QBM Final Exam Review

CH1: Overview of Molecular Biology

Molecular Biology involves the study, analysis, and manipulation of DNA & proteins. M.B. has its roots in early 1900s when geneticists and biochemists began to understand

genes & proteins. Geneticists used bacteria and fruit flies to study genes; biochemists studied and

characterized the proteins those genes encoded. 1953: Watson & Crick determine the structure of DNA & proteins

= True beginning of M.B. M.B. = the study of DNA and proteins and their interactions, and the molecular

information transfer within a cell. Recombinant DNA Technology: The process of taking a gene from one source and

joining it with another piece of DNA and inserting that DNA into bacteria for further study and manipulation.

DNA 2 strands – Antiparallel – 5’->3’ Directionality – A/T & C/G DNA copied by DNA Polymerase during S phase of cell division, and mRNA is

transcribed from DNA by RNA Polymerase. The ribosome reads this message and translates it into protein. (CENTRAL DOGMA OF MOLECULAR BIOLOGY)

DNA probes can be used to determine the presence or absence of an allele, which can indicate a predisposition to a specific disease

Probes useful for generating pedigrees, paternity testing, and forensics. Small Scale: Genes can be knocked out to study their impact, or inserted via gene therapy

to cure a disease Large Scale: Entire genomes can be sequenced and studied, and the sequence can be

compared between individuals or species.

Proteins Consist of amino acids linked by a peptide bond between the carboxyl terminus of one

amino acid and the aminos terminus of the next. Protein synthesis by the ribosome if from the N- to C-terminus.

Primary Structure: Amino Acid Sequence Secondary Structure: alpha-helices and beta-sheets Tertiary Structure: several motifs (alpha-helices, beta-sheets, etc) further folded into

complex Quaternary: Interaction between multiple polypeptide chains.

Clinical Perspective- protein markers fors are important for disease classifications and protein mutants often play a role in phenotype or disease states.

Most drugs interact with or inhibit protein function in some way, so knowing the proteins function and impact on the cell is essential.

Proteins can be mass produced for medicinal use (insulin, vaccines, and certain enzymes)

**p53 protein= tumor suppressor activated upon DNA damage or cell stress; binds to DNA and activates transcription of the damage response genes.

CH2: Lab Safety, Oversight, and Hazards

Carcinogenic = cancer-causing Occupational Safety and Health Administration (OSHA): Set rules for research

laboratories to follow. Also states that: Employers must inform employees of potential hazards Every hazardous substance has to have a Material Safety Data Sheet (MSDS)

MSDS: states a chemical’s physical and chemical properties and any associated physical hazards, health hazards, the primary route of entry, exposure limits, and whether or not it is toxic, carcinogenic, flammable, or radioactive. Also lists proper safe handling of the chemical and emergency and first aid procedures.

Centers for Disease Control (CDC) regulates researchers who work with microorganisms that can cause disease by assigning each lab a Biosafety Level (BSL)

BSL-1: Minimal potential hazards to lab personnel **BSL-2: these labs work with organisms that are of moderate potential hazard BSL-3; these labs work with pathogenic organisms that can cause serious disease.

Must be properly ventilated, access is restricted, and special protective equipment must be utilized.

BSL-4: these labs work with dangerous pathogens that cause severe to fatal disease for which vaccines or treatments are not available.

Locally, each institution is regulated by internal oversight through a specific department or Laboratory Safety Officer.

At UCF, the Department of Environmental Health and Safety provides a comprehensive health and safety program and makes the campus a safe and healthful place in which to live, learn, teach, work or visit.

DEHS also offers training classes and certifications required for specific procedures (such as handling radioactive material) and chemical and biological

waste disposal. Also respond in event of emergency or to clean up waste spills.

Personal Protective Equipment (PPE) is essential in the lab for protection from hazardous chemicals and pathogens.

Latex/Nitrile gloves (possibility of latex allergies) Plastic gloves not acceptable Special gloves for handling hot/cold, UV eye protection, specific sites for waste

disposal, and lab coats/pants sometimes required. Decontaminate benches with EtOH

CH3: Micropipettes, Centrifuges, and Spectrophotometers

Most lab equipment that comes into contact with samples must be clean and sterile before use.

Acetone NOT used to rinse glassware b/c it can interfere with reagents and damage DNA and proteins.

Autoclave – Device which uses high temperature and pressure – required to fully sterilize glassware and other equipment.

Transferring Liquids and Micropipetting

Volumes (~25ml to 2L) – GRADUATED CYLINDER Volumes (~1-25 to 25ml) – GLASS/PLASTIC PIPETTES

Mohr pipette: Has pointed tip for finer measurements b/c drops will be smaller Serological pipette Transfer/Pasteur pipette: used for quick transfers when accuracy is not required.

Micropipettes (~0.1ul to 1ml) – contains plunger, tip ejector, volume adjuster, volume setting window, and disposable plastic tip.

Accuracy depends on calibration and proper technique (tip 1cm into liquid/ perfectly vertical

Centrifugation

Centrifuge: spins samples held in rotor at very high speed and at many times the force of gravity (‘g’)

Separate samples by mass, shape, and density. Greatly increases rate of sedimentation. Can be set by adjusting their ‘g’ (also relative centrifugal force, rcf) or revs per minute

(rpm). Best to use ‘g’ Two types used in the lab:

Preparative: Used to prepare samples for further use. Separate molecules and can help to isolate/purify a sample. Used to spin down tubes quickly, used in minipreps and gel extractions of DNA, and for protein concentration. Usually reach 13,000 rpm and ~7000g. Ex- Small bench-top centrifuge

Large-capacity refrigerated centrifuge: used with larger volumes (up to 1L) for separation of large amounts of material like liters of bacteria from media. Usually reach 6000rpm and ~6500g.

