Greetings! Here are the lecture notes from today. Related information can be found in your book on the following pages. However, the information is not always as comprehensive so please look at the following slides for other details or examples.

Pg 555-557 flourescence microscopy and immunoflourescencePg 574-578 radioisotopesPg 452 Figure 7-107 RNA interference

Also, I will put a note on every slide to tell you what you should focus on when studying. Therefore you will have an idea of how you should study. I am interested in the major concepts that I present to you.

STUDY GUIDE FOR TEST (the stuff I talked about in class):Review intro biology on central dogma, mitosis, meiosis, and cell cycle.Know main categories of life and viral genomes. Pay attention to viral intermediates.Vertical vs horizontal transmissionTypes of Gene duplications and how they are causedOrthologous and paralogous genes (Primate evolution tree example)Basic gene structure (promoters, coding regions)Microarray analysis

• FLUORESCENCE MICROSCOPY

• PROTEIN LABELING AND LOCALIZATION

• DNases, RNases, Proteinases

• siRNA

•RADIOLABELING OF NUCLEIC ACIDS

•FLOW CYTOMETRY

TECHNIQUES IN CELL AND MOLECULAR BIOLOGYThe techniques presented here are used in all kinds of ways in research; thus it is important that you have a basic understanding of how they work. We will visit their use in context throughout the rest of the year.

nobelprize.org

Flourescence Microscopy

All you need to know is that you can excite molecules at a wavelength and they emit at another wavelength. The filter used on the microscope allows you to look at one flourescent emission at a time.

Immunofluorescence utilizes antibodies that have a specific target, i.e., proteins.

www.serotec.com/

NOTE: Either primary or secondary antibodies can be tagged

You can see where a protein is in a cell by using a flourescently labeled antibody that recognizes either a) your protein directly, or b) the antibody that binds to your protein (shown above).

http://www.microscopyu.com

HeLa Cells (HPV transformed)Primary Anti-tubulin mouse monoclonalSecondary goat anti-mouse conjugated to Alexa Fluor 568TO-PRO-3 targets nucleus (DNA)

This is an example of a primary antibody against tubulin and a secondary antibody with a fluorescent tag against the primary antibody. The nucleus lights up yellow because the cells have been stained with a flourescent tag that specifically binds DNA.

Fluorescence microscopy utilizes a variety of natural and synthetic compounds:

NATURAL:Phallotoxins are isolated from the mushroom, Amanita phalloides. They bind to and stabilize actin filaments by inhibiting depolymerization. Phalloidins and phallacidins are similar peptides, used more or less interchangeably to label filamentous actin.

Wheat germ agglutinin (WGA), a lectin that binds sialic acid - an important constituent of glycoproteins and glycolipids.

Green Flourescent Protein (GFP) is a spontaneously fluorescent protein isolated from the Pacific jellyfish, Aequoria victoria.

SYNTHETIC:4'-6-Diamidino-2-phenylindole (DAPI) is known to form fluorescent complexes with natural double-stranded DNA, showing a fluorescence specificity for AT, AU and IC clusters.

The molecules here are used in all kinds of ways depending on what you want to look at. For example, if you want to know where a protein is in the cytoplasm, then perhaps using phalloidin conjugated with a flourescent molecule in ADDITION to your tagged protein (use a different flourescent molecule like GFP) is a way to PROVE it is in the cytoplasm (they co-localize).

Important information is underlined.

GFP is a naturally occurring molecule found in jellyfish. It was originally identified at Friday Harbor Laboratories.

Rhodamine Phalloidin(actin)

DAPI(DNA and RNA)

Dr. Jalal VakiliVLST Corp.

This is just an example of the use of Phalloidin conjugated to the flourescent molecule Rhadamine (you do not need to know the name); DAPI binds DNA to show you where the nucleus is at.

www.alzforum.org

Plasma membrane localization of Beta-Catenin. Rhodamine immuno-fluorescence staining of methanol-fixed A-431 cells with a DAPI counterstain

Here is an example of how one would use immunoflourescence to show where a protein is located in the cell (the plasma membrane in this case).

Nuclei were visualized with mouse anti-histones (core) primary antibodies, while the Golgi complex was stained with rabbit anti-giantin antibodies. Secondary antibodies were goat anti-mouse and anti-rabbit conjugated to Texas Red and Oregon Green 488, respectively to produce red nuclei and green Golgi cisternae. The filamentous actin network was counterstained with Alexa Fluor 350 conjugated to phalloidin.

This is an example of how one would localize a protein that is thought to be in the Golgi. Note, that we know where these proteins localize, but in an experiment, that would be a hypothesis, particularly if you are working with a protein that you THINK localizes in the Golgi.

www.gurdon.cam.ac.uk

Wild type cyclin B1GFP Fluorescence

Alanine-mutantYFP fluorescence

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This is just showing you how merged images can be shown as one.

www.gurdon.cam.ac.uk

DIC = differential interference contrast microscopy This is another example of a merged image and what it would look like.

http://www.microscopyu.com

Figure 1, a triple-labeled Drosophila embryo at the cellular blastoderm stage. The specimen was immunofluorescently labeled with antibodies to three different proteins. After three corresponding images were collected in the red, green, and blue channels of the confocal system, the images could be rearranged by copying them to different channels.

