Hb-1 Week 1

Front 1

DRAW CELL CYCLE AND KNOW CELLULAR FUNCTIONS AT EACH PHASE

Back 1

Front 2

RELATIONSHIP BTWN LIFESPAN OF CELL & DISEASE

Back 2

Long lived cells can accumulate products or toxic damage over time that can result in cell death e.g. Alzheimer's

Front 3

RELATIONSHIP BTWN RATE OF CELL TURNOVER & DISEASE

Back 3

Cells w/rapid turnover have increased chance of accumulating mutations that affect growth & division, leading to diseases e.g. cancer

Front 4

HIERARCHY OF STEM CELLS

Back 4

Totipotent (can give rise to any of the cell types) ---> Pluripotent (can give rise to multiple cell types) --> Blood Stem Cells (can give rise to RBC or WBC) or Other Stem Cells (can give rise to Muscle, Nerve, Bone & Other tissues)

*As you go further down the hierarchy, the stem cells are able to give rise to less differentiated cell types

*Induced Pluripotent Stem Cells - We can reprogram stem cells and essentially get them to go backwards

Front 5

CRITERIA FOR CELL DEATH

Back 5

1. Plasma membrane breakdown

2. Cell explodes

3. Cell eaten by other cells

Front 6

CELL DEATH: PROPOSED POINTS OF NO RETURN

Back 6

1) Massive activation of caspases

2) Protracted loss of membrane potential

3) Phosphatidyl serine (PS) exposure which triggers for phagocytosis -- normally PS is on the inner leaflet

Front 7

4 TYPES OF CELL DEATH

Back 7

1. APOPTOSIS - Cells killing themselves

2. AUTOPHAGY - Cell digesting own organelles; Results in vacuolization of the cytoplasm

3. CORNIFICATION - Commonly occurs in skin; involves removal of cytoplasmic content - cell is dead but retains its shape

4. NECROSIS - Basically all other forms of cell death

Front 8

APOPTOSIS: MORPHOLOGICAL FEATURES

Back 8

*Cytoplasmic shrinking

*Rounding up of the cell

*Retraction of pseudopodes

*Nuclear shrinkage (pyknosis)

*Membrane blebbing

*Nuclear fragmentation (karyorrhexis) -- [aka Intranucleosomal DNA Fragmentation] - DNA cuts up into little pieces; ordered disassembly from inside

*Engulfment by phagocytes/macrophages

-Minor modification of cytoplasmic organelles

*Phosphatidyl serine (PS) flipping to extracellular portion of the Plasma Membrane - exposure of PS to outside of cell MARKS cell for rapid consumption by Macrophages

*Cell loses attachment to substrata it was growing on

*Cell shrivels up (without lysing)

Front 9

AUTOPHAGY: MORPHOLOGICAL FEATURES

Back 9

-Lack of chromatin condensation

-Massive vacuolization of the cytoplasm

-Accumulation of double membraned autophagic vacuoles

-Little or no uptake by phagocytic cells

Front 10

CORNIFICATION: MORPHOLOGICAL FEATURES

Back 10

-Elimination of cytosolic organelles

-Accumulation of lipids

-Extrusion of lipids in extracellular space

-Modifications of plasma membrane

-Desquamation (loss of corneocytes) due to protease activation

Front 11

NECROSIS: MORPHOLOGICAL FEATURES

Back 11

-Cytoplasmic swelling (oncosis)

-Rupture of plasma membrane

-Swelling of cytoplasmic organelles

-Moderate chromatin condensation

Front 12

ERYTHROPOIETIN

Back 12

*Key protein involved in regulating rate of Red Blood Cell Production (aka erythropoiesis)

*Thus, it is involved in regulating # of RBC

Front 13

RED BLOOD CELL PRODUCTION (aka ERYTHROPOIESIS)

Back 13

*Triggered by Hypoxia (lack of adequate oxygen supply) occurs due to decreased RBC count, decreased availabity of Oxygen to blood, or increased tissue demands for Oxygen

*Results in reduced Oxygen levels in blood

*Kidney releases erythropoietin

*Erythropoeitin stimulates Red Bone Marrow

*Enhanced erythropoiesis occurs

*Results in more RBC

*Increased Oxygen carrying ability of blood

*Return to Normal Blood Oxygen levels

Front 14

LIFESPAN OF RBC

Back 14

120 DAYS

Front 15

ERYTHROPOIESIS - BIRTH & CELLULAR STAGES

Back 15

*Process takes 7 days

*Begins with proerythroblast --> Basophilic erythroblast --> Polychromatophilic erythroblast --> Orthochromatophilic erythroblast --> Reticulocyte --> Erythrocyte

*Reticulocyte's can be used as diagnostic tool: High amt of Reticulocytes = Hemolytic Anemia b/c means more red blood cells are being made by the bone marrow. This can occur after a lot of bleeding, a move to a high altitude, or Hemolytic Anemia, condition in which RBC are being broken down (hemolysis)

Front 16

ERYPTOSIS

Back 16

Programmed cell death for the erythrocyte

- As erythrocyte ages, changes in membrane composition (i.e. PS on OUTER LEAFLET) cause it to be recognized by macrophages in spleen/liver/bone marrow

*Flipase is the enzyme that flips PS to the inner leaflet - this process requires NRG...so when there's not enough NRG provided, PS accumulates on outer leaflet and then phagocytosed

- Old cells therefore removed by phagocytosis

Front 17

SICKLE CELL ANEMIA

Back 17

- Caused by mutations in the beta-globin gene; occurs in ~1:500 African-American children

SIGNS OF SICKLE CELL ANEMIA:

- Sickle cell crisis (5-7 days) - Anemia

- Vaso-occlusion (ischemic even to organs)

- Aplastic anemia (viral attack on RBC production)

- Hemolytic crisis (increased RBC breakdown, G6PD)

- Sequestration (splenic infarction)

Front 18

NEURONS IN THE CORTEX SERVE AS AN EXAMPLE OF WHAT?

Back 18

- Long lived cells

- An instance where cell differentiation is linked to cell cycle

*There are 3 types of Neuronal Precursors

*Way in which cell develops fr the precursor all depends on cell cycle:

a. Proliferative division

b. Differentiative asymmetrical division - results in 1 neuron cell from a precursor

c. Differentiative symmetrical division - results in 2 neuron cells from precursors

Front 19

NEURONAL DEATH

Back 19

1. Acute changes in neuronal environment that induce cell death (ischemia & apoptosis)

2. Trauma (i.e. gunshot)

3. Chronic changes that affect neuron indirectly (immune mediated demyelination, e.g. multiple sclerosis)

4. Protein aggregates & neuronal degeneration (when a protein misfold & forms aggregate)

Front 20

ALZHEIMER'S DISEASE

Back 20

*most common neurological disorder

*predicted to affect 1/80 globally by 2050

*mostly affects ppl over age 65

*considered both a protein aggregate disease and a tauopathy

*Amyloid Plaques & Neurofibrilliary Tangles (NFT) -- Hallmarks of Alzheimer's

*Associated with beta-amyloid aggregates

Front 21

LIFESPAN OF A PROTEIN

Back 21

*Proteins, like cells, also degrade

*Half life of proteins ranges fr 0.2 hrs to years

*Proteins can be non-specifically turned over (degraded) by mechanisms such as PROTEOLYSIS

*Proteins can be specifically targeted for degradation by UBIQUITINATION

*There is a regulated balance btwn synthesis of proteins & destruction

Front 22

DEATH BY UBIQUITINATION

Back 22

*Ubiquitin - serves as the tag that marks a protein for degradation

*The Ubiquitin-Proteasome Pathway*

Ub itself does not degrade proteins. It serves only as a tag that marks proteins for degradation.