High-speed refrigerated centrifuge: used for large volumes needing even higher speeds for the collection of microorganisms, cellular debris, organelles, or proteins precipitated by ammonium sulfate. Sample volumes ares usually smaller (~50ml tubes). Can reach about 25000 rpm and ~60000g. Samples must be balanced within 0.25g of one another

Ultracentrifuge: useful for separating samples that require a lot of force. Operate under vacuum to prevent frictional heat; also monitored by lasers to detect unbalancing. Can reach 80,000 rpm and ~600,000g. Require samples to be balanced within 0.1g of one another.

Analytical: can be used to determine a sample’s purity, molecular weight and size, conformational changes, sedimentation and diffusion coefficients, stoichiometry, equilibrium constant, thermodynamics, and absorbance or fluorescence. Also used to perform sucrose or cesium chloride gradients to analyze or separate a sample.

Fixed-Angle Rotors: allow for pelleting of a sample Swinging-Bucket Rotors: don’t creat tight pellets. Best for sample analysis or

running cesium chloride or sucrose density gradients (5 % -> 25%) in an ultracentrifuge.

Meselson-Stahl Experiment: Utilized swinging-bucket centrifugation to analyze DNA replication.

Bacteria was grown in heavy (15-N) DNA, and switched to light (14-N) DNA, then analyzed for various generations. DNA was originally denser, but as each generation replicated the DNA became lighter. Demonstrated that DNA replication is semiconservative. Figure on pg.22

Various types of bottles used. Clear allow for better visibility, but are less resistant to chemicals and can weaken

and crack/break over time.

Spectroscopy & Spectrophotometry

Electromagnetic Radiation: a type of energy (self-propagating wave) that is transmitted through space at enormous velocities, and includes visible light, heat gamma-rays, X-rays, UV light, IR light, microwaves, and radio waves.

Spectroscopy: the study of this radiation interacting with matter; can be used to analyze molecular structures or dynamics through absorption, emission, and scattering.

Spectrometry: the measurement of the interactions of spectroscopy. Can be measured quantitatively or qualitatively.

Spectrophotometry: involves measuring a certain wavelength using prisms or gratings and measures the spectral properties of a molecule.

Spectrophotometers: device used to measure the intensity of a specific wavelength of light as it passes through a sample.

-These devices compare the amount of light transmitted through a sample to that transmitted through a colored sample.

-Components: Light source (tungsten – visible / deuterium – UV); Monochromator (selects particular wavelengths); prism or grating

(disperses light); sample compartment (holds cuvette in place); detector (uses phototubes and photomultipliers to convert photons into electrical energy)

-Plate Reader: can quickly read microliter volumes of dozens or hundreds of samples from a single plate

Colorimetry: converts a molecule to a colored compound by a chromogenic (color-forming) reaction.

Chapter 4 – DNA Quantification, Protein Quantification, and Enzyme Assays

-Only light of the correct energy for causing transitions from state to another is absorbed.

260nm: DNA

280nm: Protein

595nm: Bradford Assay / Coomassie Brilliant Blue when bound to proteins

600nm: Bacterial growth in LB Media

-Transmittance (T) = Incident light energy (I0) / transmitted light (I)

- Concentration ↑ = Absorbance↑ = Transmittance ↓

Transmittance decreases exponentially

Beer’s Law

A = 2 – log(%T) = -logT = log (1/T) = ebc = ecl AND T = 10 ^ (-A)

A = absorbance or optical density (OD600)

e = Molar extinction coefficient (L * mol- * cm-)

-tryptophans X 5500 + tyrosines X 1490 + cysteines X 125

b / l = path length (usually 1cm)

c = concentration (mol/L)

DNA Quantification: @ 260nm (UV part of spectrum)

A260 = 1 [DNA] = 50 ug/ml

Purity: A260/A280 > 1.5

A230 = organic contaminants

A320 = sample turbidity or buffer absorbance

Protein quantification: @ 280nm (due to tryptophan and tyrosine)

-Proteins lacing tryptophan or tyrosine will have little or no absorbance at 280nm

Amino Acid Analysis: Most accurate method for determining protein concentration. Protein is analyzed by amino acid hydrolysis followed by chromatography, where the protein’s identity and concentration are determined. Very expensive and takes various days, but very accurate

Colorimetric methods

-Bradford Assay: fastest and simplest method. Uses coomassie brilliant blue which changes color from red (465nm) to blue (595nm) as dye binds to

proteins (forms noncovalent/van der Waals complexes with proteins).

-Binding is relative to number of positive charges on the protein.

-Does NOT detect free amino acids or DNA; does NOT bind to proteins below 3000Da

-Requires STANDARD CURVE with each assay, HIGH variability

-Somewhat accurate and cost-effective

Lowry Method: uses copper binding to peptide bonds (little interaction with free AAs). Uses a folin reagent which becomes reduced by Cu+ and turns blue

(measured at 750nm)

-Commonly used, inexpensive, easy, reproducible, LOW variability, more sensitive than A280

-Must use STANDARD CURVE

-Sensitive to contaminants

DC Protein Assay: Modified Lowry Assay (detergent compatible)

-upper range is 1.5mg/ml

Standard Curve: allows one to accurately correlate absorbance in the spec to protein concentration in the sample. Known standards must be used, such

as BSA diluted to various concentrations and mixed with colorimetric reagent.

Enzyme overview (function, activation energy)

Enzymes are proteins or RNA that are essential to cell function by increasing the rate of a reaction. Highly specific; very tightly regulated by cells.

-Equilibrium: when the ratio of substrate to product remains the same. Equilibrium constant can be calculated by dividing the forward (Ka) reaction rate by the reverse (Kb) reaction rate. Enzymes do NOT affect equilibrium; reaction will reach equilibrium regardless of whether enzyme is present or not. (Enzymes accelerate time it takes to reach equilibrium)

Enzymes work by stabilizing the transition state (TS) of a reaction and by decreasing the activation energy.

