Exam 3 Flashcards
1
Q
What is a polyclonal antibody?
A
A collection of antibodies made by the same organism, each are produced by a different B cell, but recognized by the same antigen. All bind to a different epitope.
2
Q
What are the differences in polyclonal antibody?
A
Different Fab regions and primary structures
3
Q
What is a monoclonal antibody?
A
A collection of identical antibodies, made from a clonal B cell population. They are recognize the same epitope on the antigen. They all have the same Fab region and primary structure.
4
Q
Difference between polyclonal and monoclonal antibodies?
A
polyclonal are made by the same organism, but have different Fab regions, primary structure, and bind to different epitopes. Monoclonal are identical antibodies. They bind to the same epitope, made by the same b cell, and have the same Fab region and primary structure.
5
Q
Where does the primary antibody bind?
A
It binds to the antigen
6
Q
Where does the secondary antigen bind?
A
The Fc region of the primary antibody
7
Q
What do all antibodies produces by a given organism have in common?
A
The same Fc region (same AA sequence)
8
Q
What antibodies does the secondary bind?
A
It binds all primary antibodies from the given species regardless of the epitope that the primary antibody binds to.
9
Q
How are primary and secondary antibodies used for detection?
A
The secondary antibody is often bound to a fluorescent enzyme to allow for visual detection.
10
Q
What is ELISA?
A
Enzyme-linked immunosorbent assay, used to detect and quantify antibodies and other target proteins (usually antigens)
11
Q
What are nucleosides and nucleotides and what do they contain?
A
They are the building blocks of DNA and contain a nitrogenous base, a sugar (either ribose or deoxyribose)
12
Q
What do nucleotides contain that nucleosides lack?
A
Phosphate groups
13
Q
What bases are found in DNA only?
A
A,G,C, T
14
Q
What bases are found in RNA only?
A
A,C,G,U
15
Q
What number carbon is the phosphate group usually attached to?
A
3 or 5
16
Q
What carbon is the base attached to?
A
1
17
Q
What carbon distinguishes between ribose and deoxyribose?
A
Carbon 2, whether i has two H or an H and an OH attached.
18
Q
How many members do nucleotide rings contain?
A
5
19
Q
What are 5-membered cyclic sugars known as?
A
Furanoses
20
Q
How can we create diverse nucleotides/nucleosides?
A
By modifying the building blocks (bases, phosphates and sugars)
21
Q
What are tautomers?
A
Structures that differ in locations of hydrogens
22
Q
What are the predominant tautomeric forms?
A
amino and keto forms
23
Q
What are the rare tautomeric forms?
A
imino and enol forms
24
Q
What differs between the two tautomeric forms?
A
The capacity of hydrogen bonding.
25
What us the bond between the sugar and the base called?
The glycosidic bond
26
What are syn nucleotides?
The base is above the furanose ring (most of the base is directly above)
27
What are anti nucleotides?
The base is facing away from the nucleotide
28
How are there able to be syn and anti nucleotides?
There can be rotation around the glycosidic bond.
29
How are nucleic acid polymers formed?
when phosphodiester bonds connect nucleotide monomers together
30
How is a phosphodiester bond created?
5' end of a nucleic acid (the free phosphate group)
and the 3' end of a nucleic acid (free hydroxy; group)
31
What are the two grooves in the double helix structure?
It has major and minor grooves, that differ based on the distance between the two strands.
32
What is the backbone of DNA?
A sugar-phosphate backbone
33
What are the DNA strands in relation to each other?
complimentary and anti-parallel
34
How are the nitrogenous bases arranged in the DNA molecule?
They are located on the interior of the helix and are stacked horizontally to maximize non-covalent interactions
35
What are the three different types of DNA form?
B-form, A-form, and Z-form
36
What is B-form?
It is the first model identified, and is the most common form of DNA, is is right-handed. More stretched compared to A-form.
37
What is A-form?
More commonly found in RNA, right hand. More vertically compressed than B-form, has similar major and minor grooves?
38
What is z-form?
It is left-handed and is transient, it is induced during transcription
39
How is DNA stabilized?
There is hydrogen bonding between strands and Pi stacking within the strand
40
What is base stacking?
it is pi stacking, where there is orbital overlap of the aromatic rings in the bases. It is attractive, non-covalent interactions, and is driven by the hydrophobic effect
41
Can triplexes and quadruplexes exist?
Yes!
42
What is unique to RNA as opposed to DNA?
It can fold back on itself to form diverse secondary structures
43
What is the only form RNA can adopt and why?
It can only adopt A-form because the 2' OH cannot fit into a B-form helix.
