BIOL 103 Unit 2 Midterm

what is DNA replication? when and where does DNA replication occur?

what? replication of DNA only in preparation for cell division (meiosis/mitosis)

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when? S phase

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where? in the nucleus (eukaryotes), in the cytoplasm (prokaryotes)

direction of reading DNA

3' to 5' direction

direction of DNA synthesis

5' to 3' direction (need to add to 3’ OH group)

ori

origin of DNA replication

replication bubble

(DNA replication)

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Segment of a DNA molecule that is unwinding and undergoing replication

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a region of DNA, in front of the replication fork, where helicase has unwound the double helix

replication fork

(DNA replication)

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a Y-shaped point that results when the two strands of a DNA double helix separate so that the DNA molecule can be replicated

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A Y-shaped region on a replicating DNA molecule where the parental strands are being unwound and new strands are being synthesized

helicase

An enzyme that untwists the double helix of DNA at the replication forks.

Topoisomerase

Enzyme that functions in DNA replication, helping to relieve strain in the double helix ahead of the replication fork

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corrects "overwinding" ahead of replication forks by breaking, swiveling, and rejoining DNA strands

lagging strand

(DNA replication)

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The strand in replication that is copied 3' to 5' as Okazaki fragments and then is joined up

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The strand that is synthesized in fragments using individual sections called Okazaki fragments

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A discontinuously synthesized DNA strand that elongates by means of Okazaki fragments, each synthesized in a 5' to 3' direction away from the replication fork

leading strand

(DNA replication)

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The new continuous complementary DNA strand synthesized along the template strand in the mandatory 5' to 3' direction.

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synthesized continuously

primase

(aka RNA polymerase)

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synthesizes RNA primer

DNA polymerase

principle enzyme involved in DNA replication

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An enzyme that catalyzes the formation of the DNA molecule

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Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule

DNA polymerase 1 vs 3

DNA polymerase 3 is essential for the replication of the leading and the lagging strands whereas DNA polymerase 1 is essential for removing of the RNA primers from the fragments and replacing it with the required nucleotides

Why does DNA polymerase require a primer?

DNA polymerase needs a free 3' OH group, and primase (an RNA polymerase) does not; therefore, primase can lay down several nucleotides (creates the 3' OH end), which will then allow DNA polymerase a place where it can attach and begin synthesizing DNA

DNA ligase

an enzyme that eventually joins the sugar-phosphate backbones of the Okazaki fragments

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A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.

single stranded binding proteins (SSBPs)

holds single-stranded DNA (ssDNA) bc ssDNA is thermodynamically unstable (prone to nucleophilic attacks that damage DNA) → stabilizes ssDNA for replication

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keeps ssDNA separate so that they don’t close back in preventing replication (stops “reannealing” of DNA)

DNA replication steps

1) ***Helicase (enzyme)*** - unzips the parental double helix at the replication fork and forms a replication bubble where DNA is unzipped

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2) ***DNA topoisomerase*** - upstream of helices alleviating torsional strain (works in FRONT of helicase and allows relaxation of positive supercoils by breaking/reforming phosphodiester bonds in DNA backbone so that helicase can unzip DNA without simultaneously coiling ends tighter)

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3) ***Single-strand binding proteins (SSBP)*** stabilize unwound DNA (aided by DNA gyrase)

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4) ***Primase (RNA polymerase)*** synthesizes a short RNA primer for DNA polymerase to bind to in the 5'-to-3' direction (DNA polymerase can then synthesize DNA continuously) and on each strand on the 3'-to-5' DNA strand (DNA polymerase can then synthesize DNA but DIScontinuously)

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5) ***DNA polymerase 3*** synthesizes the leading strand in 5' to 3' direction while the lagging strand is made discontinuously by primase (RNA polymerase) making short pieces and then DNA polymerase 3 extending these to make Okazaki fragments

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6) ***DNA polymerase 1*** removes primase sections and replaces with DNA

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7) ***DNA ligase*** joins the Okazaki fragments together

in which direction do polymerases always build?

