Cell and molec unit 1 study of cells

vocab from lectures 1-8

primordial soup

mixture of early Earth's atmospheric gases (ammonia, methane, hydrogen, CO2, N2) that may have led to the formation of organic compounds

Miller-Urey experiment

experiment that simulated early Earth conditions to demonstrate the formation of organic compounds

cell theory

concept in biology that states (1) cells are the basic unit of life and structure, and (2) all cells arise from pre-existing cells

Pasteur’s Swan-Necked Flask experiment

experiment that tested the theory of spontaneous generation and supported the idea that cells come from other cells

unicellular models

organisms consisting of a single cell (often yeast or bacteria), grown in a lab to study cells

what unicellular models are useful for

1) studying processes common to all cells
2) producing molecules, such as proteins
3) studying individual cell behaviors
4) studying specific abilities

isolated cells

mammalian cells from tissue used as models for study

what are four major macromolecules?

proteins, nucleic acids, lipids, and carbohydrates

monomers

small subunits that can join together to form larger structures known as polymers

polymers

repeating chains of subunits, the macromolecules we know

proteins

• monomer amino acids

• polymer is polypeptide/protein

• workhouse of the cell - functions as enzymes, receptors, channels, hormones

• structure consists of a central carbon, carboxyl group to the right, amino group to the left, and an R-group

amino acids

• monomer of proteins

• 20 different amino acids, each with unique properties

• amino acids bind together by creating a peptide bond between the amino group and carboxyl group

  • also known as c-terminus and n-terminus

carbohydrates

• monomer monosaccharides

• polymer is polysaccharides/carbs

• function in different sugars varies depending on their branching patterns

• three main sugars

  • glucose (mono), energy source for metabolic processes

  • cellulose (poly), structural, plant cell walls

  • glycogen/starch (poly), short term energy storage

nucleic acids

• monomer is nucleotides

• polymer is nucleic acids

• main types are DNA and RNA

  • store genetic information

• structure consists of central sugar with a phosphate group and a base (variable)

base pairs

• two types: purines and pyrimidines

  • two base pairs of the same type cannot bond together

• DNA has A, G (purines) and C, T (pyrimidines)

• RNA has A, G and C, U

• hydrogen bonds form between base pairs to hold them together

DNA

main storage of genetic information in cells

RNA

transcribed DNA, doesn’t contain introns and has many different forms (mRNA, tRNA, rRNA, etc.)

central dogma

DNA → RNA → proteins

transcription, translation

hybridization

complementary base pairing, used when studying genes and trying to detect a specific sequence

Southern Blotting

detection method used for detecting sequences of DNA. uses a probe, a single-stranded DNA sequence to detect the desired gene. probes are labeled, often with radioactive molecules. probes with hybridize with target gene if it is present

Northern Blotting

detection method used for RNA rather than DNA. needed b/c sometimes DNA probe can be complementary to a wide variety of sequences in the genome. often targets specifically mRNA, allows us to see if gene is expressed or not as well

Western Blotting

detection method used for proteins rather than nucleic acids. uses an antibody rather than a probe, proves that a specific gene is being expressed. will bind to protein of interest like probes do

restriction endonuclease

an enzyme that cuts specific portions/sites on a sequence. cuts the phosphorus bonds between nucleotides, creating sticky ends between the base pairs allowing them to then base pair elsewhere

recombinant DNA (reDNA)

fractions of DNA that have been combined together, often modified plasmids.

plasmid

piece of circular DNA that is used in bacteria to store genes. can be modified to act as a “shuttle” for specific genes scientists want to implant into cells

bacteria can take plasmids up from solutions, make copies of plasmids, and produce proteins from genes within a plasmid

preparing recombinant DNA

plasmid is cut (digested) to create space for the DNA fragment, ligase then secures these bonds to hold the fragment in place

ligase

secures bonds in recombinant DNA to hold the new DNA fragment in place

transformation

natural process during which bacteria are placed in media with the plasmid of interest, with the intent that the bacteria will take up the plasmid

selection

second step during uptake of plasmids, used to weed out non-transformants. a specific visual cue or ability to survive can be used as a way to weed out, ex: putting an anti-biotic on a plate where the plasmid contains antibiotic resistance

trypsyn

an enzyme that breaks proteins into smaller chunks, used when trying to isolate and identify a specific genetic sequence

mass spec

process used to identify individual amino acids, when repeated allows for the entire sequence of a protein to be identified

n-terminal degredation

process used to cut off the n-terminal amino acid, signaling it out so it can be researched individually

cDNA library

entire genome of an organism that can be used to help identify a desired gene, contains only the coding sequences. portions of the cDNA then placed into vectors (often plasmids) so the probe can bind to the desired gene

reverse transcriptase

uses mRNA and turns it into cDNA

phospholipid

a type of lipid that is amphipathic, has a hydrophilic head and a hydrophobic tail. makes up a cell membrane, are fluid within the membrane (can move or rotate)

cell membrane

consists of two phospholipid layers that allow for selective permeability

• composition of the membrane can affect fluidity (more unsaturated = more fluid), fluidity is beneficial under certain circumstances, such as cold weather

