BIOL 311 Exam 2

Molecular Biology Techniques

* a group of techniques that are designed to manipulate, analyze, expand, express DNA/RNA etc

Genomics

* Gene transfection
* editing genes

DNA sequencing

* Oxford nanopore technologies
* MinION
* $4000 genome

Transcriptome

* all RNAs in cell and their function

Proteome

* Proteomics (huge?)

Epigenetics

* non base changes in the nuclear genome
* eg. methylation, histone phosphorylation
* lifestyle can alter your epigenetic profile

Metabolome / metabolomics

* study of smaller molecules and their actions

Secretome

* Can be used as biomarkers for cancer
* Secrete factors in exosomes important to tissue repair and physological changes
* May replace stem cell therapy in the future
* Adv. stem cells are forever; exosomes have half life
* Stem cells are well known to influence immunomodulation

Mutation causes

* ultra violet light
* chemicals
* EMS = ethyl methane sulfonate
* critical in molecular genetics

Yeast

* 1960s – 1970s L.H. Hartwell – did experiments w yeast to determine the genes in cell division
* Advantages
* Grow in a simple basic medium (easy to grow)
* Can be haploid or diploid
* Temperature sensitive mutants
* Permissive temperature = 23 C
* all yeast will grow
* Non-permissive temperature = 30 C
* temperature sensitive mutants wont survive

Restrictive nucleases (enzymes)

* 1963 - first discovered
* cleave DNA at known points
* first discovered in bacteria
* used to ward off viruses
* 600+ now commercially available
* 1978 - noble prize

Nucleic Acid hybridization

* Roles
* FISH
* antisense RNA/DNA
* southern blots
* northern blots
* cDNA microallays
* molecular beacons

Gel electrophoresis

* separates different DNA fragments using nucleases
* polyacrylamide gel electrophoresis
* 10 - 2000 bases (small)
* agarose gel electrophoresis
* 200 - 20KB bases
* applications
* band shift assay - DNA sequence of interest
* Apoptosis vs Necrosis
* apoptosis - non random DNA cleavage (ladder)
* necrosis - random DNA cleavage (smear)

Southern Blots

* gel electrophoresis technique to detect gene sequences
* quantitative - molecular weight
* qualitative - differences in abundance
* transfer paper
* applications
* Nucleic acid polymorphisms
* CODIS → forensic medicine
* eg. VNTR → Variable Number Tandem reapeats
* Bone Marrow transplant

Single Nucleotide Polymorphism (SNPs)

* 98 - 99% of our genomic DNA is non-coding

Northern Blots

* gene expression
* gel electrophoresis - transfer paper
* views mRNA, not DNA

Molecular Beacons

* nucleic acid hybridization
* “quencher”
* molecular beacons probe’s fluorescence is quenched (not fluorescent) unless bound to target RNA or DNA strand

cDNA Microarrays (Gene Chips)

* gene expression of up to 8500+ genes in a single experiment
* microscipe slide - 8600
* Q. what mRNAs are expressed in response to insulin
* evolved from northern blots

Prokaryotes

* 1981 – Humulin
* How to harvest protein of interest
* mAb ii.
* Add 6 histodines to the carboxy end of the protein
* Collect nickel affininity chromatography

Eukaryotes (mamalian cells)

* transient and stable
* how to harvest
* “tarin” the proteins secreted
* mAb
* epitope tagging

PCR

* amplify amount DNA
* forensic medicine
* heat/cooling cycle
* 95 degrees
* DNA polymerase → tag polymerase

1970s

* Freeze Fracture
* Fluorochromes/mAb

Four experiments

* freeze fracture
* cell fusion
* cell patching/clapping
* FRAP

Freeze Fracture Experiment

* RBCs
* Freeze Fracture
* saw lots of bumps on cell
* liposomes have no proteins
* conclusions
* “bumps” → integral cell membrane proteins
* different size bumps → mosaic
* E = ectoplastic
* P = protoplasmic

