Exam 1 Flashcards
(104 cards)
1
Q
Cell Theory
A
The cell is the fundamental unit of all life; all living things are made of cells
New cells only arise from preexisting cells
2
Q
Cell size
A
Very small, 0.1um-1mm
3
Q
All cells are surrounded by __
A
Plasma membrane
4
Q
Cells are filled with __
A
Concentrated chemicals
Most chemicals same for all cells
5
Q
Similarities of cells
A
-DNA is genetic material
- Same sugars and AAs
- Use ATP for energy
- Phospholipid membrane
- Central dogma of molecular biology
6
Q
Prokaryotes
A
No nucleus or organelles
- Smaller cells (0.1-10um)
- Cell wall
- inner and outer membrane
- nucleoid
- flagella
- periplasmic space (‘mitochondria’), where respiration occurs
- Cytosol
- Ribosomes
- Divide by binary fission
7
Q
Eukaryotes
A
Nucleus and organelles
- Larger (10um - 1mm)
8
Q
Eukaryotic cells exchange nutrients ____ easily with the environment
A
Less
Smaller surface to volume ratio
- Why organelles are important
9
Q
Mitochondria
A
An anaerobic archaeon took up an aerobic bacterium
10
Q
Origin of a eukaryotic cell
A
- An archaeon (a type of simple, single-celled organism) from the TACK group loses its protective cell wall, making it more flexible.
- The cell’s internal structure (actin cytoskeleton) changes, allowing it to engulf other cells (phagocytosis).
- The archaeon starts engulfing bacteria, and some of the bacteria’s DNA gets mixed with the archaeon’s DNA through horizontal gene transfer.
- A protective membrane forms around the archaeon’s genetic material, creating a nucleus. At the same time, one of the bacteria it engulfs remains inside and becomes the mitochondrion.
- The mitochondria start multiplying inside this early eukaryotic cell, helping it produce energy more efficiently.
11
Q
Homologous traits
A
Shared traits inherited by a common ancestor
12
Q
Homologs
A
Genes with similar sequences and functions
13
Q
paralogs
A
individuals with multiple copies of genes with similar sequences (gene duplication and divergence)
Similar function
convergent evolution
14
Q
Orthologs
A
Genes in two different species that have the same function (divergent evolution)
15
Q
Intragenic mutation
A
mutations that occur within a single gene
16
Q
Gene duplication
A
Can create genetic redundancy, allowing one copy to mutate and take on a new function
17
Q
Gene shuffling
A
the rearrangement of genetic material within a genome, creating new combinations of genes or gene variants
18
Q
Horizontal gene transfer
A
genes transferred across different species.
19
Q
How to break open cells
Cell in tissues
A
a. Mortar and pestle
Blender
b. Put tissues in a buffer at a specific pH with protease inhibitor
These methods could also break open organelles!
20
Q
How to break open cells
Cells in culture
A
- French press
- Sonication
- Detergents
- Osmotic stress
21
Q
French press
A
High pressure –> rapid release of pressure
Disadvantage: heat generation, expensive
22
Q
Sonication
A
a. Probe vibrates rapidly
b. Cavitation: vapor filled cavities for and collapse (lyse)
Disadvantage: Heat generation, expensive
23
Q
Detergent lysis
A
a. detergent reacts will cell membrane
b. detergent destroys cell membrane
c. intracellular components released
Disadvantage: detergent can denature proteins - bad for cells with walls
24
Q
Osmotic shock
A
Put cells in hypotonic solution
Disadvantage: wont work on cells with walls
25
Cell fractination
Separating cell componenets
26
Differential centrifugation
Based on size and density of subcellular structures
a. spin lysate at low speed
- analyze pellet for protein
b. transfer supernatant to new tube
c. spin at higher speed
- analyze pellet for protein
d. repeat
Disadvantage: difficult to isolate organelles
27
Density gradient ultracentrifugation
Velocity sedimentation
a. lysate layered on top of sucrose gradient
b. more dense or larger sediment farther then smaller or less dense
c. poke hole in bottom of tube and separate
- best for separation of large proteins like ribosomes
- shallow gradient
- most cell components could eventually reach the bottom
28
Density gradient ultracentrifugation
Equilibrium sedimentation
a. lysate is mixed into gradient
b. cell components migrate until neutral density is reached
c. gently pull of layers with pipet
- Best method for 'pure' organelles
- steep gradient (20-70% sucrose)
- components migrate until they reach a place of equal density
29
Chromatography
Stationary phase: tiny beads with specific physical properties
Mobile phase: buffer at specific pH and ionic strength
Cystolic fraction: mixture of many proteins
a. Apply sample to column
b. apply mobile phase
c. chromatogram
30
Chromatography
Size exlusion
Separation based on size
- Stationary phase: porous beads
small proteins get stuck in beads, elute slower
- if you know predicted protein sequence, computational analysis gives size in daltons
31
Chromatography
ion exchange
Based on charge
Any protein can be +/- charged!!!
