process requires chromatin remodeling, a number of DNA sequences and over 100 proteins. Just to initiate transcription.

• Promoters: The site where the transcription machinary binds to start transcription.
• The promoter typically facilitates a basal level of transcription
• Typically adjacent to the gene (upstream)
• contains a few 100 nucleotides.
• (31:30) Within those nucleotides there are number of key sequences of the promoter
• TATA Box a.k.a. Core promoter - 25-30 BP region of DNA that is bound by RNA POL. This contians a 7-8 BP consensus sequence. The consensus sequence contains a number of nucleotide sequences and the TATA sequence. This is the RNA POL "docking site".
• mutations in this sequence decrease the level of transcription. Deletion of the sequence results in loss of transcription.
• CAAT Box - these element contain the sequence CAAT or CCAAT located 70 BP upstream of the start of the gene.
  
• Mutations in this sequence=decreased transcription.
  
• (36:15) GC Box - GGGCGG and is found 110 BP upstream of the transcription start site.
  
• both the GC box and the CAAT box can serve as enhancers as well as part of the promoter.
  

• (40:00) Enhancers: Can be found on either side of a gene and can some times be great distances away.
  
• enhancers are typically bound by multiple proteins with a net effect of stimulation of transcription.
• (42:50) How do we differentiate an enhancer from a promoter?
• Promoter regions are found at fixed locations. Enhancers are not found in fixed locations (they can move around.
• You can invert an enhancer without affecting its activity. (not the case for a promoter)
  
• If you move an enhancer to a different gene, that gene will now be regulated by the enhancer.
  
• Promoters are responsible for the basal level of gene expression. Enhancers are necessary for the full expression of a gene.
  
• Enhancers can be cell type specific and promoters are not.

(47:45) How do enhancers stimulate the level of transcription?

• Factors can bind enhancers which help with chromatin remodeling.
  
• When a factor binds the enhancer it bends the DNA bringing the enhancer and promoter closer together. This can help stimulate RNA POL binding to the promoter
  

Genetics Lecture 29, 11/12; Chapter 16, Lac operons, Jacob & Monod

See the Power Point for Chapter 16 here
http://docs.google.com/Presentation?id=dhqwrndc_501cs7scpdq



Quick Review:
Last time we talked abou the lac operon and how we regulate the expression of the lac operon in the presence and absence of lactose.
Function of the lac operon is to help convert lactose to glucose and galactose.

What happens when both lactose and glucose (primary metabolite) are present?

• ANS: In the presence of glucose the lac operon is off. (fail to transcribe the lac operon because you don't need to break the lactose down)
  
• so then . . . "How is this accomplished?". It is accomplished through a process called CAP (Catabolite activator protein) and cAMP (cyclic AMP)
  
• Quick Review: RNA POL on its own has a hard time finding promoters. (That's why we have the sigma factor, it aids the RNA POL in getting to the promoter)
  
• (3:40) The CAP is always present in the cell. CAP binding to the promoter of the lac operon is necessary for RNA POL to bind a promoter. So without the binding of CAP RNA POL seldom binds to the promoter.
  
• CAP can only bind to the promoter for the lac operon when its complexed with cAMP. When cAMP is not present, CAP does not bind to promoter.
  

(7:00) cAMP levels are dependent on the level of glucose in the media.

• cAMP is a derivative of ATP
• One of the enzymes utilized in cAMP production is called adenylcyclase. What happens is that the production of adenycyclase is inhibited by glucose. Thus, when glucose is present in the media the adenylcyclase levels fall. As they fall cAMP levels fall. SUMMARY: in the presence of glucose you have low levels of cAMP, therefore, you rarely form the cAMP - CAP complex, therfore, you rarely transcribe the lac operon.
• Summary of this example: Lactose is present in this example which means lactose is able to bind to lac I bind to the repressor protein, inhibit the repressor proteins ability to bind to the operator. If RNA POL can bind, it can copy BUT the presence of glucose reslts in low cAMP and the lac operon is off.
  

(11:35 - 21:25) Schau used the following volunteers to demonstrate regulation of lac operon.
Dan - promoter
Becky - operater
Laura - structural genes
Emily - RNA polymerase
Chris - catabolyte activator protein (his hat is cAMP)
Lindsey - Lac I
Lisa - Lac I gene
Britney - Lactose
E.J. - Glucose

(15:25) Review of Monday's lecture:

• Begin with absence of lactose - no sugar at all.
• Lac I gene gives rise to Lac I protein, Lac I protein goes to operator (Lac O). RNA POL goes to promoter and attempts to copy but Lac I stops. As a result there is no transcription that happens

(16:50) Introduce a sugar - Lactose
Lac I gene gives rise to Lac I protein, Lac I protein goes to operator (Lac O). Lactose binds to Lac I and prevents it from getting to operator. RNA POL can then come in, bind to promoter and transcribe.

