Midterm 1

what is a cell

the basic structural, functional, and biological units of life

three things all cells share

- DNA as genetic material
- Surrounded by plasma membranes
- Similar mechanisms for metabolism and energy production

where did cells emerge from

A single primordial cell

Primordial soup theory

Theory that states early life was created by atmospheric reactions that formed amino acids

Stanley experiment on primordial soup theory

- Demonstrated the synthesis of organic molecules providing basic material (aka the original conditions)
- The biggest thing that was created were amino acids

Altman and Cech experiment on primordial soup theory

- Discovered that RNA can serve as a template to catalyze its own self replication
- As a bonus it also catalyzes several other reactions

The critical characteristic of the macromolecule from which life evolved

ability to self replicate

evolution of metabolism

anaerobic glycolysis --> photosynthesis --> cellular respiration

anaerobic glycolysis

- Occurs in the cytoplasm
- It's the first mechanism of metabolism but generates little atp

photosynthesis

- occurs in the chloroplasts

- came after anaerobic glycolysis

- still didn't generate a loot of atp but was an improvement

- created oxygen

Oxidative metabolism (cellular respiration)

- Upgrade from photosynthesis
- Uses the oxygen that is now present and creates a loot of ATP (their main source of metabolic energy)
- Has three main processes (that we need to know)
1)Glycolysis
- Happens in the cytoplasm
2/3)Citric cycle and oxidative phosphorylation
- Happens in the mitochondria

Endosymbiotic theory

theory that eukaryotic cells formed from a symbiosis among several different prokaryotic organisms

- Mitochondria and chloroplasts were originated by endosymbiotic events (they once were or came from a prokaryotic cell)

Carbohydrates (sugars)

simple sugars

- monosaccharides

- disaccharides

- polysaccharides

what type of carbohydrate is glucose

monosaccharide

glucose

the principal source of cellular energy

3 polysaccharides

starch, glycogen, and cellulose

starch

storage of glucose in plant cells

glycogen

storage of glucose in animal cells

cellulose

stable, good for structural components, found in plants cell wall

type of bond between two carbohydrates

glycosidic bond

three main roles of lipids

1. Energy storage

2. Major components of cell membranes

3. Role in cell signaling

fatty acids

hydrocarbon chain with carboxyl group at one end

saturated fatty acids

fatty acids with no double bonds

unsaturated fatty acids

fatty acids containing double bonds

triglycerides

backbone (glycerol) + 3 fatty acids

phospholipids

glycerol + 2 fatty acids + phosphate group

only phospholipid with a non glycerol group

sphingomyelin

where are phospholipids commonly found? what special property do they give this structure?

- cellular membranes (eg. phospholipid bilayer)

- amphipathic properties

glycolipids

two hydrocarbon chains + polar head group w/ carbs

cholesterol

amphipathic

- serve as signaling molecules

testosterone and other steroid hormones are derived from...

cholesterol?

type of bond between lipids

ester bond

nucleic acids

polymers specialized for the storage transmission and expression of genetic information

- DNA

- RNA

nucleotides

the monomers that make up nucleic acids

- Pentose sugar

• Ribose or deoxyribose (OH at bottom/no OH at bottom)

- Phosphate group(s)

- Nitrogen-containing base

• DNA: atgc

• RNA: augc

how are nitrogen bases paired?

complementary base pairing

important roles of nucleotides other than DNA/RNA

cell signaling, working as ATP, etc.

bond between nucleotides

phosphodiester bond

proteins

chains of amino acids

components of an amino acid

- amino group

- carboxyl group

- R group (side chain)

which side of a polypeptide chain is the N terminus and which is the C terminus?