-The degradation itself is carried out by a proteasome

- Proteins that are to be degraded are first tagged by conjugating them with Ub and these tagged proteins are then recognized and shuttled to the proteasome for degradation

- Involves interaction w/3 enzymes

THINK OF PROTEASOME AS THE CELL'S GARBAGE CAN

Cell cycle & Circadian Rhythm depend on ubiquitination

Front 23

LONG LIVED PROTEINS

Back 23

- Certain proteins turn over v. slowly or never turn over/get modified once formed

- e.g. elastin in the skin endure UV damage, resulting in wrinkles

- another e.g. crystallines in the eyes' lens, results in cataracts

- Hence diseases relating to those proteins are AGING DISORDERS (and occur due to accumulated damage)

Front 24

DEVELOPMENT OF THE HUMAN CORTEX

Back 24

Front 25

KEY TAKEAWAY POINTS FROM LAMBERT'S

CELL CYCLE SLIDES

Back 25

1. Cells arise from a proliferating precursor regulated by cell cycle

2. Cell death can be programmed, or happen as a result of aging/accumulation of damage

3. Lifespan of cells within the body can vary greatly

4. Balance btwn birth & death of cells MUST be maintained

5. Longer living cells manifest problems associated w/accumulation of damage (aging)

6. Creation & Destruction of cellular Proteins has similar patterns (to creation & destruction of cells)

Front 26

ISCHEMIA/REPERFUSION

Back 26

Injury arising from acute myocardial infarction, cardiac surgery, and chronic heart failure

*Infarction - Tissue death caused by local lack of Oxygen due to obstruction of tissue's blood supply

**Myocardial Infarction is the partial death of heart tissue (aka heart attack)

Front 27

TOXIC XENOBIOTICS (EXAMPLES)

Back 27

-Arsenic

-Heavy Metals (Lead, Mercury, etc)

-Salicylate (FYI: salicylate is a ubiquitous agent, used as an analgesic agent for the treatment of mild to moderate pain, common sources of unintentional and suicidal ingestion)

-Acetaminophen

Front 28

NEURODEGENERATIVE DISEASES (EXAMPLES)

Back 28

*Alzheimer's - (AD) most common form of dementia; Know the Amyloid Hypothesis

*Parkinson's - (PD) degenerative disorder of the central nervous system

*Huntington's - (HD) a genetic neuro-degenerative disorder affecting muscle coordination and leads to cognitive decline and behavioral symptoms

Front 29

Alzheimer's Disease: The Amyloid Hypothesis

Back 29

The amyloid hypothesis postulates that extracellular beta-amyloid (Aβ) deposits are the fundamental cause of the disease

Front 30

APOPTOSIS: FACTS AT A GLANCE

Back 30

*Apoptosis is GENETICALLY ENCODED

*Think of it as a "very dignified, neat, self-enclosed death" -- Cell does NOT pop open...membrane remains intact

*It is a UBIQUITOUS PATHWAY

*Enables cells to undergo HIGHLY REGULATED DEATH IN RESPONSE TO PRO-DEATH SIGNALING

*Important in:

- Normal digit development & development of brain & immune system

- Normal maintenance of tissues & cell population

*Without Apoptosis, normal development can't occur

Front 31

APOPTOSIS: MECHANISM'S 3 KEY EVENTS

Back 31

1. Initial Event - Cytochrome C's sudden release from mitochondria --> cytosol

2. Lipid Assymetry of membrane breaks down -- Phosphotidyl Serine (PS, shown in red fluorescent in video) becomes exposed on outside of cell -- labels dead cell to be rapidly consumed by macrophages

3. Although cell does NOT lyse...membrane becomes permeable to small molecules that can bind to DNA

Front 32

CORE APOPTOTIC SIGNALING COMPONENTS

Back 32

Pro-apoptotic protein ---> Anti-apoptotic protein (e.g. Bcl-2) --> Pro-apoptotic adaptor protein --> Cysteine protease --> APOPTOSIS for cells that are unhealthy/fated to die

*Pro-apoptotic Adaptor Protein - Like an "ON" switch

*Anti-apoptotic Adaptor Protein - Inhibit the Pro-Apoptotic Adaptor Proteins in normally functioning cells that do not need to undergo Apoptosis --> Thus, Cysteine Proteases remain inactive --> Apoptosis is prevented

*Cysteine Proteases - Once active, they begin cleaving proteins that regulate nuclear membrane integrity/cellular metabolism & DNA -- The "fuse is Lit" once these are activated

Front 33

BCL-2

Back 33

*Bcl-2 is the founding member -- the classic anti-apoptotic protein

*In B-Cell Lymphoma, Bcl-2 is over expressed -- first disease it was observed in (hence the name)

*Bcl-2 activity -- depends on proper localization @ mitochondria --> Bcl-2 goes btwn cytosol & mitochondria, but for it to be truly active it needs to be on surface of mitochondria

Front 34

BAX & BID

Back 34

*BAX --> belongs to Bax Family of Proteins

*BID --> belongs to BH3-only Family of Proteins

*Both are Mammalian pro-apoptotic proteins

*Pro-apoptotic Protein expression often altered in cancer -- Often downregulated in cancer

*BH3-only Pro-Apoptotic Proteins antagonize anti-apoptotic family members

Front 35

CASPASES

Back 35

*Caspases are Mammalian Cysteine Proteases

*They are the central executioners of Apoptosis

*Caspases "Karate chopping proteins" - Proteases responsible for the morphological & biochemical aspects of apoptosis

- There are 14 caspases in Mammals

*Cysteine Dependent Aspartate Directed Proteases

*Exist as inactive zymogens activated by cleavage at aspartate residues within themselves

* Have an ASPHOLE

Front 36

CLASSIFICATION OF CASPASES

Back 36

INITIATOR CASPASE - Caspases 8, 10, 9, 2

-Containing LONG N-terminal pro-domain

-Auto-activated

-DED: Caspase 8 and 10 contain a death- effector domain

-CARD: Caspase 9 and 2 contain a caspase activation and recruitment domain (CARD)

EFFECTOR CASPASES - Caspases 3, 7, 6

-Containing SHORT N-terminal pro-domain

-Activated by Initiator Caspases

Front 37

APAF-1

Back 37

*Type of Mammalian pro-apoptotic adapter protein

*Activation of caspases --> Depends on Adapter Protein termed Apoptotic Protease Activating Factors

Front 38

ACTIVATION OF CASPASES

Back 38

*3 factors together (Apaf-1, ATP, Cytochrome C) comprise the apoptosome which activates initiator caspases

- Recall: Cytochrome C is a critical mobile carrier involved in ETC & ATP production, normally retained in the mitochondria....when it leaves the mitochondria, bad things happen...like the activation of Caspases

Front 39

HOW IS CYTOCHROME C RELEASE COORDINATED?

Back 39

BH3-only pro-apoptotic proteins play key role:

*They inhibit anti-apoptotic proteins

*AND activate multi-domain pro-apoptotic proteins

*Multi-domain Pro-Apoptotic Proteins then form pores & release Cytochrome C

FYI: This is why over-expression of Bcl-2 blocks cell death

Front 40

HOW ARE BH3-ONLY PRO-APOPTOTIC FAMILY MEMBERS REGULATED SO THAT THEY CAN TIP THE BALANCE TOWARDS DEATH?

Back 40

BH3-only proteins are regulated by a variety of mechanisms including transcription and post-translational modifications

Front 41

SUMMARY OF CELL DEATH DIAGRAM

Back 41

Front 42

APOPTOSIS: INTRINSIC PATHWAY

Back 42

*The intrinsic, or mitochondrial, pathway is initiated from within the cell

*Involves mitochondrial assembly of pro-apoptotic factors - Formation of Apoptosome

*Works through CASPASE 9

*Then CASPASE 9 interacts with CASPASES 3 & 7 in the cleavage of proteins & execution of Apoptosis

Front 43

APOPTOSIS: EXTRINSIC PATHWAY

Back 43

A.k.a. Cell-Death Receptor Pathway

*Extrinsic Pathway involves delivery of death ligand to death receptor

*Begins outside the cell via activation of pro-apoptotic receptors on the cell surface (TNF Receptor, FasL Receptor) -- aka the DEATH RECEPTORS

- TNF: Tumor Necrosis Family

- Characterized by extracellular Cysteine Rich Domains (CRDs)

*Involves formation of DISC

*This pathway signals thru Adaptor Protein to activate CASPASE 8

*Then CASPASE 8 interacts with CASPASES 3 & 7 in the cleavage of proteins & execution of Apoptosis

Front 44

INTRINSIC VS. EXTRINSIC PATHWAYS COMPARED

Back 44

Front 45

NECROSIS

Back 45

Necrosis is diff. from apoptosis in that it is NOT programmed and is "messy"

*Disordered, not programmed

*No signaling

*Cell contents spill out

*Promotes tissue injury & inflammation

Front 46

APOPTOSIS VS. NECROSIS SIDE BY SIDE COMPARISON

Back 46

Front 47

Where is erythropoietin produced?

Back 47

Kidneys

Front 48

What is Erythropoietin produced in response to?

Back 48

Lack of oxygen

Front 49

How long does erythropoesis take?

Back 49

7 days

Front 50

Why is the red blood cell's life span only 120 days?

