Test 1 Foundations Review

Histology + Lab, Physiology, and Bio Chem all in 1 except the disorders and their associated genes

Human Genome Project

1990-2003 project to map out the entire human DNA sequence

Benefits: reference blueprint, gene discovery, disease insight, tools and tech catalyst, personalized medicine

Mitochondrial DNA

• circular

• 32 genes (13 for oxidative phos. 22 tRNAs, 2 rRNAs)

• 99% of mitochondrial proteins are nuclear coded and imported

• Maternal inheritance

• mtDNA mutations lead to metabolic dysregulation

Heteroplasmy

coexistence of normal and mutant mtDNA (under the threshold)

Satellite DNA

Also known as “tandem repeats” a type of repetitive DNA

Microsatellites (1-6bp) used in CODIS, prone to replication slippage

Minisatellites (10-60 bp)

Alpha Satellites (About 171 bp) at centromeres - binds CENP-A and kinetochore proteins

Telomeric repeats

Transposons

Also known as Interspersed repeats (jumping genes)

DNA sequences that can be moved via copy and paste

DNA transposons move directly as DNA

Retrotransposons move via an RNA intermediate

uses transcriptase to be transcribed into RNA and then reverse transcribed into DNA to be inserted elsewhere

LINES

(Long Interspersed Nuclear Elements)

Autonomous, encodes its own reverse transcriptase (17% of genome)

SINES

(Short Interspersed Nuclear Elements)

Non-coding about 300 bp, rely on LINE-1 machinery

majority are ALU repeats

Overwinding DNA

360 to the right (becomes 8.3bp per turn)

produces positive super coils

NOT PREFERRED

inhibitory to DNA replication and transcription

In front of the replication form

Underwinding DNA

360 to the left (becomes 12.5bp per turn)

produces negative super coils

PREFERRED

DNA is more relaxed, promotes DNA separation

Behind the replication form

DNA Gyrase

Cells actively create this under twist with enzymes

Histones

Positive (+) charged

Forms ionic bonds with DNA

5 Classes: H1 - Linker DNA / H2A,H2B,3,4 - 2 of each form an octamer core of the nucleosome

Histone proteins have N-terminal tails that can be modified via methylation, acetylation (promotes transcriptions)

Facilitates packing nucleosomes tighter

Levels of DNA packaging

• DNA Double Helix strands

• Nucleosomes (DNA wrapping around Histone octamers attached via H1) “Beads on a string”

• Chromatin fiber - Nucleosomes are compacted into 30nm fibers

• Looped Domains - Fibers are packed into loops onto a protein scaffold, loops are functional units of DNA replication

• Chromosome - loops form chromosomes, 10k fold shorter than its extended length

Euchromatin

open 10,30 nm fibers, gene rich, transcriptionally ACTIVE, early S phase

Heterochromatin

condensed 300nm structures, gene poor, transcriptionally SILENT, late S phase

dNTP’s

A, T, C, or G

act as the material needed to construct new DNA

How is DNA read and translated?

DNA is read 3’ to 5’ / translated 5’ to 3’

Primer

Nucleic acid with a free 3’-OH (hydroxyl group)

Polymerization Reaction

Free hydroxyl group on the sugar carries out a nucleophilic attack on the α phosphate of incoming dNTP

• DNA synthesizes

α DNA polymerase

contains primase, extending RNA primer by 20-30 dNTP

β DNA polymerase

Used for repair

γ DNA polymerase

Used to replicate mitochondrial DNA

has proof reading capability

δ DNA polymerase

Elongates Okazaki fragments of the lagging strand

has proof reading capability

ε DNA polymerase

Elongates the leading strand

has proof reading capability

Helicases

pulls apart DNA helix to separate strands using MCM (Minichromosome Maintenance complex)

RPA

keeps DNA helix unwound after helicase

Topoisomerase

Relaxes supercoiling

Type 1 - Nicks one of the strands, unwinds, seals the nick

Type 2 - Nicks both strands staggered, unwinds DNA with one turn, seals the nick

DNA Ligase

Catalyzes the final phosphodiester linkage between Okazaki fragments (uses ATP from oxidative phos.)