Free Energy ΔG: measure of difference in energy between substrates and products.

- ΔG>0 = thermodynamically unfavorable, nonspontaneous (exothermic)

- ΔG<0 = thermodynamically favorable, spontaneous (endothermic)

Active Site: a cleft or crevice on the surface of the enzyme which enhances the binding of the substrate which interact with the active site via multiple weak forces (occasionally reversible covalent bonds).

-Active sites are highly conserved; changing single amino acid can affect specificity or reaction rate.

-Cofactors (Metal) & Coenzymes (organic): help promote the reaction, stabilize the transition state, or perform a mechanical function

-Conserved amino acids indicate important functions performed in the cell

Enzyme-substrate models

Lock & Key Model: substrate and the active site of the enzyme fit together like a key into its lock (complementary). This model does not perfectly explain transition state stabilization.

Induced Fit Model: the binding of substrate induces conformational changes in the enzyme which bring the substrates together and stabilizes the transition state.

Enzyme kinetics overview (rate, inhibitors)

Enzyme kinetics is the study of an enzymes rate.

Vmax: when the enzyme is saturated with substrate and catalyzes the reaction at its maximum rate.

-Enzyme’s rate is its inherent velocity, and depends on the concentration of the substrates, temperature, pH, and the presence of cofactors.

-Rate decreases as reaction equilibrium, substrate is depleted, product inhibits forward reaction, or enzyme is inactivated.

Enzymes must be studied during INITIAL RATE PERIOD

Km, Michaelis Constant: is equal to the rates of breakdown of ES complex over its rate of formation - measure of STABILITY of the ES complex.

-High Km = weak substrate binding

-Low Km = strong substrate binding

Inhibitors

Competitive inhibitors: bind to the same site on the enzyme that the substrates bind (active site), thus competing with the substrate. Can be affected by

substrate concentrations (out-competed)

Noncompetitive Inhibitors: bind to a second site on the enzyme, causing conformational changes in the enzyme that prevent enzyme-substrate

interactions. Not affected by substrate concentrations

Enzyme Assay- can be used to determine an enzymes rate. Measured by their activity (units)

-A unit of restriction enzyme is defined as the amount of enzyme required to cleave 1ug of DNA in 1 hour in 50ul total volume

CDNB Assay: CDNB absorbs light @ 340nm when acted on by GST

-CDNB + GSH --(GST)--> 1-(S-glutathionyl)-2,4-dinitrobenzene

This assay can be used to track the kinetics of the reaction and the Km of the substrates, and GST if often tagged to other proteins which allow

researchers to track the target protein by quantifying GST.

-Usually tracked with spectrophotmeter

Coupled Assay: the reaction of interest is linked to one that is measurable

-Pyruvate Kinase-Lactate Dehydrogenase Assay

Kinase Assay: used to study protein kinases, which phosphorylate other proteins. Can also be used to study signaling pathways or screen enzyme inhibitors

Radiolabeled ATP is mixed with a protein kinase and a target can be phosphorylated with the radioactive 32P from the ATP. Phosphorylated, radiolabeled proteins can be detected and quantified by a scintillation counter or run on a gel and detected with autoradiography.

-Fig. on pg38

Chapter 5 – Measurements, Concentrations, Dilutions, and Buffers

1 Dalton = 1 gram / mole

Milliliters to microliters conversion

1ml = 1000ul

Molarity, millimolar, micromolar, conversions between the three

1 M = 1 mole/L = 1000 millimoles/L

1mM = 1000 micromoles/L = 1000uM

1uM = 1000nM

Percent composition (v/v and w/v)

Weight/weight: both solute and solvent are expressed in the same physical mass units

Volume/volume: both the solute and the solvent are expressed in the same volume units

Weight/volume: the amount of solute is expressed in physical mass units, while the solution is expressed in volume units.

Dilutions (MV = MV)

M1V1 = M2V2

Buffers – be able to prepare a buffer as you did in lab or on the homework

PRACTICE PREPARING BUFFERS

Chapter 6 – Laboratory Data, Experimental Design, and Research Ethics

Protocol writing: list of steps that should be followed to obtain the desired results.

-When designing a protocol, it is best to use your previous work or published protocols as a guide.

-Everything needed to reproduce the experiment exactly should be included in the protocol. Also note any deviations from standard protocol. Not allowed to refrain from publishing entire protocol to harm competition.

Seminars, conferences, poster sessions

Overall goal of each of these is to present scientific data and research progress to colleagues for feedback, networking, discussion, or to further advance the field.

Seminars: usually held at program or department level, and usually involves graduate students and postdocs (some principal investigators PIs occasionally).

-Researcher presents another’s work, such as current paper of interest from a top journal.

-Setting gives graduate students chance to practice reading papers and presenting to others. Those in the audience are brought up to date on a specific area

of research

Invited Seminar Series: involves department or entire campus. Allows a large group of scientists to see current research from an internal or external researcher, who may be given an honorarium (payment).

-Allows researchers a chance to present their data to a group of colleagues, interact with and teach students, network/get feedback, and promote their

research

-May be used as part of an interview process for a position at the institution

Conferences/Symposiums: gatherings of experts in a field, sometimes once a year, which features posters and invited lecturers. Allows scientists to present their research to colleagues, learn new techniques, and discuss new theories and the direction of the field.

-Can be conferences regarding specific new techniques (crystallization) or regarding a single topic (TB, HIV, Cancer)

-Lectures given by PIs

Poster Sessions: can be an on-campus display of one’s research to other faculty and fellow students. Can also be at a conference.

-Allows researchers who are not presenting to still show their work, and gives grad students/postdocs the opportunity to show their data to others.

Grants- money to perform research, must apply for them. Apply to NIH or obtain private donations.