Back 50

The components of the cell wear out, and without a nucleus, they are unable to replace them

Front 51

What is the incidence of sickle cell anemia?

Back 51

1:500 African American children

Front 52

True or False: "The longer a cell spends in G1, the more likely it is to give rise to differentiated daughter cells."

Back 52

True

Front 53

What causes Sickle Cell Anemia?

Back 53

Mutation of a single nucleotide (SNP) from A-->T on the beta globin gene.

Results in Glutamic Acid --> Valine substitution at position 6

Front 54

THE CENTRAL DOGMA

Back 54

1. DNA can replicate itself ---> REPLICATION
2. DNA can be transcribed into RNA, a message that allows original gene to be amplified ---> (REVERSE) TRANSCRIPTION
3. RNA can move out of nucleus to be translated into protein --> TRANSLATION

Front 55

GOAL OF DNA REPLICATION

Back 55

To replicate genomic contents of a single cell in order to allow # of cell divisions it takes to form an organism

(which is about 7*10^13 divisions)

Front 56

5 CHALLENGES ASSOCIATED WITH DNA REPLICATION

Back 56

1*Must reproduce 6.4 * 10^9 base pairs in the 4-6 hours that cell division allows...which is roughly size of 2 DVD's ~300,000 bp/s so must be highly efficent

2*Must be of high fidelity to avoid errors (Error rate = 1/10^9 or 1/10^10 bp incorporated)

3*DNA is highly coiled in the nucleus & associated w/proteins to form chromatin --> largely inaccessible to replication enzymes

4*Polymerases necessary for DNA replication only work in 1 direction but DNA replication occurs in 2 --> Challenge is replicating both DNA strands simultaneously

5*DNA replication has to be limited to once/cycle --> Challenge is stopping replication after it has been initiated

Front 57

MECHANISM OF DNA REPLICATION

(Think in terms of conservation)

Back 57

*DNA replication is SEMI CONSERVATIVE

This means there's a parental strand and a new daughter strand after replication

*Uses Watson-Crick base pairing to duplicate DNA

Front 58

ORIGIN OF REPLICATION

Back 58

Site within DNA where replication is initiated

Other Things to Know about Origin:

1. Characterized by specific DNA sequences recognized by group of proteins called Origin Recognition Complex (ORC)

2. ORC leads to further protein recruitment to assemble pre-replication (pre-RC)

3. In humans, DNA recognition sequence can vary widely

4. E.coli has a single replication origin (replicon) while humans have up to 100,000 replication origins in a single cell

5. Replication origins are typically A-T rich because easier to separate than G-C due to having 2H bonds instead of 3

Front 59

WHAT IS A REPLICON?

Back 59

A replicon is a DNA or RNA molecule, or a region of DNA or RNA, that replicates from a single origin of replication.

*For most prokaryotic chromosomes, the replicon is the entire chromosome

*For eukaryotic chromosomes, there are multiple replicons per chromosome

Front 60

THE REPLICATION FORK

Back 60

Forms when DNA strands have been sufficiently unwound to allow replication to take place

*Located AHEAD of parental DNA, and BEHIND replicated DNA

Front 61

STAGES OF DNA REPLICATION

Back 61

1. Unwinding DNA to form replication fork

2. Stabilization of single stranded DNA

3. Initiation/Priming

4. Elongation (includes proofreading & editing) of the Leading Strand (5' --> 3')

5. Lagging strand synthesis (3' --> 5') -- it is discontinuous

6. Licensing: Ensuring that each replicon only replicates once/cycle

Front 62

HOW REPLICATION FORK IS FORMED

Back 62

- DNA strands separated in ATP-dependent fashion by helicases

- They bind to single strand & move thru DNA like a zipper

Front 63

PROBLEMS WITH DNA'S INTRINSIC TWIST

Back 63

DNA has an intrinsic twist (360 degrees for every 10bp) but is overwound (supercoiled) in order to facilitate packaging in nucleus --> creates problems when strands are separated by helicase

Front 64

ROLE OF TOPOISOMERASES

Back 64

DNA becomes overwound ahead of the replication fork. Its supercoils can delay/interfere w/advance of replication fork

This DNA Twist problem is alleviated by topoisomerase enzymes - they relax the supercoiling in DNA by making breaks/nicks that lessen tension

Topoisomerases are often indicated in cancer

Front 65

WHY DOES SINGLE STRANDED DNA (ssDNA) NEED TO BE STABILIZED & HOW IS THAT ACHIEVED?

Back 65

*Newly separated ssDNA will try & satisfy unpaired bases - ends up forming hairpin helices that restrict passage of DNA polymerase

*SSBP = Single Stranded Binding Proteins

- Stabilize newly formed ssDNA w/o covering the bases

Front 66

CHARACTERISTICS OF DNA POLYMERASES

Back 66

1. Enzymes that catalyze polymerization of deoxyribonucleotides into a DNA strand (like a giant hand that wraps around DNA as it moves thru the hand)

2. Magnesium ion used for catalytic activity

3. Can only add free nucleotides to 3' end

4. Cannot form new strands

5. Synthesize DNA only in 5' --> 3' direction (required for accuracy)

6. Misincorporation rate = 1*10^6 (Note: Lower than actual rate of fidelity of DNA replication)

7. Have proofreading ability (exonuclease activity that removes errant base pairs), therefore bringing the fidelity rate up to 1/10^9 or 1/10^10)

Front 67

3 MAJOR POLYMERASES ASSOCIATED W/DNA REPLICATION (IN HUMANS)

*There are 15 total but 3 major ones

Back 67

1. Polymerase alpha = RNA PRIMASE

2. Polymerase delta = Lagging strand synthesis

3. Polymerase epsilon = Leading strand synthesis

Front 68

POLYMERASE ALPHA DETAILS

Back 68

(aka RNA PRIMASE)

-Synthesizes an RNA Primer (removed later on)

-Then acts as DNA pol to elongate strand for approx 20 bp before delta & epsilon take over

*UNLIKE OTHER DNA POLYMERASES, POLYMERASE ALPHA CAN INITIATE A NEW STRAND

Front 69

POLYMERASE DELTA DETAILS

Back 69

LAGGING STRAND SYNTHESIS

-Highly processive (means it can synthesize a lot)

-Proofreads 3' --> 5'

-Exonuclease activity

Front 70

POLYMERASE EPSILON DETAILS

Back 70

LEADING STRAND SYNTHESIS

-Highly processive

-Proofreads 3' --> 5'

-Exonuclease activity

-Related to polymerase delta

Front 71

MAJOR POLYMERASES IN PROKARYOTES

*There are 5 major ones, but 3 we need to know

Back 71

1. DNA POL I -- DNA repair & removal of primers

2. DNA POL II -- DNA repair

3. DNA POL III -- Main polymerase in bacteria

Front 72

PROCESSIVITY OF POLYMERASE DELTA & EPSILON

Back 72

- Polymerases fall off DNA strand, thereby curtailing their ability to extend strands

- To extend processivity, Polymerase Delta and Polymerase Epsilon associate with protein PCNA ("clamp proteins")

*FYI: PCNA = Proliferating Cell Nuclear Antigen

Front 73

THE LAGGING STRAND ISSUE & SEMI DISCONTINUOUS SYNTHESIS

Back 73

DNA polymerases extend DNA template ONLY in 5' --> 3' direction

This is OK for one DNA strand but not the other, given that the replication fork moves in 1 direction. That is why you end up with Semi Discontinuous Synthesis

Front 74

HOW SEMI-DISCONTINUOUS SYNTHESIS IS ACHIEVED

Back 74

1. Priming of leading strand

2. Initiation of leading strand synthesis

3. Priming of lagging strand

4. Synthesis of first Okazaki fragments

5. Priming/Synth. of second Okazaki fragments

Front 75

CLEAN UP PROCESS ON LAGGING STRAND

Back 75

-RNA Primers have to be removed before the polymerase reaches it (i.e. before next round of synthesis)

-DNA Polymerases repair/fill in missing segment

-DNA Ligases join Okazaki fragment to new strand (seal together)

Front 76

THE REPLISOME

Back 76

The replisome is a complex molecular machine that carries out replication of DNA.

The replisome is composed of a number of proteins including helicase, PCNA, topoisomerase, primase, DNA polymerase I, and ligase.