5’ to 3’ (need to add to OH end of nucleic acids)

leading vs. lagging strand

leading strand synthesized continuously bc synthesis follows the direction of the helicase opening DNA

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lagging strand synthesized discontinuously bc direction of synthesis is opposite the direction of helicase

how were Okazaki fragments discovered?

***2 theories on synthesis of lagging strand:***


1. 3’ to 5’ synthesis on lagging strand
2. 5’ to 3’ synthesis of lagging strand in fragments

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***Okazaki lab*** developed experiment to test these 2 competing theories:


1. treated bacteria with a brief “pulse” of **radioactive deoxyribonucleotides** (cells completely used these for DNA synthesis within a few seconds
2. lab quickly killed the cells to extract the DNA, denature it, and separate it by size


1. leading strands labeled with diff sizes bc depends how far the replication is away from the ori
2. lagging strands labeled but *relatively* ALL really tiny (bc they are fragments)

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***RESULTS of experiment:*** leading strand pieces could be any size depending how far along they are from the origin at the time of labeling BUT the Okazaki fragments were CONSISTENTLY small → created a peak in radioactively labeled DNA that was super small sizes (okazaki frags) and a set of data that was about equal sizes of DNA (leading strands bc extracted the DNA after different amounts of time passed for leading strand synthesis)

structure of DNA helix

anti-parallel double helix (one strand is 5' to 3' and other strand runs 5' to 3' but in opposite direction, so it seems like it's 3' to 5')

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backbone: deoxyribose sugar @ 3' end, phosphate PO4 @ 5' end

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base-pairing rules: A=T, G=C

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\*NOTE: DNA is always synthesized in the 5'-to-3' direction, meaning that DNA polymerase can only add nucleotides to the 3' hydroxyl group of the growing strand

why is DNA more stable than RNA

the difference in stability comes down to the chemistry of the pentose at the 2’ carbon → DNA is DEOXYribose (there is just an H at end of the carbon) where as RIBOSE contains an OH group at the 2’ carbon

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“deoxy” = no oxygen at 2’ carbon → the oxygen is reactive chemically and leads to nucleic acid fragmentation/degredation

in DNA vs. RNA, what is important about the:


1. C 1’
2. C 2’
3. C 3’
4. C 5’

1. C 1’: nitrogenous base attached (both)
2. C 2’: OH (ribose) vs. H (deoxyribose) attached
3. C 3’: OH group for adding nucleotides (SAME in both ribose and deoxyribose bc need to add nucleotides in BOTH RNA and DNA here)
4. C 5’: phosphate group here

DNA replication in eukaryotes vs. prokaryotes

MECHANISMS are same (leading/lagging strand, proteins that carry out replication, etc)

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BUT prokaryotes only have ONE replication bubble form when replicating their circular DNA whereas eukaryotes have MULTIPLE replication bubbles needed to efficiently replicate long linear DNA

gel electrophoresis

a technique to separate DNA based on size with electric current

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REMINDER: DNA gets a negative charge (bc of all the phosphate groups)

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procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

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load DNA by the CATHODE (- electrode) and DNA will migrate to the ANODE (+ electrode) when the electrical field is switched on

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use a DNA LADDER as a standard/known metric (know exactly how long DNA ladder is) to compare to the actual DNA length

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DNA is cut by restriction enzymes before being loaded into the wells → longer chains of DNA = harder to move through gel pores = shorter distance traveled VS shorter chains of DNA = easier to move through gel pores = longer distance traveled

end replication problem

helicase opens DNA completely → opens terminal end also →→→ DNA can't replicate at beginning of very first LAGGING strand because primase can't attach to form a primer at end of lagging strand → DNA polymerase can't attach to primary so can't continue to replicate → DNA gets shorter with each replication because of inability to replicate at end

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THUS with normal replication and NO telomerase, the lagging strand will get shorter with each round of replication

senescence

process by which a cell ages and permanently stops dividing but DOES NOT DIE

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shortened telomeres are prone to DNA damage → if corrected, cell can continue to live (without dividing); if damaged greatly, the cell may go through programmed death called apoptosis

telomerase

an enzyme that catalyzes the lengthening of telomeres in eukaryotic germ cells

how does telomerase work?