Proposals- must have 3 goals. Greater chance of getting funded if researchers show that the work is relevant, that current published works support their line of thought and that preliminary data supports their hypothesis.

NIH review process- Each grant proposal is reviewed by a panel of scientists who give it a score/percentile. If the score is high enough and the funds are available, the PI will receive the money they requested.

-Some projects that use human/animal/certain pathogen as subjects will require Institutional Review Board (IRB) approval before the grant is funded.

R01: most sought-after grant; can provide a lot of money for 3-5 years New investigators given special consideration.

Research Publication

Relevant Title

Authors

Abstract- describes what was performed & the conclusions that were drawn.

Methods Section- (sometimes at very end of paper/written up separately in another journal). Includes everything one would need to reproduce the experiments performed. Steps should never be omitted. Only include deviations from standard protocols.

Results Section- contains all of the data and figures, and includes the specifics of the paper. Usually very technical and detailed. Never should data be edited or falsified, or spliced together from multiple images.

Discussion- where the authors analyze and discuss the data presented in the results section, and bring all of the data together into a narrative. Usually contains most interesting and exciting material in the paper as the author’s conclusions, implications, and future direction.

References/Citations- also includes financial support list, gratitude to contributors who are not authors, and a competing financial interest’s statement.

Impact Factor: total number of citations for each article in the following two years by other research publications

Authorship Order: the PI of the lab who earned the grant that supported the work has the final say in authorship. Usually:

-First Author: Graduate student or postdoc assigned to the project by the PI

-Second Author: Close collaborator on the project

-Middle Authors: Lab technicians or other research scientists who contributed to the paper.

-Second to last Author: Supervising technical collaborator (possibly another PI)

-Last Author: PI of the lab, usually the corresponding author of the paper

Harvard Authorship Guidelines on pg.59

References/Citations: cite any statements made in the introduction to add validity to your claims. Methods section may also include citations (for protocols), as may the discussion section. Sources (even your own) should never be used word-for-word without quotes and citations.

Peer Review: where article is analyzed and criticized by scientists in the field. Usually three anonymous peer reviewers; all reviewers questions must be answered, may sometimes require further experiments. Final decision is often up to the journal editor, who can send the article to a fourth reviewer for further comments.

Experimental Design – review the steps

Scientific Method: Ask a question; identify the subject for experimental investigation; evaluate the current state of knowledge by reading scientific literature on the subject, formulate a hypothesis, select the biological system to be used, identify the variables and controls, test the hypothesis, come to a conclusion. Also, possibly formulate a new hypothesis and test the new hypothesis.

-If there is enough supporting (and no contradictory) evidence from many experiments, a scientific theory can be proposed.

Variables: what is measured or varied in an experiment

Independent Variable: that which is manipulated (time, initial amounts, etc)

Dependent Variable: changes in response to the independent variable and is observed and measured

Controls: Potential variables that must remain constant. Act as a baseline from which to measure changes in the dependent variable.

-Good controls are the key to high quality scientific research.

Positive Control: are used as the normal test and should produce expected, measurable results.

-Ensure the experiment worked as designed

Negative Control: should not be observed, and if they are seen it indicates that the sample or reagents are contaminated.

Experimenter bias & Prevention

Experimenter bias, at the least, involves a researcher subconsciously biasing their data.

To prevent bias, scientists use good controls, statistically analyze data, report all data (good & bad) and any manipulations thereof, undergo peer review process and experiment duplication, use double-blind experiments, and hold to scientific rigor.

Scientific rigor: ensures the data and conclusions are supported across all tests, and withstand any experiment they are put through.

Double Blind Experiment: tool used to prevent experimenter bias. In a double blind experiment, the experimenter does not know which is the control group and which is the experimental group

-Are the key to combating anecdotal evidence.

-Not practical for day-to-day experiments in a research laboratory.

**Above all else, scientists must be 100% ethical, have absolutely no bias, and adhere to scientific rigor when performing experiments.**

Chapter 19 - Cell Culture, Flow Cytometry, and Animal Research

A researcher who wishes to start a cell/animal project will: begin with full literature review, followed by project design, bioinformatics analysis of the gene of interest, and then DNA manipulation or protein purification. Next step would be to us cell culture to study in vitro, and after possibly in vivo.

Model systems: system that will best fit experiment, often requires tradeoffs. (advantages/disadvantages of each???)

Cell Culture Examples

Bacteria: inexpensive and efficient; proteins may not express, fold properly, or be properly modified

S.cerevisiae (yeast): inexpensive and easy to work with; can be used to study eukaryotic systems not found in bacteria, such as mitosis

Baculovirus expression vector system (BEVS): uses virus to insert genes into an insect cell line. Cells can express large proteins in high amounts and contain proper post-translational modifications. More expensive, take longer to grow (3-4 days).

Mammalian cell lines: effective because systems being studied are usually in their native environment, resulting in proper expression and folding. Much more difficult to work with—require CO2 incubators, media must be changed every few days, and are not easily stored long-term

Plants are also an alternative

Cell culture: an in vitro method that involves the incubation of biologically derived material in an artificial physical and chemical environment, outside of a living organism.

Major disadvantage: artifacts may appear—mechanisms or behaviors that only exist in a cell culture (ex. abnormally high amount of hydrogen peroxide)

Cell culture Media (In special CO2 incubator): pH ~7.4, phenol red dye as pH indicator; Possibly salts, vitamins, carbon sources, amino acids, proteins, growth factors=Fetal bovine serum (FBS); Media must be changed frequently (~2 days); Require sterile techniques to prevent bacterial contamination

Cells in culture may be modified or have genes inserted/removed

Transfection: the process of inserting DNA into eukaryotic cells (like transformation in bacteria); resulting cells can have genes inserted or removed = pathways/disease states can be studied.

