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59 Cards in this Set

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  • Back
How does RAs-GTP work?
GTPAses are enzymes including RAS
RAs works as part of the biochemical pathway linkin growth factor receptors in the plasma membrane of animals cells to regulation of cell cycle
What are the general principles of macromolecular assembly?
1. Large structures assemble from subunits
2. Specificty vcomes from multiple weak bonds on complementary surfaces
3. Subunits in symmetrical structures have identical or quasi-equivalent bonds between subunits (helices, icosahedrons)
4. New properties emerge along aseembly pathways
5. Regulation is imposed at mulitple steps along pathways
What are the advantages of assembling large structures from small protein subunuts?
!. Conseres space in the genome, one small gene can encode all of the pieces of a large structure
2. Allow synthesis of error free components: errors in protein synthesis since errors occur every 1/3000 residues
3. Quality control: defective subunits can be discarded
4. Subunits are easy to recycle
5. Multiple modes of regulation are available
Decribe the bonds in macromolecular structures?
*multiple weak bonds on complementary surfaces
*No covalent bonds between subunits
*Hydrophobic effect usually predominates
*Hydrogen bonds, electrocstatic bonds
What was Crane's hypothesis?
macromolecular structures in cells are assembled from small subunits
How do new properties emerge along assembly pathways? (Use actin as an example)
1. Association of subunit sbrings together partial binding sites: formation of an action trimer nucleus has all of the binding sites to grow an actin filament
*Conformation change creates a binding site: interactions with an actin filament and WASp cause a conformational change allowing Arp2/3 complex to grow an actin filament branch
What is the formula for formation of a nucleus for the growth of an actin filament?
That is the velocity of nucleus formation is a function of the actin concentration raised to the 3.4 power
What was the first virus assmebled in the labratory? How
Tobacco mosaic virus from purified protein and RNA
HOW does TMV assemble?
RNA allows elongation at neutral pH and determines the length
How does the Tomato Bushy stunt virus form?
180 identical subunis, subunits pack differently at the 12 five fold vertices than at the six fold vertices, this is called quasi equivalent packing
What is the structure and assembly of DNA tumor viruses?
pentameric subunits interact at both 5 fold and 6 fold vertices by virtue of flexible C-terminal tails
How does the bacteriphage T4 assmble?
from 49 gene prodcuts along three different pathways (tail, head, tail fibers)
What is resolution vs magnification?
Magnification makes an image bigger, but doesn't necessarily mean you can see more, resolution is the ability of the eye to distinguish between two things as being seperate
What is the formula for resolution?
.6*wavelngth/ NA
NA=numerical aperture=nSina
Where n is the refractive index of the mdeium between the objective and the specimen
n of air=1
n of microscope=1.515
What is bright field for?
Based on Absorption stains required and fixed non living cells
What is phase contrast based on?
Refractive index differences, used for living and fixed cells
What is DIC based on?
Gradient of refractive index...fir both fixed and living
What is fluorescnce for?
based on emission by fluorescent molecule for living and fixed
What is dark field base?
light scattering for living and fixed
What is polarization based on?
birefrigence used for fixed and living
What do amplitude objects do?
cause changes in amplitude
What do phase objects do?
Cause changes in phase (shifts amplitude over) almost all living cells are phase objects not amplitude objects (unless they are pigmented)
What is the wave theory of light?
light waves are oscillating sine waves of a specifc amplitude and wavelength
1. amplitude is directly proportional to brightnes
2. wavelength, within a narrow range of the electromagnetic spectrum, is detected as color by the eye
3. The phase of the sine waves of light relative to each other are also important
What is the essence of the phase microscope?
it manipulates the light as it passes through the microscope so that in essence it converts phase changes into amplitude changes that the eye can detect
What are P,S, and D?
P-the wave your sees as coming from the particle in focus (=D+S)
S=the light from the surroundign medium
D-the light deviated by diffraction in the particle, due to refractive index differences in the specimen (particle), this wave has been retarded in phase relative to S wave, by the 1/4 wavelength (which is characteristic of the biological samples)
What did Zernike realize about the D wave?
if he could impart another 1/4 wave retardation to the D wave then it would be 180 degrees out of phase with S and thus interfere with the S wave to produce a P of a different amplitude this makeing the P wave visible
Adding a condenser annulus and an objective phase plate, was able to manipulate the retardation of the D waAve thus increasing its retardation to 1/2 a wavelength
D and S now completely destructively interfer with each other, producing a P wave of smaller aplitude and thus P is now visible against the brighter bakcground of S
What does DIC do?
gives cells a shadowcast and 3-d appearance
What is protoplasm?
refers to the contents of a living cell, the matter contained within the plasma membrane
What is cytoplams?
all the stuff in a cell that is not the plasma membrane and the nucleus
What is cytosol?
the part of the cytoplsm that does not appear to contain membranous or particulate subcellular components
What is the cytoplasmic fraction?
