Molecular Biology - Exam II

In vivo

Study of tissue within a living organism (e.g. use of live model organisms)

Model organisms

Organisms used in In vivo studies that often…

• Have well-characterized genetics

• Grow and reproduce quickly (short life cycle)

• Can be maintained in a laboratory setting

In vitro

Outside a living organism (e.g. in a test tube or petri dish)

In cell culture

In cells that are grown in a dish and passaged (e.g. used in study of cancer, viruses, proteins, and cellular differentiation)

• Primary cell culture: cells that have been removed from an animal tissue and grown in culture

• Cell line: cells that can be grown in cell culture and passaged multiple times

Passaging

Removal of calls growing in a dish filled with tissue culture media to another dish; enzymes involved in cell-cell-contact-protein digestion often used to remove cells

Cell culture media

An aqueous solution of salts, growth factors, and nutrients that will support cell growth

Ex vivo

In or on tissue that came recently from an organism

Organoids

Miniature organs grown in Ex vivo culture via self-organization of stem or progenitor cells

In silico

Computational simulations of biological processes

Light microscopy

• Uses light to illuminate the sample

• Up to 1000x magnification

• 0.2 mm - 200 nm diameter

Transmission electron microscopy (TEM)

• Uses electrons to “illuminate” the sample

• Up to 100,000x magnification

• Can resolve structures smaller than 1 nm

• Resolution higher than light’s

Scanning electron microscopy (SEM)

• Generates microscopic images with 3D quality

• Shows sample’s surface topography

Magnification

How big a microscopic sample appears

Resolution

Clarity and contrast of a microscopic sample

SDS-PAGE

Gel electrophoresis in which proteins are driven through a gel matrix and organized primarily by molecule size; progress is often tracked using dye

Western blots

Small scale study of proteins; allow detection of specific protein samples via…

1. Transfer of proteins from SDS-PAGE gel to piece of paper (nitrocellulose)

2. Paper is incubated with protein that will block non-specific binding of the antibody

3. Antibody, which is covalently attaches to an enzyme that produces a light reaction, recognizes specific protein on blot

4. Light reaction can be detected via camera of film to reveal protein band

Proteomics

Large scale study of proteins; provides a list of proteins within a sample by…

1. Digestion of proteins in a SDS-PAGE gel

2. Separation of peptides

3. Use of mass spectrometer to identify and gather data on proteins in sample

Non-ionic detergents

Triton X-100 and digitonin; permeabilize (make holes in) the plasma membrane or nuclear envelope to facilitate movement

Immunocytochemistry

Utilizes fluorophores attached to antibodies in order to recognize specific proteins in cells; done by…

1. Permeabilization of cell membrane via Triton X-100

2. Penetration of antibody through new holes in plasma membrane

3. Fluorescent tag (fluorophore) identifies antibody’s (therefore protein’s) location in the cell

Immunohistochemistry

Utilizes stained antibodies to recognize specific proteins in tissues; done by…

1. Staining primary and secondary antibodies with DAB (brown dye)

2. Identifying the location of antibodies (therefore proteins) in tissue

Sanger Sequencing

DNA sequencing method used to determine the exact nucleotide sequence of a targeted segment by…

1. separation of DNA strands

2. introduction of a DNA primer

3. complementary strand synthesis via DNA polymerase w/ insertion of a fluorescent marker

4. disassociation of new strands, creating segments of different sizes

5. separation of segments via gel electrophoresis

Polymerase chain reaction (PCR)

Creates exact copies of a target DNA sequence

Fluorescence In Situ Hybridization (FISH)

Analyzes and detects the location of a specific DNA or RNA sequence on a chromosome; labels complementary DNA fragments with fluorescence

RNA ISH

Localizes RNA by…

1. adding “probe” to specimen

2. denaturing probe and target

3. hybridization

4. binding of labeled antibodies

5. fluorescence => labeling/detection

RT-PCR

Converts RNA into DNA in order to replicate it for study

Single-cell RNA sequencing

Measures gene expression in individual cells using RNA by…

1. Collecting sample

2. Dissociating and isolating single cells (using oil)

3. Isolating RNA in each cell

4. Sequencing RNA

Bulk RNA sequencing

Measures gene expression in many cells using RNA; often used to compare genomes and find a mutation

Dissociation

Mechanical dissociation:

• Mortar and Pestle

• Scissors

• Syringe

Enzyme dissociation:

• Papain

• Collagenase

• Trypsin

• Dnase

• Dispase

Endogenous

Growing or originating from within an organism

Exogenous

Growing or originating from outside an organism

Recombinant DNA technology

Uses bacterial DNA to make a specific DNA segment by…

1. Linking a vector DNA (derived from bacterial genome) is linked to target DNA segment to create a plasmid

2. Bacterial cells are expanded and used to make many copies of plasmid

3. Plasmid DNA is isolated from bacteria for use in other cells

Recombinant plasmid

Requires…

• Origin of replication (ORI)