Eukaryotic cells can be transfected via CaCl2 or other cationic polymers; electroporation; heat shocking; viruses (adeno-associated virus AAV) or retroviruses (HIV or lentivirus); lipofection using vesicles called liposomes that fuse with the cell membrane;nanoparticles; gene gun.

T-cell engineering: T-cell receptors are modified to target specific cell lines.

RNA interference: can be used to mediate sequence-specific degradation of a target mRNA, thus shutting down translation/expression of a specific protein.

Microinjection: technique that involves adding or removing nuclear material (enucleation) or injecting molecules into a cell with a small glass pipette.

Molecules can be inserted into cell

Nucleus can be completely removed/new one added.

Useful for plant manipulation as well

Immunohistochemistry/Immunocytochemistry (what do they tell you?): technique very similar to a western blot, where antibodies recognize and bind to an antigen. In both techniques, antibodies recognize a specific protein while it is still in its native cellular environment. Researchers can use this technique to identify where proteins are found in the cell or tissue and track their expression and localization.

Immunohistochemistry IHC – tissues

Immunocytochemistry ICC – cells

Pulse-Chase (purpose): Used to determine metabolic activity by tracking radiolabeled amino acid through a cell. Cell is exposed to radioactive molecule (Pulse), and after a few minutes exposed to a non-radioactive molecule (Chase); this radioactivity can be tracked throughout the cell; can follow metabolite, protein, or nucleotide through cellular compartments.

Electrophysiology & Patch Clamp (purporse): Electrophysiology is the study of the electrical properties of a cell

Microelectrodes attached to an organ; Patch clamp allows scientist to suck up a small section of the plasma membrane in a pipette, which contains an electrodxe that measures changes in current as ion channels in the cell open & close.

Readings are recorded on an oscilloscope.

Cell sorting – FACS/flow cytometry purpose, data analysis from histogram

Cell Sorting: the isolation in bulk of individual cells or populations of particular cells from a mixed population.

Bacteria & Eukaryotes can be selected based on ability to survive:

-Eukryotes transfected to make them resistant to infection, cytotoxins, or antibiotics

Cells can also be separated on basis of physical properties (RBC from WBC via centrifugation).

-Also separation by surface charge, electrophoresis, affinity chromatography, agglutination, or phase partitioning.

Flow Cytometry (for eukaryotic cells): allows a researcher to separate cells based on size, health, or other properties such as cell stage or number of chromosomes.

-Technique uses a vibrating nozzle to produce small droplets from a cell sample that contains several cells. The droplets pass through a laser that detects various cell properties. A charge can be added to a drop, which allows a deflector to divert the sample to several collection tubes. Samples often passed through a flow cytometer various times to ensure proper sorting. Figure on pg227

-Forward scatter = size / side scatter = granularity (health)

-Forward & side scatter can be recorded and presented as histogram. Individually, the histograms represent the size and health of the cells, and together the entire cell population can be analyzed based on both properties = Scatter plot

Fluorescence-activated Cell Sorting (FACS): improvement to flow cytometry; allows one to sort cells based on light scattering and fluorescence.

-Offers researchers a wide range of properties upon which to sort cells

-Cell-binding fluorescent dyes can be very specific to cell types or properties (also to DNA, organelles, or proteins. GFP-tagged proteins can also be detected via FACS

-FACS can be used diagnostically to determine cell ploidy or immunophenotyping to screen cell surface markers (such as CD markers in the Luminex Assay) to determine a patients disease state.

-Scatter plot figure on pg.228

In situ Organ perfusion: technique involves infusing a compound (like formaldehyde and sucrose) through a fine hollow needle inserted into the artery carrying blood to the organ, and performing subsequent analysis on the blood being transported from that organ.

-Can be used to study any organ (or interaction of a drug with an organ)

-Organ is removed from animal, maintained at constant temperature and pH in apparatus, and fluid is passed through the organ by gravity/pump that resembles heart flow.

Whole-body autoradiography (WBA): Entire slices of an animal are made. Animal is fed a radio-labeled drug, killed, frozen, and then sliced. Whole-body autoradiography is then used to trace the localization of the drug in the slice to determine where it takes effect.

-Can be used to track tumor growth, drug localization, or tumor/drug interactions

Animal Studies (advantages and disadvantages): used when experiments require more than in vitro cell cultures to obtain data.

C. elegans (roundworm), Drosophila (fruit fly), & zebrafish: commonly used due to their simplicity

-C. elegans has same basic organs and body plan as all animals in only 959 (hermaphrodite) or 1031 (male) cells, and is transparent (visualization). Nervous system has been completely mapped.

-C. elegans gene expression easy to disrupt via RNA interference

-Drosophila very useful for studying genetics

-Zebrafish very useful for studying development

Mice, rats, guinea pigs: ease of use, low cost, large litters

-Mice genome has been sequenced; 99% gene homology with humans; similar gene order as well.

-Mouse techniques perfected; genome easy to manipulate

Animals bred specifically for experimental purposes – under scrutiny of Institutional Animal Care and Use Committee (IACUC)

-Animals only used if in vitro models not suitable for experiments.

Disadvantages: animals don’t always produce human-like patterns of pathology, or respond in the same way to a particular treatment.

-Animal models = 66% prediction of human toxicity

-Rodents even less predictive

Transgenic animals: have manipulated genomes or carry genes from another species. Process involves injecting the gene or interest into an animal zygote and implanting this embryo into a surrogate female.

-Animals designed to mimic a human disease state

NOG Mouse: severely immunodeficient mouse that has been designed to lack T cells, B cells, NK cells, and has defective macrophage, dendritic cells, and complement functions.

RNA interference (RNAi): can be used in vivo. siRNA injected into a living animal instead of a cell culture. Can be used to study the role of specific genes in animals, as silencing expression can cause observable changes.

Cloning: process of creating an exact copy of a living organism. Dolly the sheep in 1996; Mouse in 2008; Pyrenean ibex in 2009 (had been extinct), survived briefly.