This complex moves along dsDNA like zip of a zipper

**To facilitate this, lagging strand must bend back

Front 77

KEY POINTS OF DNA REPLICATION

Back 77

1. DNA Replication is a HIGH FIDELITY, HIGHLY EFFICIENT process that allows complete duplication of human genome in matter of hours

2. Major characteristic of DNA Replication is the REPLICATION FORK, which can arise on up to 100,000 origins within the cell

3. A lot of different proteins are involved in DNA replication -- they form a "machine" known as the REPLISOME to facilitate replication

4. Replication is asymmetric, w leading & lagging strands that need to be coordinated

5. Replication occurs in a # of STAGES: (1) Unwinding (2) Strand separation (3) Priming (4) Elongation (5) Clean Up

6. Replication coordinated w/cell cycle --> LICENSING -- this ensures duplication of DNA content takes place only once per cell division

Front 78

DNA REPLICATION IN THE CELL CYCLE (LICENSING)

Back 78

- DNA replication in cell is confined to 4-6 hrs during "S Phase"

- 100,000 replicons replicate DNA at this time, but each cannot replicate more than once due to Licensing of DNA Replication Origins

- Licensing helps prevent GENETIC INSTABILITY

Front 79

GENETIC INSTABILITY

Back 79

Uneven distribution of genes; It is a hallmark of cancer

Front 80

HOW IS LICENSING ACHIEVED?

Back 80

- ORC proteins bind to Origin throughout cell cycle

- Cdc6 is recruited. Mcm proteins (i.e. helicase) recruited too. They form pre-RC.

-CDK activates pre-RC to initiate bidirectional replication forks

-Cdc6 destroyed upon initiation until next cycle. No new initiation can occur without it - thus no more than 1 replication/cycle

Front 81

WHAT ABERRANT CONDITIONS ILLUSTRATE NEED FOR CONTROLLED, HIGH FIDELITY DNA REPLICATION?

Back 81

1. Genetic disorders (e.g. Ataxia Telangiectasia) - decreased fidelity results in increased mutation rate

2. Uncontrolled proliferation (e.g. Cancer, Autoimmune Disorders- can also be promutagenic)

3. Bacterial & Viral Replication

THESE DISEASES CAN BE TARGETED W/DRUGS THAT AFFECT DNA REPLICATION (FOR THERAPEUTIC PURPOSES)

Front 82

EXAMPLES OF DRUG CLASSES THAT TARGET DNA REPLICATION FOR THERAPEUTIC PURPOSES

Back 82

1. DNA Damaging Agents -- i.e. Cisplatin, popular in treatment of cancers

2. Topoisomerase Inhibitors -- if you interfere w/topoisomerase inhibitors, DNA will be under stress & replication stops

3. Anti-Metabolites -- we can design small molecules that will fit into the ATP pocket of the enzyme and interfere w/its activity

4. Nucleoside Analogs - main drug used in HIV is a nucleoside analog (derivative of one of the 4 bases) that prevents RNA synthesis from proceeding forward

Front 83

OKAZAKI FRAGMENTS

Back 83

Short lengths of DNA synthesized btwn RNA primers on the lagging strand of the replication fork

Front 84

4 IMPORTANT CONDITIONS FOR A CELL TO PROLIFERATE CORRECTLY

Back 84

- correct & accurate duplication of genome

- correct & accurate increase in organelles

- sufficient nutrients & metabolites for replication

- mitotic machinery functioning correctly

Front 85

CELL GROWTH & DIVISION - WHAT'S THE DIFFERENCE?

Back 85

Growth w/no cell division --> ONE BIG CELL

Cell division w/no growth --> BUNCH OF TINY LITTLE CELLS (molecule involved is WEE1 - mutation would make cells shrink)

Cell division + Growth = PROLIFERATION, YAY!

Front 86

THE CELL CYCLE DEFINED

Back 86

Eukaryotic cell cycle is orderly series of events whereby a single cell replicates its contents & divides into 2 cells. Cell Cycle can be seen as master regulator of DNA synthesis

Consists of these stages:

INTERPHASE

G1: Cellular contents, EXCLUDING chromosomes, are duplicated

S: Each of the 46 chromosomes is duplicated

G2: Cell double checks duplicated chromosomes for errors, making any needed repairs

MITOSIS

M: Mitosis

CYTOKINESIS

Cytoplasmic Division

Front 87

G0 (G Zero Phase)

Back 87

G0 = Quiescent state; Resting phase

Many mammal cells, such as the neuron, remain permanently or semipermanently in G0.

Some cells enter G0 temporarily & divide infrequently, like hepatocytes (liver cells)

Front 88

Regulatory Checkpoints in Cell Cycle

Back 88

*The regulatory checkpoints lie at or near the points of transition within interphase

*The 1st checkpoint is located at the end of the cell cycle's G1 phase, just before entry into S phase, making the key decision of whether the cell should divide, delay division, or enter a resting stage

*The 2nd checkpoint is located at the end of G2 phase, triggering the start of the M phase (mitotic phase). In order for this checkpoint to be passed, the cell has to check a number of factors, such as DNA damage via radiation, to ensure the cell is ready for mitosis.

Front 89

DNA CONTENT THROUGHOUT CELL CYCLE

(for animal cells)

Back 89

*n = haploid

*2n = diploid

G1: 2n

S: Cells go from 2n --> 4n

G2: 4n

M: 4n

After Cytokinesis: 2n

Front 90

WHY WOULD A LIVER CELL WANT TO DIVIDE INFREQUENTLY & ENTER G0?

Back 90

-Hepatocytes exposed to lots of toxins

-Temporary exit into G0 limits the pool of regeneration going on in the liver by controlling the proliferation of hepatocytes

Front 91

CYCLIN-DEPENDENT KINASES (CDK'S) - WHAT ARE THEY?

Back 91

-Family of small protein kinases (~30kDa)

-Regulate large # of cellular functions including transcription/mRNA processing/cellular differentiation

-Level of CDK's remains fairly constant, but their activity during cell cycle changes, depending on level of activators present (activators are known as cyclins)

Front 92

KINASES

Back 92

Carry out phosphorylation, use ATP

"KINdly put a phosphate on"

Front 93

CYCLINS

Back 93

Family of proteins that form complexes w/cdk's to yield an active kinase

Cyclin expression levels are regulated by expression of the previous Cyclin

-In other words...Cyclin's interaction w/CDK control the synthesis of subsequent Cyclin/CDK's

Front 94

CYCLINS & CDK'S AND THE MATCHING CELL CYCLE PHASE

Back 94

G1

-Cyclin D with CDK 4, CDK 6

G1/S

-Cyclin E with CDK 2

S

-Cyclin A with CDK 2 (again)

M

-Cyclin B with CDK 1

Mnemonic/Peg List:

*Brad Pitt's Dick is 4-6 inches long --> G1, Cyclin D, CDK 4/6

*Brad Pitt's Sh*t is the size of 2 Elephants --> G1/S, Cyclin E, CDK 2,

*Sh*tting with 2 A*ssholes is hard --> S, Cyclin A, CDK 2

*1 Motherf*cking B*tch --> M, Cyclin B, CDK 1

Front 95

D-TYPE CYCLINS

Back 95

There are 3 in mammals:

-D1, D2, D3

Front 96

Maturation Promotion Factor (MPF)

Back 96

Cyclins w/respective CDK form the MPF

Cyclins can be divided into 4 basic categories based on their period of expression during cell cycle (i.e. M-Cdk is trigger mitosis machinery and S-Cdk is trigger DNA replication machinery)

Progression thru each phase of cell cycle is combined efforts of cyclin (Regulatory subunit) and CDK's (Catalytic Subunit)

Front 97

Important checkpoints in cell cycle

Back 97

1. G1/S Checkpoint

2. G2/M Checkpoint

3. Spindle Checkpoint

Front 98

CDK INHIBITORS

Back 98

-Cyclin-dependent kinase inhibitor - proteins that perturb cyclin/CDK interaction

-They slow down or arrest progression of the cell cycle through this interference

--> Often important in cell cycle checkpoints

--> Referred to as Tumor Suppressor Proteins (bc mutations of these proteins associated w/tumors)

--> Nomenclature derives from their motility on SDS-PAGE (gel-electrophoresis)

e.g. p16, p53

Front 99

p16

Back 99

-Important in transition from G1/S

-Frequently mutated in cancers

-Associated w/prevention of melanoma & esophageal cancer

Front 100

p53

Back 100

"GUARDIAN OF THE GENOME"

-p53 mutated/missing in 50% of tumors

Serves 3 Major Purposes:

(1) Activate DNA repair mechanisms

(2) Arrest cell cycle at G1/S checkpoint

(3) Induce Apoptosis in cells where DNA damage wasn't corrected

-basic functions of p53 regulated thru interactions with mdm2....via UBIQUITINATION

-p53 comes into play in 3 instances:

1. DNA Damage

2. Cell cycle abnormalities

3. Hypoxia

-DNA Damage activates cell cycle checkpoints via the activity of p53

Front 101

Hypoxia

Back 101

Condition in which the body or a region of the body is deprived of adequate oxygen supply

Front 102

WAYS IN WHICH DNA DAMAGE CAN OCCUR

Back 102

-Ionizing Radiation

-UV light

-Oxidation from metabolites

*Damage can vary from 10^4 to 10^6 lesions per cell per day

Front 103

3 POSSIBLE FATES FOR CELL W/SIGNIFICANT DNA DAMAGE

Back 103

1. Senescence - irreversible dormancy (cell won't be able to divide again, and may lose most/all of its functionality too; not the same as G0)

2. Apoptosis - programmed cell death

3. Cancer

Front 104

WHAT DETECTS DNA DAMAGE/ACTIVATES THE CHECKPOINTS?

