Exam 1 Lecture Notes

Zoology 570 with Amann/bement at University of Wisconsin - Madison

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By: Mandi Prichard
Textbook:

Molecular Cell Biology (Lodish, Molecular Cell Biology)
Created: 2011-09-26
Size: 95 flashcards
Views: 36

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Philosophy of Cell Biology

"You can never turly know what's going on but can bring in other approaches in attempt to figure it out"

traditional biochemical view of the cell (1960's)

cell is a bag of enzymes based on the observation that complex chemical reactions could occur with materials obtained from ground up cells

microscopy

the technical field of using microscopes to view samples and objects that cannot be seen with the unaided eye (objects not within the resolution range of the normal eye). Well-known branches: optical, electron, and scanning probe microscopy.

resolution

ability of a microscope to identify 2 objects separated by distance x as distinct

key limit resolution

minimum distance is the wave length of source of illumination (light microscopy)

theoretical limit of resolution

minimum distance is half the wavelength of source illumination, usually ~250nm

Sample Prep for microscopy

1. Fix sample

2. Permeabilize

3. Stain

4. Section

super-resolution microscopy

type of light microscopy that exceeds supposed limit of resolution of LM, with ~10-100nm

Limitations of electron microscopy

1. Cannot be used with living systems

2. requires a vacuum

3. expensive and time consuming

4. not appropriate when our sample is large or a greater picture is needed

fixing samples

process of applying a chemical that kills cells and "freezes" everything in place; provides a "snapshot" of what was occurring.

permeabilization

the use of a non-ionic detergent to "poke holes" in the membranes of a sample

staining

soaking sample in "stains" to give contrast; usually bind to cell structures and differentially absorb or reflect light for LM stains or electrons for EM stains; allow us to see general structures like organelles

sectioning

slicing a sample thin enough so as to allow a source of light or electrons to penetrate sample

probe

a reagent that allows us to follow the distribution of a specific cell constituent in cells

reporters

usually florescent molecules (fluoropheres) which allow for the simple detection of the probe

fluoropheres

florescent molecules, excited by a particular wavelength of light that emit a different wavelength of light, usually detected with aid of a fluorecence microscope

Fluorescein, Alexa 488,

fluoropheres excited by blue light, emit green light

Rhodamine

fluorophere excited by green light, emits red light

Cy5

fluorophere excited by red light, emits far red light

flurescence microscope

a device which can illuminate samples with different wavelengths of light as well as collect different wavelengths

immunofluorescence

practice of using antibodies directly or indirectly to a fluorophere to detect specific proteins; permits visualization of multiple proteins using different fluoropheres however cannot be used in living cells - requires permeabilization

direct immunofluorescence

fluorophere is directly attached to antibody that binds to the protein so sample can be viewed immediately; used less as it compromises protein function

indirect immunofluorescence

(most used) - fluorophere bound to an antibody which binds to the protein-binding antibody

direct protein tagging

purify protein of interest and chemically attach fluorophere, then microinject fluorescent protein into cells; permits imaging of protein dynamics and can work with 2 or more proteins at once.

disadvantages of direct protein tagging

attachment of fluoropheres can compromise protein function
not easy to purify proteins, esp. when needed in entirety
limited life span of protein in cell, degrades
microinjection is slow and difficult; limits number of cells in experiments

Fluorescent proteins used in fusion

GFP (green fluorescent protein), excited by blue emits green; RFP (red fluorescent protein) excited by green, emits red light

fluorescent fusion protein in cells

link the DNA encoding fusion protein to DNA encoding protein of interest; transfect cells with artificial gene, once in cells, cell treats it as native DNA and replicates it

disadvantages of fluorescent fusion protein

1. attachment of fluorescent protein may cause problems for the protein of interest

2. no real control over amount of protein that will result from transfection

Visualization/detection of specific nucleic acids

construct probes that are complementary to nucleic acid of interest, and attach a reporter and use in situ hybridization to permit probe to hybridize to NA of interest

in situ hybridization

technique for getting probe to target nucleic acid, requires killing cells, cannot look at dynamics of particular NA

plasma membrane

the lipid bilayer which surrounds the cell; controls what goes in and out of the cell

membrane transport proteins

permit passage of nutrients into the cell and metabolic wastes out of it; maintain proper ionic composition and pH of the overall cell

cell junctions

specialized areas of plasma membrane that contain proteins and glycolipids that form specific structures between cells that strengthen tissues and allow the excahnge of metabolites between cells

membrane receptor proteins

proteins that bind specific signaling molecules such as hormones, growth factors and neurotransmitters leading to various cell responses.