The matieral obtained after cells are homogenized and unbroken cells, cell debris, and nuclei are remvoed by centrifugation. not synonymous with othera
What is the soluble fraction?
any fraction produced that remains soluble after some treatment (NOT cytosol)
What are the three types of light microscopy?
bright field, phase, DIC
What is Fluorescence?
Absorption of a photon raises an electron to an excited state, a longer wavelength photon is emitted when the elecgtron falls back to the ground state
problem: few biological molecules are fluorescent
blue-->green (fluorescein)
Uses a filter and a dichroic mirror
What do fluorescent dyes need to be bound to?
protein: needs to be microinjected
lipid: can be injected or fused to cell
nucleic acid: in situ hybridization in fixed cells
ligand for mellular molecule: i.e. antibody isolated from animals immunized with target molecule
WHat are Fluorescent fusion proteins?
GFP-cDNA for green fluorescent protein
(GP) from jellyifsh can be fused to genes for many proteins and expressed in live cells
*variants of are yellow, cyan, and red
How does fluorescent antibody staining work?
inject bunny with purified antigen,, bost with purified antigen, bleed to obtain serum, purify antibodies, label antibodies with fluorescent dye
What does fluorescent phallloidin bind to?
actin filaments
What is confocal microscopy?
allows detection of only the fluorescent signal emanating from the place of focus-the microscope rejects signal coming from slightly above or below the plane of focus\, in conventional, fluorescence microscopy, the focused image can be contaminated with out focus fluorescence, this not too big of a problem with single cells normally, but it cann be sever in thick specimans
What is deconvolution?
micropscope is told via computer to start at the top of a specicman and taqke images until the whole speciman is represented in a series of slices
the z stack is than deconvulved by software algorithms that look at each slice . by knowing something called the pint-spread-function of the microscope the algorithms determine the out of focus ligt in the target slice and remove it ,
the slices are then summed together to genreate a complete image of the speciman in which the out of focus light has been removed
What is the point of deconvolution
a computational method to remove out of focus light from micrographs,
Compare resolution of an electron micrscope to light microscope
The EM image has about 200x the resolution of the LM image
How is specimen prepared for transmission EM?
tissue washed and dehyrdated, put into medium and into vial, , plastic is polymerized in oven, when plastic is hard the block is trimmed and section
How does scanning EM works?
similar to DIC in that images appear to have depth
How do you figure out what microscope was used
Does it contain color or grey levels?
If contains grey the LM or EM
if colors-it is fluorescnce or a stained (both LM)
-everything else blakc (fluoresence LM
If #D its either DIC or SEM or TEM
LM can not resovle more than .200nm
What is gel electrophoresis?
run on gel
What are restriction enzymes?
enzymes that cut DNA strnads at specific base sequences, ie,e they are restricted to a specific sequence, and DNA will not be cut, read in 5 to 3' direction
Some cut leaving overhanging ends
Some leave blunt ends
What are ligases?
enzymes that anneal the two DNA ends
What is PCR?
amplifies DNA sequences, heat to seperate ends, hybridize primers, replicate
What is the likelihood that two individuals sgare by chance the same DNA?
1 in 10 billion
What are the differnt ways to break up cells and tissues?
1. high frequency sound, 2. use a mild detergent to make holes in the plamsa membrane 3, force cels through small hole using high pressure 4. shear cells between a close fitting rotating plunger and he thick walls of a glass vessel
What are the 2 types of centrifuges?
fixed angle rotor, swinging arm rotor
What is differential centrifugation?
repeated centrifigutation at progessively faster speeds will fractionate cells homogenates into components-- (1 whole cels, nuclei, cytoskeletons), 2 mitochodnria lysosomes, peroxisomes, 3 microsomes 4 ribosomes, viruses, large macromolecules
What is the basic idea of columb chromatography?
A glass clumn is filled with a resin that has an affinity for or a certain effect on specific molecules in a complex mixture
sample is applied to the top and it flows by gravity and is replaced by a continuous supply of buffer from a reservior, during passage through the resin, components of the sample interact with the resin and this varies their rate of movement throguh the column thus separating one from the other
What are the three main classes of chromatographhy used in bio?
ion exchange, affinity chromatography, gel filtration
How does ion exchange chromatography work?
charged particles at top, go through charged column, some stick, rinse with competitior like CL- or NA+
what is gel filtration chromatography?
proteins sort by size, biggest out first
Whatr is affintiy chromatography?
protein to be purified with bind to receptor
What is PI
PH at which a protein cariies no net charge