Back 104

*Two serine/threonine kinases are instrumental in detecting DNA Damage

*If DNA Damage is detected, cell cycle checkpoint is activated via activity of p53

Front 105

ATAXIA TELANGIECTASIA (ATM)

Back 105

Kinase protein mutated in AT --> responds to double strand breaks & chromatin disruption and works thru activity of p53 --> p21

Disease associated w/cerebellar degeneration (causes pt to shiver/shake)

Front 106

ATAXIA TELANGIECTASIA & RAD3 RELATED PROTEIN (ATR)

Back 106

Kinase that get's activated by stalled replication forks

Also works thru activity of p53 --> p21

Front 107

p53 & DNA damage response for G1 arrest

Back 107

-DNA damage activates ATM/ATR pathways

-Leads to activation of p53

-p53 transactivates p21 Wafl/Cip1 (a CDK inhibitor)

-Results in G1 arrest

*Targeted disruption of p21 Wafl/Cip1 gene shown to compromise G1/S checkpoint

Front 108

p53 & G2/M arrest

Back 108

-Progression of cells from G2 to Mitosis is driven by Cyclin B and CDK 1

-p53 shown to induce G2/M arrest by inhibiting activation of cyclin B/CDK1, again thru p21

-Cyclin B/CDK1 activation prevented by repressing cdc25C --- on account of phosphorylation by Chk1

Front 109

WHAT IS A TATA BOX?

Back 109

TATA BOX:

-A DNA sequence recognized by components of the General Transcription Factors (most notably, site at which RNA polymerase binds DNA)

-Located within promoter, upstream of transcription site

Front 110

WHERE IS THE TATA BOX LOCATED?

Back 110

LOCATED WITHIN PROMOTER, UPSTREAM OF TRANSCRIPTION SITE

(at about -25)

Front 111

What is Gene Regulation?

Back 111

Fundamental process by which gene pdts (RNA, and finally protein) are controlled

*Remember - All cells have the SAME DNA...what defines their identity is the 'suite of genes' that are active vs. inactive

Front 112

Why is Gene Regulation important? (i.e. What processes depend on Gene Regulation?)

Back 112

Turning ON & OFF of genes (or up & down of genes) is the goal of many pathways.

Gene Regulation is Key to:

1. Embryology & Development (regulation of proliferation/differntiation)

2. Terminal cell differentiation (Identity) & therefore function

3. Cellular response to disease & injury

Front 113

What is the pdt of transcription?

Back 113

RNA, ultimately.

-But 1st, DNA transcribed into Pre(hn)-mRNA

-Then RNA processing (splicing) takes place to create mRNA

-Then mRNA leaves the Nucleus & goes to Cytoplasm (Nuclear export) for translation

Front 114

GENE EXPRESSION ON THE TRANSCRIPTIONAL SIDE IS INVOLVED IN THE REGULATION OF...?

Back 114

RNA levels in the cell

Gene Regulation is able to regulate RNA levels by:

1. Controlling access to basic resources (i.e. DNA as a template)

2. Production of the product from basic resources

3. Regulating the lifetime of the pdt

Front 115

What does mRNA concentration (in the cell) reflect?

Back 115

Balance btwn Synthesis & Degradation

Front 116

At what point can RNA be degraded?

Back 116

After transcription, when it leaves nucleus --> cytoplasm via Nuclear Export

That way it does not get translated into a protein

Front 117

STAGES OF TRANSCRIPTIONAL GENE REGULATION

Back 117

1. Gene Activator protein binds to chromatin

2. Chromatin Remodeling (with Chromatin Remodeling Complex)

3. Covalent Histone Modification (with Histone Modification Enzymes)

4. Additional Activator Proteins bind to gene regulatory region

5. Assembly of pre-initiation Complex at the Promoter (once general TF & RNA pol come)

6. Transcription Initiation

Front 118

WHAT IS TRANSCRIPTIONAL SYNERGY?

Back 118

Gene Activators & Gene Repressors - their combined effect on Transcription exceeds the sum of the individual effects

Front 119

What % (approximately) of human proteins are involved in regulation of gene expression?

Back 119

5-10%

Front 120

Levels of Chromatin Organization

Back 120

1. DNA wraps around histone proteins, forming nucleosomes

2. The nucleosomes arranged like "beads on a string" are Euchromatin (genes under *Active* transcription)

3. DNA coding *Inactive* genes ("turned off" or "silent") are more tightly packaged as Heterochromatin

Front 121

HOW IS CHROMATIN'S OVERALL STRUCTURE AFFECTED BY CELL CYCLE PHASE?

Back 121

Chromatin's overall structure depends on the stage of the cell cycle:

-During interphase, chromatin is structurally loose to allow access to RNA and DNA polymerases that transcribe and replicate the DNA.

-In & just before entering mitosis/meiosis, the chromatin packages more tightly to facilitate segregation of the chromosomes during anaphase

Front 122

HOW IS CHROMATIN'S LOCAL STRUCTURE AFFECTED BY GENE EXPRESSION?

Back 122

The local structure of chromatin depends on the genes present on the DNA:

*DNA coding active genes are actively transcribed ("turned on") & more loosely packaged (euchromatin)

*DNA coding inactive genes ("turned off")

are found associated with structural proteins and are more condensed/tightly packaged (heterochromatin)

Front 123

What are the 2 major mechanisms used in Chromatin Remodeling?

Back 123

1. COVALENT HISTONE MODIFICATION

2. ATP-DEPENDENT CHROMATIN REMODELING (i.e. SWI/SNF)

Front 124

WHAT ARE COVALENT HISTONE MODIFICATIONS?

Back 124

Acetylation, methylation*, phosphorylation, and ubiquitination:

These processes affect packing of chromatin & affinity of DNA for protein.

*this methylation is different from DNA methylation

Mnemonic: Histone Acetylation makes DNA Active (b/c it relaxes DNA coiling, allowing for transcription)

Front 125

WHAT DOES ATP-DEPENDENT CHROMATIN REMODELING REFER TO?

Back 125

Move, eject, or restructuring of nucleosomes

Front 126

WHAT ROLE DOES ACETYLATION PLAY IN CHROMATIN REMODELING?

Back 126

*Acetylation = transfer of an acetyl group

*It is a type of Covalent Histone Modification

Acetylation removes the (+) charge on the histones, thereby decreasing the interaction of the N termini of histones w/negatively charged phosphate groups of DNA.

As a consequence, the condensed chromatin is transformed into a more relaxed structure that is associated with greater levels of gene transcription.

Front 127

WHAT IS THE KEY ENZYME IN ACETYLATION?

Back 127

Acetyl-Coenzyme A

Front 128

WHAT AMINO ACID RESIDUE ON THE N-TERMINUS OF HISTONES IS ACETYLATED DURING ACETYLATION?

Back 128

LYSINE

Recall: Lysine is a + charged amino acid (a base)

Front 129

WHAT ROLE DOES METHYLATION PLAY IN CHROMATIN REMODELING?

Back 129

Methylation of histones does the OPPOSITE of Acetylation

*Histones can be methylated on lysine (K) & arginine (R) residues but methylation is most commonly observed on lysine residues of histone tails

Mnemonic: Histone Methylation Mostly Makes DNA Mute (bc usually represses DNA transcription - tho not always)

Front 130

WHAT IS EPIGENETIC REGULATION OF GENE EXPRESSION?

