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immunobiotechnology > Lecture 4 > Flashcards

Lecture 4 Flashcards

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1
Q

Which Ig have 4 constant domains?

A

IgM and IgE

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2
Q

Which Ig have 3 constant domains and a hinge region?

A

IgG, IgA and IgD

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3
Q

What do restriction enzymes recognise on dsDNA in order to cut it?

A

Palindromic sequence

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4
Q

What area of dsDNA must the restriction enzymes recognize the DNA?

A

major and minor grove

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5
Q

What kind of ‘ends’ of dsDNA do restriction enzymes produce?

A

Blunt ends
Sticky ends: 5’ overhang
sticky ends: 3’ overhang

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6
Q

What are the two types of light chains?

A

kappa and lamda

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7
Q

Which of the light chains have more constant region in the germline DNA that the other?

A

Lamda: 5 constant regions i.e. each of the J segment has its own constant region in the germline DNA
Kappa: only 1

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8
Q

How does somatic recombination occur in light chain DNA?

A

V-J join by DNA rearrangement.
VJ segment joined with a constant region,

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9
Q

How does somatic recombination occur in heavy chain DNA?

A

D-J joined by DNA rearrangement.
V-DJ joined.

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10
Q

Give the approximate number of ‘nt’ in V and J segments?

A

V segment: ~300nt
D segment: 10-12nt
J segment: up to 40nt

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11
Q

In what chromosome are the genes for kappa and lamda light chain?

A

Lapda: chr 22
Kappa: chr 2

Heavy: 14

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12
Q

What is the full form of RSS?

A

Recombination signal sequence

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13
Q

What are recombination signal sequences?

A

Recombination signal sequences are conserved heptamer and nonamer sequences flanking the gene segments of V, D and J regions.

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14
Q

What are the heptamer and nonamer spaced out by?

A

12 or 23 bp

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15
Q

What is the significance of the 12/23 rule?

A

12 or 23 bp represent one or two turns of the double helix. The rule is that a 12/23 pairing would allow a rearrangement to take place

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16
Q

What protein recognizes the ‘heptamer-23 bps-nonamer’ and ‘heptamer-12 bps-nonamer’?

A

RAG-1/2 (recombination activating genes)

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17
Q

Explain sequence of events up till the formation of hairpin structure during somatic recombination?

A

-protein complexes RAG 1/2 recognize and bind to the 12bps and 23 bps
-protein complexes bind to each other bringing the segments together
-DNA is cleaved to create hairpin
-

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18
Q

How is the hairpin formed?

A

Cleave one DNA strand at the 5’ end of the heptamer, leaving a
free 3’-OH group of the coding segment.
The free 3’-OH group then hydrolyze the phosphodiester bond of the other strand to form a hairpin

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19
Q

What happens after the formation of the hairpin?

A

-Ku70:Ku80 bind to it and stablize it
-The DNA hairpins are opened by DNA-PK and Artemis
-Additional bases added by TdT (terminal deoxynucleotidyl transferase) or substracted by exonuclease
-DNA ligase joins the ends of gene segments

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20
Q

What are the 2 joints produced by somatic recombination?

A

coding joint and signal joint

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21
Q

After joining of 2 segments in somatic rearrangement, what are N nucleotides and P nucleotides?

A

N nucelotides: non-template nucleotides because they are not added according to the template

P nucleotide: palindromic nucleotide because the two ends are derived from the ‘palindromic’ sequences

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22
Q

What is meant by junctional insertion?

A

insertion of nucleotides during the joining of segments in somatic rearrangement.

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23
Q

What is the criteria for junctional insertion to be functional/productive?

A

has to be multiple of 3 to prevent frameshift

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24
Q

Which of the CDR (complementarity determining regions) most variable?

A

CDR3 because it is which
corresponds to the junction between the V segment and the J or DJ segments