• Antibiotic resistance: allows positive selection in bacteria

• Promoter

• Target gene (YFG)

CRISPR-Cas9

DNA-dependent endonuclease that can cut DNA in a specific region and insert new DNA at cut site

GFP fusion proteins

Utilizes gene encoding green fluorescent protein (GFP) by…

1. Placing GFP gene in a plasmid vector with target gene

2. Transfecting plasmid into cell

3. Allowing plasmid to be transcribed and translated into fusion protein (containing target protein and GFP)

Transfection

Introduction of nucleic acids into eukaryotic cells

Lipid delivery agent

Utilizes membrane-enclosed vesicles containing target plasmid to induce membrane fusion and release of substance into cytoplasm

Electroportation

Cell is subject to short electric shock in order to temporarily permeabilize the membrane and allow target plasmid into cell

Transduction in plasmid delivery

Use of viral vectors to package exogenous nucleic acids in viral capsids, using cell surface proteins as receptors to enter cells

Adenoviral vector

• Does not integrate with genome

• Does not replicate during cell division

• Quick, transient, high-level expression

Lentiviral vector

• Integrates into genome

• Transfects most cell types, but with varying efficiency

Signal transduction

The process by which an extracellular signal induces a cellular response

Cell surface receptors

Transmembrane receptors that bind to ligands on the outside of a cell’s plasma membrane and transduce the signal within the cell

Intracellular receptors

Cellular receptor within the target cell which requires the small, hydrophobic ligand to diffuse through the plasma membrane

Autocrine

The cell producing the ligand also accepts and responds to the message

Paracrine

Ligand must travel a short distance (i.e. to a neighboring cell) from the cell in which it was produced to the target cell

Endocrine

Ligand must travel a long distance (i.e. through the bloodstream) from the cell in which it was produced to the target cell

Hormones

Regulatory substances (steroids) produced in an organism and transported through the bloodstream (i.e. endocrine) to stimulate specific cells by…

1. Diffusing into the cell vis facilitated diffusion

2. Binding to intracellular receptor

3. Movement of signal-receptor complex into the nucleus

4. Binding of signal-receptor complex to promoter/enhancer region of DNA to activate gene transcription

Estrogen receptor signaling

1. Estradiol (E2) diffuses through membrane

2. E2 binds to estrogen receptor α (ERα)

3. E2 and ERα migrate into the nucleus to activate transcription

Receptor tyrosine kinases

Extracellular, transmembrane receptors that bind to growth factors and hormones; activate kinases (enzymes that phosphorylate proteins) by…

1. Awaiting signal molecule binding as monomeric, inactive receptors

2. Binding to signal molecule and stimulating kinase activity

   1. Ligand-mediated dimerization: dimeric ligands induce dimerization and activation

   2. Receptor-mediated dimerization: monomeric ligands are used; receptors must be dimerized in order to activate

3. Transphosphorylating tyrosines on partner receptor to activate it

Cytoplasmic kinase cascade

Kinases activate other kinases, which allow post-transcriptional modifications via phosphorylation of amino acids

• Cytoplasmic kinases are generally Ser and Thr kinases

G-protein coupled receptor signaling

Transmembrane receptor couples with G-protein to induce a response by…

1. Binding of ligand to GPCR (receptor)

2. Association of G-protein complex with GPCR, activating it and forming GTP

3. Conformational change of G-protein, releasing α subunit

4. Binding of α subunit to target protein, activating it

5. Hydrolysis of GTP, inactivating the α subunit and causing dissociation from target protein

6. Re-association of α subunit with β and γ subunits for future G-protein use

Second messengers

Diffusible, non-protein molecules that relay signals from an effector molecule to activate target inside the cell; significantly amplify signals

Cyclic AMP (cAMP)

Second messenger produced by adenyl cyclase and derived from ATP; used to activate certain kinases

IP₃

Small, water-soluble sugar phosphate molecule capable of rapid diffusion throughout the cell that bind to their specific receptors in the smooth ER

DAG

Lipid molecule that recruits and activates effector proteins across the plasma membrane bearing its specific binding domain

Ca²⁺

Ions that bind to various target molecules, triggering many specific cellular responses; associated with the smooth ER

Apoptosis

Cellular response to kill cells that are unnecessary, damaged, or dangerous in which…

1. Cell volume shrinks

2. Cells lose cell-cell contacts

3. Actin and chromatin are chopped into smaller pieces

4. Cell membrane forms blebs

5. Cell is phagocytosed

Extrinsic pathway (apoptosis)

