BIO-333 Quiz 2
Method used to make copies of DNA sequences and separate a gene of interest from its entire genome.
Why is DNA cloning important/useful?
Scientists can now use a small amount of DNA to make millions of copies.
An enzyme that cuts/cleaves a short, specific nucleotide. They "restrict" the transfer of DNA between strains of bacteria. This a way to protect the cell from invaders.
Why are restriction enzymes useful in lab?
Each enzyme will cut a particular DNA molecule at the same sites. Thus for a given sample of DNA, a particular restriction enzyme will reliably generate the same set of DNA fragments.
How is bacteria's own DNA protected from restriction enzymes?
Bacteria prevent eating away their own DNA by masking the restriction sites with methyl groups ( CH3 ). Methylation of DNA is a common way to modify DNA function and bacterial DNA is highly methylated.
True or False: Only foreign DNA can be cut up.
False. Any foreign DNA or RNA can be cleaved by restriction enzymes.
Restriction enzyme HaeIII characteristics
- Cuts blunt end
- Palindromic (meaning: symmetrical around central point); cut both strands of double-stranded DNA/RNA
Restriction enzyme EcoRI characteristics
- Staggered/over-hang cut
- Staggered cuts have sticky ends
- Over-hang/sticky ends allows for easier hybridization
Small, circular DNA molecule that replicates independently of genome. Used as a vector for DNA cloning.
Cells take up DNA molecules from their surroundings and express genes present on that DNA.
- DNA fragments spread across gel according to size
- DNA fragments are negatively charged
- Fragments move down towards positive electrode
- Larger fragments move more slowly
Inserting the DNA fragments into a carrier, or vector. Because this union involves “recombining” DNA from different sources
Another piece of DNA that can be copied inside cells. Delivers random DNA fragment to new organism.
A collection of cloned DNA molecules that represents the entire genome of a cell.
Experimental technique in which two complementary nucleic acid strands come together and form hydrogen bonds to produce a double helix; used to detect specific nucleotide sequences in either DNA or RNA.
Steps of PCR
1. Heat to separate strands (melt hydrogen bonds)
2. Cool to anneal primers (1 forward, 1 backward)
3. DNA synthesis
Enzyme used in PCR
Sanger sequencing (Dideoxy sequencing)
Technique that uses DNA polymerase, along with special chain-terminating nucleotides called dideoxyribonucleoside triphosphates to make partial copies of the DNA fragment to be sequenced. Dideoxy sequencing reactions ultimately produce a collection of different DNA copies that terminate at every position in the original DNA sequence.
3' OH allows strand extension at 3' end
3' H prevents extension at 3' end
Fluorescent probe advantage
Allows identification of location of particular gene
Whenever active, studies expression/ activity pattern
- Uses fluorescent or enzymatic activity
What happens after ddNTP is added?
The chain no longer grows due to prevention of addition at 3' end.
Defense mechanism that bacteria use to attack/disrupt invading viral DNA (fight infection)
Used to identify target sequence
Cleaves DNA/makes double-stranded break
Protein-studded, fatty film so thin that it cannot be seen directly in the light microscope. Every cell on Earth uses such a membrane to separate and protect its chemical components from the outside environment. Without membranes, there would be no cells, and thus no life.
Advantages of cell membrane
- Act as selective barriers
- Separates cell from surroundings
- Self-healing (quickly reseals if punctured)
Eukaryotic membrane vs Bacteria
Euk: double membrane
How can the cell membrane transmit information?
By information going directly into membrane (Telling in person) OR by using a receptor (Talking via cellphone)
What organisms have membrane-bound organelles?
Why are lipids in the lipid bilayer called amphipathic?
Hydrophilic head and hydrophobic tail
Phospholipid head characteristics
- Dissolve readily in water
- Can form hydrogen bond with water
Phospholipid tail characteristics
- 2 tails
- 1 tail is saturated (no double bond); 1 tail is unsaturated (double bond)
- "Fears" water
- Forms free-energy cage-like structure around water
Significance of unsaturated chain in phospholipid tail
Double-bond causes a kink, which increases the fluidity because the molecules will be more spread apart and molecules can get in easier.