1. telomerase that contains an RNA primer binds to 3’ G-rich tail
2. telomeric DNA is synthesized on the G-rich tail via reverse transcription
3. telomerase is translocated (moves further along) and steps (1) and (2) are repeated
4. telomerase is released; primase and DNA polymerase fill gap
5. primer removed; gap sealed by DNA ligase

why can cancer cells keep dividing even as telomeres shorten with replication?

they are able to maintain the length of their telomeres by expressing telomerase - since the telomeres don’t shorten the cells do not senesce like non-cancer cells

comparison: in vivo DNA replication vs. PCR DNA replication

how do we design primers in PCR?

* primers must be 5 nucleotides long and amplify the ENTIRE target region for PCR (therefore must be OUTSIDE the PCR region)
* need 2 primers for PCR: 1 to run replication forward and 1 to run replication backward

how long is the final PCR product?

runs from primer to primer (see image example)

PCR definition + process

***definition:*** a technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides (allows molecular biologists to make many copies of a particular gene)

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NOTE: NEED TO KNOW SEQUENCE OF DNA for PCR rxn (because need to make the primer sequence so that it is complementary to the "flanking" (end) sequences)

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***reaction tubes contain:***

1\. oligonucleotide primers for each end of target sequence

2\. free nucleotides (A, T, C, G)

3\. heat-stable TAQ DNA POLYMERASE (Taq = thermus aquaticus) - the key to automation

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***steps of PCR (each step = approx. 30 sec):***

1\. @ 95ºC: DENATURATION - denature ("separate") DNA strands so that it opens up (basically like a replication bubble)

2\. @ 55ºC: HYBRIDIZATION/ANNEALING - now that DNA is opened, cool the temperature down to allow the primer to anneal ("attach") to the DNA at the ends (aka "flanking" the target DNA)

3\. @ 72ºC: ELONGATION - polymerization proceeds; this is the optimal temperature for the Taq polymerase to use the free nucleotides to synthesize DNA starting at the primer

4\. this cycle (steps 1-3) are REPEATED → leads to EXPONENTIAL growth of DNA (bc with each replication, there becomes double the DNAs to build off of)

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PCR only needs abt 25-30 cycles bc of exponential growth (@ 25 cycles, already DNA is at approx. 30mill copies of the target DNA if began with ONLY 1 copy of target DNA originally)

1. why do we see only a short band on our gel when the original strands of DNA are so long? (AKA how is our PCR product so short when we started with 2 long pieces of DNA)
2. what happens to the primers?

1. The original longer strands are still present, but you’ll have \~1x10^12 short bands by the end of 40 rounds of PCR (it’s exponential), so you aren’t likely to see them
2. primers get INCORPORATED INTO the DNA (thus need to start PCR with TONS of extra primers bc can’t reuse them

2 kinds of cell division signals keep the balance of cells in our body:

1. ***stimulative*** - a “go” signal must be present to divide
2. ***inhibitive*** - a “stop” signal must not be present to divide

what are 4 checkpoints of the cell cycle, and what is being checked at each?

1. G1: are internal and external conditions favorable? is DNA damaged? → near end of G1 (BUT not all the way at end)
2. S: is there any DMA damage? (did DNA replicate properly) → at end of S
3. G2: is DNA replicated? is DNA damage repaired? → at end of G2
4. M: are chromosomes attached to mitotic metaphase spindle → between meta and anaphase

in the G1 cell cycle checkpoint:


1. what are the key questions/events
2. what are the regulatory processes leading to stimulation
3. what are the regulatory processes leading to inhibition

1. key questions/events:


1. key question: rest or divide (can DNA synthesis begin?


