Cell Bio Exam 3

In situ hybridization can reveal

when and where a gene is expressed

How can in situ hybridization can be used to locate genes on isolated chromosomes

• hybridize a probe, that is specific for the gene of interest, tagged with a fluorescence chemical or antigen

• incubate the cells in a solution containing a secondary probe or antibody linked with a reporter enzyme

in situ hybridization can also be used to study

specific gene expression

reporter genes allow specific proteins to be

tracked in living cells

most used reporter gene

GFP

goal of reporter genes

to determine which cells or where protein X gets expressed

sequence for protein X can be replaced with sequence for

reporter protein Y, GFP

promoter fusion

study gene resolution (transcriptional level)

protein fusion

study protein localization (translational fusions)

goal of reporter genes

pattern of a gene expression; reporters with various combinations of the regulatory regions associated with gene X can be constructed

GFP can be used to identify specific cells in a

living animal; join gene encoding GFP to the regulatory DNA sequences that direct the production of a particular Drosophila larva protein

GFP fluorescence at different wavelengths help revels

connections that individual cells have within a specific tissue or organs

RNAi

study of mutants can help reveal the function of a gene and/or protein; RNA interference inhibits the activity of specific genes

RNAi is involved in

RNA-induced silencing complex (RISC)

dsRNA

complementary to mRNA; dicer cuts the dsRNA into small interfering RNA (siRNAs) or microRNAs (miRNAs)

RNA induced silencing complex (RISC) contains

Argonaute proteins

RNAi is useful approach for

future gene therapy

RNAi is involved in RISC in three main processes

1) making of the small RNA

2) siRNA/miRNA join Argonaute (AGO) protein to form RISC

3) Represses expression through mRNA cleavage, degradation, and/or translational repression

1) making of the small RNA

siRNAs are processed from double stranded RNAs (dsRNAs) by Dicer/Dicer-Like enzyme

2) siRNA/miRNA joins Argonaute (AGO) protein to form RISC

siRNA/miRNA acts as a guide, directing RISC to complementarily match a target mRNA

RNAi can be programmed to silence

any nucleic acid sequence

RNA interference can be programmed to target virtually

any nucleic acid sequence for silencing

RNAi can be useful for gene therapy

FDA approved patisiran, first RNAi-based drug to treat hereditary transthyretin (hATTR) amyloidosis

Patisiran works by silencing

transthyretin (TTR) protein

targeted gene replacement

a known gene can be deleted or replaced with an altered version

steps to targeted gene replacement

1) prepare altered version of the gene

2) altered gene will replace the corresponding normal gene through homologous recombination

3) embryonic cells with the altered gene

4) after multiple breeding, you have transgenic mice

CRISPR can be used to study gene function in variety of species

system promotes the precise and rapid replacement of a target gene:

1) endonuclease Cas9 enzyme

2) guide RNA

Agrobacterium vector

agrobacterium-mediated genetic transformation is a powerful and effective method for generating transgenic plants

Agrobacterium tumefaciens inserts

foreign DNA into the genome of cells of numerous plant species

Agrobacterium binary vector system is derived from

natural tumor-inducing (Ti) plasmids

Summary of exploring gene function

• in situ hybridization can reveal when and where a gene is expressed

• report genes allow specific proteins to be tracked in living cells

• the study of mutants can help reveal the function of a gene

  • RNA interference (RNAi) inhibits the activity of specific genes

  • A known gene can be deleted or replaced with an altered version

  • genes can be edited with great precision using the bacterial CRISPR system

• Transgenic plants are also important for both cell biology and agriculture

all membrane lipids are

amphipathic

cell membranes consist of lipid bilayer in which proteins are

embedded

cell membranes act as ________ barriers

selective

all cell membranes prevent molecules on one side from freely mixing with

those on the other side

some bacteria have only the plasma membrane and no other membrane

true

in addition to the plasma membrane, eukaryotic cells also have internal membranes that enclose individual organelles

true

phosphoglycerides (most abundant)

i. glycerol backbone

ii. tails - two hydrophobic fatty acyl chains; commonly 16 or 18 carbons; can be saturated or unsaturated

iii. head - 4 major head groups: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylinositol (PI)