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25
What are the four 'change of one bp' mutation?
1. Silent 2. Neutral 3. Deleterious 4. Positive
26
What is silent mutation?
Change in nucleotide does not change the amino acid, due to the degeneracy of the codons, e.g., CGG to AGG, both codes for Arginine
27
What is neutral mutation?
Change in nucleotide lead to change of an amino acid of similar properties, e.g., AGC to ACC, serine to threonine. No effect of the protein function most of the times – not absolute.
28
What is deleterious mutation?
Change into a stop codon, render the protein completely useless, e.g., AAA to TAA (UAA), from Lysine to Stop
29
What is positive mutation?
Change results in promoting the antibody to have a higher affinity, no specific rules. When this happens, the it is called affinity maturation
30
What is meant by higher affinity?
Higher affinity means the two molecules have a higher on-time than off-time. This would lead to the two molecules have more time with each other to produce a higher functional consequences such as proliferation and clonal expansion
31
How is antibody diversity ensured in other animals?
Cows and sheep: somatic recombination Chicken and rabbit: gene conversion (from pseudogenes)
32
What is gene conversion?
Gene conversion is the process by which one DNA sequence replaces a homologous sequence. it means that the sequences are similar such that they produce similar structures. In these case, it is to ensure, among other features, that the final product can assume an Ig fold.
33
Describe active gene and pseudogene.
An active gene can be transcribed and translated into a protein product. A pseudogene looks like a gene, but it cannot produce a product. (pseudo: not genuine)
34
What are isotypes?
Isotypes are molecules that are coded from different genes but have similar functional and structural properties.
35
What are the number of disulphide bonds in the hinge region?
IgG: 2 IgA and IgD: 1
36
What is N linked glycosylation?
N-linked glycosylation is putting the carbohydrate on the Asn on the tripeptide sequence of Asn-X-Ser or Asn-X-Thr, where X is any kind of animo acid except proline.
37
What is glycosylation?
the covalent addition of sugar moieties to specific amino acids. Glycosylation would affect functions or proteins and their structural stability
38
What two Ig are produced by naiive B cells?
IgM and IgD
39
In naiive B cells, during the production of antibodies, what is the function of RNA splicing?
1. dictates whether membrane-bound or secreted antibody is produces 2. dictates whether IgM or IgG is produced
40
What 2 forms do all antibody classes exist in?
Membrane-bound forms and secreted forms
41
What is the function of membrane bound antibodies?
Membrane-bound forms are necessary to communicate to the B cell for downstream development, such as affinity maturation and class switching to IgG, IgA, etc.
42
Which Ig is expressed in very low levels in blood?
IgD
43
what is the lymphatic system?
The lymphatic system is a network of tissues, organs, vessels, and nodes that play a crucial role in maintaining fluid balance, immune function, and the absorption of fats and fat-soluble nutrients in the body. It works in conjunction with the circulatory system to help maintain homeostasis and protect the body from infections and diseases.
44
What is the amount of lymphocytes in lymph and blood?
Blood: lymphocytes account for 40% Lymph: over 90% of leukocytes are in lymph are lymphocytes
45
Is the passing of blood to lymph selective for lymphocytes
Yes
46
What is the affarent and efferent lymphatic?
Affarent: incoming Efferent: outgoing
47
How are lymphocytes (T cells) trafficked to lymph node?
through high endothelial venules (HEV)
48
What is the structural difference between high endothelial venule and a normal venule?
HEV: tall and plump normal venule: flat
49
what are high endothelial venules?
High endothelial venules (HEVs) are specialized post-capillary venules found primarily in lymphoid organs such as lymph nodes, tonsils, and Peyer's patches in the intestine. They are named "high endothelial" due to the distinctive cuboidal or columnar shape of the endothelial cells that line these vessels, which differs from the flat endothelial cells found in regular venules.
50
What is the major role of HEV?
Lymphocyte Entry into Lymphoid Tissues: HEVs play a critical role in the recruitment of lymphocytes, particularly T cells and B cells, from the bloodstream into lymphoid organs where immune responses are initiated. Interaction with Lymphocytes: The expression of addressins on the endothelial cells of HEVs allows for interactions with lymphocytes expressing corresponding adhesion molecules, enabling lymphocyte extravasation.
51
Describe the process of migration of lymphocytes through the HEV
1. They slow down by rolling. 2. They establish firm adhesion. 3. They crawl by remodelling the actin filaments. 4. They squeeze through between adjacent endothelial cells
52
How do plasma cells physically change when activated?
before activation: small, dense nucleus, little cytoplasm --> indicating low metabolic activity after activation: large cytoplasmic content with large amount of RER --> high metabolic activity
53
For the migration of lymphocytes through HEV, what molecules are used for rolling and firm adhesion?
Rolling: Lymphocytes: CD34 Endothelial cells: L-electin Firm adhesion: lymphocytes: a4'B'1/a4'B'2 integrins endothelial cells: VCAM-1/ ICAM!
54
What holds the pentameric IgM together?
J chain and internal disulphide bonds
55
What is clonal expansion?
