Cell Biology Exam 1 (Topics 1-2)

Common Mass Spectrometry Methods

(ESI) electrospray ionization and (MALDI-TOF) matrix-assisted laser desorption ionization-time-of-flight

Problems of RNAi

-does not efficiently inactivate all genes -certain tissues can be resistant to RNAi -RNAi sometimes produces off-target effects by inactivating related genes

Two-Dimensional Gel Electrophoresis Step 1:

Sample is dissolved in a small volume of a solution containing a nonionic (uncharged) detergent, beta-mercaptoethanol and the denaturing agent urea (solution solubilizes, denatures and dissociates all the polypeptide chains but leaves their intrinsic charge)

Fluorescence Resonance Energy Transfer (FRET)

Technique for monitoring the closeness of two fluorescently labeled molecules (and thus their interaction) in cells, the energy transfer from the labeled proteins by illuminating the first protein and measuring the emission from the second

Secondary Culture

a cell culture where the cells are derived from a previous culture, as with immortalized cell lines

molecular cloning

isolation and incorporation of a piece of DNA into a vector so it can be replicated and manipulated into a living organism

Light and Electron Microscopy Mutual Benefits

light microscopy can be used to very accurately locate specific molecules within a cell and the electron microscopy then allows the tagged molecule to be examined in its cellular context

Very High Speed Centrifugation

pellet contains ribosomes, viruses, large macromolecules

Low Speed Centrifugation

pellet contains whole cells, nuclei, and cytoskeletons

Gel-Filtration Chromatography

separates proteins based on size (porous beads let the bigger molecules through faster, while the smaller molecules get stuck in the pores and filter out last)

Two-Dimensional Gel Electrophoresis Isoelectric Focusing

separating polypeptide chains in a pH gradient which takes advantage of the variation in the net charge of a protein with the pH of the surrounding solution because the proteins separate to their characteristic isoelectric point where the protein has no net charge

fluorescence microscopy

uses a fluorescent dye that emits fluorescence when illuminated with ultraviolet radiation

Applications of of CRISPR/Cas9

1. Gene editing 2. Gene regulation 3. Base editing 4. RNA targeting 5. Chromatin topology 6. Chromatin imaging

Transmission Electron Microscope (TEM)

A microscope that uses an electron beam to study the internal structure of thinly sectioned specimens.

mass spectometry (MS)

- used as a detector to definitively identify solutes - measures mass to charge ratio - solutes show fragment pattern, which is compared to the published reference spectra -composed of the ion source, mass analyzer and a detector

CRISPR/Cas9 advantages

-relatively easy to design guide RNA (follows standard base pairing conventions -the gene to be controlled does not have to be modified (exploits DNA sequences already present in the genome -numerous genes can be controlled simultaneously (Cas9 expressed once with many guide RNA expressed in the same cell)

Affinity Chromatography

-uses specific interactions to slow down select molecules -can make use of receptor-ligand, enzyme-substrate, and antigen-antibody interactions to separate proteins (can create a nearly pure form from such specific interactions)

Transgenic Organisms

animals and plants that have been genetically engineered by gene deletion or gene replacement

Complementation Test DNA

used to ascertain whether a mutation falls in the same gene or in different genes, take a homozygous individual for the mutant gene in question and mates it with a homozygous individual for the other mutation (if they are on the same gene, all offspring will show the mutation but if not, the resulting offspring could show a normal phenotype because they retain a normal copy meaning they complement each other and restore a normal phenotype)

when is fluorescence microscopy most often used?

used to detect specific proteins or other molecules in the cells and tissues (FISH) -> reveals the cellular distribution and abundance of specific expressed RNA molecules in sectioned materials or in whole amounts of small organisms

electron microscopy

uses electrons instead of light, the shorter wavelength of electrons gives greater resolution, shooting electrons with shorter wavelengths to achieve a higher resolution in the picture

Complex Animal RNAi introduction

uses recombinant DNA techniques to make transgenic animals that express the RNAi under the control of an inducible promoter

Northern Blot

uses specific single stranded RNA probe to detect RNA expression level in the cell

Southern Blot

using specific single stranded DNA to detect DNA expression level in the cell

Restriction Nuclease Target Sequences

usually 4-8 nucleotide pairs which increases specificity of gene sites

Culture Shock

when culture conditions cause excessive stimulation from cell proliferation and the cell activates a poorly understood protective mechanism which stops cell division

Primary Culture

Cells are cultured directly from the tissues of an animal and have a limited life span.