• asymmetric, different composition of molecules on either side of membrane

unsaturated fats

type of lipid that contains double bonds in the tail, causes there to be “kinks” or bends

saturated fats

type of lipid that lacks any double bonds in the tail, tail is therefore completely straight and doesn’t have any bends

amphipathic

term used to describe a molecule which has varying polarity (example is phospholipid)

lipid rafts

portions of the membrane that contain a higher concentration of molecules, shows that distribution of molecules throughout a membrane is NOT uniform

membrane proteins

proteins embedded in or on the edge of membranes that serve unique and important functions, such as cell signaling, channels

integral proteins

proteins embedded in the membrane, three main types: membrane associated, lipid-linked, and transmembrane (across the membrane) . have stronger bond/connection to the membrane and are more difficult to remove

peripheral proteins

proteins attached to the membrane through ionic bonds, much easier to remove than integral proteins

study of membrane proteins

gel electrophoresis cannot be used for studying integral membrane proteins. instead, put into a solution with detergent (another solution that is amphipathic), which surrounds hydrophilic portions of the membrane, allowing the hydrophobic portion to be better studied

transport proteins

transmembrane proteins that move molecules across the membrane through facilitated diffusion or active diffusion

simple diffusion

movement of molecules down or with the gradient, doesn’t require any energy. can be either passive (no protein needed) or facilitated (requires a channel protein)

active diffusion

movement of molecules against their concentration gradient, requires an input of energy (usually ATP or through co-transport)

concentration gradient

the difference in amount of a molecule on either side of a membrane, moves with the gradient if it travels to where there is a lower concentration

gated protein channels

a specific stimuli is needed, often the presence of the molecule, to trigger the channel to open

coupled transport

the transport of two molecules in the same or opposite direction, one being passive and one active, with the passive transport providing energy for the active transport (ex: glucose and sodium leaving the gut)

symport

coupled transport where both molecules are going in the same direction

antiport

couple transport where the molecules are traveling in opposite directions

endomembrane system

consists of the plasma membrane and most membrane-enclosed organelles in eukaryotes. endosomal and secretory pathways occur in the system, material (usually proteins) moves between the various locations in this system

secretory pathway

the secretion, or expulsion, of molecules through a cells membrane, sending them to their designated location in the cell

endosomal/lysosomal pathway

when cells take in large amounts of molecules to break them down for nutrients

order of the secretory pathway

rough ER → Golgi apparatus → vesicles, leaves cell

free ribosomes

ribosomes that float around in the cytosol and aren’t bound to a membrane, often relocate themselves to membrane-enclosed organelles so proteins can be translated into the organelle

n-terminal signal sequence

a signal sequence n the n-terminal end of protein that binds to the signal recognition particle, stops translation so the ribosome can move to a membrane

SRP receptor

SRP molecule binds to the receptor, which is embedded in the membrane. leads protein to translocation channel so translation can continue into the membrane

signal recognition particle (SRP)

recognizes signal sequences and has a binding site where the signal sequence fits, translation stops when the protein binds to the receptor

translocation channel

channel that offers an opening into the ER lumen from the cytosol, allows the ribosome to translate into the channel so the protein ends up on the other side of the membrane

signal peptidase

part of the membrane that cuts off the signal sequence off from the rest of the protein, cuts the peptide bond

ER lumen

site of many post-translation modifications

stop-transfer sequence

similar to the signal sequence, but is in the middle of the protein rather than on the n-terminal. stops translation halfway through to allow half of the protein to be translated on each side. used for the creation of transmembrane proteins

types of proteins that use secretory pathways

secreted proteins (hormones, signaling proteins), integral membrane proteins (channels), proteins involved in secretion - such as the SRP receptor itself

types of proteins that don’t use secretory pathways

cytoplasmic proteins, nuclear proteins, mitochondrial/chloroplast proteins

transport vesicle

“bubble” type molecule that transports other molecules between organelles

SNARE proteins

proteins that help vesicles fuse to the target membrane, wrap around each other to bring the protein closer to the membrane. creates an opening in the membrane for proteins to enter the membrane and become a transmembrane protein

constitutive secretion

secretion that is continuous, no stimuli is required for secretion of the molecules

regulated secretion

secretion that requires a signal/conditions

phagocytosis

membrane wraps around large molecules to bring it into the cell

endocytosis

membrane buds inward to allow large molecules to enter the cell

clathrin

material that coats the bubble created during endocytosis, like a “net”. supports the structure of the bubble and helps with the pulling inward during endocytosis.

things a cell might endocytose

signal factors bound to receptors, extracellular molecules, its own surface proteins/lipids

early endosome

first stage in classical pathway of endocytosis

late endosome

second stage in classical pathway of endocytosis

lysosome

final stage in classical pathway of endocytosis, breaks down materials of molecules so they can be recycled and reused in the cell

LDL

low density lipoprotein, a dietary lipid. delivered to other cells via the bloodstream. more hydrophobic

LDL binding receptor

receptor embedded in the membrane that acts like a “hook”, will grab LDL out of the bloodstream

LDL binding receptor N-terminal mutation

a mutation on the n-terminal end of the receptor, which prevents any LDL from binding to the receptor at all. mutation on the extracellular domain. mutation causes negative health effects such as elevated cholesterol and heart attacks at a young age.