1. is cell big enough that its SA:V ratio (which decreases as cell gets bigger) is no longer efficient?
2. is there room for cell to divide?
3. are there enough nutrients to support BOTH cells after division?
2. if DNA is undamaged AND enough resources are available for cell to keep growing and dividing → growth signals move cell through rest of G1 and into S phase

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2. regulatory processes (stimulation): growth factors stimulate signals inside cells that cause G1 cyclin concentrations to rise → cyclins bind to appropriate CDKs → drives cell cycle into S phase

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3. regulatory processes (inhibition):


1. p53: if DNA is damaged → p53 initiates cell signaling pathway to turn p21 on → stops progression to S phase by INHIBITING cyclin-CDK complex (allows for DNA repair OR if damage is too much then p53 initiates apoptosis)
2. Rb: prevents cells from entering S phase in the ABSENCE of growth factors (when growth factors are present, they activate cyclin-CDK due to phosphorylation of Rb → inhibits Rb function and stimulates cell into next part of cycle)

in the S cell cycle checkpoint:


1. what are the key questions/events
2. what are the regulatory processes leading to stimulation
3. what are the regulatory processes leading to inhibition

1. key questions/events:


1. key question: is DNA ok AFTER replication?
2. if DNA synthesis progresses WITHOUT errors → growth signals stimulate cell to move into G2 to mature

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2. regulatory processes (stimulation): growth factors stimulate signals inside cells that cause S cyclin concentrations to rise → cyclins bind to appropriate CDKs → drives cell cycle into G2 phase

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3. regulatory processes (inhibition): ATM and BRCA1 (don’t need to know)

in the G2 cell cycle checkpoint:


1. what are the key questions/events
2. what are the regulatory processes leading to stimulation
3. what are the regulatory processes leading to inhibition

1. key questions/events:


1. key question: is the cell fully equipped to divide?


1. was DNA copied correctly? (if not: were all errors fixed?)
2. are there enough CYTOPLASM materials for cell to divide evenly AND have both new daughter cells FUNCTION properly?
2. ALL chromosomes checked that they are fully replicated and contain no other types of damage


1. if damaged → caught/fixed at checkpt
2. once corrected → growth signals stimulate cell to enter M phase

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2. regulatory processes (stimulation): growth factors stimulate signals inside cells that cause G2 cyclin concentrations to rise → cyclins bind to appropriate CDKs → drives cell cycle into M phase

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3. regulatory processes (inhibition):


1. p53: checks DNA damage AFTER replication → if damaged, p53 protein initiates cell signaling pathway to STOP cell cycle progression until damage is repaired OR if damage is too much to fix → p53 initiates apoptosis)

in the M phase cell cycle checkpoint:


1. what are the key questions/events
2. what are the regulatory processes leading to stimulation
3. what are the regulatory processes leading to inhibition

1. key questions/events:


1. key question: are ALL sister chromatids attached to spindle fibers and lined up EVENLY in the MIDDLE of microtubules?
2. for mitosis to proceed correctly, the 2 sister chromatids should both be attached to the mitotic spindle and lined up in the MIDDLE (end of METAphase) → if so: cell enters ANAphase

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2. regulatory processes (stimulation): APC/C (don’t need to know)

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3. regulatory processes (inhibition): MAD proteins

what molecules are part of stimulatory pathways to allow cells to progress through check points

active CDKs

why are cyclins called cyclins?

different cyclins (proteins) are synthesized and QUICKLY DEGRADED by the cell depending on the phase of the cell cycle it is in

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a balance bwn expression (transcription/translation) and degredation

what pathway is an example of growth factors stimulating cell cycle progression?