General Steps of Protein Purification

1. Collect Intestinal Tissue from Experimental model 2. Mince Tissue and digest it into protein mix 3. Mix protein homogenate with protein assay to test if protein of interest is in the homogenate 4. Test whether the assay succeeded 5. Chromatography columns-> assay -> columns -> repeat

CRISPR/Cas9 Critical Requirements

1. Guide RNA provides the gene sequence specificity 2. Cas9 functions as the restriction enzyme to cut the DNA 3. Donor DNA that the experimenter designed provides the template for DNA repair

PCR General Steps

1. Heat to separate strands 2. Cool to anneal primers 3. DNA Synthesis (PCR is the method of choice for cloning relatively short DNA fragments)

By putting new DNA into animals we can...

1. Over express a gene 2. Knockout a gene

Hybridoma Cell Line Production Process

1. Propagate a clone of cells from a single antibody-secreting B lymphocyte to obtain a homogenous preparation of antibodies in large quantities 2. Fuse B-lymphocyte cells from immunized organism to the transformed B-lymphocyte 3. Propagate the hybridoma cells to create the permanent and stable source of a single type of monoclonal antibody

Equilibrium Binding Experiments

2 proteins are mixed at a range of concentrations, allowed to reach equilibrium, and the amount of bound complex is measured (half of the protein complex will be bound at a concentration that is equal to Kd), these experiments often involve the use of radioactive or fluorescent tags as one of the protein partners

restriction endonuclease

A bacterial enzyme that recognizes a specific DNA nucleotide sequence and that cuts the double helix at a specific site within the sequence.

Velocity Sedimentation

A method of separating subcellular components in a dilute salt solution. The tube is centrifuged, and the components sediment throughout the tube based upon size and shape when layered over a solution containing sucrose gradients (the bands must be protected from convective mixing to succeed)

Scanning Electron Microscope (SEM)

A microscope that uses an electron beam to scan the surface of a sample, coated with metal atoms, to study details of its topography, gives insight into its outer 3D shape

Polymerase Chain Reaction (PCR)

A technique for amplifying DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides (polymerase enzyme adds nucleotides to the 3' end of a growing strand of DNA but it requires a DNA primer which is a short nucleotide sequence that provides the 3' end from which synthesis can begin) PCR utilizes hybridization usually to synthesize the primer -at the start of each PCR cycle, the two strands of double stranded DNA are separated and a different primer is annealed to each which mark the right and left boundaries of the DNA to be amplified

SDS-PAGE

An electric field applied to a solution containing a protein molecule causes the protein to migrate at a rate that depends on its net charge and on its size and shape (uses a polyacrylamide gel as the inert matrix where proteins migrate and the gel can be prepared with specific pore sizes and the proteins are dissolved in sodium dodecyl sulfate, which is a powerful negatively charged detergent which causes proteins to unfold into extended polypeptide chains, individual protein molecules are released from associations to be analyzed separately)

Immortalized Cell Line

Cell line capable of an unlimited number of cell divisions, human cells generally coaxed to infinite proliferation by providing a gene that encodes the catalytic subunit of telomerase

Equilibrium Sedimentation

Centrifugal technique using a sucrose gradient to separate cellular components based on their buoyant density regardless of size and shape with the gradient and centrifugation separating the dense components (components of highest buoyant density are located at the bottom of the tube) - so selective that it can even separate macromolecules that contain different isotopes

What method is most often used to fractinate proteins?

Chromatography

Column Chromatography

Chromatography in which the substances to be separated are introduced onto the top of a column containing a matrix to separate the mixture based on various characteristics

CRISPR Acronym

Clustered Regularly Interspaced Short Palindromic Repeat

epistasis analysis

Comparing the phenotypes of different combinations of mutations to determine the order in which the genes act.