N: amino end

C: carboxyl group end

4 categories of amino acids

1. acidic with carboxyl

2. charged basic

3. nonpolar

4. uncharged but polar

primary protein structure

the sequence of amino acids forming a polypeptide chain

secondary protein structure

Polypeptides form alpha helices and beta pleated sheets

tertiary protein structure

- Bending and folding results in a macromolecule with specific 3D shape

- structure is determined by interactions between R groups

- creation of domains

domains

- they give the specific function that the protein will have

- usually hydrophilic aa side chains will remain on the outside and hydrophobic aa side chains will localize in the center

quaternary protein structure

Two or more separate polypeptide chains coming together and interacting

regulation of proteins

allosteric regulation and post-translational modification

allosteric regulation

Inhibitor binds to protein → substrate can't bind → protein inactive

post-translational modification

Protein is phosphorylated (or undergoes other chemical changes) so it will no longer have its functions

in vitro - cell culture

Growing specific cells on an isolated surface

in vitro - organotypic culture

3D cultures that mimic the environment of the cells while dividing

2 ways to obtain cells for in vitro

- primary cells

- cell lines

in vitro - primary cells

- cells come directly from the organism

- these cells will eventually stop dividing like all cells are supposed to

in vitro - cell lines

Cell with mutations that allow them to duplicate constantly

• Note: these cells aren't "healthy" cells so they won't exactly mimic primary cells

light microscopy

Uses visible light to illuminate an object and we can magnify with lenses to see it

- If tissues are alive they will be transparent (use contrast or staining)

fluorescence microscopy

use proteins with fluorescence to see the life cells

super resolution fluorescence microscopy

gives increased optical resolution of where the location of the fluorescence is than the normal fluorescence microscope

types of electron microscopes

- scanning electron microscope (SEM)

- transmission electron microscope (TEM)

scanning electron microscope (SEM)

The electrons are scattered and don't pass through the specimen

• We see a 3D image of the surface of the specimen as the electron beam moves

transmission electron microscope (TEM)

Uses short electron waves that can pass through the cells (or object)

• Allows us to see the inside structures

flow cytrometry

Cells pass one by one through cytometer and light is passed through each cell

- Light will be scattered in different directions and it lets us understand size and complexity

methods to detect proteins

- immunolabelling

- immunoprecipitation

- subcellular fractionation

- electropheresis (also for nucleic acids)

- immunoblotting

- mass spectrometry

protein immunodetection (immunolabelling)

use of antibody techniques to identify the location of proteins

direct immunofluorescence

Uses primary antibodies that will attach to the protein of interest with fluorescence

indirect immunofluorescence

Uses first primary antibody but then adds fluorescent secondary antibodies that attach to the primary antibody

- This will produce more fluorescence signal than the direct method (if using fluorescence microscopy)

- We can also use the secondary antibody batch to recognize the same primary antibody in different specimens

immunoprecipitation

the use of antibodies to cause other molecules, such as proteins, to precipitate, which allows them to be collected by centrifugation

1) You have a mixture that contains your protein

2) Use an antibody against that protein

- The antibody is bound to beads for easy collection?

- The antibody will attach to only that protein in the mixture

3) Collect antigen-antibody beads

4) Release the protein you wanted

subcellular fractionation

Done to isolate specific components of the cell often through centrifugation

methods to further purify subcellular fractionation results

- Velocity centrifugation

- Equilibrium centrifugation

electropheresis

DNA, RNA, or protein fragments are separated according to their size using an electric current

if band A is further than band B in electropheresis then the molecules in band A are (smaller/larger) than the molecules in band B

smaller

immunobloting (western blot)

Detects proteins in a sample of cellular extract (takes place after electrophoresis)

- after extraction of protein, use immunolabelling to visualize the protein

mass spectrometry

Identifying the proteins by their mass

methods to detect nucleic acids

- in situ hybridization

- restriction endonucleases

- PCR

- RT-PCR

- sequencing

- transcriptomics

- recombinant DNA

- electropheresis

in situ hybridization

a molecular technology technique that uses probes (the DNA/RNA sequence) to recognize sequences

- Hybridization of fluorescent probes (FISH) to specific cells can be analyzed with fluorescent microscopy

restriction endonucleases

Cleaving DNA molecules at unique sites using an enzyme found in bacteria

- We can then use these enzymes (endonucleases) to cleave the DNA

polymerase chain reaction (PCR)

Amplifies a single or a few segments of DNA

1) Denature

- Breaks the H bonds and opens up the double helix

- Increase of temperature

2) Annealing

- Insertion of primers to base pair

- Decrease of temperature

3) Extension

- Providing new nucleotides to grow the DNA

real time PCR (RT-PCR)