(18:00) Presence of lactose and CAP - cAMP complex.
Lac I gene gives rise to Lac I protein, Lac I protein goes to operator
(Lac O). Lactose binds to Lac I and prevents it from getting to
operator. CAP helps RNA POL find the promoter.

(19:10) Presence of lactose and glucose and CAP - cAMP complex.
Lac I gene gives rise to Lac I protein, Lac I protein goes to operator
(Lac O). Lactose binds to Lac I and prevents it from getting to
operator. CAP helps RNA POL find the promoter BUT glucose reduces levels of cAMP and without it you can not get RNA POL to the promoter.

side note: Lac I repressor protein (when present and functioning) binds to operator and inhibits RNA POL from transcribing genes

(23:15) 3 different processes we NEED to KNOW

• absence of sugar
• presence of lactose
• presence of lactose and glucose.

• For each of the above conditions we should know
  
• Is the operon transcribed?
• How does the regulation of the operon take place?
• In regards to regulation include talking about cAMP and CAP and lac I repressor protein

(25:40) Additional work by Jacob and Monod

• Quick Review: The understanding of how this regulation takes place relied on these two constituitive mutants: lac I- and lac O-
  
• When these above mutations were orginally identified, nobody knew if they were mutations in cis-acting elements or trans-acting factors. We (as a genetics class) know that Lac I is a trans-acting factor and Lac O is a cis-acting element. Jacob and Monod did not know this as a result they developed the "Cis-trans test".
• (28:10) "Cis-trans test", used to determine the nature of these mutations. How they did the test:
• The lac I mutant strain in the cell constiuitively expresses the lac operon.(despite presence or absence of lactose. Normally it is sensitive to lactose, however)
  
• Sooo . . . They placed a wildtype (WT) copy of the lac I gene onto a plasmid (a piece of extra chromosomal DNA). Then they introduced this plasmid into the lac I- strain. What this accomplishes: The WT copy of the lac I gene on the plasmid will produce or give rise to the WT lac I protein. It will NOT correct a mutatnt lac I DNA sequence in the cells DNA.
  
• Important: The presence of the plasmid with the lac I gene can correct a mutation in a trans-acting factor. Not in a cis-acting element.

• (33:35) What will the presence of WT lac I protein do to the lac I mutant strain?

• We would expect that this would correct the constituitive phenotype. So the lac I mutant strain with the WT copy of lac I on a plasmid, making protein, will no longer be constituitive.
• Now that we have a normal protein it will require lactose to bind to it to prevent it from binding to the operater. After performing the test the conclusion is that lac I produces a trans-acting factor - a molecule that is a protein that is produced to regulate the expression of another operon.
  
• Summary: This cell will only express the lac operon when lactose is present

(36:55) Now they want to know about lac O.

• The lac O mutant strain in the cell constiuitively expresses the lac operon
• Is lac O a cis - acting element or a trans acting factor?
• Introduce a WT copy of the lac O sequence on a plasmid, this can produce WT copies of the lac O protein. However, the problem is, lac O does not produce a protein.
  
• The presence of the plasmid does not correct the mutant lac O sequence. Therefore, the lac I repressor cannot bind to the mutant lac O sequence.
  
• The presence of the plasmid does not correct the constituitive phenotype.
  
• Thus we conclude that lac O is a cis-acting element.
• (plasmid will correct a trans-acting factor but not a cis-acting element.)
  

(43:25) Recap of Jacob and Manod "Cis-trans test"

• cis acting element - sequence of DNA that is acted upon by a trans-acting factor to regulate the expression of a gene or operon.
  
• trans acting factor - protein that regulates the expression of a gene or an operon by acting ON a cis-acting element. Binds to DNA and either turns on or off the expression of a gene.
• In the lac operon, the binding of lac I (the repressor protein) to the operator turns off transcription. When you mutate either of these elements you mutate the protein and it won't bind to the operator if you mutate the operator the normal protein can't bind - same effect. The end result is constituitive expression - we always express the lac operon.
  
• (46:50) What Jacob and Monod started this test, all they knew was that they express constituitively. They wanted to know was it trans-acting factor or cis-acting element.
  
• They take the mutant strains, started with lac I. They have an abnormal copy of the lac I gene.
  
• They go to a different cell and get a normal copy. They put this normal copy into the lac I mutant strain. It will be transcribed and you will make WT copies of the lac I protein. So instead of a cell that only has "bad" versions of lac I protein that can't bind to the operator, we have a cell that also has normal versions of the protein that can bind to the operator. When that happens the cell that was always expressing the lac operon is now only going to express the lac operon when lactose is present - so there is a change in the phenotype of the cell. BECAUSE that works for lac I we say that lac I is a trans-acting factor. If it DID NOT work we would have called it a cis-acting element.
  
• (50:00) They then repeat the process for lac O.
• They take a mutant copy of lac O and put in a normal version (from some other cell). It is a cis-acting element.