Cell Bio Lecture 19

How are transgenic plants made using recombinant DNA techniques optimized for plants?

1. Leaf discs cut out from plant

2. Leaf discs incubated with AGROBACTERIUM, which carries the recombinant plasmid

3. The wounded plant cells attract bacteria, which inject their DNA. Only cells expressing the selectable marker survive and proliferate into a callus

4. Manipulation of growth factors to form shoots, these shoots grow into adult plants W THE ENGINEERED GENE

What is Agrobacterium?

These are the bacteria that carry a recombinant PLASMID with a selectable marker and the desired gene.

Selectable marker allows for the filtering out of other cells that do not have the selectable marker

What was made to combat vitamin A deficiencies?

Development of “Golden Rice” HIGH LEVELS OF β-CAROTENE in endosperm

How can large amounts of mRNA be produced from an inserted gene?

By utilizing a PLASMID with a HIGHLY ACTIVE PROMOTER

How can we experimentally move from protein to gene?

1. A purified protein is analyzed via mass spec to obtain a partial amino acid sequence

2. Sequence is ran through DNA database, we design primers, and clone the gene by PCR from a sequenced genome

3. Gene is inserted into an expression vector which can then be used to reproduce the protein

What are cell membranes?

These are barriers that separate cells from their environments

Also in eukaryotes, they enclose organelles, preventing free mixing of molecules

What are the functions of plasma membranes?

1. Receiving information via RECEPTOR PROTEINS

2. Facilitating the import and export of small molecules via TRANSPORTERS AND CHANNEL PROTEINS

3. Provides flexibility for expansion and cell growth, MOVEMENT AND EXPANSION

What does a cell membrane consist of?

Lipid bilayer and embedded proteins

What makes up the lipid bilayer?

Phospholipids

Hydrophilic head and TWO hydrophobic tails

What is the most common phospholipid in cell membranes?

Phosphatidylcholine

• Hydrophilic head

  • Choline linked to a phosphate group

• Glycerol

  • linking head and tail

• Hydrophobic tails

  • 2 hydrocarbon tails, one with a KINK (C=C)

What characteristic do all membrane lipids share?

They are all AMPHIPATHIC

Hydrophilic + hydrophobic parts

What bonds form between polar molecules?

Electrostatic bonds and hydrogen bonds

How do nonpolar molecules interact with water?

Water molecules surround the nonpolar molecule without forming any bonds with it.

Are fat molecules hydrophobic or hydrophilic?

Highly hydrophobic, TAGS (triacylglycerols) make up fats and are nonpolar due to their hydrocarbon structure

Why do phospholipid bilayers enclose themselves?

To be more ENERGETICALLY FAVORABLE, polar sides should interact with water while the non-polar sides are shielded from the water

How does the lipid bilayer behave?

Behaves as a two-dimensional fluid, LIPID MOLECULES MOVE AROUND

How do the lipid molecules move?

• Lateral movement ← and →

• Flexion

• Rotation

• RARELY DO THEY FLIP FLOP

Which molecule is responsible for stabilizing the membrane?

CHOLESTEROL

Also amphipathic, hydroxyl group with polar heads, hydrophobic region with the tails

KEEPS MEMBRANE IN BALANCE

How does Cholesterol play a role in regulating membrane fluidity and stability at different temperatures?

• High temperatures

  • cholesterol reduces fluidity by preventing phospholipids from moving too freely

• Lower temperatures

  • prevents membrane rigidity

  • Prevents phospholipids from packing too closely together

Where does membrane assembly begin?

ER ENDOPLASMIC RETICULUM

• ER membrane

  • Cytosolic side and ER lumen side

  • Biosynthetic enzymes synthesize and insert phospholipids to cytosolic side

What does the enzyme SCRAMBLASE do?

Scramblase is responsible for the transfer of RANDOM PHOSPHOLIPIDS from one monolayer to another (cytosol to ER side

What do FLIPPASES do?

Responsible for maintaining ASYMMETRIC phospholipid distribution in cell membranes

What do flippases in the GOLGI do?

Transfer phosphatidylserine and phosphatidylethanolamine to the CYTOSOLIC MONOLAYER

How are phospholipids and glycolipids distributed asymmetrically in the lipid bilayer of the cell plasma membrane?

• Noncytosolic monolayer (Outer membrane)

  • Phosphatidylcholine

  • sphingomyelin

  • glycolipids

• Cystolic monolayer (Inner membrane)

  • Phosphatidylserine

  • phosphatidylethanolamine

  • phosphatidylinositol

What are the different functions of plasma membrane proteins?

• Transporters

  • Na pump

• Anchors

  • Integrin proteins

• Receptors

  • insulin receptors

• Enzymes

  • Cyclase

What are the different ways that membrane proteins associate with the lipid bilayer?

• Integral Proteins (Interact in some way with the inner lipids)

  • Transmembrane - spans through both ends of the bilayer

  • Monolayer-associated

  • Lipid linked

• Peripheral membrane proteins (found only on the surfaces of the bilayer)

  • protein attached

What structure does a polypeptide usually form to cross the lipid bilayer?

a-Helix structure

about 20 amino acids are needed to span the membrane

5 a-helices are needed to form a channel