Rb pathway

Rb pathway

(example of how growth factors STIMULATE cell division)

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1. growth factor is received by growth factor receptor (EXTRAcellular receptor) → causes conformation change in the receptor
2. conformation change initiates an INTRAcellular signaling pathway → results in signal sent to nucleus
3. *in the nucleus:*


1. BEFORE signaling pathway reaches: active Rb protein is BOUND to transcriptor regulator → inactivates transcription factor → NO transcription (without signal)
2. WITH signal: CDK-cyclin in the nucleus PHOSPHORYLATES Rb (adds 2 phosphate groups to Rb) → changes the shape of Rb → Rb can no longer stay bound to transcription regulator → THUS the phosphorylation of Rb DEactivates Rb function
3. transcription now occurs bc transcription regulator binds to DNA
4. *back outside of nucleus:*


1. translation of transcribed RNA into protein
5. RESULT of growth factor deactivating Rb pathway: CELL PROLIFERATION (the protein that is translated results in a protein that stimulates the cell cycle from G1 into S phase)

what family of proteins phosphorylate other proteins?

KINASES

what family of proteins DEphosphorylate other proteins?

PHOSPHOTASES

is Rb an oncogene, proto-oncogene, or tumor suppressor gene?

TUMOR SUPPRESSOR → requires BOTH alleles of Rb to be mutated for complete loss of function

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(Rb is normally part of INHIBITORY pathway so when Rb is mutated, can lead to tumors)

p53 pathway

NOTE: in ABSENCE of DNA damage, p53 EASILY gets degraded (in PROTEOSOMES)

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1. DNA gets damaged (can be from error in replication or X-rays, etc)
2. now that DNA is damaged, protein kinases are ACTIVATED → activation of protein kinases leads to phosphorylation of p53 → adding the phosphate group STABILIZES/ACTIVATES p53
3. active p53 binds to the REGULATORY region of p21 gene


1. this results in the transcription of p21 into p21 mRNA
2. p21 mRNA gets translated into p21 protein (this is a CDK INHIBITOR protein)
4. the CDK inhibitor protein (translated from p21 gene) binds to an ACTIVE G1/S cyclin-CDK or S cyclin-CDK → causes a conformation change in the cyclin-CDK complex
5. RESULT of p53 pathway: p21 protein INACTIVATES the cyclin-CDK complex that it binds to (stops the stimulatory signals from moving the cell into S or G2 phase so that p53 can repair DNA damage or initiate apoptosis)

is p53 an oncogene, proto-oncogene, or tumor suppressor gene?

TUMOR SUPPRESSOR → requires BOTH alleles of p53 to be mutated for complete loss of function

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(p53 is normally part of INHIBITORY pathway that gets activated by DNA damage (in which case it HALTS the stimulative effects of CDKs) so when p53 is mutated, can lead to tumors)

flow cytometry

technique to look at DNA content at a point in time

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*method of flow cytometry:*


1. DNA in cells stained with propidium iodide (a dye with to fluorescent tag)
2. sample of stained DNA is loaded into a narrow chamber in the flow cytometer (to separate the DNA molecules as much as possible)
3. a laser light source hits the flow cytometer (and thus hits the stained DNA in it - each DNA molecule passes through the beam of light one at a time)
4. light is scattered differently depending on the density of the DNA content and recorded → fluorescence emitted from stained cells detected while forward/side-scattered light from all cells also detected


1. light scattering/emission (level of fluorescence aka DNA density) is measured (plotted on x-axis) as well as the number of cells WITH that specific fluorescence level (plotted on y-axis)
2. the amount of fluorescence per cell is determined by the amount of DNA in that cell → more DNA = higher fluorescence for that cell

anchorage dependence

the requirement that to divide, a cell must be attached to a solid surface (require solid anchorage to a solid surface)

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cells must be attached to divide - w/o solid anchorage the cells die

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aka monolayer cells: if cells must be anchored to each other (anchorage dependence) + cannot divide on top of each other (can't divide past certain density - density dependent inhibition) → on a surface these cells will only be 1 layer of cells connected to each other

density dependent inhibition

on a surface: cells continue dividing until entire surface is covered - once they run out of space (no more density available to divide), they HALT cell cycle

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The phenomenon observed in normal animal cells that causes them to stop dividing when they come into contact with one another.

if given 2 different skin cell lines to grow IN CULTURE (one normal and one cancerous) how could you figure out which is which?