Back 130

Gene silencing - prevents expression of a certain gene; can occur either during Transcription or Translation

Mechanisms of Epigenetic Silencing:

Chromatin remodeling (i.e. histone deacetylation, histone methylation) and DNA methylation

Front 131

WHAT ARE SOME EXAMPLES OF THE MECHANISMS OF EPIGENETIC SILENCING (TRANSCRIPTIONAL)?

Back 131

*Chromatin Remodeling - specifically Histone deacetylation and Histone methylation

*DNA Methylation

Front 132

WHAT IS DNA METHYLATION?

Back 132

Process where a methyl group is added to the cytosine or adenine DNA nucleotides

(NOTE: DIFFERENT FROM HISTONE METHYLATION)

Front 133

WHAT IS CONSTITUTIVE TRANSCRIPTION?

Back 133

Constitutive Transcription - when genes are transcribed (and thus expressed) all the time (i.e. for glyclosis & housekeeping proteins)

UNLIKE regulated transcription

Front 134

WHAT IS REGULATED TRANSCRIPTION?

Back 134

When rate of gene transcription is controlled, for example, by helping or hindering RNA polymerase binding to DNA

-Exists b/c some proteins only need to be produced at specific times or by certain cells. (Don't need to be constitutively expressed)

-Regulation of transcription carried out by transcription factors

Front 135

What are Transcription Factors?

Back 135

*TRANSCRIPTION FACTORS: sequence-specific DNA-binding proteins that function in controlling the rate of transcription

*Make contact w/sides of the bases exposed in major groove of B-DNA (bc that is where they base edges are most accessible)

*Key feature of Transcription Factors is that they have DNA Binding Domain(s) which are often further characterized by a recognition helix

Front 136

What are Enhancers?

Back 136

Regions of DNA that alters gene expression thru the binding factors of Transcription Factors

*Opposite of Silencers, basically

Front 137

What are Silencers?

Back 137

Region of DNA where negative repressors bind

Front 138

Where are Enhancers/Silencers located on the eukaryotic gene?

Back 138

Enhancers & Silencers may be located close to, far from, or within the introns of the gene whose expression it regulates. These regions may be 1000's of bp away from promoter region!

*But in lecture/diagrams, most often we see the Enhancers upstream of the transcription start site

Front 139

How do Transcription Factors contribute to transcription regulation?

Back 139

Function in controlling the rate of transcription

*Often, they activate transcription

*But they ONLY interact w/specific genes (that's why most eukaryotes have > 1,000 TF's)

*Transcription Factors most often modified/activated via phosphorylation

*Activated Transcription Factors bind to Enhancer Regions, often upstream of transcription start site. Once bound to DNA, the TF will move towards transcription start site to interact with Transcription Complex.

*Once formation of Transcription Complex is complete, transcription can begin

Front 140

6 TYPES (OR EXAMPLES) OF DNA BINDING DOMAINS

Back 140

*Their names describe their structure*

1. Helix-turn-helix: repressor proteins, regulators of developmental processes)

2. Zinc Finger: Zn ion stabilizes finger-like structure (specific triplet of base pairs)

3. Leucine Zipper: Leucine every 7th residue; Zips up to dimeriZe proteins

4. Winged Helix: 4 helices & 2-strand beta sheet

5. Winged Helix turn Helix: 3-helical bundle & 4 strand beta sheet

6. Helix loop Helix: Two helices connected by a loop (associated w/ some transcription factors)

Front 141

WHAT DO COACTIVATORS DO?

Back 141

Because enhancer/repressor regions are sometimes located far from the Promoter, a protein that rearranges DNA's structure to bring them closer is sometimes needed -- thats what Coactivators do!

Coactivators bind to Activators or Transcription Factors, not to DNA directly (?)

Front 142

TRANSCRIPTIONAL CONTROL IN PROKARYOTES

Back 142

Transcription is where most regulation occurs in prokaryotes b/c translation is coupled.

Classic example is Lac Operon

Front 143

LAC OPERON - WHAT IS IT AND WHAT DOES IT SIGNIFY?

Back 143

-Consists of 3 genes needed for digestion of lactose (to be utilized for NRG in absence of glucose)

-Represents the idea of an OPERATOR region that binds to a REPRESSOR: A tetrameric Repressor protein (I gene) is localized to Operator to prevent RNA pol read-thru

-In absence of glucose & presence of lactose, Allolactose will bind to repressor subunit, inducing a conformational change that reduces the repressor's affinity for the lac operon. cAMP (signals starvation) forms CAP-cAMP, which promotes RNA pol attachment.

-In presence of glucose, no CAP-cAMP forms & poor read-thru of RNA pol -- thus, digestion of lactose will not occur

This makes sense b/c Glucose is preferred as an NRG source over Lactose

Front 144

WHAT IS A DNA MICROARRAY & WHAT DOES IT HELP DIAGNOSE?

Back 144

- Collection of microscopic DNA spots attached to a solid surface (looks like a chip)

- Measures expression levels of large # of genes simultaneously

-Useful in diagnosing cancer - for example if you were to hybridize the spots w/mRNA's from Head & Neck Cancers

Front 145

THE VARIOUS TYPES OF RNA'S & THEIR CLASSIFICATION

Back 145

1. Coding RNA

- mRNA

2. Non-coding RNA = ncRNA

- Transcription RNAs (rRNA & tRNA)

- Small RNAs (such as miRNA, siRNA, piRNA, snoRNA, snRNA)

- Long non-coding RNA (such as XIST and HOTAIR)

Front 146

What is miRNA?

Back 146

MicroRNA (22-24bp in length) - involved in Translational control, and can work as both Positive & Negative regulators of cellular function; Can control transcript degradation, translation repression, potentially transcriptional repression as well. Very old.

-Human genome encodes > 1,000 miRNA's that target ~60% of our genes! Different sets for different tissues

-Type of non-coding, small RNA

Front 147

What is siRNA?

Back 147

Small interfering RNA (22bp in length) - RNA interference

-Useful in treating Choroidal neovascularization (CNV); Vascular Endothelial Growth Factor (VEGF) and its receptor are targeted using siRNA. These molecules control growth of new blood cells.

-Type of non-coding, small RNA

Front 148

What is Choroidal Neovascularization (CNV)?

Back 148

Choroidal neovascularization (CNV),

an age-related growth of new blood vessels in retina that leads to blindness

It is a common symptom of macular degeneration

Front 149

What is piRNA?

Back 149

PIWI-interacting RNA - Transposon silencing

-Type of non-coding, small RNA

Front 150

What is XIST?

Back 150

Xist - On the X chromosome; acts as major effector of the X inactivation process

-Type of non-coding, long RNA

Front 151

What is HOTAIR?

Back 151

HOX Antisense Intergenic RNA - faculty silencing

-Type of non-coding, long RNA

Front 152

Problems w/using siRNA in Therapeutics

Back 152

1. Ensuring good, dose-dependent delivery to targets

2. Off-target effects

Front 153

How Does Genetic Information Get Amplified to Organismal Phenotype?

Back 153

Genome structure and corresponding pathways of “amplification” of genetic information facilitate multi-level expansion of the information available to influence and regulate Cellular Function & Organismal Phenotype.

Front 154

KEY STRUCTURAL FEATURES OF A HUMAN GENE

Back 154

Typical human gene has Exons, Introns, and a Promoter

Front 155

β-globin (Beta-globin)

Back 155

Very small gene (only 3 exons) involved in hemoglobin disorders

- (e.g. Beta-thallassemia, Sickle Cell Disease)

Front 156

BRCA 1

Back 156

Gene that has 24 exons

Associated w/inherited BREAST and OVARIAN CANCER susceptibility

Front 157

Beta-myosin heavy chain (MYH7)

Back 157

Large gene (40 exons)

Mutations on this gene associated w/inherited Hypertrophic Cardiomyopathy

Front 158

KEY CHARACTERISTIC OF GENETIC CODE

Back 158

The genetic code is DEGENERATE. Because there are 61* possible combinations for triplets of nucleotides (i.e. codons), an Amino Acid can have multiple corresponding codons.

*64 – 3 (for the STOP codons which don’t code for an Amino Acid) = 61 triplets

Front 159

START CODON & MATCHING AMINO ACID

Back 159

AUG

- Encodes for Methionine (Met) when located in KOZAK SEQUENCE (GCCPuCCAUGG)

Front 160

STOP CODONS

Back 160

Mnemonic:

UGA = U Go Away

UAA = U Are Away

UAG = U Are Gone

Front 161

CODON USAGE IN NUCLEAR VS. MITOCHONDRIAL GENES – THE SAME OR DIFFERENT?