extracellular matrix

mixture of fibrous proteins and polysaccharides that provide a bedding on which sheets of epithelial cells or small glands lie

endosomes

a single bilayer, act as sites of accumulation and sorting for material coming from outside cell or from golgi

endocytosis

the process by which cells absorb proteins and such by engulfing them; used by all cells of the body as most important substances are large polar molecules that can't pass thru the hydrophobic lipid bilayer

lysosomes

single lipid bilayer, major site of macromolecular degredation, usually via enzymes which are effective at a low pH and poorly active at neutral pH

phagocytosis

a process in which large, insoluble particles are enveloped by the plasma membrane and internalized; may be degraded by lysosomes

peroxisomes

organelles found in virtually all eukaryotic cells; involved in catabolism of very long chain fatty acids, branched chain fatty acids, D-amino acids, polyamines, and other important biological molecules

endoplasmic reticulum

single lipid bilayer, an extensive nitwork of closed flattened, membrane-bound sacs called cisternae; number of important functions, primarily responsible for synthesis of lipids and proteins; also involved in calcium regulation

smooth endoplasmic reticulm

primarily responsible for synthesis of fatty acids and phospholipids; cells generally have very little of this organelle

rough endoplasmic reticulum

cytoplasmic ribosomes bound to the rough ER synthesize certain membrane and organelle proteins and virtually all proteins to be secreted from the cell

Golgi (complex/apparatus)

a series of flattened membrane vesicles (cisternae) with three defined regions (cis, medial and trans) where proteins are sorted and modified depending on their structures and final destinations

nucleus

largest organelle, surrounded by a double membrane, contains genetic information and is the site of replication and transcription

nuclear pores

ringlike complexes composed of specific proteins (nucleoporins) through wich material moves between nucleus and cytosol

mitochodria

double lipid bilayer, site of ATP production in aerobic metabolism, have a very small amount of DNA,

cytosol

aqueous viscous gel surrounding organelles, site of thousands of enzymatic reactions as well as signal transduction and cytoskeleton

actin filaments

helical polymers made up of a protein called actin, ~4nm in diameter, concentrated in cell cortex, the region just inside the membrane. Active jobs include promotion of "cell crawling", cytokinesis, and maintaining cell shape

microtubules

hollow tubes, ~25nm in diameter, made up of protein called tubulin; extend from a focus point outside nucleus to place on cell periphery

roles of microtubules

participation in "cell swimming" (cilia/flagella), transport of organelles & distributing/separating chromosomes during mitosis/meiosis

intermediate filaments

fibrous structures, ~10nm in diameter,made up of numerous proteins called intermediate filament proteins, such as laminas, keratins and vimentin; underlay and wrap around outside nuclear envelope & extend out to plasma membrane

phases of cell cycle

G1 (Gap 1 - cell growth)

S (DNA synthesis or replication)

G2 (cell growth)

M (meiotic or mitotic phase)

phases of mitosis

prophase (DNA condenses, nuclear envelope breaks down, spindle assembly)

metaphase (spindle assembly complete)

anaphase (chromatid separation and cytokinesis begins)

telophase ( chromatid sep & cytokinesis finish, envelope reforms, DNA decondenses)

cell fractionation

separation of cells into different fractions based on differences in physical or chemical properties of cell constituents

homogenization

disruption of starting material in fractionation to free cell materials from each other, usually done physically, ex. crushing, putting in a blender

centrifugation

fractionate sample based on basis of mass and density, rather crude method of separation

chromatography

cell homogenate is run over a column loaded with a matrix and reacts with matrix differentially thus spending different amounts of time on the column

genetics

discipline concerned with analysis and manipulation of genes and analysis of the results of those manipulations

forward genetics

1. subject organisms to mutagenesis

2. screen for phenotypes of interest

3. identify and sequence changed gene

4. deduce amino acid sequence of protein encoded by gene

conditional mutant

only manifest phenotype of interest under certain conditions

Cdc (Cell division cycle) mutants

fission yeast mutants that arrest in different phases of cell cycle (e.g. G1, S, G2, M), numbered in order of discovery

Cdc2

a temperature sensitive fission yeast movement that arrests in the cell cycle at 37° but behaves normally at 25°, allowed for deduction that Cdc2p and MPF were the same cyclin-dependent kinase