could look for these differences in growth patterns (density dependent inhibition, anchorage dependence, etc - whether cells continue to grow with infinite resources)

benign tumor

mass of abnormal cells with specific genetic/cellular changes such that the cells are not capable of surviving at a new site and generally remain at site of tumor’s origin

malignant tumor

cancerous tumor with cells that have significant genetic and cellular charges and are capable of invading and surviving in new sites

metastisis

spread of cancer cells to locations distant from their original site

benign vs. malignant tumors

benign = noncancerous (pre-cancerous) VS. malignant = cancer has capability to spread

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Tumors can be benign (noncancerous) or malignant (cancerous). Benign tumors tend to grow slowly and do not spread. Malignant tumors can grow rapidly, invade and destroy nearby normal tissues, and spread throughout the body.

key principles of cell signaling (fill in image)

paracrine signaling

targets NEIGHBORING cells nearby

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targets cells locally, or within the vicinity of the emitting cell

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ex: neurotransmitters

autocrine signaling

this kind of signaling is when a cell emits a signal and affects the very same cell via a receptor

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autocrine signals are produced BY the emitting cells FOR that same cell via its own receptors

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ex: immune cells

contact-dependent signaling

(aka juxtacrine signaling)

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this signaling targets adjacent cells that are touching each other

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targets adjacent cells that are touching the emitting cells

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ex: notch signaling

endocrine signaling

this kind of signaling targets cells at a distance - they travel through the blood to reach all parts of the body

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targets cells at a distance. The signals travel through the blood to reach all parts of the body

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ex: hormones

will all ligands bind to any receptor in the body?

NO - ligands have GREAT SPECIFICITY to receptors (only specific ligand shape binds to specific receptor site) → THUS EVEN different cell types CAN have DIFFERENT responses to the SAME ligand

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(ex: acetylcholine (Ach) ligand only binds to specific Ach receptor BUT can result in muscle contraction in muscle cells that have ligand-gated ion channel OR decreased heart rate in heart cells that have GPCRs with same receptor site as muscle ligand-gated ion channel)

extracellular/intERcellular ligand properties

* polar (or charged)
* hydroPHILIC (water soluble) - require a CHANNEL protein to enter cell

intRAcellular ligand properties

* nonpolar (or small polars)
* hydroPHOBIC (water INsoluble) - do NOT require a channel protein to enter cell (simple diffusion through membrane)

the way most proteins in signaling transduction cascades are activated is by:

KINASES (phosphorylation cascades)

cells often receive MULTIPLE signals → what determines WHICH cells receive the signals and HOW they interpret them?

1. different cell types have a different cell surface receptor
2. different cell types have different cytoplasmic signal transduction proteins and pathways

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*cells respond to signals based on the signals present, the receptors they have or don’t have, and the unique relay molecules and pathways they use*

how can intracellular signaling molecules be switched on/off?

1. ON: phosphorylation by kinases; OFF: dephosphorylation by phosphotases
2. ON: GTP binding; OFF: GTP hydrolysis into GDP

G-protein coupled receptors

a type of receptor that has an extracellular receptor site (for ligand to bind) + collection of proteins inside the cell membrane + G-protein with 3 subunits inside cell/outside membrane where GDP stays bound to when GPCR inactivated/where GTP binds when GPCR activated

activation of GPCR

when a signaling molecule binds to a receptor, it causes a conformational change (shape change), which allows it to associate with the alpha subunit of the G protein → the alpha subunit then exchanges GDP (bound to g-protein in inactive form) for GTP which results in an activated alpha subunit (which signals further proteins downstream) → this activation is TEMPORARY (G alpha will hydrolyze GTP to GDP after some time, making G alpha inactive once again)

generic Ras pathway

1. extracellular ligand binds to receptors of RTK → causes RTK to dimerize and activate phosphorylation of tyrosines
2. activated (phosphorylated) tyrosine activates a bound protein → now-activated protein (bound to RTK) removes GDP on a Ras protein (a type of G-protein) and replaces it with GTP → activates Ras protein
3. the activated Ras protein activates a MAPKKK (protein kinase) → *initiates phosphorylation cascade*