- Depends on buffer pH and ioselectric point
Anion exchange - positive stationary phase
32
if pH > pI
Negative charge
33
if pH < pI
+ charge
34
Chromatography
Affinity
A substrate anolog bound to the beads
35
SDS
Sodium dodecyl sulfate
- strong anion detergent
- denatures proteins and gives them a uniform negative net charge
- all proteins get same mass/charge ratio allowing for separation based on size only
36
B-mercaptoethanol
Breaks disulfide bonds
37
SDS poles
DNA starts at negative pole and migrates towards positive pole
38
2D SDS page
Step 1: isoelectric focusing
Proteins are separated by electrophoresis in gel with a pH gradient
- proteins migrate until they reach a portion of the gel where they have no net charge (pI)
39
2D SDS page
step 2: SDS page
Stain gel
- each spot represents a single protein
- helps separate protein that might have the same mass
40
Antibodies
Produced in B-cells in response to foreign antigens
- a single B-cell makes 1 type of antibody (IgG)
41
Polyclonal antibody
Group of antibodies that recognize the same antigen
- different B-cells bind to a differe epitope
- a single antigen may have many epitopes
42
Advantages of polyclonal antibodies
-Cheap, quick, easy
- Multiple binding sites, increased signal
43
Disadvantages of polyclonal antibodies
can be non specific
44
Monochromal antibodies
Normal somatic cells can be fused with tumor cells to form a heterokaryon
- derived from clones of single B cell producing a single type of antibody
Advantages: highly specific and easy to purify
Disadvantages: takes longer
45
Western blotting
Detecting protein on a membrane after SDS page
a. protein transferred to membrane
b. needs two antibodies
- primary AB recognizes protein of interest
- secondary AB recognizes primary AB (contains label)
Multiple 2nd can bind to one primary, amplifying the signal
46
Protein interactions
every protein binds with something
47
Protein tagging
Adding an epitope tage to your protein through genetic engineering
ex: GFP, GST
48
Pull down assays
depends on affinity purification
a. protein binds to column via tage (GST)
b. proteins from cell lysate added, see what sticks to your protein
c. bait/prey complexes eluded from column (with glutathione)
d. detect interacting western
49
Co-immunoprecipitaion
Depends on antibodies (stuck to inert beads) against your protein
a. precipitate your protein with the antibody linked beads and see what sticks
b. detect interactions with western
50
Chromatin immunoprecipitation (ChIP)
Use antibodies to immunoprecipitate your protein and the DNA its bound to
b. use PCR or sequencing to identify what DNA is bound to your protein
51
Resolution
Smallest distance between 2 points in an object that is still perceived as separate in the image
52
Standard light microscopy
not a high max resolution ~ 0.2um
- many cellular structures smalelr
visible light 400-700nm - not refracted by smaller cell structures
53
Sample prep
Free cells
Slide --> image
54
Sample prep
Cells in tissue
too thick for light to pass through
fix with chemicals to maintain morphology --> section in thin slices --> image
55
Electron microscopy
electrons refracted by smaller structures
56
TEM
high resolution, 2nm
only image fixed or section samples
analogous to compound microscope
57
SEM
High resolution 3-20nm
intact tissues/ cells
cant see inside cells
Analogous to dissecting scope
58
Eukaryote DNA
Numerous linear chromosmes
59
Prokaryote DNA
One small circular chromosome
60
Chromatin
DNA and associated proteins
61
DNA winds around ___
Histones - Small basic proteins with N-terminal tails that can be modified
- protein complex of eight small proteins
-allows chromosomes to condense very tightly
-
62
DNA wrapped around one histone complex is called a ___
Nucleosome
-150 base pairs per nucleosome
- DNA wraps around histone 1.6 times
63
