Download Lecture 10/09

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 immunoflourescence
Pg 574-578 radioisotopes
Pg 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 transmission
Types of Gene duplications and how they are caused
Orthologous and paralogous genes (Primate evolution tree example)
Basic gene structure (promoters, coding regions)
Microarray analysis
TECHNIQUES IN CELL AND MOLECULAR BIOLOGY
The 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.
• FLUORESCENCE MICROSCOPY
• PROTEIN LABELING AND LOCALIZATION
• DNases, RNases, Proteinases
• siRNA
•RADIOLABELING OF NUCLEIC ACIDS
•FLOW CYTOMETRY
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.
nobelprize.org
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).
HeLa Cells (HPV transformed)
Primary Anti-tubulin mouse monoclonal
Secondary goat anti-mouse conjugated to Alexa Fluor 568
TO-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.
http://www.microscopyu.com
Fluorescence microscopy utilizes a variety of natural
and synthetic compounds:
NATURAL:
Important information is underlined.
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).
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 Vakili
VLST 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.
Plasma membrane localization of Beta-Catenin. Rhodamine
immuno-fluorescence staining of methanol-fixed A-431 cells with
a DAPI counterstain
www.alzforum.org
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.
Wild type cyclin B1
GFP Fluorescence
Alanine-mutant
YFP fluorescence
Merged Image
time
0 min
3 min
This is just showing you how merged images can be shown as 6one.
min
www.gurdon.cam.ac.uk
This is another example of a merged image and what it would look like.
DIC = differential interference contrast microscopy
www.gurdon.cam.ac.uk
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
http://www.microscopyu.com
areas
of a developing embryo.
Small intestine cross-section. Texas Red-X conjugated to wheat germ
agglutinin (WGA), a lectin that binds to N-acetylglucosamine and Nacetylneuraminic (sialic acid) residues. Alexa Fluor 488 conjugated to
phalloidin and DAPI.
http://www.microscopyu.com/galleries
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
–Biotinyation There 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
Recombinant method
DNA encoding Protein X
Fuse to synthetic sequence
Chemical modification
Biotin molecule
Linkage Chemistry
Express protein
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 epitopes
Tandem 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
Ni
Ni
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 tags
Green fluorescent protein (GFP)
But there are other modified kinds too!
UV photon
Green 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.
DNASES
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
There are DNases that degrade
Nick Translation
DNA and they function on either
single stranded or double
stranded molecules.
RNASES
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
There are RNases that degrade RNA.
Some recognize single stranded RNA,
some recognize double stranded
RNA, and others DNA/RNA duplexes.
2006 Nobel Prize Winners
for their discovery of RNA interference – gene silencing by
double-stranded RNA
Craig Mello
Andrew Fire
This is one example of why
RNase is important. . .
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.
Dicer is an RNase III
Argonaute is part of RISC
and has endonuclease
activity, particularly
against the
complementary mRNA!
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 proteases
Metalloproteases
Aspartic 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 Cytometry
This 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.