BIOL 2003 UMN

Explain how CRISPR is used to alter gene sequences

·       RNA made of tracrRNA and crRNA creates sgRNA

·       Spacer region of sgRNA complimentary matches DNA of gene

·       sgRNA bind to Cas9, brings it to target gene, Cas9 breaks double stranded gene

·       2 different ways to repair: ends join but small deletion OR if homologous DNA is added then mutation can be put into gene

Describe the method of dideoxy DNA sequencing

* fluorescently labeled ddNTPs are used
* a fluorescence detector measures the 4 different types of ddNTPs as they emerge from the gel

List three approaches to identify a drug that alters a protein’s function

* Test a ton of drugs (grocery store approach
* See if other proteins that have a similar structure are already known to react with a certain drug and test it
* If 3d structure is known, on a computer you can predict if certain compounds/drugs will bind to it

Distinguish light and electron microscopy

* Light: image is produced with beam of light, less resolution, less magnification
* Electron: image is produced with beam of electrons, higher resolution, higher magnification

Define resolution

being able to observe two things next to each other and realize they are distinct

Define contrast

differentiating one structure from another

Define magnification

ratio of the actual size of an object and the size of a picture produced by a microscope

Explain how fluorescence microscopy is used to identify particular cellular organelles and proteins

* Component of interest is marked with a fluorescent molecule
* The molecule absorbs light at a particular wavelength range and then emits a longer wavelength range
* Particular cellular organelles and proteins can then be observed under a fluorescence microscope

Describe how antibodies are used to label specific proteins

recognize short specific amino acid sequences in proteins

Describe how GFP (green-fluorescence protein) are used to label specific proteins

next to coding sequence of protein, insert DNA sequence that encodes GFP, and when gene is expressed it creates a fusion protein

Explain how subcellular cell fractionation is carried out

* Sample of cells are broken apart
* Parts of cells are separated and put into tubes via centrifugation or chromatography
* Able to separate due to different densities of different components

Describe the methods of Western blotting to identify a specific protein within a mixture of proteins

a sample of a “fraction” is put into a well on op of a gel matrix, then the sample is coated in SDS, then it is put into a electric field where proteins move to the bottom, this produces bands based on the particular protein and its mass, the proteins are then blotted into a thin membrane, the proteins are exposed to primary and secondary antibodies, if dark band is present then the fraction contains protein of interest

Describe the methods of immunoprecipitation to identify a specific protein within a mixture of proteins

a sample of a fraction is exposed to a primary antibody which recognizes gene of interest, protein AG is like a secondary antibody and binds to the primary antibody and has agarose beads attached to it, low speed centrifugation, the protein of interest is in the pellet while other proteins are in the supernatant, sample is exposed to agent and gets released from primary antibody, the sample is then subjected to SDS-PAGE and gets stained, if protein of interest is present dark band will show

three tenets of the cell theory

* All living organisms are composed of one or more cells.
* Cells are the smallest units of life
* New cells come only from pre-existing cells by cell division

Ribosomes

synthesize polypeptides

Cytoplasm

site of metabolism ( includes cytosol)

Pili

allow bacteria to attach to surfaces

Flagella

allow bacteria to swim

Nucleoid

where DNA is found

Plasma membrane

encloses cytoplasm

Cell wall

support and protection

Glycocalyx

outer gelatinous covering

Chromatin

DNA and proteins

Nucleolus

site for ribosome assembly

Nuclear envelope

double membrane that encloses nucleus

Nuclear pore

passageway for molecules in and out of nucleus

Nucleus

where genetic material is organized and expressed

Centrosome

where microtubules grow and centrioles are found

Rough ER

protein sorting and secretion

Smooth ER

detoxification and lipid synthesis

Mitochondrion

ATP synthesis

Cytoskeleton

protein filaments that provide shape and help with movement

Peroxisome

where H2O2 and other harmful molecules are broken down

Golgi

modification, sorting, and secretion of lipids and proteins

Cytosol

site of many metabolic pathways (fluid)

Plasma membrane

controls movement of substances in and out of cell and site of cell signaling

Ribosome

site of polypeptide synthesis

Lysosome

where macromolecules get degraded

Microtubules

cell shape, organization of organelles, chromosome sorting

Intermediate filaments

cell shape, provide cell mechanical strength, anchorage

Actin filament

cell shape, cell strength, muscle contraction, movement of cargo

Peroxisome

where H2O2 and other harmful molecules are broken down

Vacuole

storage or waste removal

ER

calcium storage, protein synthesis, lipid metabolism

Mitochondria

make ATP, semiautonomous

Chloroplast

carry out photosynthesis, semiautonomous

Explain how the proteome is responsible for cell specialization

Even though DNA is identical in all cells different proteomes make it so only certain particular sets of genes are expressed in certain cells

significance of surface area-to-volume ratio

\-The larger the cell, the smaller the ratio, the less nutrients and waste export

\-Want smaller cell, and larger ratio

List the steps of the pulse chase assay and describe how this technique can be used to follow the sorting of proteins through a cell

o   Radioactive amino acid injected, and then nonlabelled leucine is injected (called chase)

o   at different times remove samples of pancreatic cells

o   stain samples with osmium tetroxide (heavy metal that sticks to membranes)

o   cut samples into thin slices and add layer of radiation sensitive emulsion

o   examine under transmission electron microscope

-by removing samples at different times you can see how the proteins are in various locations at times and can “follow” them

Explain endosymbiotic theory as it relates to the origin of mitochondria

lived inside a-proteobacteria host cell, then evolved and became its own organelle