44
What are ribozymes?
RNA that can act as enzymes because of their structural flexibility
45
What are the different secondary structures RNA can form?
Loops, Hairpin, Bulge, Duplex
46
How is the secondary and tertiary structure of RNA important?
The structures from by the secondary and tertiary folding give functions, like binding amino acids, interacting with ribosomes, enzymes, etc.
47
Is RNA more or less stable than DNA and why?
RNA is less stable than DNA. There are free hydroxyls in the environment that will induce the deprotonation of the 2'-OH'. There is then a Intra NA on the adjacent phosphate and then cleavage of the phosphodiester backbone.
48
What do the nucleic acid methods provide?
The ability to manipulate, modify, or determine the sequence of DNA/RNA
49
What are the three methods discussed?
Cloning, PCR, and Sanger Sequencing
50
What is cloning?
A technique used to make many identical copies of DNA.
51
What type of DNA is used for cloning?
Circular DNA called Plasmids
52
What enzymes are used in cloning?
Endonucleases, they cleave within the DNA
53
What is produced from cloning?
Recombinant DNA
54
How do restriction enzymes/ endonucleases work?
They cleave by recognizing specific sequences.
55
How do Type 2 restriction enzymes cleave?
They cleave within the recognition sequence
56
What does the cleavage of DNA via restriction enzymes result in?
They generate 5' overhangs, 3' overhangs or blunt ends
57
How is DNA incorporated into plasmids?
The gene of interest and the plasmid are cleaved with the same restriction enzyme, then they are annealed to promote hydrogen bonding between the complimentary sequences on the sticky ends. Finally DNA ligase is used to covalently join the fragments by forming a new phosphodiester bond.
58
What does DNA polymerase do?
It extends the DNA strand during replication.
59
What are the requirements for DNA polymerase to work?
There needs to be a template strand that will be duplicated and a primer that will be extended. There also needs to be dNTPs which are added to the growing strand.
60
During replication, what is formed and what is released?
A new phosphodiester bond is formed with the 3' OH and a diphosphate group is released.
61
What is a primer and how does it work?
A primer is a short single-stranded nucleic acid sequence that binds anti-parallel to the template strand. It is then extended on the 3' end as dNTPs are added via DNA polymerase.
62
What is the new strand in relation to the Template strand in DNA replication?
It is reverse and complimentary to the template strand.
63
What is reverse transcription?
Synthesis of DNA from RNA, and it allows all genes actively transcribed from mRNA.
64
How does reverse transcription work?
mRNA is converted to DNA using reverse transcriptase. RNA dependent DNA polymerases are then used with dNTPS to created the complimentary DNA strand. This product is a RNA-DNA hybrid. Ribonuclease H is then added to cleave the RNA so only the cDNA is left.
65
What does ribonuclease H do?
Cleaves the RNA in the DNA-RNA hybrid strand during reverse transcription.
66
What is PCR?
It is used for DNA amplification:
generates multiple copies of a
specific DNA target segment
67
What are the three temp. phases of PCR?
Denaturation, Annealing, Extension
68
What happens during the denaturation phase of PCR?
The DNA is exposed to high temps. in order to separate the strands
69
What happens during annealing in PCR?
The temp. is lowered to allow for the primers to anneal to each strand.
70
What happens during the extension phase of PCR?
The temp. is raised again so the primer can be extended and the new DNA strands can be synthesized
71
What else happens at high temps and how is it accounted for during PCR?
Protein denaturation occurs, so thermostable enzymes are used
72
How does the number of strands grow in PCR?
They grow exponentially
73
What is Sanger Sequencing?
It is used to determine the sequence of a DNA molecule and is known as the chain termination method.
74
What are the requirements of Sanger Sequencing?
DNA polymerase to extend the chain, a template strand, a primer, dNTPs and ddNTPs
75
What is the difference between dNTPS and ddNTPS?
dNTPs have an H at the 2' and an OH at the 3', while ddNTPs have H's at both positions
76
How does Sanger Sequencing work?
Each ddNTP has a different color flourescent dye, and are incorporated during replication, which terminated replication. This results in different sized strands, which are then separated via capillary electrophoresis.
77
How does Agarose Gel Electrophoresis work?
It separates DNA by size. The negative charge on the sugar-phosphate backbone pulls it through the gel. Smallest molecules will run the farthest in the gel.
78
How are the segments able to be seen in an agarose gel run?
They are stained with intercalator dyes such as ethidium bromide.
79
What are intercalators?
They are flat aromatic molecules that slip in between the bases, this stacking of rings stabilizes the interactions
80
Why is EtBr only fluorescent when bound to DNA?