Flow of genetic material general

DNA -> Transcription (mRNA) -> Translation/Protein Synthesis (tRNA) -> proteins

Recombinant DNA

DNA produced by combining DNA from different sources

DNA polymerase

Enzyme involved in DNA replication that joins individual nucleotides to produce a DNA molecule, involved in the synthesis and repair of DNA strands, often used to create exact copies of existing DNA molecules in a lab setting and can even regenerate modified DNA with things like fluorescent or radioactive tags

Protein Purification Step 1:

Extracting a protein from the cells (cells can be broken up in various ways: osmotic shock, ultrasonic vibration, forced through a small hole, ground up in a blender etc) this breaks up many membranes into small fragments that immediately reseal into vesicles but if done correctly, leaves the organelles largely intact

GFP fusion protein

GFP gene introduction behind an endogenous gene --> fusion protein --> permits visualization of cellular gene products in vivo(WITHOUT antibody or fixation) viewed by fluorescence microscopy (CHROMOPHORE FORMED POST-TRANSLATIONALLY FROM PROTRUDING SIDE CHAINS OF 2 AA RESIDUES IN A SERIES OF AUTOCATALYTIC STEPS)

Replicative cell senescence

Phenomenon observed in primary cell cultures as they age, in which cell proliferation slows down and finally halts (reflects the progressive shortening and uncapping of the cell's telomeres)

bright field microscopy

Shines light through sample and magnifies the image with a series of lenses.

Epitope

Small, accessible portion of an antigen that can be recognized, antigenic determinant on a modified gene for tagging

Western Blot

The most common confirmation test for detecting protein expression level using antibodies after an SDS-PAGE by transferring the proteins to an sheet of nitrocellulose paper or membrane then soaking it in the tagged/fluorescent antibody to reveal the protein of interest

Dideoxy Sequencing

This method uses DNA polymerase, along with special chain-terminating nucleotides called dideoxyribonucleoside triphosphates to produce a collection of different DNA copies that terminate at every position in the original DNA sequence to determining the nucleotide sequence of a DNA fragment

How are a majority of proteins studied?

Tissues are not generally the preferred source of cells, more generally we use specific cell types grown in a culture (provide a more homogenous population of cells from which to extract materials)

Immunoprecipitation (IP)

To isolate proteins from complex mixtures and detect protein-protein interactions. Create a cell extract--add antibody for protein of interest--add large, insoluble beads that bind to the antibodies--centrifuge and remove soluble extract--elute protein of interest off of the insoluble beads. *Co-IP: Add antibodies for protein A and then immunoblot for protein B. Will only be able to see B if they the proteins interact.

Tandem mass spectrometry (MS/MS)

Using two mass spectrometers in tandem to determine the amino acid sequence of a single protein contained within a mixture of proteins, typically uses an electrospray ion source and then a quadrupole or ion trap to filter the other peptide identities out and identify the single peptide in a mix of many (also useful in detecting a precisely mapping post-translational modifications of proteins such as phosphorylation or acetylation)

CRISPR/Cas9

a bacterial system that can be used either to produce a mutation in a specific gene or to correct a mutation that is already present, uses a guide RNA sequence to target specific double-stranded DNA which it then cleaves and the gene coding key for this experiment (bacterial Cas9 protein) because it provides the environment to cleave the DNA

CRISPR

a collection of RNA sequences that tells Cas9 exactly where to cut

EcoR I

a common restriction enzyme produced by E. Coli. Cleave between G and A in following sequence, C-T-T-A-A-G- (reading 3' to 5')

Two-Dimensional Gel Electrophoresis

a method that separates the proteins in a sample into individual spots, combines two different separation procedures

Homogenate

a mixture of all of the components of the cell, but no intact cells, the thick slurry formed in step 1 of protein purification

Green Fluorescent Protein (GFP)

a protein that fluoresces green and is widely used in genetic analysis, two proteins of interest are labeled with different fluorescent proteins such that the emission spectrum of one fluorescent protein overlaps the absorption spectrum of the second (this causes an energy transfer if the proteins come very close to each other the energy of the absorbed light is transferred from one protein to the other to cause fluorescence)

Bacterial Artificial Chromosome (BAC)

another type of vector; allows for easier replication/manipulation as the number of genes is reduced to a smaller size or increased to a very large number

Monoclonal Antibody

antibody produced in a laboratory to attack antigens and to destroy cells; each antibody recognizes only a single type of antigenic site

Optical Sectioning with Light Sheet

based on wide field fluorescence microscopy, except the excitation light source is only a thin sheet of light where the eyepiece only sees the plane of fluorescent labels in the light

Hybridization

breaking the hydrogen bonds connecting complementary strands of DNA, but keeping the strand intact to study by heating the DNA sequence to 90*C before reattaching them by lowering the temperature

How do we trigger homologous recombination

by damaging the DNA strand and causing a break

Proteolytic Enzymes

catalyze the hydrolysis of peptide bonds (digests proteins in the extracellular matrix with agents that bind the Ca2+ on which cell-cell adhesion depends