Reverse Transcriptase

Makes DNA from an RNA template

Heat Stable DNA Polymerase

From thermophilic bacteria, withstands 70-78C

Bacterial Topoisomerase Inhibitors

Antibacterial

Clinical Use: UTI treatment

Examples: Quinolones, Nalidixic Acid, Ciprofloxacin

Type 1 Topoisomerase Inhibitors

Anticancer

Examples: Irinotecan, Topotecan

Type 2 Topoisomerase Inhibitors

Anticancer

Examples: Etoposide, Doxorubicin

Joining Okazaki Frags Sequence

1. FEN1 removes primer (flap endonuclease)

2. DNA Polymerase δ fills in the gap

3. DNA ligase seals the two together

PCNA (Proliferating Nuclear Antigen)

“Sliding Clamp”

Keeps the DNA polymerase from dissociating, stabilizes it

DNA proof reading

1 in 400k errors

High fidelity rate (not many errors)

If there is an error, 5’ to 3’ transcription pauses and gets put in reverse (starts going 3’ to 5’) to correct the mistake

End Replication Problem

When RNA primer is removed from 5’ end of newly synthesized DNA, DNA polymerase can’t fill in the gap

This leads to progressive shortening of chromosome ends after each division

Responsible for aging

Telomeres

Aglet of the chromosome

Short DNA repeats (TTAGGG x1000) folded into a 3-D structure

Confers stability at the end (T-Loop Cap)

Hayflick Limit

The number of times a cell can divide

The cells state at the end of the Hayflick Limit is called ‘Replicative Senescence’

TERC

(Telomerase RNA Component)

Provides the telomere template 3’AAUCCC5’ to guide the repeats

TERT

(Telomerase Reverse Transcriptase)

Uses an RNA template from TERC to add DNA to the end of the chromosomes

Imetelstat

Telomerase inhibitor, reducing tumor size in xenographs

Dyskeratosis Congenita

telomeres get shorter much quicker, endures premature aging and death

Telomerase Mechanism

• Telomerase binds to 3’ end of chromosome w/o the gap using template RNA sequence

• Using reverse transcriptase, extends the parent strand by 6 nucleotides

• Relocates to the end

• Repeats this extension several times

• Polymerase α and Polymerase δ are now able to complete lagging strand synthesis

Endogenous DNA Damage

Damage from INSIDE the cell

Examples:

• Oxidative damage (ROS from metabolism)

• Spontaneous base deamination, AP sites, methylation

• Replicative errors

Exogenous DNA Damage

Damage from OUTSIDE the cell

Examples:

• Genotoxic chemicals (PAH’s, Alkylating agents)

• Ionizing Radiation (x-rays, gamma rays)

• Ultraviolet radiation (thymine dimers)

Direct DNA repair

Repairs mechanism without excising DNA base or backbone that reverse damage in place

NOT common

Base Excision Repair Steps

Single Stranded Repair

corrects small non helix distortion

problems that effect single bases

Steps:

1. DNA glycosylases recognize damaged bases

2. Hydrolyze N-glycosidic bond to remove the base leaving an AP site

3. AP endonuclease cleaves the strand at the AP site making a 5’ end, and dRP Lyase removes sugar phosphate group

4. Short Patch or Long Patch

5. Short Patch - DNA Pol β removes 5’ phosphate and fills in correct base and DNA Ligase III seals the nick (commonly used)

   Long Patch - DNA Pol δ/ε creates a flap that is removed by FEN-1 and the nick is sealed by DNA Ligase I

Short Patch vs. Long Patch

If the 5’ end is a clean cut = Short patch (1 nucleotide replaced)

If 5’ end is oxidized or blocked = Long patch (2-10 nucleotides replaced)

PARP-1

(Poly-ADP Ribose Polymerase 1) recognizes and binds to single strand breaks

PARP-1 inhibitors trap it on DNA, Blocking repair = Cancer therapy

PARP-1 primarily acts as a signal amplifier and recruiter

Transition-Coupled Nucleotide Excision Repair

used to remove heavy distorting lesions like thymine dimers due to UV radiation

Steps:

1. RNA Pol Stalling - RNA polymerase stops working when it hits a lesion

2. Lesion Recognition - CSA and CSB are alerted by this

3. DNA Unwinding - TFIIH unwinds DNA around lesion

4. Damage Verification - XPA and RPA come in and verify, then stabilize complex

5. Dual Incision - XPG (3’) and XPF-ERCCI (5’) cut around lesion (20-30 nt)

6. DNA Synthesis and Ligation - DNA polymerase use template strand to fill gap, DNA Ligase seals it

CSA and CSB are a lot faster and prioritized so transcription can resume ASAP

Global Genomic Nucleotide Excision Repair

used to remove heavy distorting lesions like thymine dimers due to UV radiation

Steps:

