Cell Bio Ch. 10 Lecture, Cell Bio Ch. 16 Lecture (WIP), PCB3023 Ch. 18/20, PCB3023 Ch. 17

Endogenous DNA study concepts

mutations, gene organization, copy number

endogenous RNA transcripts study concepts

levels, splice forms, regulation

endogenous protein study concepts

levels, regulation, localization

over-express

Increase expression of a given gene

knockdown

delete the gene

site-directed mutagenesis

mutate the gene

Methods to purify genomic DNA

aCGH, sequencing, PCR cloning

aCGH

array comparative genomic hybridization

cytogenic technique used to detect chromosomal copy number variations across genome with high resolution

sequencing uses

identify mutations

PCR uses

forensics, genetics, mutagenesis

denaturation

DNA heated to 95 degrees C to separate the strands.

annealing

DNA is exposed to primers, then cooled to 50-55 degrees C allow primers to hybridize to complementary sequences

Extension

mixture is incubated with DNA polymerase and the four dNTPs to synthesize DNA, repeated multiple times to make more DNA, at 70 degrees C

Taq polymerase

Heat-stable polymerase from thermophilic Thermus aquaticus, not denatured by heat treatment, doesn't have to be readded

STR

short tandem repeats

made of stretches of CACA or GTGT found in various loci in the genome

they are highly variable, people will inherit 2 different numbers of repeats from their parents at each locus, making them highly unique per person

How are STRs used in forensic science

PCR use primers recognizing unique sequences around an STR locus, create a pair of amplified DNA band (one maternal and one parental)

the DNA's length and position in gel electrophoresis changes on the number of repeats

the more loci examined, the more accurate

how to purify total RNA

real time PCR, sequencing, RT-PCR: Cloning

converting mRNA to DNA

1) lyse cells and purify mRNA

2) hybridize with poly(t) primer

3) make DNA copy with reverse transcriptase

4) degrade RNA with RNase H

5) synthesize a complementary DNA strand using DNA polymerase, with RNA fragment as a primer

What type of DNA can PCR clone

genomic and cDNA clones are possible

How can PCR detect viral genome presence in a nasal sample

1. nasal swab taken

2. RNA extracted from virus

3. Reverse transcription + PCR amplification of cDNA

4. Gel electrophoresis with control sample of non-infected person

Ways to isolate endogenous proteins

SDS-PAGE, Western blotting + RPPA, subcellular fractionation

SDS-PAGE electrophoresis

Proteins are heated with SDS and mercaptoethanol, then loaded into a vertical polyacrlymide gel

small proteins move faster

proteins can be detected with a coomassie stain

Western blotting

procedure that uses labeled antibodies to detect specific antigens in a mixture of proteins separated according to their molecular weight

Western blotting steps

1) SDS-PAGE

2) Transfer onto nitrocellulose membrane (blotting)

3) Specific antibodies binds to the protein of interest

4) incubate with secondary antibody that carries a tag for detection purposes

5) visualize

antibody structure

Two parallel pairs of polypeptide chains

One pair of heavy chains

One pair of light chains

Each chain contains:

Constant segments

Variable segments

How to visualize protein localization in cells?

1. fix cells with formaldehyde for 30 minutes at room temp.

2. Block with goat serum in phosphate buffered saline and triton x-100

3. incubate with antibodies that target your protein of interest in PBS and Triton X-100

4. Visualize under fluorescence microscope

What does RPPA stand for?

Reverse phase protein array

What type of method is RPPA?

Quantitative high throughput method

What is the sensitivity of RPPA?

Femtogram sensitivity, nanograms of starting protein

What is required for RPPA to work effectively?

High quality validated antibodies and robotics

How many antibodies have been validated for use in RPPA?

200 antibodies validated

How many samples can be processed per slide in RPPA?

Up to 1000 samples per slide

Restriction nucleases

bacterial enzymes which cut at specific nucleotide sequences in DNA (4-8bp)

Types of restriction nucleases and the cuts they do

HaeIII = Blunt

EcoRI = Sticky

AluI = Blunt

NotI = Sticking

HindIII = Sticky

SmaI = Blunt

gel electrophoresis process

- incubate DNA with restriction enzymes at 37C for 1 hour

- Run reaction products on agarose gel to identify products

- Visualize with ethidium bromide in gel - a dye that intercalates into the DNA double helix and fluoresces under UV light

Hybridization

Denaturing double stranded helixes with heat or high pH separates them, but they reform when the conditions are returned to normal

how does hybridization used to screen for genetic disorders?

it is used to find genetic similarity between dna/rna sequences

What tests have DNA:DNA base pairing?

southern blotting, CGH

what tests have RNA:RNA base pairing

northern blotting, in Situ hybridization, microarrays

DNA ligase

Enzyme that joins DNA fragments together using ATP

plasmid vector components

1) promoter elements

2) origin of replication

3) antibiotic selection markers

DNA cloning steps

1) recombinant DNA is created using DNA ligase

2) recombinant DNA is placed in plasmid vector, by cleaving it with restriction enzymes, than joining it with DNA ligase

3) recombinant DNA is placed into bacterial cell

4) bacteria is cultured to create millions of copies

How to express protein in large amounts?