Back 161

Different!

Front 162

Significance of GT & AG sequences

Back 162

-Sequences at Exon/Intron borders

-Associated w/RNA splicing

*GT & AG are SPLICE SITES*

Front 163

Polyadenylation Signal

Back 163

“AATAAA”

Important signal for addition of polyA tail, which protects mRNA from degradation

Front 164

Length of Poly A tail is significant because…

Back 164

Determines half-life of mRNA

Front 165

What is a CAAT Box?

Back 165

- Binds transcription factors to regulate amount of transcription

- Located in Promoter at ~ -80

Front 166

TATA Box binds to what?

Back 166

TFIID – Transcription Factor II D

*Determines the exact start site (not in all promoters)

Front 167

What is the Initiator (INR)?

Back 167

INR compliments the TATA Box to localize the transcription start site

Front 168

Challenge with large genes (such as Dystrophin)

Back 168

The larger the gene (in terms of kb), the more difficult it is to screen for

e.g. Dystrophin, associated w/muscular dystrophy, is one of the longest and most difficult ones (~2500 kb)

Front 169

Pseudogenes

Back 169

*Pseudogenes are Non-functional copies of a gene

- Usually not expressed

- Often occur in gene families such as

α and β-globin genes

- Cause misalignment via erroneous chromosomal pairing during meiosis

Front 170

α-Thalassemia - What is it & what causes it?

Back 170

What is it? Blood disorder that reduces production of hemoglobin

What causes it? Deletion of 1 or more alpha-globin genes on Chromosome 16 which is the result of....

Misalignment of homologous pair & recombination btwn the α1 and α2 gene results in deletion/duplication of α-globin genes

*Illustrates effects of Unequal Crossing Over

*Most common in Southeast Asians

Front 171

What is the result of Unequal Crossing Over?

Back 171

If homologous chromosomes misalign during crossover….

· One pdt will contain an insertion

· And the other pdt will have a deletion

Front 172

How are Clustered Human Repetitive DNA Sequences arranged?

Back 172

-As arrays of short repeats organized tandemly in Head-to-Tail Configuration

-Clustered Human Repetitive DNA Sequences comprise ~10-15% of human genome (evolutionary remnants)

Front 173

What does “satellite DNA” refer to?

Back 173

The tandem repeats of Clustered Human Repetitive DNA Sequences, collectively

Front 174

Length of the Tandem Repeat Arrays

Back 174

*Variable both in terms of total length, and the nucleotide repeat that comprises the array

Front 175

Applications of Tandem Repeats?

Back 175

· Routinely used in designing tools for Molecular Pathology

· Also used in forensics, paternity testing, etc

Front 176

What is noteworthy about LONGER tandem repeats (e.g. α satellite DNA)?

Back 176

· Found at centromeres of human chromosomes

· Important for attachment of chromosome to the microtubules of the spindle during cell division

Front 177

What are SINEs?

Back 177

SINEs = Short Interspersed Nuclear Elements

· Include Alu sequences that are ~300bp

· Make up ~10% of DNA

Front 178

What are LINEs?

Back 178

LINE = Long Interspersed Nuclear Elements

· L1 Family

· Up to 6 kb long

· Accounts for ~20% of human DNA

Front 179

Familial Hypercholesterolemia (FH) is associated with what mutation(s)?

Back 179

*Unequal Crossing Over with ALU repeats*

-Result: 1 pdt with a deleted exon & other product has an extra exon

- Affects the LDL (Low-Density Lipoprotein) Receptor gene, which leads to FH

Front 180

What types of mutations are Alu & LINE Sequences associated with?

Back 180

· Insertional Activation: Some Alu & LINE generate copies of themselves & integrate at other genomic sites

· Recombination & Unequal Crossing Over: Causes disease

Front 181

What is the size of mitochondrial (mt) genome?

Back 181

37 genes (16,569 bp)

Front 182

Key Features of the Mitochondrial (mt) Genome & Structure

Back 182

· Closed, circular

· NO INTRONS - all coding sequences are contiguous

· Located in Mitochondrial Matrix

· Exist in thousands of copies per cell

· Has 2 strands: (1) a GUANINE-RICH heavy strand (2) a CYTOSINE-RICH light strand

· Contains 13 polypeptide subunits, 22 tRNA genes and 2 rRNA genes

Front 183

Issues with mitochondrial genome are typically characterized by...

Back 183

Fatigue & muscle weakness

Recall: Where do we have a high amount of mitochondria? MUSCLE!

Front 184

FEATURES OF MITOCHONDRIAL GENETICS

Back 184

*Maternal Inheritance

*Mutation rate is higher than Nuclear DNA's

*Replicative Segregation - No segregation btwn meiosis & mitosis

*Mitochondrial DNA replicates by increasing # of genomes in proportion to mitochondrial mass; Does NOT ensure each genome replicates same # of times; Can lead to changes in representation of alleles in daughter mitochondria

*Heteroplasmy - Mixed population of normal & mutant mtDNA will be received by daughter cell

Front 185

IS THERE ANY EVIDENCE OF COOPERATION BTWN MITOCHONDRIAL & NUCLEAR GENOMES?

Back 185

Yes - Protein subunits (gene products) synthesized from Nuclear DNA pairing up with protein subunits from Mitochondrial DNA

Front 186

IS MAJORITY OF DNA TRANSCRIPTIONALLY ACTIVE OR INACTIVE?

Back 186

INACTIVE

Most of the DNA in the chromain is in form of highly compacted, highly methylated, DNase-resistant heterochromatin

Front 187

What base pair is usually methylated in DNA?

Back 187

Cytosine

Front 188

What are HATs and HDACs?

Back 188

HAT = Histone Acetyltransferases

HDAC = Histone Deactylases

*Lysine is typically the A.A. involved

When Histone is Acetylated, it has no charge, and thus lower electrostatic affinity for DNA

--> ENHANCED TRANSCRIPTION

When Histone is Deacetylated, it is + charged and has high affinity for DNA

--> REPRESSED TRANSCRIPTION

Front 189

CpG island

Back 189

CpG island

= Cytosine* - Phosphate Linkage - Guanine

Often found at the 5' regulatory regions of genes, involved in DNA methylation

*Cytosine receives the methyl group

Front 190

DNA methyl transferase

Back 190

Enzyme that methylates the Cytosine in DNA Methylation

Front 191

DNA Methylation - associated with Silenced transcription or Active Transcription?

Back 191

DNA METHYLATION --> SILENCED TRANSCRIPTION

Front 192

What factors are associated w/causing exogenous DNA Damage?

Back 192

1. UV Light exposure

2. Ionizing radiation

Front 193

What factor is associated w/causing spontaneous DNA Damage?

Back 193

Cellular Metabolism

Front 194

What factor is associated w/causing DNA Damage as a result of replication errors?

Back 194

Chemical Exposure

Front 195

What problems w/Cellular Metabolism might cause SPONTANEOUS DNA DAMAGE?

Back 195

1. Deamination

2. Base Loss

3. ROS - Reactive Oxidative Species

Front 196

How does Deamination result in Spontaneous DNA Damage?

Back 196

*Involves spontaneous loss of an amine (NH3) -

-can result in formation of a non-standard base like Uracil, Hypoxanthine, Xanthine)

-or changes in Pyrimidines or Purines

Front 197

How does Base Loss result in Spontaneous DNA Damage?

Back 197

*Spontaneously occurs at rate of 10,000 per cell per generation

Front 198

How do Reactive Oxygen Species (ROS) result in Spontaneous DNA Damage?

Back 198

H2O2 & Oxygen can contain/generate oxygens with unpaired electrons (Free Radicals) which can damage DNA

Front 199

What 2 vitamins may have protective effects against ROS?

Back 199

Anti-oxidants Vitamin C & E

Front 200

How does Ionizing Radiation result in (Exogenous) DNA Damage?

Back 200

*Ionizing Radiation = Gamma or X-rays

*Mediated via hydrolysis of water which breaks down into ROS

Front 201

How does UV light/radiation result in (Exogenous) DNA Damage?

Back 201

*DNA absorbs UV light (260nm)

*Can photoactivate nucleotides causing distortions in DNA helix

*Most reactive nucleotides are adjacent pyrimidines which become covalently linked to form PYRIMIDINE DIMERS

Front 202

What are Pyrimidine Dimers?

Back 202

*Most commonly, Pyrimidine Dimers formed are Thymine dimers

- Adjacent T's link together & create a cyclobutane

- This formation distorts structure

*Bad b/c they prevent normal base pairing during subsequent replication

Front 203

How do Carcinogens cause (exogenous) DNA Damage?