1. activated MAPKKK phosphorylates MAPKK through hydrolysis of ATP
2. activated MAPKK phosphorylates MAPK through hydrolysis of ATP
3. activated MAPK phosphorylates a transcription regulator
4. RESULT of Ras pathway: TRANSCRIPTION FACTOR turned on (activated through phosphorylation via MAPK cascade) → CELL DIVISION induced

mitogen

a substance that induces or stimulates mitosis.

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small bioactive protein or peptide that induces a cell to begin cell division, or enhances the rate of division

mitogen-activated protein kinase

(MAPK)

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mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival

what do MAPKs do?

MAP kinase enters the nucleus and phosphorylates SPECIFIC transcription factors that promote cell proliferation

generic MAPK pathway

NOTE: there are diff versions of these kinases that can lead to specific biological responses BUT these individual processes that use MAPKs have in common a similar PROCESS (transduction PATHWAY) from signal to response

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stimulus → MAPKKK → MAPKK → MAPK → biological response

example specific MAPK pathway (overview)

growth factors/mitogens/GPCR agonists (stimulus) → Raf (MAPKKK) → MEK 1/2 (MAPKK) → ERK 1/2 (MAPK) → growth/survival/differentiation/development (biological response)

Ras-ERK pathway

OVERVIEW of pathway: RTK → Grb2 → Ras-GEF → Ras-MapKKK (Raf) → MAPKK (MEK1/2) → MAPK (ERK1/2) → RSK → ERK1/2 + RSK to nucleus → activate transcription factors of cyclin D1 → cell proliferation response

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1. ***outside cell:***


1. mitogen growth factors bind to 2 spots on RTK as (EXTRAcellular) ligands to activate RTK units and cause them to dimerize
2. ***inside cytosol:***


1. conformation change (dimerizing) activates (phosphorylates) tyrosines on RTK which activate Grb2-Sos protein complex (bound to RTK tyrosines) → active Grb2-Sos activates Ras (G-protein bound to cell membrane) by removing GDP and binding GTP)
2. Activated Ras activates (phosphorylates) Raf (type of MAPKKK)
3. Activated Raf phosphorylates MEK 1/2 (type of MAPKK)
4. Activated MEK 1/2 phosphorylates ERK 1/2 (type of MAPK)


1. NEGATIVE FEEDBACK at this step: activation of ERK 1/2 goes back to INHIBIT MEK 1/2 and Raf
5. Activated ERK 1/2 phosphorylates RSK
6. BOTH RSK and ERK 1/2 *translocate* (move) to NUCLEUS
3. ***inside nucleus:***


1. activated RSK phosphorylates CREB and Fos
2. activated ERK 1/2 phosphorylates Pos and Elk-1
3. CREB/Fos/Elk-1 are proteins that regulate transcription factors for transcription (and thus translation/EXPRESSION) of Cyclin D1 protein

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RESULT of Ras-ERK 1/2 pathway: expression of cyclin D1 (a cyclin associated with G1/S transition of cell cycle) → CELL PROLIFERATION

housekeeping gene definition + how it is used in biotech

genes that are consistently expressed across tissues, essential, carrying out cellular maintenance, and conserved across species

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cellular maintenance genes which regulate basic and ubiquitous cellular functions. In many RT-qPCR reactions, these genes are used as internal CONTROL GENES without proper validation (helps determine if the same amount of total protein is added to each lane → thus allows western blot/RTPCR/PCR to be QUANTITATIVE)

common housekeeping gene for PCR/RTPCR/Western Blot

GADPH

apoptosis

programmed cell death

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mechanism to eliminate unwanted or damaged cells

why would we want apoptosis?