1) cut dna with nuclease

2) insert dna into an expression vector with a strong promoter

3) introduce recombinant DNA into cells

4) cell will now overexpress mRNA, which will result in overexpressed protein

How to produce a chimeric protein that is fluorescent

fuse dna of target with a green fluorescent protein

Purpose of signaling pathways around the cell

- sense nutrients

- avoid poisons/toxins in environment

- avoid predators

- communicate with other cells

- coordinate animal physiology and behavior

signal transduction

conversion of signals that carry information from one form to another is called signal transduction

endocrine signaling

hormones into the blood, very extensive

paracrine

local mediators, but not in blood

autocrine: self-produced local mediators

neuronal

long distances, but directed

contact dependent

Cell to cell by contact, no signal released

Receptors

cells contain many, each receptor type is activated by one signal

Classes of receptors

cell surface receptors and intracellular receptors

cell surface receptors

interact outside the cell, with molecules that are too large or hydrophilic to cross the membrane

intracellular receptors

receptor inside the cell, that interacts with small hydrophobic molecules

Large hydrophilic ligands

- binds to cell surface receptors

- ex. epidermal growth factor, platelet-derived growth factor, nerve growth factor, transforming growth factor-B

Small hydrophobic ligands

binds to intracellular receptors ex. cortisol

Signal can induce different response in different cells?

signal enters into target interior via intracellular signaling molecules

the molecules act in sequence to change effector proteins

this relay system varies from cell to cell in response to the same signal

Acetylcholine

in heart pacemaker cells, it decreases firing rate

in salivary gland cells, it induces secretion,

in skeletal muscle cells, it induced contraction.

Low levels of receptors

a receptor is typically expressed at 10k-20k molecules/cell

0.0001-0.002% of total protein

Actin > 10^7 molecules/cell

what effects speed of cell response to signals

Fast responses are caused by altering activity of pre-existing proteins inside the cell

Slow responding proteins are caused by the signal inducing changes in gene expression, resulting in the need to synthesize new proteins

fast response example

acetylcholine stimulates muscle contraction within milliseconds, and salivary gland secretion in a minute

slow response

EGFR stimulates epithelial cells to grow

binding of ligand to receptor

- specific and reversible

- multiple weak interactions

- ligand has no function independent of receptor

What does R mean in RAIDM?

Relay: spread signal in the cell

What does A mean in RAIDM

Amplify:

Make signal stronger

What does I mean in RAIDM?

Integrate:

Detect signals from multiple pathways and integrate those prior to relaying signal

what does D mean in RAIDM?

Distribute:

Distribute signal to more than one effector protein

What does M mean in RAIDM?

Module via feedback:

Regulate activity of upstream components (inhibition or activation)

Positive feedback

Intensifies change, can cause an explosive response

negative feedback

a mechanism of response in which a stimulus initiates reactions that reduce the stimulus

Amplifiers

second messengers, that usually allow a kinase to be activated

ex. cAMP, IP3, DAG, Ca++

signaling by protein phosphorylation

surface receptor activation generally activates protein kinases

PO4 state leads to on or Off

Signaling by GTP-binding proteins

a large group of GTP binding proteins act as molecular switches to regulate signal transduction.

ex. trimeric G proteins and RAS

GEF (guanosine exchange factors)

promote exchange of GDP for GTP, turning protein on

GAP (GTPase activating protein)

GAPs stimulate hydrolysis of GTP to GDP, switching protein off

classes of cell surface receptors

1. Ion-channel-coupled receptors

2. G-protein-coupled receptors

3. Enzyme-coupled receptors

What is the largest family of cell receptors?

G-protein coupled receptors (GPCRs)

How many GPCRs are there in humans?

More than 700

What types of molecules do GPCRs respond to?

Hormones, local mediators, neurotransmitters

What is notable about the structure of ligands for GPCRs?

The structure of ligands is varied; proteins, small peptides, derivatives of

amino acids/fatty acids

Why are GPCRs considered attractive targets for therapeutic development?

They are used to treat diseases

What fraction of all drugs target GPCR signaling pathways?

1/3rd

What is GPCR?

A large class of proteins.

How many times do GPCRs span the membrane?

7 times.

What are GPCRs linked and coupled to?

G proteins.

G proteins

- 20 types

- GTP binding protein that acts as a switch (GTP bound = on, GDP bound = off)

- Made up of 3 subunits (alpha, beta, and gamma)

Gi, Gs

Gi = inhibitory

Gs = stimulatory

They activate/repress ion channels or membrane bound enzymes

G protein alpha subunit

- can activate or inhibit production of second messengers

- has intrinsic ATPase activity, which happens seconds after activation

What happens when GPCR is stimulated?

It activates G proteins by encouraging the alpha subunit to release GDP and pick up GTP.

How does the alpha subunit of G proteins turn itself off?

By hydrolyzing bound GTP to GDP, then it binds itself back with the beta-gamma complex

How does Gi protein couple receptor activation?

it couples them to K+ channel opening in the plasma membrane of heart pacemaker cells

What happens when Acetylcholine binds to GPCR in heart pacemaker cells

results in activation of G

What happens with the activated Beta-gamma complex?

it opens a K+ channel in the plasma membrane

- increases k+ permeability

- makes membrane harder to activate + slow the heart rate.

-

How is the alpha subunit inactivated?

Its bound GTP is hydrolyzed, which returning G protein to its inactive state, which allows the K+ channel to close

Adenylyl cyclase

- Can break down ATP to generate cAMP

PLC

Phospholipase C

- breaks down phosphatidylinositol bisphosphate (PIP2)

what happens when PIP2 is broken down?

IP3 and DAG are created as second messengers

What happens when IP3 is generated

it stimulates the release of Ca++

what does cAMP exert most of its effects on?

- Protein Kinase A (PKA) or Serine/Threonine Kinase

cAMP

phosphorylates enzymes to turn them on and/or phosphorylate proteins that regulate gene expression