When it is free in solution it is quenched, which disables the fluorescent ability
81
What are some functions of carbohydrates?
Energy, structure, and information stransfer
82
What other molecules can carbs be linked to?
proteins and lipids
83
What are the two broad classes of carbs?
Aldoses and ketoses
84
What are enantiomers?
They are molecules that are non-superimposable mirror images of each other, varying at the arrangement of atoms at the chiral centers.
85
How do we identify D or L isomers?
If there are multiple chiral centers, they are assigned to the farthest away from the carbonyl carbon.
86
What are diasteriomers?
They are molecules that differ at some but not all chiral centers.
87
What are epimers?
They are molecules that differ at only one chiral carbon.
88
Is the formation of cyclic sugars spontaneous?
Yes, it is more stable. Where the an alcohol on the chain reacts with the aldehyde or ketone group on the chain.
89
What structure does the attack of the OH on an aldehyde or ketone produce?
A hemiacetal or hemiketal
90
What is the difference between alpha and beta in the rings?
In alpha, the OH and the CH2OH groups are on opposite sides, and in beta, the OH and the CH2OH are on the same group. They are also known as anomers
91
What is the anomeric carbon?
It is the new chiral center formed from the attack of the OH group.
92
How do you locate the anomeric carbon?
Locate the O in the ring, and then look at the carbons directly next to it. Whichever has an OH group attached is the anomeric carbon
93
What determines the anomeric form of the sugar?
The orientation of the carbonyl
94
What determines the size of the ring?
Which carbon the OH group is attacked to that does the NA.
95
How many members does a pyranose ring have?
6 (includes an O)
96
How many members does a fyranose have?
5 (includes O)
97
How can you tell if a sugar has a ketose or aldose based on the ring?
A ketose will have a CH2OH attached to the anomeric carbon
98
How can monosaccharides be modified?
The aldehyde can be oxidized to yeild an aldonic acid and the primary OH can be oxidized to form uronic acid. Aldehydes can also be reduced to form an alditol
99
Can alditols form rings?
No, there is no carbonyl
100
How is a deoxy sugar formed?
An OH is reduced, so one carbon is missing an OH.
101
How are amino sugars formed?
An amine is substituted for an OH. This group can also be acetylated to form N-acetyl sugar
102
What is a glycoside?
A sugar joined to another non-sugar molecule via a glycosidic bond. Can have alpha and beta fomrs
103
What is a glycosidic bond?
joins a sugar together with another molecule through the anomeric carbon. This bond is formed through a reaction with hemiacetal/hemiketal and a nucleophile
104
What is a disaccharide?
Two sugars linked by a glycosidic bond
105
When are you able to determine the overall from of a disaccharide?
if the second sugar has a free anomeric carbon
106
When is a sugar reducing?
When an anomeric carbon is free
107
When is a sugar non-reducing?
When there is no free anomeric carbon.
108
What is a homopolysaccharide?
It is a chain of 100's of repeating sugar units. They can differ based on the alpha/ beta orientation of the glycosidic bond which can change the function of the molecule.
109
What is the difference between cellulose and amylose?
Amylose is two alpha while cellose is one alpha one beta.
110
What is amylose?
A homopolysaccharide used in energy storage. It is loosely packed with a helical structure.
111
What is cellulose?
A homopolysaccharide this used for structure, it is tightly packed and dense
112
What are polysaccharides?
They can be disaccharide heteropolymers or branched.
113
What are proteoglycans and glycoproteins?
Sugars that are conjugated to proteins
114
What is an N-linked glycoprotein?
When the sugar is linked to the -NH2 of an Asn
115
What is an O-linked glycoprotein?
When a sugar is linked to the -OH of a Ser/Thr
116
What can be done to proteins in viruses to promote their parasitic activity?
Glycosylated
117
What is a lipid?
any molecule more
soluble in organic solvent than
in water
118
What are the important roles of lipids?
Energy storage, cell membranes, and signaling
119
What are fatty acids?
carboxylic acids with
hydrocarbon chains (4 to 36 carbons
in length)
120
what are most lipids derived from?
fatty acids
121
What is a saturated FA?
When there are no double bonds in the chain (fully saturated with hydrogens)
122
What are unsaturated FAs?
Have double bonds (1-mono) (>1- poly) can cause a kink in the chain
123
How do we name fatty acids?
1. number carbons starting at carbonyl.
2.Count number of double bonds
3. count from the last carbon to the first encountered double bond
124
What orientation are double bonds in FA's usually?
Cis!
125
What is Tm trend?