-Original cloning: involved removal of the nucleus from donor, transfer of a new nucleus from donor, egg then transplanted into surrogate mother

-New process: donor cells transferred to cell culture, genetically engineered into pluripotent stem cells by transfection of certain genes or demethylation of DNA, and the cells are then injected into a donor egg and surrogate mother. (This process is EXTREMELY INEFFICIENT)

Knock-out: involves deleting a gene; extremely useful in determining that gene’s function in the cell.

Knock-in: involves inserting a gene

Knock-down: involves partial gene disruption; may be needed if a gene is embryonic lethal if missing.

-All three techniques begin with basic DNA manipulation (PCR, cloning, transformation, transfection, cell culture) and use some advanced techniques to knock in or knock out a specific gene.

-Procedure: Mouse embryonic stem cells manipulated via homologous recombination so that they gain/lose gene of interest. Cells then selected for and injected into a mouse blastocyst, which is injected into a surrogate mother. Resulting mouse is a Chimera (contains cells from two different mice).

-Crossing chimeras will result in the production of a knockout mouse that lacks the gene of interest.

Cre-Lox mouse (Conditional knockout): involves knocking out a target gene only in a specific cell type. Use Cre recombinase (from a bacteriophage) to cause recombination between two LoxP sites (a specific sequence of DNA). Genes are turned off via inversion or complete removal.

First, Cre mouse is produced that contains Cre recombinase in its genome, with a cell-specific upstream transcription factor binding site. It is then cross with a LoxP mouse, which has the gene of interest flanked by LoxP sites. The resulting Cre-LoxP mouse will express Cre recombinase only in cells that contain the correct transcription factor. Cre recombinase will seek out the LoxP sites and remove the gene. A reporter system (GFP, LacZ) can be used to verify that the gene has been turned off

Optogenetics: Algae express protein channelrhodopsin embedded in the cell membrane that uncoils and opens up channels in response to light, which turns on flagella (in the algae). This gene and a specific promoter are inserted into a retrovirus and injected into the brain of a mouse; the virus then inserts the new gene into every neuron in the vicinity. Because of the promoter the gene will only be expressed in specific neurons. When light strikes the neuron, channelrhodopsin opens and the neuron fires.

-Other rhodopsin variants can be used to actually inhibit neuron firing

NOTES FROM LECTURE

• BONUS: what are HeLa cells? Cervical cancer cell line taken from Henrietta Lacks (?)

Chapter 20 - Bioinformatics and Computer-Based Research

Bioinformatics overview: A large amount of molecular biology research today relies on some form of computational analysis

The computational branch of molecular biology is called bioinformatics

-This branch ties together molecular biology, physics, chemistry, biochemistry, computer science, math, statistics, informatics, and artificial intelligence.

-Uses algorithms and databases to analyze DNA sequences, gene expression, genomes, protein structures, protein interactions, and drug interactions

-In silico: techniques performed in a computer

Genome sequencing techniques: roots of bioinformatics is in genome sequencing of the 1970s

RNA genome of bacteriophage MS2 sequenced in 1976

Frederick Sanger sequences genome of bacteriophage ϕ-X174 (had previously sequenced insulin in 1955

Haemophilus influenza: 1.8 million bp; first non-viral genome to be sequenced (1995); scientists at Johns Hopkins University

Human Genome Project: started in 1990 by James Watson at NIH, funded by Dept. of Energy. Taken over by Francis Collins in 1992.

Goal was to sequence 3 billion chemical base pairs that make up human DNA, identify 20,000-25,000 human genes, and store all this information in databases. Also, to improve DNA data analysis, transfer related technologies to private sector, and apply knowledge gained to other fields.

Cost $3 billion in public funds, $300 million by Celera- $100 per bp

End Result: near-complete sequence of the human genome stored in a computer database accessible to all. Only regions not sequenced were centromeres and telomeres = highly repetitive and difficult to sequence by current methods.

Two approaches used: chromosome walking & shotgun sequencing. Together, two approaches helped to produce the complete human genome in the early 2000s

Chromosome walking: genomic DNA carried in a clone is sequenced, and a fragment of this sequence is used as a probe to isolate the next clone, which is sequenced next. Only 500-1000 bp of DNA could be sequenced at a time (very challenging)

Shotgun sequencing: put into practice in the human genome by Craig Venter. Human genome is mechanically sheared, polished to add adenines to each piece of DNA, incorporated into plasmids via TA cloning, and sequenced using plasmid-specific primer. Sequenced fragments are then pieced back together on a computer.

Human genome laid the foundation for using genomic info for drug discovery, led to new field of study – systems biology: the study of entire biological systems and their interactions between components.

Genomics: the study of an organism’s entire genome.

NCBI: National Center for Biotechnology Information – “National resource for molecular biology information, NCBI creates public databases, conducts research in computational biology, develops software tools for analyzing genome data, and disseminates biomedical information – all for the better understanding of molecular processes affecting human health and disease”

GenBank: database that contains DNA sequences amounting to billions of base pairs and DNA from thousands of different organisms, from viruses to humans. Journals expect researchers to submit their DNA and protein sequences to GenBank prior to publication.

PubMed: searchable database of thousands of scientific journals. First place that one should visit when looking for peer-reviewed scientific data on a topic, or when performing a full literature review for a research project.

BLAST: Basic Local Alignment Search Tool; tool for comparing any sequence with which you are working to the entire GenBank database.

Can identify what gene the sequence is, or any similar genes in the database.

Also useful for finding similar sequences among various species

-If two sequences are ~30% identical or more (whether within a species or across species), it usually indicates they have a similar function

Sequence Alignment: sequences from blast search are further analyzed by looking for conserved nucleotides (BLASTn - nucleotide) or amino acids (BLASTp - protein) across species

-Alignment provides information useful for evolutionary analysis or can be used to determine which amino acids play an important role in a proteins function. Identical amino acids across species usually indicate that they serve an important function as they have been conserved since the last common ancestor.