1. development/morphological changes in the cell (that lead to programmed cell death)


1. blebbing
2. nuclear fragmentation and condensation
3. cell shrinkage
2. cells damaged beyond repair
3. infection

Bcl-2 VS. Bax

Bcl-2: “pro-survival” - inhibits Bax

Bax: “pro-apoptosis”

Bcl-2 BAX pathway OVERVIEW (in mammalian cells)

(don’t need to know details beyond this flashcard)

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*relates to Ras-ERK1/2 pathway*

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pathway → Ras → MEK → ERK1/2 → cell proliferation

BUT ALSO

pathway → Ras → MEK → ERK1/2 → Bcl-2 —| BAX

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(ERK drives the expression of both cell proliferation proteins AND Bcl-2, pro-survival signals of the cell)

how to push a cancer cell towards apoptosis with Bcl-BAX pathway

ERK activity needs to be inhibited so that Bcl-2 can be turned off and BAX can be turned on → this is so that the cell can receive the apoptotic message in the absence of survival signals

what are the goals when treating a patient with cancer?

1. stop the cancer cells from proliferating
2. cause the cancer cells to die (undergo apoptosis)
3. cause minimal harm to the healthy cells

how does PD184352 prohibit cell proliferation?

PD184352 is an inhibitor of ERK1/2 (think: Ras-ERK pathway) → if we inhibit ERK, downstream proteins do not get activated, resulting in reduced gene expression of cyclin

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Inhibition of ERK1/2 by treatment of cells with PD184352 reduced the expression of proteins involved in cell proliferation (Cyclin D1, Cyclin A) PD184352 (ERK1/2 inhibition) also increased expression of p27kip1. No changes to the expression of ERK1/2 occurred in PD184352 treated cells

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Cyclin D1 = G1→S transition

Cyclin A = S→G2 transition

p27kip1 = tumor supressor protein

1. In a normal cell, ERK signals ____ and ____
2. ERK inhibition in a proliferating cell would ____ cell division
3. ERK inhibition in a cell that received an apoptotic signal would ____

1. cell proliferation; survival
2. reduce
3. cause the cell to undergo apoptosis

multipotent

a stem cell that can differentiate into multiple types of specialized cells in an adult

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cells that can generate only a LIMITED number of cell types

pluripotent stem cell

a stem cell that can differentiate into most, but not all, cells in a multicellular organism

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can generate MOST cell types (just not as much as totipotent)

totipotent stem cell

a stem cell that can differentiate into any cell type in either an embryo or adult

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can generate ANY cell type

induced pluripotent stem cell

a differentiated cell that becomes pluripotent when new genes are introduced in the laboratory

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INDUCED bc this is the result of a LAB - taking multipotent and turning them into PLURIpotent stem cells

stem cells

beginning of cellular process

embryonic vs. adult stem cells

embryonic: PLURIpotent → cells can generate MOST embryonic cell types

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adult: MULTIpotent → cells can generate a LIMITED number of cell types

cancer vs. stem cells


1. where cell originates from
2. mortal or immortal
3. ability to move from one place to another (Y/N)
4. genetic abnormalities (Y/N)
5. ability to differentiate (Y/N)
6. telomerase expression (Y/N)

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developmental biology

the study of how stem cells differentiate into different cell types to form tissues and organs

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in cancer, fully differentiated cells differentiate into altered cell types to create the tumor environment

during embryonic development, what processes are taking place?

1. apoptosis + mitosis
2. cell migration → morphological changes
3. differentiation
4. cell signaling → causes phenotype change

specific vs. general transcription factors

general: line up the RNA polymerase on the promoter

specific: bind to the enhancers (or silencers)

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specific TFs physically interact with the polymerase and general TFs to increase (or decrease) the rate of transcription

the DNA is the ___ in all cells

SAME

a gene will be “off” in a cell because the cell doesn’t have the transcription factors to initiate transcription → WITHOUT adding the transcription factors, how can we turn the cell ON?

we can turn it “on” in the cell if we link the coding region DOWNSTREAM of a cell’s promoter that HAS the transcription factors already to drive the expression in this cell type