KASH proteins

integral membrane proteins that bind intermediate filaments and microtubules

Sun proteins

integral memebrane proteins that bind to nuclear lamina which is bound to the inner nuclear membrane by both these and other proteins

lamina

a meshwork of proteins that sits beneath the inner nuclear membrane and acts as a strengthening and stabilizing force

chromatin

the combination of DNA & proteins that make up the contents of the nucleus; packages DNA into a smaller volume to fit in nucleus, strengthens the DNA to allow mitosis/meiosis & prevent DNA damage, control gene expression & DNA replication

histones

chief protein components in chromatin; highly alkaline proteins found in eukaryotic cell nuclei that package and order the DNA into structural units called nucleosomes

core histones

H2A, H2B, H3 and H4: 2 copies of each associate with each other to form an octamer, putting DNA into the "beads on a string" (~10nm diameter)

histone H1

allows formation of 30nm fiber called solenoid

DNA replication

process of DNA synthesis that occurs during S phase of the cell cycle and is semi conservative

Origin of Replication Initiation (ORI)

specific regions of DNA where replication begins; often thousands in eukaryotes to help cell effect replication in a short amount of time, between 100-3000bp, sequences are not often well conserved

Origin Recognition Complex

6 subunits (ORC 1-6), binds to ORI's and acts as a landmark indicating "start replicating here"

CDC6p

binds to ORC and Cdt1 in DNA replication

Cdt1 (Cyclin depedent transcript 1)

binds to MCM complex in DNA replication

MCM complex (mini-chromosome maintenance)

comprised of 6 subunits (named MCM2-7) that possess helicase activity; unwinds helix forming two separate strands

Cdc45p

binds to MCM complex and activates helicase activity during DNA replication

RPA (Replication Protein A)

binds to a single stranded DNA and keeps it single stranded during DNA replication

DNA Polymerase Alpha (DNAPA)

binds to template strand, reads 5' to 3', has primase activity (can install RNA that is complementary to template strand (usually ~10-30 nucleotides); acts as a primer

PCNA (Proliferating Cell Nuclear Antigen)

wraps around DNA and binds toDNAPD and DNAPE

DNA Polymerases Delta and Epsilon (DNAPD/E)

DNA polymerases that lack primase activity; much more accurate than DNAPA and have ability to "edit" newly synthesized DNA (can recognize and replace incorrect nucelotides)

Leading strand

the strand of template DNA on which DNA synthesis is continuous because the DNA polymerase can keep synthesizing as long as MCM separates DNA

Lagging strand

DNA is discontinuous because new DNA PA has to keep landing as MCM complex separates DNA

Transcription

the generation of RNA from DNA template;

Process of transcription

Helicase unwinds/"unzips" DNA by breaking the H-bonds
RNA nt paired with cmplmtry DNA bases.
RNA sugar-phosphate backbone forms w/ help from RNA pol.
H-bonds of the untwisted RNA+DNA helix break, freeing the newly synthesized RNA strand.
RNA further processed (addition of a 3' poly-A tail and a 5' cap), exits thru to cytoplasm via nuclear pore

promoter

the promoter is ~50-200bp and have a region called the core promoter (runs from ~-1 to ~-50nt from gene)
has a TATA box @ about -25nt from gene
usually 7-9nt from gene, transcriptional machinery will begin to assemble

Transcription Factors (TFII...)

a protein that binds to specific DNA sequences, thereby controlling the flow (or transcription) of genetic information from DNA to mRNA

TFIID

binds directly to TATA box during transcription

TFII A,B, & F

bind to TFII D and RNA polymerase II

TFII E

binds to polymerase II, binds to TFIIH

TFIIH

binds to polymerase II and DNA, TFIIE, has helicase and kinase activity

Addition of 5' Cap

specially modified nucleotide (7-methylguanosine) is attached to the 5' end of the transcript

generated and added to transcript by the capping enzyme complex (CEC), which associates w/ pol II and as 5' end emerges, CEC caps it.

necessary for export from nucleus and to

Splicing

removal of introns from the transcript and linking of exons, loops introns which cuts them out and is eventually degraded

Exons will be represented in final (mature) transcript
much is done by "spliceosomes", structures made of protein and small nuclear RNA (catalytic part is RNA, not protein)
some splicing, however, does not require spliceosomes but is done by the transcript itself (self-splicing)

Alternative Splicing

provides cell ability to generate multiple mRNA's and thus multiple proteins from a single gene

3' end processing

immature transcript is cleaved to generate 3' end and then usually polyadenylated (long runs [10-200] of A's are added

About this deck

By: Mandi Prichard
Textbook:

Molecular Cell Biology (Lodish, Molecular Cell Biology)
Created: 2011-09-26
Size: 95 flashcards
Views: 36

About StudyBlue

“I have used this website for three exams, and I see a huge difference in my test results.”
Naj