Phylogenetic Tree: can be used to display sequence data visually and determine relationships between species. Can be created with DNA or protein sequences.

ExPASy: Expert Protein Analysis System – site contains tools and software used to analyze DNA and protein sequences. Some of the tools allow for protein identification and characterization (Mw, pI), DNA to protein conversion, similarity searches, post-translational modification and topology prediction, and more..

Sequence analysis – pI, Mw, regions of homology, percent identity, phylogenetic tree, etc.

Secondary structure analysis: Most basic technique for secondary structure prediction is to use alignments with known sequences or structures.

Chou and Fasman (1974): their techniques can be used when no homologous structures exist. Their analysis of proteins showed that certain amino acids were more likely to be involved in alpha-helices, B-sheets, and turns than others. Approx. 50% accurate

Garnier-Osguthrope-Robson (GOR): analyzes a larger segment of amino acids (17 AAs total) to predict protein structures. Approx 75% accuracy.

Ultimate goal of bioinformatics (with regards to protein structure) is to be able to predict exact tertiary structures from the primary sequence.

Ramachandran Plot: this plot analyzes the phi and psi angles around the alpha-carbon of an amino acid. Certain combinations of phi and psi tend to be found in specific secondary structures, and some AAs are limited to certain phi and psi angles by steric hindrances.

-Alpha-helix: phi = -60 and psi = -50

-B-sheet: typically have positive psi.

-Plots useful for predicting secondary structures, but also used as a refinement tool to ensure all angles are within acceptable limits when solving proteins structures.

Helical Wheel: used to predict an alpha-helix. Proteins can be analyzed to look for pattern of alternating residues to not only discover new alpha-helices, but predict their location and possible function in the overall protein structure.

-Globular proteins and leucine zippers often have alpha-helices with alternating hydrophobic and hydrophilic residues. It is more favorable for hydrophobic AAs in a protein to be buried internally away from the solvent

Hydrophobicity Plot: AAs can be polar or nonpolar; this plot assigns a number to each amino acid based on how hydrophobic or hydrophilic it is (Fig on pg240). A sequence can be analyzed to look for hydrophobic or hydrophilic stretches of amino acids.

-Hydrophobicity information can be useful in predicting transmembrane proteins which have long stretches (~20) of hydrophobic AAs followed by stretches of hydrophilic amino acids (Figure on pg241). Hydrophobic AAs are buried in the plasma membrane, while the hydrophilic AAs are exposed to the solvent on inside/outside of cell.

Disorder Plot: another type of secondary structure analysis, which looks for regions of high movement or disorder. Often, such a stretch of AAs are important in protein function, ligand binding, protein-protein interactions, and protein activation.

Protein Data Bank (PDB): once the tertiary structure of a protein is solved, its coordinates are deposited into the Research Collaboratory for Structural Bioinformatics Protein Data Bank. These structures are free and accessible from any computer. Currently contains over 10k protein structures, including small molecules, DNA, RNA, and more.

Structural modeling: involves taking unknown sequences and predicting tertiary structures, or taking known structures and modeling protein activation, protein-protein interactions, ligand-binding, and much more.

-Accurately predicting tertiary structures is very difficult because of large number of structures possible from even a handful of AAs

Molecular Mechanics: most common technique for accurately predicting tertiary structures from scratch (ab initio).

-This technique uses the atomic nuclei and force fields to predict structures and interactions.

-Fast; fairly accurate; able to handle millions of atoms

-Still not a perfect representation of what is occurring at molecular level.

-Quantum Mechanics: takes into account the electrons of the atoms and wave-particle duality, along with other complex properties. Produced structures are very accurate, but require a lot of computing power, and are still size-limited.

Energy minimization/Simulated annealing: Once tertiary structure is solved, it is further optimized through this technique. Temperature of molecule in the computer is increased, whence it will begin to vibrate greatly, and as it “cools” it will hopefully find the energy minimum, or the most favorable (and likely) structure.

Molecular Dynamics: another aspect of structural modeling that allows one to model molecular interactions, like a protein with a binding partner. (protein with binding partner, myoglobin with oxygen, etc..)

Virtual screening (Structure-based design): was an answer to lack of diversity produced by medicinal chemistry and the high cost of high-throughput screening. It is an efficient way to screen thousands of potential protein inhibitors on a computer.

-Cost effective; reduces false-positives; increases the number and diversity of potential inhibitors; helps to find new classes of drugs.

-Take a known structure, mark the site to which a potential inhibitor should bind, search a database for drugs that would bind to the site, and score possible hit.

-Once potential inhibitor is identified, it can be modified, synthesized, or purchased and tested in vitro to see if it inhibits the target.

Proteomics: the study of all of the expressed proteins in an organism, including isoforms and modifications. Purpose is to produce comprehensive protein databases of gene function and help to lay the groundwork, along with genomics, for systems biology (studying entire organism).

NOTES FROM LECTURE:

• Carsonella ruddii (proteobacteria) – 159,662 (182 genes) = Smallest genome we’ve found

• 2.86/3.1 Gb sequenced (92%) of Human Genome

• Benlysta (treatment for Lupus) one of the first drugs to come out of the genomics revolution.

• If two proteins have 30% identity/homology = very good chance both have same function

• RecA (e.coli) & RAD51 (Humans) only have 15% homology but structurally there is a lot of homology; both have very similar functions

• Φ-X174 (5,368 bp) infects E. coli

• Long stretches (~20) of nonpolar/hydrophobic AAs can indicate that the protein is transmembrane

• NMR can capture movement; Although X-ray crystallography is usually better, it cannot capture movement

• Issue with virtual screening is that the structure of the drug targets have not been resolved and are also often times transmembrane proteins

• Dorzolamide (1995), an anti-glaucoma drug that targets carbonic anhydrase, is the result of structure-based drug design

• Creating life 2014 – synthesized functional yeast chromosome from scratch (272kb)

Chapter 21 - Mass Spectrometry, Microscopy, NMR, and Crystallography

Mass spectrometry (MS): allows one to determine the mass of a molecule with extreme accuracy. Useful for identifying, verifying, and quantitating metabolites, proteins and peptides, oligos, and much more.