Cell fusion experiment

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* conclusion: integral membrane proteins are fluid within the plane of the membrane
* fluorochromes
* mAbs

Cell patching/capping experiement

* same conclusion as cell fusion: integral membrane proteins are fluid within the plane of the membrane

FRAP experiment

* con A → binds to carbohydrate groups
* binds to all the integral membrane proteins
* only ab 50% of the proteins can move laterally in the cell membrane
* “fluid”

1972

* “fluid mosaic model”

RBC advantages

1. No dissociation (EGTA/Protease)
2. Plentiful and easy to procure
3. Pure populations
4. Few other membranes
5. Few cell membrane proteins
6. RBC – ghosts -> inside out vesicles

RBC proteins

1. Band 3 – anion exchange
2. Glycophorin
3. Spectrin – fibrous protein dimer or tetramer
a. Cytoscrobe ion protein

3 catagories of Phospholipids

1. Phosphoglycerates
2. Sphingolipids
3. Sterols

Phosphoglycerates

* Eg. Phosphatidyl serine
* Phosphatidyl choline

Sphingolipids

* Sphingomyelin -> nerve cell
* “excitable” cells
* Eg. Neurons
* Confers high resistance

Sterols

* eg. Cholesterol
* 50% is in cell membrane

How are phospholipids synthesized

\- endoplasmic reticulum

\- uses enzymes in the cytoplasm

\- inserted in the “cytosol leaflet”

\- flipped to the “lumen leaflet”

Types of Channels - fast

* ligand - gated
* voltage - gated

cell junction

* adherents junction
* desmosomes
* tight junction
* gap junction
* neurochemical synapse

Aderents junction

* adheres cells together
* E-cadherin
* CA2+ dependent molecule (EGTA + protease - Ca2+ chelator)
* cis and trans mobility
* adheres via E-cadherin - F actin (cytoskeleton)

Desmosomes

* “spot weld”
* epithelial cells
* find lots in skin
* intermediate filaments
* skin -eg. keratins
* hemidesmosomes (1/2)

Tight junction

* liquid barrier
* eg. kidney cells, bladder cells, cap endothelial cells
* apical region
* lanthanum hydroxide → TEM stain

Gap Junction = electrical junction

* pass small molecules from cell to cell
* connexon = 6 connection proteins
* two types of connexons
* rectifying = pass ions in one direction only
* non rectifying - pass ions in both directions

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Neurochemical synapse

* release of neurotransmitter
* chemical
* slower than electrical (2 msecs)
* modulate
* electrical
* faster than chemical
* little modulation

Receptors

* molecules (typically proteins) that interact with a ligand and change the behavior of the cell
* 1. transient - change the enzyme behavior - cytoplasm
* 2. longer term - nuclear genome → increase protein abundanceplas

ligand

* natural hormone or synthetic analog that binds to a receptor
* 1. antagonist → receptor is turned on
* 2. antagonist → receptor is turned off

Hydrophobic ligands

* membrane soluable
* steroid receptors
* eg. steroids, retinoids, thyroxine
* receptors → in cytoplasm

Hydrophilic ligands

* not membrane soluble
* receptor on outer membrane

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1. ligand gated ion channels


1. eg. nicotinic acetyl-choline receptor
2. Na+/K+ channel
3. super fast
2. receptor mediated endocytosis


1. 20 - 60 minutes - super slow
2. endocytosis of a needed factor

Receptors - distance traveled by ligand

1. Plasma membrane attach proteins/receptors
2. Synaptic nm distance travelled
3. Autocrine – cell releases a ligand that interacts with its own receptor


1. Cultured cells care about cell density
2. Autocrine secretion of growth factor?
4. Paracrine – nm -> um
5. Endocrine – travels through blood stream
6. Pheromones - oldest


1. organism to organism

Lipid solubility of the ligands

1. hydrophobic
2. hydrophilic
3. receptor associated kinases
4. GPCR = 6 protein coupled receptors