Selected B cells undergo clonal expansion, leading to the generation of a larger population of B cells producing antibodies with improved affinity for the target antigen. This amplification of high-affinity B cell clones enhances the overall immune response against the specific antigen.
56
Why does IgM have low affinity but high avidity?
IgM is made first among the different classes of antibody. Hence it generally has low affinity for each antigen binding site. By multiplying the effective binding sites, the avidity (effective total binding strength) goes up and is therefore more effective in the activation of complement
57
What is the significance of IgM forming pentameric structure?
IgM is made first among the different classes of antibody. Hence it generally has low affinity for each antigen binding site. By multiplying the effective binding sites, the avidity (effective total binding strength) goes up and is therefore more effective in the activation of complement
58
How does IgM overcome the limitation of low affinity?
By forming pentameric structure
59
State the differences between VDJ recombination and isotype switching
1. VDJ uses the heptamer nonamer and the 12 or 23 bp spacer. Isotype switching uses the signal sequences (called switch signal sequences) 2. VDJ recombination could be unproductive due to the joints would affect the translation frames. Isotype swtiching is always productive as the rearrangement takes place in intronic regions. 3. VDJ recombination takes place during development, signal from antigens not involved. Isotype switching is in response to antigen stimulation, as well as signals from other sources, such as cytokines from T cells.
60
What is the role of cytokines in regulating the expression of antibody isotypes?
Inhibit and enhance Augment: enhance-- from a low level to higher level induce: from nothing to a positive level
61
What is the difference between heavy chain genes of human and mouse?
Human heavy chain gene has the duplication cassette
62
IgA can exist as ___________ in serum and _____________ in secretary fluids
monomer dimer
63
In a dimer IgA, what is present?
J chain and a secretory component
64
What is the purpose of the secretary component in IgA?
1. it is responsible for the transport of sIgA (secretary IgA) into secretary fluids such as tear, sweat, intestinal lumen and saliva 2. it makes the IgA more resistant to proteolytic enzymes in harsh environments such as gastrointestinal tract
65
For sIgA, what ensures that it does not get digested by proteolytic enzymes?
secretary component
66
What is the function of IgA?
binding to bacteria, viruses and toxin to prevent them from attachment to epithelial cells
67
Describe the process by which IgA is transported across epithelia
Transcytosis of IgA: 1. binding of IgA to receptor 2. endocytosis 3. transport to apical face 4. release of IgA dimer
68
Where is IgA usually found in the body?
Gut Respiratory tract salivary glands lactating breasts tear glands
69
Which Ig is transported across the placenta into the bloodstream of the fetus?
IgG
70
What receptor is used to transport IgG across placenta?
Neonatal Fc receptor (FcRn) takes 2 FcRn for the transport of IgG.
71
Where is FcRn usually found in the human body and why?
gut and liver of adults. it is responsible to bind to the IgG and resorb back to the blood by endocytosis. In the lysosome, it protects the IgG from degradation. The IgG can be released back into the blood plasma.
72
Why is the half-life of serum IgG longer than other immunoglobulins?
FcRn protects the IgG from degradation in the lysosome during endocytosis (from gut and liver to blood)
73
At what condition does the transcytosis of IgG from maternal blood to the fetus via FcRn occur?
acidic pH
74
What is the layer of cells forming the interphase between the maternal blood and the embryonic extracellular fluid called?
Syncytiotrophoblast
75
Describe the common mechanism by which toxins work?
1. Toxin binds to cell receptor 2. endocytosis of toxin-receptor complex 3. dissociation of toxin to release toxic chain, which acts on the cell
76
How many components do toxins have and why?
Many toxins have two subunits, one for binding to a receptor to mediate its entry into a cell. In the endosome, due to low pH, the two subunits separate and the toxic subunit enters the cytoplasm and deliver its damaging effect
77
What are the steps of neutrlization for viral infection?
1. aggregation 2. blocked attachment -steric interference -capsid stabilization -structural chamges 3. blocked uncoating -capsid stablization -fusion interference
78
How do antibodies prevent the action of bacteria?
Antibodies can block the addherence (adhesins) of bacteria to host cells -colonisation of cell surface by bacteria adhesions -some bacteria become internalized and propagate in internal vesicles -antibodies against adhesins block colonization and uptake
79
What is the main function of IgG?
Activation of the classical pathway of complement via IgG binding to a bacterial surface
80
What is the main function of IgE?
during first exposure, B cell produce IgE after exposure to allergen, IgE attach to mast cell and lead to degranulation. During second exposure, allergen attach to IgE on mast cells, leading to degranulation
81
What proteins bind to the hairpins after RSS cleavage?
-Ku70:Ku80 -XRCC4 -DNA-dependent protein kinases
82
Additional bases are added by tdT, what are they subtracted by?
exonuclease
83
The addition of nucleotides by tdT is similar to which other molecular processes?
DNA replication and repair
84
Signal from which sources leads to isotype switching?
-antigens -cytokines from T cells
85
How many disulphide bonds in the hinge region of IgG, IgA and IgD?
IgG: 2 IgA: 1 IgD: 1

immunobiotechnology (8 decks)

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