plasmalogens

one fatty acyl chain attached to glycerol by an ester linkage and one attached by an ether linkage

sphingolipids (derivative of sphingosine —> amino alcohol with long hydrocarbon chain)

i. fatty acyl chains connected by amide bond

ii. sphingomyelins (SM) —> contain a phosphocholine head

iii. others contain a sugar or oligosaccharide head —> glycolipids

ex) glucosylcerebroside (GlcCer) has a glucose heat group

sterols

membrane components

i. animals (cholesterol)

ii. fungi (ergosterol)

iii. plants (stigmasterol)

structure

i. head group —> single polar hydroxyl

ii. tail —> conjugated four ring hydrocarbon and short hydrocarbon chain

typical bio membrane composed of three classes of ampipathic lipids

phosphoglycerides, sphingolipids, and sterols

membrane lipids form _______ in water

bilayers

biological membranes:

i. vary in lipid composition

ii. impermeable to water soluble molecules and ions

iii. have viscous consistency with fluidlike properties

membrane lipids will spontaneously form ________

liposomes

phospholipid bilayers spontaneously close in on themselves to form _______

sealed compartments; avoids the exposure of the hydrophobic hydrocarbon tails to water, which would be energetically unfavorable

plasma membrane: single bilayer

cystolic and exoplasmic leaflets

vesicles and some organelles: single bilayer

internal aqueous space is equivalent to the outside of the cell

nucleus, mitochondria, and chloroplast organelles: two bilayers

enclosed by two membranes separated by a small intermembrane space

phosphatidylcholine is the most common phospholipid in cell membranes; has five parts

i. hydrophilic head, consists of choline linked to a phosphate group

ii. two hydrocarbon chains which form the hydrophobic tails

iii. glycerol which links the head to the tail

.lipid bilayer formation and study of phospholipid bilayers

a) treatment with organic solvent mix of chloroform and methanol (selectively solubilizes the phospholipids and cholesterol)

b) mechanical dispersal of extract in water (lipids spontaneously form liposomes)

c) planar bilayer formation over a small hole in a partition separating two aqueous phases (used to study permeability to solutes)

cholesterol tends to stiffen cell membranes

fluidity of bilayer depends on its composition:

i. eukaryotes, cholesterol fits into the gaps between phospholipid molecules in a lipid bilayer

ii. prokaryotes, the degree of fatty acid saturation determines membrane fluidity

phospholipid synthesis in the ER membrane

1) two fatty acids synthesized on fatty acyl CoA; hydrocarbon tails anchor the molecule to the membrane

2) phophatase converts phosphatidic acid into diacylglycerol

3) phosphotransferase transfers a polar head group

4) transport proteins (flippase and floppase) use ATP energy to catalyze movement of phospholipids from the cytosolic leaflet to the exoplasmic leaflet or vise versa

in eukaryotes, membrane lipids formed at cytoplasmic leaflet of

ER membrane

Golgi sends lipids, in vesicles, to the plasma membrane and lysosomes

flippases in the golgi move lipids from cytosol side to the luminal side of the membrane or the outer leaflet of bacteria

flippases and floppases maintain the

asymmetric distribution of phospholipids

phosphatidylserine and phophatidylethanolamine flipped to

cytosolic side

phosphatidylcholine and sphingomyelin will remain

in non-cytosolic monolayer

membranes retain their orientation during transfer between cell compartments

membranes are transported by a process of vesicle budding and fusing; vesicles bud from golgi and fuse with plasma membrane

cytosolic face will always face cytosol and non-cytosolic face will always face extracellular space or lumen space T or F

T

flippase

in the golgi and plasma membrane; outer —> inner; requires ATP; specific to PS and PE; maintains asymmetry

floppases

in golgi and plasma membrane; inner —> outer; requires ATP; specific; export lipids

scramblase

in ER, PM, and golgi; bidirectional movement; doesn’t require ATP; non specific; disrupts asymmetry