Activation of apoptosis from outside the cell

1. Fas ligand of killer lymphocyte binds to Fas protein of target cell

2. Adaptor proteins (Apaf-1) and inactive casp-8 molecules associate with Fas protein

3. Aggregation of casp-8 molecules, therefore activating them and a caspase cascade

4. Dissociation of killer lymphocyte

Intrinsic pathway (apoptosis)

Activation of apoptosis from inside the cell

1. Cytochrome c releases from injured mitochondrion and binds to adaptor proteins (Apaf-1)

2. Aggregation of Apaf-1 and association with inactive casp-9

3. Activation of casp-9 and caspase cascade

Stages of the cell cycle

1. G₁: organelles replicate, cell grows and carries out normal metabolic functions

2. S: DNA replication and chromosome duplication

3. G₂: cell grows and prepares for division

4. M: mitosis, including prophase, prometaphase, metaphase, anaphase, telophase, and cytokinesis

Cell cycle checkpoints

1. G₁: is environment favorable?

2. G₂: is all DNA replicated properly? is environment favorable?

3. Metaphase: are all chromosomes attached to the spindle?

G₀

Quiescence; state in which cells have exited the cell cycle, but can reenter when acted upon by a stimulus

Senescence

A permanent state of quiescence or G₀

Cyclin-dependent kinase (Cdk)

Kinases that regulate cell cycle entry by direct phosphorylation of targets; activated by binding to cyclins

Cyclin

Protein partner for Cdks whose levels are regulated during the cell cycle

Ubiquitin-proteosome cycle

Cycle that degrades Cdk-cyclins throughout the cell cycle in order to regulate its progression

Activation of cdks

• Inactive cdk must be phosphorylated once (by CAK) in order to be activated

• Active cdk can be further phosphorylated (by Wee1 kinase) in order to be inactivated

• Diphosphorylated (inactive) cdk can have a phosphate group removed (by Cdc25 phosphatase) in order to be activated

Active cdk/cyclin complex provides positive feedback to activate more complexes

p27

A cdk inhibitor that binds to the cdk/cyclin complex and inhibits its activity; must be removed for cell cycle to progress

p53

Tumor-suppressing transcription factor that directly impacts the binding of DNA or its conformation

• Elephants have multiple copies of this gene, making cancer extremely rare

Gated protein transport

Proteins move from the nucleus to the cytoplasm via nuclear pore complex, which acts as a “selective gate”

RanGTP

Regulates nucleocytoplasmic transport; interacts with RanGAP in the cytoplasm and the chromatin-associated RCC1 in the nucleus, creating a Ran-GTP gradient and concentrating Ran in the nucleus

Nuclear localization signals (NLS)

Target proteins for passage through the nuclear pore complexes

Transmembrane protein transport

Proteins move from the cytosol to an organelle across membranes via unfolding and “snaking” through a translocator

Co-translational import into ER

1. Ribosome translates mRNA

2. N-terminal signal peptide is recognized by signal recognition particle (SRP)

3. SRP is recognized by SRP receptor on ER surface

4. SRP receptor brings ribosome to translocator

Vesicular protein transport

Proteins move from the ER to the plasma membrane (or vice versa) via membrane-bound vesicles formed to “transport cargo”

Rough ER

Organelle containing ribosomes for protein synthesis; serve to…

• synthesize integral membrane proteins, secretory proteins, and soluble proteins

• fold and oligomerize proteins (with the help of chaperones) into their tertiary and quaternary structures and run a “quality control check” (ERAD pathway)

• initiate glycosylation or proteins and lipids

• transport proteins to Golgi complex for further processing

N-linked glycosylation

Linking of a sugar to the N-terminus of a protein, specifically the amino acid asparagine; starts in the ER and is modified in the Golgi

O-linked glycosylation

Linking of a sugar to the O-terminus of a protein, specifically the amino acids serine and threonine; occurs entirely in the Golgi

Chaperones

Proteins that undergo a change in conformation (typically requiring ATP) to facilitate protein folding; occurs during co-translational import into the ER

ERAD pathway

Checks the quality of proteins to ensure they’re normally folded before leaving the ER; if released into the cytoplasm misfolded, proteosomes (organelles containing digestive enzymes) will disassociate them back into ammino acids

KDEL and KKXX

Amino acid sequences that signify a protein’s return to the ER; are recognized by their own specific receptor complex

ER entry signaling

Proteins require a signal sequence and ER retention signal

Golgi entry signaling

Proteins require a signal sequence and a Golgi retention signal

Lysosomes entry signaling

Proteins require a signal sequence and a Mannose 6-phosphate

Nucleus entry signaling

Proteins require an NLS

Mitochondria entry signaling

Proteins require an MTS

Peroxisomes entry signaling

Proteins require a PTS