Back 203

There are several types:

1. Aduct Formers (cigarettes)

2. Alkylating Agents

3. Cross Linking Agents

Front 204

What are Aduct Formers?

Back 204

Carcinogens that covalently attach to DNA nucleotides.

-Bulky

-Interfere w/replication & transcription

-Can lead to Base Substitutions

e.g. BENZO[A]PYRENE (Cigarette Smoke)

Front 205

What are Alkylating Agents?

Back 205

Carcinogens that are electrophilic chemicals

-They form covalent bonds w/electron rich atoms in DNA

-Usually involves adding methyl, ethyl or other large entity to one of the DNA bases, which disrupts stucture & pairing

Front 206

What are Cross Linking Agents?

Back 206

Carcinogens that can bond to 2 positions on DNA strand, forming crosslinks either inter or intra-strand

-The ones that facilitate inter-strand cross-links are most harmful b/c it inhibits replication & transcription completely

Front 207

What therapy/treatment option is associated w/Ionizing Radiation?

Back 207

Gamma irradiation

Front 208

What therapy/treatment option is associated w/Alkylating Agents?

Back 208

Cytoxan

Front 209

What therapy/treatment option is associated w/Crosslinking Agents?

Back 209

Cis platin

Front 210

What are Type II topoisomerase inhibitors?

Back 210

Type II topoisomerases cut both strands of the DNA helix simultaneously in order to manage DNA tangles and supercoils.

Type II topoisomerase inhibitors poison that enzyme & prevent it from working. They are often used as anticancer & antibacterial therapies

*e.g. Etoposide inhibits re-ligation of DNA strands; induces double strand breaks

Front 211

What Errors in DNA Replication commonly lead to DNA Damage?

Back 211

1. Polymerase Error

2. DNA Base Tautomers

3. Microsatellite Instability

Front 212

What is meant by "polymerase error?"

Back 212

Misincorporation of a nucleotide by polymerase (occurs in 1/1,000,000 bases)

That frequency is further reduced by "proofreading" and 3'-->5' exonuclease activity

Front 213

What are DNA Base Tautomers?

Back 213

Alternate forms of standard bases

*Rare, but the consequences are severe if happens during replication

Front 214

What is "Microsatellite Instability?"

Back 214

Occurs during Replication of repetitive DNA (known as microsatellites)

Can be thought of as "Slippage" of the primer strand relate to the DNA being copied

-Produces mismatches or bulges in DNA

-May lead to deletion/expansion of repeated sequences

Front 215

Possible Consequences of Damaged Genome on Cell Replication

Back 215

1. If DNA backbone IS intact -- replication can be error prone --> Translesion Synthesis

2. If DNA backbone is NOT intact -- replication can result in Double Strand Break, which is more difficult to repair

-Mitosis in the setting of a double strand break can cause aberrant chromosome segregation (end up w/aneuploidy or translocations/deletions)

Front 216

What might happen at the cell cycle checkpoints in response to DNA Damage?

Back 216

Cell Cycle Arrest: Halting normal progression of cell in S or M phase to give it more time to repair damage

Cell Death: via Apoptosis, to remove cells that have acquired deleterious mutations

Front 217

DNA Repair Pathways & their Primary Targets

Back 217

1. BER = Base Excision Repair

Targets: Deamination, Depurinations

2. NER = Nucleotide Excision Repair

Targets: UV Photoproducts, Adducts, Cross-links

3. Translesion Synthesis (TS)

Bypasses the Above, It is a tolerance process that allows the DNA replication to 'skip' past DNA lesions such as thymine dimers

4. Mismatch Repair (MMR)

Targets: Replication errors

5. Homologous Recombination (HR)

Targets: Dbl Strand Breaks, Adducts, Cross-links

6. End Joining

Targets: Dbl Strand Breaks

Front 218

EXCISION REPAIR MECHANISMS

Back 218

*include BER, NER, MMR

*They excise damage on 1 strand, using undamaged complimentary strand as a template for repair

*very specific

Front 219

DOUBLE STRAND BREAK REPAIR

Back 219

*includes HR, End Joining

*They can't use a complementary strand for a template - that is why double strand breaks are considering the most deleterious form of DNA damage

*less specific, lower fidelity processes than Excision Repair

Front 220

How does Base Excision Repair (BER) work?

Back 220

Consists of recognition & repair of single-base alterations that occurred as a result of Deamination

-Employs specific DNA glycolysases (i.e. Uracil glycosylase for Uracil) and its specifity makes it the least flexible

Front 221

How does Nucleotide Excision Repair (NER) work?

Back 221

*Primary repair mechanism for UV photoproducts but can also repair damaged bases not recognized by a specific glycosylase

- Employs a large multiprotein complex (>10 proteins). Primary element of an active NER complex is XPA.

-NER recognizes damage and cuts 5 bases 3' of the damage....then cuts 23 bases 5' of the damage. The intervening ssDNA (~28 bases long) is then removed.

-Gap filled by DNA pol & ligase

Front 222

What is XPA?

Back 222

Xeroderma Pigmentosum group A gene product

Front 223

What diseases are associated w/defects in NER repair?

Back 223

1. Xeroderema Pigmentosum

2. Cockaynes Syndrome, Trichothiodystrophy

Front 224

What is Xeroderma Pigmentosum (XP)?

Back 224

Patients are sensitive to UV rays from sunlight & more susceptible to skin cancer

*Due to defects in NER pathway genes needed for transcription-coupled repair & global genome repair

*1:1,000,000

Front 225

What is Cockaynes Syndrome/Trichothiodystrophy?

Back 225

Patients may be sensitive to UV, and NER compromised - but not always

*Developmental defects, hearing loss

*Due to mutations in transcription-coupled NER or general defects in transcription

Front 226

Features of Transcription Coupled Repair

Back 226

1. NER damage recognition usually coupled to transcription

2. NER of damage in actively transcribed regions & in template regions is MORE EFFICIENT than non-transcribed "silent" regions

3. RNA polymerase encounters DNA lesion, stop and initiates formation of an NER complex. That NER complex repairs damage.

*Global Genome Repair - a transcription-independent mechanim that is less efficient, and slower

Front 227

TRANSLESION SYNTHESIS

Back 227

-not a true repair pathway, but still a way for replication to progress despite DNA damage

-synthesis bypasses damage in template strand

-occurs in a "non-templated" manner & is therefore error prone

-specialized DNA polymerases involved - pol eta, zeta, and mu

Front 228

What is Ataxia-telangiectasia (AT)?

Back 228

-variable autosomal recessive disease associated w/immune deficiencies, cerebellar ataxia, and telangiectasias of the conjunctiva (easy to spot in Ocular Exam, prominent blood vessels on the sclera)

-mutation in ATM gene (11q22.3)

-incidence of 1:40,000

-pts. are highly sensitive to irradiation & prone to lymphomas/leukemias

Front 229

What is ATM?

Back 229

The gene mutated in Ataxia-telangiectasia (AT)

It is a large gene w/66 exons & 150kb that codes for a 3056 amino acid protein which regulates cellular responses to DNA damage

Front 230

DNA Mis-Matching ("Slippage")

Back 230

1. Backward slippage -- INSERTION

2. Forward slippage -- DELETION

Mis-matching more likely to occur in regions w/highly repetitive sequence

Front 231

DNA Mis-match Repair

Back 231

Front 232

What is Hereditary Non-Polyposis Colon Cancer (HNPCC)?

Back 232

*aka Lynch Syndrome

*disease associated w/defective mismatch repair

*early (but variable) age of diagnosis ~45 yrs

*tumor site: proximal colon

*also associated w/extracolonic cancers, particularly endometrium & ovarian

Front 233

What are the Genetic characteristics of HNPCC?

Back 233

-Autosomal dominant inheritance

-Genes belong to DNA mismatch repair (MMR) family

-Genetic heterogeneity

-Most commonly attributed to defects in MSH2 (30%) & MLH1 (30%)

-MSH6 mutation (6%) as the cause is much rarer

Front 234

How can molecular pathology help us detect Colorectal cancers?

Back 234

Microsatellite Instability (MSI) Analysis: Would show shift peaks that are abnormal & indicative of high instability

Front 235

MSI Classification in Colorectal Cancers (CRC)

Back 235

-microsatellite stable (MSS)

-microsatellite instability low (MSI-L)

-microsatellite instability high (MSI-H)