Illustrates the power of using flourescent molecules to look at gene expression. The proteins are transcription factors expressed in different areas of a developing embryo.

http://www.microscopyu.com/galleries

Small intestine cross-section. Texas Red-X conjugated to wheat germ agglutinin (WGA), a lectin that binds to N-acetylglucosamine and N-acetylneuraminic (sialic acid) residues. Alexa Fluor 488 conjugated to phalloidin and DAPI.

One can use flourescence microscopy to look at tissues, such as a cross section of the small intestine.

Protein tagging•Recombinantly created

–Synthetic epitopes/Antibody tags•Flag•CTag

–Chemical binders•His tag

–Fluorescent tags•Green fluorescent protein (GFP)

•Chemical modification–BiotinyationThere are two main ways to tag proteins:

a) Create a tag by adding a sequence in frame with your protein, so when it is expressed it gives you a tagged protein (see next page).

b) Chemically modify it (just to let you know but do not memorize)

Protein Tagging

DNA encoding Protein X

Fuse to synthetic sequence

Express protein

Recombinant method Chemical modification

Linkage Chemistry

Biotin molecule

These are just visual examples.

Synthetic epitopes

• Recombinant epitope targeted by antibody– Flag tag

Antibody can be conjugated to fluorescent tag or bound to a solid particle

SO. . . You can track it with fluorescence or purify it!

Some tags allow you to bind them to beads in order to purify them.

Synthetic epitopesTandem Affinity Purification (Tap) Tags

A Tap tag is one such tag that allows you to purify the protein with beads.

Chemical Binding epitopes

• PolyHistidine tag – Protein binds to resin conjugated to nickel

Ni

Ni

NiNi

Ni

Multiple histidines at the end of the protein let you bind the protein to a Nickel column so you could purify it away from other proteins.

Fluorescent tagsGreen fluorescent protein (GFP)

But there are other modified kinds too!

UV photonGreen photon

Proteins can be tagged with the naturally occurring protein GFP. Thus you can visualize your protein in a cell. A flow cytometer can detect it as well.

Exonuclease I (ExoI) from E. coli degrades ssDNA in a 3'=>5' direction

S1 nuclease is an endonuclease that is active against ssDNA and ssRNA

DNase I, is an endonuclease acts on ssDNA and dsDNA, chromatin and RNA:DNA hybrids

Applications:Degradation of DNA template in transcription reactions Removal of contaminating genomic DNA from RNA samples DNase I footprinting Nick Translation

DNASES

There are DNases that degrade DNA and they function on either single stranded or double stranded molecules.

RNase A is sequence specific for ssRNAs

RNase I degrades all ssRNAs

RNase H cleaves the RNA in a DNA/RNA duplex to produce ssDNA

RNase P is unique from other RNases in that it is a ribozyme and functions to cleave off extra sequence of RNA on tRNA molecules

RNase VI is non-sequence specific for dsRNAs

RNase III is specific for dsRNA

RNASES

There are RNases that degrade RNA. Some recognize single stranded RNA, some recognize double stranded RNA, and others DNA/RNA duplexes.

2006 Nobel Prize Winnersfor their discovery of RNA interference – gene silencing by

double-stranded RNA

Craig Mello Andrew Fire

This is one example of why RNase is important. . .

Dicer is an RNase III

Argonaute is part of RISCand has endonuclease activity, particularly against the complementary mRNA!

This is one example of why RNase is important. . . RNA interference! It is activated by dsRNA and targets complementary mRNA. Thus, you can use it to “knockout” a gene within cells.

WHY DID THAT EVOLVE?A) Innate immunity to incoming viruses that have a dsRNA intermediate

Family: Orthomyxoviridae (Genus Influenza)Family: Picornaviruses (Rhinoviruses and Poliovirus)Family: Reoviruses ( )

NOTE: the genome is a perfect dsRNA molecule

B) Protection against double-stranded RNA from mobile genetic elements

The life cycles of viruses often include dsRNA, and so it played a role in the evolution of DICER and RISC in RNA interference.

Proteases

Details forthcoming! Basically, they degrade proteins.

Serine proteases Threonine proteases Cysteine proteasesMetalloproteasesAspartic acid proteases Glutamic acid proteases

Just know that you can add a proteinase and degrade protein in your samples. This is important if you are wanting to study just the nucleic acids or something like that.

Radioisotopes and Autoradiography

32P-ATP, GTP, TTP, CTP, UTP is available for incorporation into nucleic acids(sometimes referred to as “labeling”)

35S available for incorporation into proteins

Nucleic acids and proteins can be labeled with radioisotopes. We can use this procedure to a) track molecules in a cell, b) look at the molecules on a autoradiograph (film), or c) detect their presence in a sample. Alpha phosphate should be used in order to incorporate it into a strand of DNA or RNA.

Flow CytometryThis technique allows us to

measure:a) Flourescent cells in a sample

(example – GFP expressing cells or cells stained with DAPI that binds DNA)

b) The size and shape of the cell

One can even sort cells depending on if they are flourescent or not.

I drew a graph on the board of how this is used to measure a cell cycle profile. The y axis is cell number (total number of cells put through the machine) and the x- is DNA content. The stain is DAPI.