exam 2

Use of recombinant DNA?

-determine protein funcation

-study genome organization

-genetically modify animals

Restriction enzyme

DNA endonuclease that recognizes specific DNA sequences and preform hydrolysis of the backbone

Which are prefered? Sticky or blunt ends

Sticky , Base pairing stabilizes interaction, makes it easier to ligate

how is recombinant DNA made

cloning vector-> restriction enzyme cleaves bond→ the gene of interst is inserted and ligated → recombinant DNA

• whole process is also called molecular cloning

components of a cloning vector

Origin of replication: plasmid replicates inside bacteria, with this million of DNA is copied

Restriction enzyme: hold different cutting enzymes for DNA , where DNA of interest is inserted

Antibiotic resistance(selection method): DNA is grown on antibiotic medium, only one w/ plasmid survive and grow

each bacterium takes up __ recombinant DNA molecule to construct a genomic library

one

cDNA process

• lyse tissue or cultured cells and purify MRNA

• poly T primer is added

• DNA copy gets made with reverse transcriptase ( DNA/RNA double helix)

• RNA partially degraded withe RNase

• DNA pol synthesizes a new strand of DNA to join

Whats type of library is used for eukaryotic protien in bacteria

cDNA library, cDNA is already processed, the presence of other components would make it difficult for bacteria to express protein

Nucleic acid hybridization

• used to detect specfic DNA/RNA sequence

• Denature DNA

  • Heat → double-stranded DNA separates

• Add probe

  • Short labeled DNA/RNA sequence (complementary)

• Annealing (hybridization)

  • Probe binds ONLY to matching sequence

• Detection

  • Fluorescent/radioactive signal shows where binding occurr

• High temp → only perfect matches bind

• Low temp → mismatches can bind

In situ hybridization tell you,,,

where gene/mRNA is

RNA- seq tells you,,,

how much gene expression

reporter gene tell you,,,

when/where gene is active

GFP fusion tell you,,,

where protein goes (intercellular location)

shotgun approach

• used to determine nucleotide sequence of entire genome

• repetive sequences created problems

process: multiplie copies of genome → random breakage → a common end and start is found -> those strands are assembled

SNPs

• single difference in nucleotide

• 1 in every 1000bp

• two unrelated individuals maybe have 3×10^6 ( 3 million) nucleotide differences in their genomes

Transgenic model organisums can be used to study gene function, what are the types?

Gene replacement:Replacing a normal gene with a modified version (mutated, tagged, or altered) , used when studying effects of specific sequences ex 5bp amino acid

gene knock out: Completely inactivating (removing) a gene, tests what happens when a entire protein is not active

gene addition: Adding an extra copy or expressing a gene in a new context

You want to know which genes are actively expressed in liver cells but not brain cells. What do you use?

cDNA library or RNA-seq → only expressed genes (from mRNA) are analyzed.

what is cDNA vs Genome ibrary

Genome library : DNA fragments that represent the entire genome

cDNA:represents specific genes at specific time

You want to study regulatory regions controlling gene expression. Genomic or cDNA library? Why?

Genomic library → contains promoters and regulatory DNA (cDNA does not)

You want to visualize where a gene is expressed in an embryo. What method?

In situ hybridization

You want to determine if two DNA sequences are complementary. What principle is used?

Nucleic acid hybridization.

Why is antibiotic resistance essential in cloning vectors?

To select only bacteria that took up the plasmid

After 10 PCR cycles, how many copies theoretically exist?

2¹⁰ = 1024 copies

Why do ddNTPs terminate DNA synthesis?

No 3’ OH → no elongation

Why is NGS faster than Sanger sequencing?

Parallel sequencing of millions of fragments.But due ti shorter reads their high error

You discover a new protein but don’t know its function. What is an approach?

gene knockout ( remove gene and visualize changes) , localization (where does this act)

functions of plasma membrane

receiving info, import and export of small molecules capapctiy for movement and expansion

proteins constitute _% of plasma membrane mass

50

lipid bilayer

• main component of membranes

• consists of phospholipids

• hydrophilic head, hydrophobic tale

• double bond in tail created kink

• ampihatic (hydrophillic & hydrophobic )

the lipid bilayer is the most…

energetically favorable way for phospholipids to exist in water

heads maintain contact w/ water while tails and toward eachother and stay dry

how is membrane fluidity demonstrated?

fusion of cells → incubation→ with time the lipids mix and have lateral membrane fluidity

How is the rate of diffusion measured in cells?

FRAP: fluorescence recovery after photo bleaching

• light shines of section of cell causing photo bleaching

• use recovery of color to make conclusions on how fast cells are moving

why is membrane fluidity important?

• allows interactions between protiens

• membrane fusion and budding

• membrane repair

• etc

membrane fluidity regulated

• length of hydrocarbon tails (longer tails make a more rigid membrane)

• degree of saturation ( saturated fatty tails make a more rigid membrane)

• cholesterol (in animals cells): more rigid membrane

fatty acids can be…

saturated or unsaturated

• saturadted has no double bonds between hydrocarbons, have max hydrogens, increase rigidity

• unsaturated at least one double bond, reduce rigidity

enzymes of the ER membrane

preform scrambles

• distributes phospholipids randomly

• randomly redistributes phospholipids between both leaflets

enzymes of the golgi apparatus

preforms flippase

• distributes phospholipids aymmetric

• requires ATP

• selectively moves specific phospholipids from the outer leaflet to the inner leaflet

types of membrane protiens

• transporters and channels

• anchors

• receptors

• enzymes

transmembrane regions are formed by…

short a-helices

• single & multi pass

• proteins pass about 11-13 times

multi-pass transmembrane proteins can form

aqueous pores to allow passage of water soluble molecule across lipid bilayer

• hydrophilic side chains form aqueous pore

• hydrophobic side chains interact with phospholipid tails

eukaryotic cells are coated with sugars, why?

provide cell movement and recognition, creates proper signals for cells to go through

Why do phospholipids spontaneously form bilayers instead of random aggregates?

Because it minimizes free energy:

• Hydrophobic tails avoid water (hydrophobic effect)

• Hydrophilic heads interact with water

• Bilayer eliminates exposed hydrophobic edges → more stable than micelles for phospholipids

• Sealing into vesicles (liposomes) removes edge instability completely

Why do flat lipid bilayers tend to form closed vesicles?

hydrophobic tails are exposed to water→ energetically unfavored

Membrane bends to eliminate edges → Forms sealed compartments

If fluorescence does NOT recover after photobleaching in a FRAP experiment, what does that suggest?

• Proteins/lipids are not mobile

• Membrane may be:

  • Anchored to cytoskeleton

  • Highly rigid (e.g., lots of cholesterol or saturated fats)

  • Proteins confined to domains

👉 FRAP = measures lateral diffusion

Why are β-barrel proteins common in bacterial outer membranes?

• Form rigid, stable pores

• Allow passive diffusion of small molecules

• Ideal for transport across outer membrane