LDL receptor C-terminal mutation

a mutation on the c-terminal end of the receptor, which prevents LDL from being internalized, or being brought into the cell. mutation on the intracellular domain. mutation causes negative health effects such as elevated cholesterol and heart attacks at a young age.

oligo dT column

column used during the formation of a cDNA library, filters out mRNA from the rest of RNA (rRNA, tRNA, etc.). filters using chains of thymine to catch the poly A tails on mRNA.

chromatography

sorting method used during the purification of “factor x” to split the proteins into different fractions to make analyzing them easier

proteolysis

method used once factor x has been purifying, breaks the protein into smaller chunks so n-terminal degradation and mass spec can be performed

bioassay

analytical method used to determine if a specific component, genetic sequence, or chemical is present. used w/ “factor x” to see if it is present in the fractions created by chromatography

purifying factor x sequence

obtain total proteins from mouse brain → proteins are isolated and divided using chromatography → bioassay to determine if factor x is present in each fraction

obtain purified form of factor x → use proteolysis to break the protein into smaller portions → use n-terminal degradation to break off the amino acid on the n-terminal end → use mass spec to identify which amino acid has been broke off → repeat this process to get a partial protein sequence for factor x

site directed mutagenesis

process in which rcDNA (recombinant DNA) is used to intentionally create mutations at specific points

done by inserting the incorrect base pair at one point into a plasmid, strength of the surrounding bonds holds the incorrect base pair in place. transformation occurs, leading to possible chance of the mutation staying rather than being corrected.

mutagenic oligonucleotide

a short stretch of nucleic acid which creates the mutation in the plasmid

ligand gated ion channels

example of something where point site mutations are useful as these gated channels are difficult to study, as they are embedded in the membrane.

actin filaments

• also known as microfilaments, but commonly just called actin

• mainly peripheral

  • models show it mostly on the surface

• the smallest of components present in the cytoskeleton

• stability jobs: associated with integral membrane proteins

  • specifically involved with intracellular and transmembrane proteins, helps to maintain cell structure

• movement jobs: shape changes (axon growth) or muscle contraction

  • allows muscles to move, works with myosin

  • present in the axons of neurons

bi-directional assembly of actin filaments

• has two main components: G-actin (globular) and F-actin (filamentous)

• forms a helical structure, is polar - has a clear positive and negative end

  • polymerization is possible on BOTH ends, but significantly (5-10x) faster on the positive end

  • can only depolymerize from the negative end

• three stages of formation: nucleation, elongation, and steady state

  • nucleation (formation): minimum 3 g-actin required for start of polymerization, then becomes f-actin, more actin continue to add

  • elongation (growing): the continuous addition of actin to the chain, leading to net growth, and therefor the chain getting longer

  • steady state: addition and removal of actin has no net change so the overall length of the actin remains the same (treadmilling)

treadmilling in actin formation

continuous addition and removal of actin, leading to zero net change in the length of the overall filament.

• when actin binds to the plus end, it has an ATP molecule attached

• soon after addition of a filament, hydrolysis of ATP occurs, releasing a phosphate, and the energy in the bond

• the ADP molecule remains attached to the filament

• when an actin depolymerizes, the ADP molecule goes with it, detaches, and is exchanged for an ATP molecule

• this process continues

necessary for a steady state

cofilin

protein that speeds up dissociation, or discourages polymerization

makes it more likely that ADP G-actin will fall off

profilin

protein that encourages the addition of more filament

speeds up the replacement of ATP, therefore increasing the probability of filament addition

ARP 2/3 complex

binding protein that enables the nucleation of filament into branches

myosin

molecule that helps with muscle contraction. has multiple bands (H zone, I band, A band, and Z lines)

• when muscles contract the filaments are squeezed together:

  • H zone, I band, and Z lines shorten

  • A band and myosin stay constant

• A bands contain both filaments

• I bands contain only thin filaments

• Z line is a vertical line that goes across the bands

tropomyosin

molecule that blocks the myosin binding site, preventing it from binding to actin, is like a thread that wraps around actin

• presence of calcium will overwrite tropomyosin, changing the shape and allowing myosin to bind

troponin

molecule that contains three parts, attaches to tropomyosin

dystrophin mutation

when not present muscle cells will waste away, prevents muscle contraction

• linked to muscular dystrophy

microtubule assembly

shares many similarities with the assembly of actin filaments

• consists of monomers known as tubulin, are a protein/gene

  • has two different types, alpha and beta → these come two separate molecules work together to form microtubules

• has a tube-like structure, is hollow on the inside

• like actin filaments, is polar, has a positive and negative end

  • although, loss and addition of tubulins is favored on the positive end

• balance between polymerization and depolymerization depends on the concentration of GTP and if it is bound to the tubulin

  • lots of tubulin bound to GTP → polymerization

  • less tubulin bound to GTP → depolymerization

  • dynamics overall very similar to actin filaments

  • can depolymerize at either end, unlike actin which only depolymerizes on the negative end