How does cholesterol affect fluidity?
Fills the gaps between phospholipid molecules, thus creating a stiffness and decreasing fluidity
Where does membrane assembly occur?
In the endoplasmic reticulum
Why does the hydrophobic cage-like structure require free energy but hydrophilic interaction does not?
Think about it:
It requires less effort to messy up a room, but takes more effort to clean.
Therefore, it takes less effort for the heads to dissolve in water, but it takes more effort to form a more ordered (clean) structure around the water
How do lipid molecules move?
Within their own monolayer- because they are two-dimensional
Why is "flip-flopping" rare?
Because it requires an insane amount of energy whereas lateral diffusion does not, so it can more readily happen and faster.
What happens to lectin production in the case of an injury or wound?
More lectin is produced
Lectins are known to play important roles in the innate immune system. Lectins such as the mannose-binding lectin, help mediate the first-line defense against invading microorganisms. Other immune lectins play a role in self-nonself discrimination and they likely modulate inflammatory and autoreactive processes.
Short chains of sugars attached to proteins in plasma membrane. Cell recognition and cell binding
Comprise of protein and carbohydrate chains that are involved in many physiological functions including immunity. Many viruses have glycoproteins that help them enter bodily cells, but can also serve to be important therapeutic or preventative targets.
Provides hydration and swelling pressure to the tissue enabling it to withstand compressional forces.
Which types of molecules most easily pass through membrane?
Small, nonpolar molecules
Why types of molecules have the most trouble passing through membrane?
Ions (can't pass through)
- High to low
- Higher concentration on outside (want to go where less concentration)
- No energy required
- Along gradient (transports more readily)
- ATP required
- Against gradient
- Pumps used
- Higher concentration on inside (low to high)
Ligand-gated ion channel
Opens/closes in response to a chemical messenger/stimulus (ex: neurotransmitter)
Opens due to physical distortion of a cell membrane (ex: pressure on skin)
Opened/closed by membrane potential (ex: Na+/K+ pump)
How does K+ move through membrane?
Inside to outside (high concentration to low concentration)
How does Na+ move through membrane?
Outside to inside (high concentration to low concentration)
Difference in voltage
Voltage + Concentration
For every Na+ in, how many k+ come out?
Pump moves solutes (2) in same direction across membrane
Pump moves solutes (2) in opposite directions across membrane
Transporter moves only one type of solute across membrane
Na+/K+ Pump: Concentrations in cytsol
K+ is high; Na+ is low
Pump used by muscle cells
- Na+ enters cytosolic side
- ATP is cleaved, creating a P group
- Binding sites exposed
- K+ binding triggers release of the P (phosphate) group
True or False: Action potential size always changes
False. It never changes. it's all or nothing.
How does electric impulse move to another neuron?
Electric impulses can't. They have to first convert to a chemical signal in order to move to the other nerve.
3 mechanisms for protein transport into organelles
- Nuclear pores (need signal)
- Across membrane
- Via vesicles (exports)
How is transport across membrane by translocators different than moving through nuclear pores and via vesicles?
Unlike the transport through nuclear pores, the transported protein must usually unfold for the translocator to guide it across the hydrophic interior of the membrane. They remain folded for the other two mechanisms
Why do proteins have to unfold to enter mitochondria and chloroplast?
Unfolding allows for the signal sequence to be exposed and cleaved off by signal peptidase. The protein cannot get in between the translocators if it is folded; it wouldn't fit.
What happens to proteins that fail to fold back after being unfolded?
They won't be able to function. Chaperone proteins bind and try to help the proteins fold back correctly.
So, if they aren't able to retain their original shape, chaperone proteins escort them to transporters where they will be delivered to the cytosol to be degraded.
Mediate contact between receptors and clathrin molecules
Act to shape the vesicle by forming at the plasma membrane
- 3-legged structures (3 heavy chains and 3 light chains)
Forms a ring around the neck of the clathrin-coated pit and causes the neck to constrict and pinch off its parent membrane