In MS, molecules are converted into a charged, gaseous state and analyzed by a detector. Gaseous ions are separated according to their mass-to-charge ratio (m/z) and the output is a plot of the relative abundance of the ions at each m/z ratio.

Electrospray Ionization: used to analyze larger molecules (like proteins). Sample is sprayed with a charged solvent and detected.

Liquid Chromatography Mass Spectrometry (LC-MS): injects liquid directly into the mass analyzer.

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF): uses a laser to gently ionize large molecules. Smaller molecules diffuse more quickly, and reach the detector first. The time it takes for the molecule to reach the detector is measured and converted into molecular weight.

-Mass spectrometers can detect isotopes if their resolution is high enough = better mass accuracy achieved

-MS can be used to identify proteins: proteins are digested and subjected to one of various types of MS. The resulting spectra will give the Mws of any fragments in the mixture, which can be compared to a table of known Mws of protein sequences.

Monoisotopic mass vs. average mass

Inverted Microscope: has optics below the stage, unlike a traditional microscope (with the optics on top of the stage).

-Especially useful for cell cultures since cells tend to be on the bottom of tissue culture flasks

Fluorescence Microscopy: allows researchers to view aspects of the cell not visible with a normal microscope

-Molecule is labeled with GFP or a dye, which is excited by a specific wavelength to fluoresce.

-Fluorescent Abs or chemicals that target a specific cell structure can be used, or genes that encode proteins that are tagged with GFP can be transfected into cells. Proteins can be tracked as they move throughout cell

Confocal microscopy: similar to fluorescence microscopy, but uses laser to greatly improve the image quality and resolution.

Electron microscopy: allows researchers to view samples at up to 10 million fold magnification. Electrons, which have very small wavelengths, are focused to resolve an image.

Transmission Electron Microscopy (TEM): electron beam goes through the sample. Higher magnification.

Scanning Electron Microscopy (SEM): beam bounces off of a gold-plated sample. More useful as it only reveals surface of a sample

Cryo-electron Microscopy (Cryo-EM): many copies of a sample are frozen and placed under an EM. Hundreds of thousands of pictures are taken of the sample from various angles, and these images are reassembled into a single image.

-Has reached 4 Angstrom resolution (enough to trace a polypeptide chain.

-Resolution not as high as NMR and X-Ray crystallography, but works well in conjunction with these other techniques to provide low resolution information.

Rotary Shadowing: technique allows one to view individual proteins and macromolecules without the need for advanced procedures, but at much lower resolution.

Atomic force microscopy: technique developed to visualize small molecules and proteins. Device is similar to a tuning fork that vibrates near molecules, causing a vibration that can be measured.

-Technique has been used to visualize molecular structures and myosin walking along actin

Circular Dichroism (CD): allows one to get an idea of the secondary structure features of a protein. CD measures differences in the absorption of left-handed polarized light vs. right-hand polarized light, which arise due to structural asymmetry in alpha-helices and B-sheets.

-Not adequate for determining tertiary structure

NMR: used to determine tertiary structure. Technique utilizes very strong magnetic fields to determine the relative positions of certain atoms in the protein that have nonzero spins and thus magnetic and angular momentum. Interactions between these various atoms and their neighbors can be measured in the presence of the magnetic field and the atomic structure can be determined

-Allows researchers to study molecules directly in solution without the need for a crystal, which produces more mobile, flexible structures. (Advantage over crystallography)

-NMR structures are very accurate. Limited to 30kDa in size.

X-Ray Crystallography: can be performed on anything, so long as a crystal of the molecule is available. Crystals generally produced by purifying the protein of interest via chromatography, concentrating it to 10mg/ml, and placing it over a reservoir of precipitant that allows for the water in the droplet to slowly evaporate, promoting crystal formation.

Asymmetric unit: most basic unit for a crystal.

Unit cell: the smallest unit of repeat within a crystal.

-Crystal is placed in front of a very powerful X-ray beam. Rays generally travel through, but can also interact with electrons of the protein and scatter. The diffraction pattern is collected on film/by camera. Crystal is also rotated to collect data from all sides to determine structure.

Space group: orientation of the crystal’s subunits; broken down into 7 major groups based on symmetry: cubic, hexagonal, rhombohedral, tetragonal, orthorhombic, monoclinic, and triclinic + 230 subclasses. Only 32 protein crystal classes

Constructive interference (in phase) vs. Destructive interference (out of phase): incident X-ray strikes a specific electron and the deflected beam results in a diffraction spot vs. no beam/electron interaction = no spot detected. Data is then used to create an electron density map

Imaginary space: created by the resulting diffraction image; is unfocused; equivalent to the light that strikes the lens of your eye before being focused. Diffraction spot intensity provides information regarding electron density in a protein.

Fourier Transform: converts imaginary space to real space. Data from hundreds of images is integrated into single data set = structure can be determined.

Phase problem: scientist must determine the phase at which the light wave struck the crystal to solve the structure. Majority of “image” info is carried in the phase, not intensity.

-Molecular replacement: known structure used to solve unknown structure.

-Multiple isomorphous replacement: heavy metals are soaked in the crystal and their diffraction is located in the data

-Anomalous dispersion: (requires synchrotron) selenomethionine or other molecules are used to measure differences in the crystals diffraction at 180 degrees.

-END RESULT: electron density map which shows where X-rays interacted with electrons. Protein structures can be built into the electron density map and refined.