Na+ pump

transporter; actively pumps Na out of cells and K in

K+ leak channel

ion channel; allows K ions to leave cells thereby influencing cell excitability

integrins

anchors; link intracellular actin filaments to extracellular matrix proteins

platelet-derived growth factor

receptor; binds extracellular PDGF and generates intracellular signals that direct the cell to grow and divide

adenyly cyclase

enzyme; catalyzes the production of the small intracellular signaling molecule cyclic AMP in response to extracellular signals

membrane protein synthesis beings on _________

cytosolic ribosomes

proteins destiend for membrane are inserted directly or by help of _____

SRP

ribosome “________” by SRP resume translating after encountering ______ in the membrane

paralyzed; FtsY

membranes proteins associate with the lipid bilayer in many different ways

transmembrane proteins extend across the bilayer as a single or multiple alpha helices or a beta barrels; membrane proteins are either anchored by amphipathic alpha helices or attached to either lipid or membrane proteins

a polypeptide chain usualyl crosses the lipid bilayer as a

alpha helix

a transmembrane hydrophilic pore can be formed by multiple amphipathic alpha helices

i. integral membrane protein found inside the membrane are hydrophobic

ii. those that are exposed to the cytoplasm or extracellular fluid tend to be hydrophilic

iii. bacteriorhodopsin acts as proton pump; polypeptide chain of this protein crosses the lipid bilayer in 7 alpha helices

integral membrane proteins found inside the membrane are formed by

hydrophobic amino acids

non polar amino acids

glycine, alanine, valine, cysteine, proline, leucine, isoleucine, methionine, tryptophan, phenylalanine

polar amino acids

serine, threonine, tyrosine, asparagine, glutamine

+positive charge amino acids

lysine, arginine, histidine

-negative charged amino acids

aspartic acid, glutamic acid

integral membrane proteins that are exposed to the cytoplasm or extracellular fluid tend to be formed by

hydrophilic amino acids

membrane proteins can be solubilized in detergents

• detergent disrupts lipid bilayer and interacts with membrane spanning hydrophobic portion of the proteins

• actions bring the proteins into solution as protein-detergent complexes

strong ionic detergents like SDS not only displace lipid molecules from proteins but also unfold the proteins, HOW?

binding strongly to the polypeptide backbone through its hydrophobic tail

plasma membrane reinforced by underlying cell cortex

spectrin dimers are linked end-to-end to form longer tetramers; this network is attached to the plasma membrane by attachment proteins and transmembrane proteins

mutations is spectrin cause hereditary defects of the erythrocyte, including

elliptocytosis and sphereocytosis

a cell can restrict the movement of its membrane proteins

proteins can be tethered and restricted:

a) to the cell cortex inside the cell

b) to extracellular matrix molecules outside the cell

c) to proteins on the surface of another cell

d) diffusion barriers

membrane proteins are restricted to particular domains of the plasms membrane of epithelial cells in the gut

true

the recognition of cell-surface carbohydrates on neutrophils by membrane proteins allows these immune cells to migrate out of the blood and into infected tissues

specialized transmembrane proteins (selectins) recognize particular sugar groups on the surface of neutrophils circulating in the blood; the neutrophils stick to the endothelial cells that line the blood vessel wall; neutrophils slip between the endothelial cells, into the tissues at the site of infection

each cell membrane has its own characteristic set of transporters

transporters bind solutes and undergo conformational changes, moving molecules slowly

channels form open hydrophilic pores allowing

rapid, passive diffusion of ions

lipid bilayers are _____________ and most uncharged polar molecules

impermeable to ions

transporters and pumps

mainly transport solutes

channels mainly transport

ions

solutes cross membranes either

passively or actively (only carried out by transporters called pumps)

some small, nonpolar molecules like CO2 can move

passively down their concentration gradient

concentration differences of ions across a cell membrane also create a

membrane potential

both the __________________ and the __________________ influence the passive transport of charged solutes

concentration gradient and membrane potential (referred to as electrochemical gradient)

water moves across cell membranes down its concentration gradient

osmosis

cells use different tactices to avoid osmotic swelling

a) amoeba accumulates water in contractile vacuoles and expel it periodically

b) plant cells use their hard covering, also true of bacteria

c) animal cells pumping out ions

passive transporters move a solute along its electrochemical gradient

conformational changes in a transporter mediate the passive transport of a solute (like glucose)

pumps actively transport a solute against its electrochemical gradient

coupled to a source of metabolic energy: ATP hydrolysis, ion gradient or light energy