As the number of double bond increases, the Tm decreases, as the double bonds cause kinks, which prevent tight packing of these chains and interactions to form a solid.
126
How does Tm vary with length?
Longer the chain, higher the Tm, this is because there are more interactions that can occur between chains that need to be broken
127
What are the two backbone molecules in lipids?
Glycerol and sphingosine
128
How many FA chains can be attached to a glycerol?
2-3
129
How many FA can be attached to Sphingosine?
1, as the backbone has a long tail, and a HG
130
What does glycerol look like?
A three carbon chain with OH on each C.
131
What does Sphingosine?
An 18 carbon amino alcohol with an unsaturated hydrocarbon chain
132
How can be lipids be classified?
Based on the backbone, type of head group and the linkage
133
How are phosphate HG linked to the backbone?
Phosphodiester bond
134
How are sugar HG attached to the backbone?
Glycosidic
135
What is a triaglycerol?
A glycerol with 3 fatty acid tails
136
What is a glycerophospholipid?
They contain two fatty acids and a polar head group
137
How do you name glycerophospholipids?
First part of the name describes the fatty acid tails. The second part of the name describes the group and linkage
138
What are phospholipases?
Enzymes that hydrolyze glycerophospholipids. They are important for recycling and lipid signaling
139
What is ceramide?
Sphingosine and a fatty acid
140
What is sphingomyelin?
Sphingosine with a fatty acid and PC or PE
141
What is cerebroside?
Sphingosine and a fatty acid with a monosaccharide HG
142
What is ganglioside?
Sphingosine and fatty acid with oligosaccharide
143
What are the three types of lipids in cell membranes?
Glycerophospholipids, sphingolipids, cholesterol
144
What is the four ring structural core called?
Steroids
145
What is cholesterol the precursor for?
Many signaling lipids, aka steroid hormones and bile acids
146
What is the first step of steroid hormone synthesis?
The cleavage of a sidechain by cholesterol desmolase
147
What do dehydrogenases do?
Oxidation and reduction in catalysis
148
What is do hydroxylases do?
Add an OH to a steroid
149
How do steroids regulate gene expression?
They bind to steroid receptors, a type of transcription factor
150
How are steroid receptors named?
They are named by their partner hormone.
151
Why can steroid receptors show promiscuity and what makes some hormones more likely to bind than others?
There are structural similarities between hormones, especially functional groups
152
What are isoprenoid lipids?
They are polymers of isoprene units and are complex, branch structures. They have conjugated systems of alternating double bonds
153
How are lipids arranged in the bilayer and why?
Heads out (hydrophilic) and tails in (hydrophobic) driven by the hydrophobic effect
154
What lipids are in the outer leaflet?
* Phosphatidylcholine
* Sphingomyelin
* gangliosides
155
What lipids are in the inner leaflet?
* Phosphatidylethanolamine
* Phosphatidylinositol
* phosphatidylserine
156
What are the general charges of the leaflets?
Outer-neutral
Inner-negative
157
What factors impact membrane fluidity?
membrane composition (types of lipids)
Fatty Acid Tm
and Temp.
158
What is lateral diffusion?
Lipids switching spots in the same leaflet. Quick.
159
What is transverse diffusion?
Lipids switching between leaflets. Slow, requires enzymes to occur
160
What are the functions of membranes?
* Defines boundary of cells,
organelles
* Selective transport
* Signaling and information
transmission
* Energy storage
161
What are the three classes of membrane proteins?
Integral, Lipid-anchored, and peripheral
162
What are integral membrane proteins?
They are proteins that are tightly packed into the bilayer. Can be trans membrane or monotopic. Detergent is needed to disrupt
163
What are lipid-anchored proteins?
They are proteins that are covalently attached to a lipid in the bilayer. They are located on the periphery
164
What are peripheral membrane proteins?
They are proteins that are non-covalently/ loosely associated with the bilayer
165
How are integral membrane proteins predicted?
By primary structure, which can also predict the number and secondary structures and the transmembrane regions
166
What are the three types of lipid-linked proteins?
Fatty-Acid linked
Isoprene-linked proteins
GPI-linked proteins
167
What are Fatty-Acid linked proteins?
They are proteins that are covalently modified to include fatty acids at particular amino acids
168
Which amino acids are associated with palmitoyl?
Cysteine/ serine
169
Which amino acids are associated with N-myristoyl?
N-terminal glycine
170
What are Isoprene-linked proteins?
Proteins covalently attached to isoprenes, and are attached at the C-terminal cysteine
171
What are GPI-linked proteins?
Glycosylphosphatidylinositol is an anchor
* connects c-terminus of protein to
membrane