1. Damage detection - XPC scans DNA for helix-distorting lesions

2. DNA unwinding - Helicase unwinds around lesion

3. Damage Verification - XPA and RPA come in and verify and stabilize

4. Dual Incision - XPG (3’) and XPF-ERCCI (5’) cut around lesion (20-30 nt)

5. DNA Synthesis and Ligation - DNA polymerase use template strand to fill gap, DNA Ligase seals it

XPC is a lot slower, it has to scan DNA on its own looking for the lesions

Mismatch Repair

Steps:

1. Replicative errors that escaped proof reading

2. MSH2-6 complex binds to mismatch

3. MLH1 endonuclease and PMS2 bind to recruit helicase and endonuclease

4. This then removes multiple nucleotides surrounding the mismatch

5. Gap is filled by DNA polymerase δ and sealed by ligase

Xeroderma Pigmentosum

Defects in global genomic NER

Clinical Presentation: Extreme UV sensitivity, freckling, premature aging of skin

High cancer risk

Cockayne Syndrome

Defects in Transcription-Coupled NER

Clinical Presentation: growth failure, neurodegeneration, photosensitivity

No cancer risk

Lynch Syndrome (HNPCC)

(Hereditary Nonpolyposis Colorectal Cancer)

Autosomal Dominant

caused by defects in mismatch repair genes

can also be caused by EPCAM (sits next to MSH2 so just by association)

Certain EPCAM mutations silence MSH2

Nonhomologous End-Joining

Double stranded break repair

used for “Shotgun wounds”

1. Ku proteins and DNA-PK form a complex at the break ends

2. Nucleases trim DNA to make blunt ends (This loses nucleotides)

3. DNA Ligase IV seals the break

Process is quick and easy but error prone due to loss of bases

preferred in G1 phase when there is no sister chromatid to copy

No polymerase because theres no way to know what was lost

simple cut, seal, and hope for the best

Homologous Recombination

Double stranded break repair

1. MRN finds to the end of the break - causes autophosphorylation of ATM

2. ATM activates BRCA1 at damage site

3. BRCA1 recruits EXO1, BLM, and DNA2 which carry out end resection, exposing ssDNA

4. RPA coats ssDNA to stabilize it

5. BRCA1 and PALB2 help RAD51 replace RPA

6. DNA polymerase extends and repairs the gap

7. Gap is filled and ligated

Basically once RAD51 replaces RPA as the coat, it causes strand inversion into the homologous template, so that polymerase can come in to extend and repair the gap

REPAIR IS ERROR FREE but slower…

ATM

(Ataxia Telangiectasia Mutated)

• Acts as a master regular of the repair pathway

• actived at DSB sites by the MRN complex

• Autophosphorylates to become active

• halts cell cycle to allow for repair

• if damage is severe, initiates apoptosis

Ataxia Telangiectasia

Caused by mutation in ATM

also by defective DSB repair

Symptoms:

• cerebellar ataxia

• delayed motor skill devlopment

• slurred speech

• SPIDER VEINS

• risk of cancer

• pregressive neurological dysfunction

Double Strand Break Repair

most lethal DNA break type

can lead to cancer

immediate and accurate repair is crucial

2 Types:

• Nonhomologous End Joining

• Homologous Recombination

Hereditary Breast Cancer

Associated with Homologous Recombination

Caused by mutations in BRCA1/BRCA2

Homologous recombination fails, cant initiate RAD51

forced to use NHEJ (the other repair)

Bloom Syndrome

Associated with Homologous Recombination

Caused by mutations in BLM helicase

HR proceeds but with defective recombination intermediates

causes genomic instability

Clinical Presentation:

Proportional dwarfism

SUNBURN!

Male infertility

Consinguinity

Marriage between close relatives

Double line on a pedigree chart

Icreased incidence probability

Factors affecting expression of disease causing genes

• New Mutations (ex. Achondroplasia/Dwarfism)

• Delayed Onset (ex. Huntingtons)

• Penetrance - proportion of individuals carrying mutation that also express it

  (Anticipation - larger # of repeats = earlier age of onset)

• Variable Expressivity (ex. polyaxial polydactyly can vary from extra digits to little nubbins)

• Pleiotropy - mutations in a single gene can result in defects of several organ systems (ex. Marfan Syndrome FBN1 mutation)

• Locus Heterogenecity - Same phenotype caused by many different genes (ex. HNPCC)

• Allelic Heterogeneity - several mutations in the same gene cause the disease phenotype (ex. Cystic Fibrosis has many different causes)

SRY Gene

This gene triggers development of the Testis - TDF (Testis Determining Factor)