Transformed Cell Lines

cell lines that are obtained from cancer cells. These cells can grow without attaching to a surface and can reach much higher cell densities

What is a normal imperfection that interferes with matrices in chromatography?

cellulose (causes uneven flow of solvent through the column that limits the resolution of conventional column chromatography)

Hybridoma cell lines

created to make monoclonal antibodies (production of unlimited quantities of identical antibodies and increases the specificity and convenience of antibody-based methods)

Perturbation

disruption in the genome and observing the changes in the associated phenotype

What happens in the SDS-PAGE?

each protein molecule binds large numbers of the negatively charged detergent molecules which mask the proteins intrinsic charge and causes it to migrate toward the positive electrode separating generally based on size because similar sizes bind similar numbers of detergent, smaller proteins move faster and farther down the gel than larger proteins

DNA ligase

enzyme that chemically links DNA fragments together, usually requires the inout of energy from a molecule like ATP

Restriction Nucleases

enzymes (proteins) that can cut DNA at specific sequences defined by the local nucleotide sequence, enabling the mixing of DNAs from different sources cut with the same enzyme (also called "molecular scissors")

GFP structure

fluorochrome shielded within the interior of beta barrel proteins

Optical Sectioning laser scanning confocal

focuses the light on a single focal point to observe only one spot with the maximum amount of light (better for thicker specimen)

epistatic

genes that mask the expression of other genes

Differential Interference Contrast Microscopy

highlights edges where there is a steep change of refractive index

Cas9 protein

holds the guide RNA, helps it basepair to DNA and cuts the genomic DNA

phase contrast light microscopy

in which phase alterations of light transmitted through the specimen are translated into brightness changes

RNA Interference (RNAi)

introduction of double-stranded RNA into a cell to inhibit gene expression, the RNA is processed and hybridized with the target-gene RNA thereby reducing the expression

Co-immunoprecipitation (Co-IP)

is a popular technique to identify physiologically relevant protein-protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein

Organoids

miniaturized and simplified version of an organ produced in vitro in three dimensions that shows realistic micro-anatomy (derived from pluripotent embryonic stem cells)

Ion-Exchange Chromatography

molecules separated based on net surface charge (beads can carry either a positive or negative charge)

High Speed Centrifugation

pellet contains microsomes, small vesicles

Medium Speed Centrifugation

pellet contains mitochondria, lysosomes, peroxisomes

homologous recombination

process that results in genetic exchange between homologous DNA from two different sources (mother and father) which creates variability in germ cells

Chromatin Immunoprecipitation (ChIP)

proteins are covalently cross-linked to DNA in living cells which are broken open, and the DNA is sheared into small fragments and then purified by antibodies directed against a given transcription of the cross-linked DNA -> then the DNA is sequenced and all the sites occupied by the transcription regulator in the cell sample can be mapped across the cell's genome (responsible for specifying the particular pattern of GENE EXPRESSION)

Hydrophobic Chromatography

separates proteins based on hydrophobicity (beads have hydrophobic side chains to retard proteins with exposed hydrophobic regions)

Plasmid Vectors

small DNA circles that are physically separated from chromosomal DNA and can replicate independently, used for DNA cloning as a self-replicating genetic element

reverse transcription - PCR

synthesis of DNA from an RNA template using DNA primers for the chosen mRNA, then heating and cooling occurs for PCR amplification

Cell Fractination

takes cells apart and separates the major organelles from one another, separate cell components sometimes by size and density in a centrifuge (the fractinations are impure but will be further purified)

Recombinant DNA technology

the ability to manipulate DNA with precision in a test tube or an organism

Two-Dimensional Gel Electrophoresis Step 2:

the narrow tube of gel w/the separated proteins is soaked in SDS and placed on an SDS-PAGE slab, and then the electrophoresis is carried out so that we have the pH gradient separation horizontally and the SDS size migration running vertically

DNA Cloning

the production of multiple copies of a specific DNA segment and the amplification of a gene sequence

How to turn on/off a gene w Cas9

the protein complex will bind to a transcription activator or repressor to either turn on or off the gene that it is modifying

dark field microscopy

the specimen is lit from the side and only the scattered light is seen

Why do we uses SDS?

to denature and linearize proteins which allows them to run according to size, and to break down the disulfide bond connecting proteins