Explain endosymbiotic theory as it relates to the origin of chloroplasts

lived inside cyanobacteria host cell, then evolved and became its own organelle

three pathways for protein sorting in a eukaryotic cell

* Remain in cytosol
* Sort while being made into proteins (cotranslational)
* Sort after being made into proteins (post translational)

Describe the fluid-mosaic model of membranes

• Mosaic because many different things: lipids, carbs, protein molecules

• Fluid because proteins and lipids can move within the membrane

peripheral membrane proteins

o Transmembrane: proteins that are physically embedded in the hydrophobic portion of phospholipid bilayer

o Lipid-anchored: proteins that have an amino acid that is covalently bound to a lipid

integral membrane proteins

noncovalently bound to polar heads of phospholipids or to integral membrane proteins that stick out from the membrane

Explain how lipid composition affects membrane fluidity.

shorter fatty acyl tails are less likely to interact and make membrane more fluid, more double bonds create kinks in the fatty acyl tails and make neighboring tails harder to interact with making the membrane more fluid

Explain how temperature affects membrane fluidity.

more or less cholesterol, which affects the stability of membranes

Describe the movements of lipids within a membrane

quick because of membrane fluidity, lateral diffusion

Flippase

aids movement of phospholipids between 2 leaflets that compose cell’s membrane

how the Frye and Edidin experiment demonstrated the lateral mobility of membrane proteins

Fused mouse and human cells, lowered temperature, added fluorescent labeled antibody in mouse in protein in plasma membrane which can’t move laterally. At 0C cell unable to move laterally, at 37C, cell can move laterally

how some membrane proteins are restricted in their mobility

because they are located in the membrane, are bound to cytoskeleton, or attached to molecules outside of the cell

Describe how phospholipids are synthesized in eukaryotic cells

enzymes in cytosol or cells from food make fatty acid building blocks, then on smooth ER it is synthesized

Describe how membrane proteins are synthesized in eukaryotic cells

synthesis begins in ER, hydrophobic transmembrane segment is made, protein remains in membrane

Describe the benefits of glycosylation

helps protect cells, plays a role in cell surface recognition

how glycosylation occurs in eukaryotic cells

when a carb is covalently bonded to a protein or lipid

glycolipid

carb to lipid

glycoprotein

carb to protein

Passive transport

requires no energy, travels with a solute gradient/down

Simple diffusion

diffusion of a solute through membrane without transport protein

Facilitated diffusion

diffusion of a solute through a membrane with the help of a transport protein

Active transport

requires energy, travels against solute gradient/up

Describe the relative permeability of solutes to move across a phospholipid bilayer

• High permeability: gases, tiny uncharged molecules

• Moderate permeability: water, urea

• Low permeability: polar organic molecules

• Very low permeability: ions, charged polar molecules

Concentration gradient

difference in concentration of a substance from one point to another

Ion electrochemical gradient

determines the direction that ions will flow through an open ion channel

Osmosis

movement of water through a membrane to create equilibrium (goes to where there is less water)

Channel

form an open passageway for direct diffusion of ions/molecules across membrane, most are gated

Transporter

aka carriers, conformational change switches the access of the solute from one side of the membrane to the other

Explain how the methods and results of Agre demonstrated the presence of a water channel in the plasma membrane of certain cells

• Identified a protein that was more abundant in red blood cells, bladder and kidney cells,

• Water can passively diffuse, some cell types allow water to move across the membrane much faster than predicted

Uniporter

a single solute (ion/molecule) moves in one direction

Symporter

2 solutes move in the same direction

Antiporter

2 solutes (or more) move in the opposite direction

Primary active transport

uses a pump, pump directly uses energy to transport solute

Secondary active transport

uses a pre existing gradient to drive transport

structure and function of the Na+, K+-ATPase

• Structure: transmembrane protein

• Function: actively transports Na+ and K+ against their gradients using energy from ATP hydrolysis, helps maintain osmotic equilibrium

diagram the steps it goes through to pump ions across a membrane (Na+, K+-ATPase)

3 Na+ bind from cytosol, ATP is hydrolyzed, ADP is released, phosphate (P) covalently binds and switches conformation to E2, 3 Na+ released outside cell, 2 K+ bind from outside of cell, P is released and pump switches back to E1, 2 K+ are released in cytosol.

Exocytosis

material inside cell is packaged into vesicles and excreted into the extracellular medium

Receptor mediated endocytosis

a solute binds to a receptor in plasma membrane and taken into a cell, plasma membrane invaginates (folds inward) to form a vesicle that brings substances into cell

energy

Ability to promote change/do work

types of energy

kinetic and potential

Chemical potential energy

when bonds are broken or rearranged, energy can be released and used to do work

two laws of thermodynamics.

• Energy cannot be created or destroyed

• Transfer of energy from one form to another increases the entropy of a system

Free energy

amount of energy available to do work

Entropy

unusable energy/disorder

Exergonic

energy release

Endergonic

energy input

Coupled reaction

endergonic and exergonic reaction joined together

relationship between exergonic and endergonic reactions and ATP

ATP drives endergonic reactions because it needs the input of energy. The energy to synthesize ATP comes from endergonic reactions

why do cells need ATP

Cells need ATP so that reactions can occur.

enzyme

Protein catalysts in living cells

describe how enzymes lower the activation energy for a chemical reaction

Enzymes strains/brings close together chemical bonds in the reactant molecules to make it easier to achieve a transition state, position reactants together to facilitate bonding, and change the local environment