TDF upregulates SOX9, developing the testis

Only located on the Y gene

AZF Genes important for spermatogenesis (USP9Y, DDX3Y, DAZ)

Microdeletions in specific regions may cause azoospermia (little to no sperm)

PAR region

location where the SRY gene is located

On the super small chance SRY crosses over to the X, it will result in an XY female with ambiguous genitalia

May also result in an XX male

Manifesting Heterzygote

Carriers may show signs of disease

Random inactivation of an X chromosome

Slide Preperation Steps

1. Fixation - preserves and hardens by cross-linking proteins (Formaldehyde)

2. Dehydration - water is replaced by alcohol

3. Clearing - Alcohol is removed by toulene/xylene

4. Infiltration - Toulene/xylene is replaced by paraffin wax

5. Embedding - Wax hardens

6. Trimming - sections the tissue block into slices

Microtome

sections tissue blocks into 5-10um slices

Cryostat - special microtome for frozen sections

Frozen Sections

Cryostat is its special microtome

Uses: Onco-surgery and histochemical studies

Transmission Electron Microscopy

• Fixation - Gluteraldehyde

• Uses heavy metals (Osmium Tetroxide) to impart electron density = better visibility

• Mounted on copper grid

• Stained with lead citrate and uranyl acetate

Staining

Stains are either acidic or basic compounds

• Acidic Dyes stain acidophillic structures (Basic/cationic - ex. mitochondria, collagen) (Dye ex. Acid Fuschin)

• Basic Dyes stain basophillic structures (Acidic/anionic - ex. DNA/RNA) (Dye ex. Methylene blue)

Basophilia

Affinity of tissue components for a basic dye

Acidophilia

Affinity of tissue components for an acidic dye

PAS

special dye for carbs

conter stain with hematoxylin to show nuclei

Lipid Staining

Use Oil Red O or Sudan Black

Elastic Fiber Stain

Use Orcein/Resorcin

Dark FIeld Microscopy

Used for unstained and transparent material

Objects deflect light and are seen in dark background

Special condenser with a shield in the middle

Fluorescence Microscopy

Uses UV light

Fluorescent stains absorb UV light and then emit it back

more specificity

Phase Contrast Microscopy

Unstained

Good for cultures and living tissues

APpears either light or dark

BAD resolution

Confocal Microscopy

Narrow laser beam, small focal plane of depth

sharp focus

tissues optically sectioned

Polarizing Microscope

Shows repetitive and crystalline structures (ex. collagen, myofibrils, cellulose)

Birefringement Specimen

Electron Microscope

• Electron beams have short wavelength = high magnification and resolution

• Transmission EM is for 2-D structures

• Scanning EM is for 3-D structures

• Magnification up to 400,000x

• Metallic fimalent emit electron beams (no light used)

• Electro magnet acts as lens

• Electron Lucent appears white

• Electron dense appears dark

Cryofracture/Freeze Etching

Used to prepare for electron microscope

Section is fractured and frozen via platinum vapor

Uses: study cell structure and organelle

Insitu Hybridization (ISH)

Basically a gene air tag

Probe - strand of RNA complimentary to what you are looking for

Probe is tagged with radio isotopes or enzyme markers

target is identified via fluorescence or autoradiography

Glycocalyx

Carbohydrate coat on external cell surface

used for cell to cell recognition

Endocytosis

material brought from outside the cell, into the cell

Exocytosis

secretory granules made inside the cell are released out (ex. collagen)

Phagocytosis

particles/bacterium from outside. brought inside

Pinocytosis

Fluid from outside the cell, brought inside

Receptor Mediated Endocytosis

Receptors outside bind a ligand and a vesicle forms to bring it inside the cell

Nissil Substance

Abundant ribosomes and RER in a neuron visible at light microscope level

Neurons produce a lot of protein

Rough Endoplasmic Reticulum

Synthesis of membrane package proteins (secretory, lysososmal, membrane proteins)

Smooth Endoplasmic Reticulum

Uses (vary on cell type):
Steroid hormone synthesis (adrena cortex)

Drug detoxification (liver)

Muscle contraction (skeletal muscles)

Autophagy

The act of fusion with autophagosomes (lysosome + INTRAcellular material)

Heterophagy

The act of fusion with heterophagosomes (lysosome + EXTRAcellular material)

What is actin (1) and microtubules (3) made out of?

globular proteins

What is intermediate filaments (2) made out of?

Fibrous protein subunits

gives the cell tensile strength

Filament type for: Epithelium Cells

Cytokeratins