Follow the amount of DNA being duplicated during PCR in real time

- During step 3, fluorescence is added

- The more DNA means more fluorescence

- There is usually a threshold before you can properly see the fluorescence

sequencing

Process to determine the precise order of nucleotides

- Uses chain-termination dideoxynucleotides to create small stands of the DNA

- After, they go through electrophoresis and a machine reads the termination nucleotides

- Each will emit a different color

- We use the color to figure out the DNA sequence

next generation sequencing

The DNA molecules will go through PCR, hence amplifying each molecule, then will follow the sequencing process

- used for rapid/increased DNA sequencing

transcriptomics

Converts RNA sequences into DNA sequences

- This is useful in the case of doing PCR

- To do RNA PCR you need it to convert to DNA first

- Once it has been converted it can also be sequenced

- Kind of like reverse transcription

bacterial transformation

introducing a gene into a bacteria in order to multiply it

1) mRNA produces cDNA (copy DNA) with reverse transcriptase

2) cDNA is cleaved appropriately

3) cDNA is ligated to a vector

- It can then be inserted into e. Coli and be multiplied and studied

cell transfection

introducing a gene into an animal cell

- Similar to bacterial transformation except the pores of the cell membrane are opened up in order to introduce the plasmid DNA

RNA interference

A way to stop translation through mRNAs

1) Double stranded RNA is cleaved into siRNA (silence induced RNA)

2) siRNA associates with RISC (silencing complex of proteins)

3) RISC complex goes to mRNA and cleaves it

- Thus inducing gene expression

nuclear envelope

Lipid bilayer that surrounds the nucleus

• has an inner and outer membrane

nuclear envelope is interrupted by…

nucelar pores

nuclear envelope is continuous with…

the RER

nucelar pore complex

Structure on the nuclear envelope that controls what goes in and out of the nucleus

structure of nuclear pore complex

nucleoporins, spokes, cytoplasmic filaments, cytoplasmic and nuclear rings, nuclear basket, and FG repeats in the lumen

components of nuclear basket

Nuclear ring

Backet filament

Terminal ring

what type of molecule are nucleoporins

proteins

where are cytoplasmic filaments found

attached to the cytoplasmic ring

what are FG repeats in the lumen of the nuclear envelope for

Hydrophobic amino acids to form a nonpolar section to control movement in and out of the nucleus

nuclear localization sequence (NLS)

signal that informs the pores which molecules to bring into the nucleus

• Labeled so that proteins that are supposed to be inside the nucleus don’t leave

nuclear export sequence (NES)

sequence to export protein from the nucleus

3 things exported from the nucleus

Ribosomal subunits

RNA

Transcription factors

2 things imported into the nucleus

Nuclear proteins

Ribonucleoproteins

How does import to nucleus work?

1. NLS is recognized by importins

2. Improtin binds to cargo protein 

3. Importin complex binds to proteins in the cytoplasmic filaments 

4. Through sequential binding, the complex is translocated through the pore

5. At the nuclear site, the protein RAN-GTP binds to importin

6. Importin changes conformation and cargo protein is released

7. Importin-RAN complex is exported through nuclear pore

8. RAN GAP stimulates RAN to hydrolyze the GTP into GDP

   1. This action triggers it to release importin back into the cytoplasm

9. RAN-GDP is imported back into the nucleus with NTF2

10. RAN GEF stimulates RAN-GDP to release GDP and pick up GTP

How to export from the cell?

1. NES is recognized by exportin

2. RAN-GTP binds to the complex to stabilize it

3. Complex binds to nuclear pore proteins and is transported through the pore

4. RAN GAP triggers release of cargo protein and GTP hydrolysis (GTP → GDP)

5. Exportins are recycled for reuse through the nuclear pore complex

RAN GAP

stimulates hydrolysis of GTP

• GTP → GDP

• Attached to the cytoplasmic filaments

RAN GEF

stimulates exchange of GDP

• GDP → GTP

• Note that it’s exchange and not an addition of a phosphate

• Is in the nucleus