Nuclease-protection assay
Isolate nuclei and treat with DNase
- 'naked' DNA (not bound to protein) is cut
- Gel electrophoresis determines the size
64
Euchromatin
Loose
Gene expression
Long DNA link between nucleosomes
65
Heterochromatin
Condensed
Little/no expression
M-phase
66
Interphase contains __
Eu and heterochromatin
67
Each chromosome occupies
Specific region in nucleus
68
Loop domains
Originally found in lampbrush chromosomes from amphibian oocytes
- regions of active transcription
69
FISH
a. random cuts made in gene
b. A probe complementary to your gene is made in test tube
c. fluorescent signals are analyzed to determine gene presence
70
Histone N-terminal tail modification
Lysine acetylation is done by __
histone acetylases (HATs)
71
Histone N-terminal tail modification
Lysine deacetylation is done by ___
Histone deacetylases (HDACs)
72
Histone N-terminal tail modification
HATs
Reduce strength of DNA binding by making N-terminal tail uncharged
Euchromatin
73
Histone N-terminal tail modification
Lysine methylation by __
Histone methyltransferases (HMTs)
74
Histone N-terminal tail modification
Lysine demethylation by __
demethylases (HDMs)
75
Histone N-terminal tail modification
HMTs
Add 1-3 methyl groups per lysine
Charge not affected
Heterochromatin
76
Histone N-terminal tail modification
phosphorylation; kinases ___
Add phosphate
77
Histone N-terminal tail modification
phosphorylation; phosphatases___
Remove phosphate
78
___ or ___ proteins (TFs) bind to DNA and recruit either code writers or code erasers
Enhancer, repressor
79
Histone chaperones
can insert / remove histone cores
80
Chromodomain proteins bind to
H3K9Me3 --> heterochromatin
81
Bromodomain proteins bind to
acetylated lysines --> euchromatin
82
DNA methylation (5-meC)
Occurs at CpG sequences --> usually leads to heterochromatin formation and transcriptional silencing
83
RNA polymerase sythesizes from
5' to 3'
84
RNAP 2
makes mRNA (99% of genes encode proteins)
85
Txn factors
Bind promotors, recruit RNAPs to transcriptional start site
86
snRNAs
components of the splicesome
87
RNAP 1
makes most rRNAs
88
Cleavage factor
cleaves mRNA from RNAP 2
89
RT-PCT
Used to study gene expression
creates complementary DNA (cDNA)
Amplify cDNA with PCT
detect product with gel electrophoresis
90
Protein synthesis initiation in eukaryotes
Initiator tRNA scans mRNA (5'->3') for AUG past the 5' cap
91
Protein synthesis initiation in prokaryotes
mRNAs are polycistronic
- ribosomes scan for Shine-Delgarno sequence
92
mRNAs are translated by
polyribosomes
93
Damaged/misfolded proteins are tagged with ___
Ubiquitin and sent to the proteosome for degradation
94
Large proteins require ___
chaperones for correct folding
95
Trigger factor
Found in bacteria, bind to nascent proteins emerging from ribosome
Folds about 70% of proteins during translation (no ATP)
96
Hsp70 system
All organisms
- Co-translational
- shields hydrophobic regions
- Constitutive: for routine folding of new proteins
-Use ATP to help fold proteins into correct conformation
- Stress-induced: Heat, cold.. anything that causes proteins to denature
97
Hsp60 system (chaperonin)
All organisms
- Post translational
- Large barrel-like structure helps fold proteins after translation is complete
- uses ATP
98
E2 - Ub transferase
Tags target proteins
- 5 genes
99
E3 Ub
Recognizes target protein
100+ genes
100
E1
Activates ubiquitin with ATP
101
What determines degredation
- misfolded, denatured, damages
- TEST motif- phosphorylated by a kinase
- Degron (destruction box) - short AA sequence bound by different E3s
- N-end rule: some proteins are cleaved after translation
102
26S proteasome
Found in cytosol / nucleus
-2.5 MDa
- ATP dependent
103
19S cap on proteasome
-binds polyUb
- unfolds target proteins via ATP hydrolysis
(unfoldase ring)
104
20S cylinder
Protease, cleave AA's
