module 03 section 02 (innate immune system) Flashcards
what did Susumu Tonegawa discover?
he eludicated the mechanism for making millions of different antibody molecules with different specificities without requiring an unreasonably large number of genes
when do Igs ungergo “somatic recombination” ?
what part of the Ig does this refer to?
heavy and light chains undergo somatic recombination of genetic material during the early stages of B- and T-cell maturation
what does somatic recombination allow for?
a high degree of diversity among Igs and T-cell receptors
in addition to somatic recombination, what contributes to the diversity of Igs?
the fact that each class of Ig has a separate pool of gene segments from which the final Ig in synthesized
what are the 4 classifications of genes contained in the light chains?
- leader sequences
- variable gene segments
- joining gene segments
- constant gene segments
light chains can be classified into two groups, what are they? and what are these groups based on?
kappa, lambda - based on small differences in polypeptide sequence
the lambda light chain locus is found on what chromosome?
22
describe the lambda light chain
there are several J and C segments, each interacting with only its corresponding segment (e.g. J lambda1, 2, 3, or 4 and C lambda1, 2, 3 or 4)
the kappa light chain is found on what chromosome?
2
describe the kappa light chain
each Jk can join with the same Ck (as there is only one Ck)
describe the process of kappa light chain rearragement (5 steps)
(1) DNA rearrangement -somatic recombination: V-J joining (regulated by RAG)
(2) transcription and rna processing (splicing)
(3) mulitple adeinin nucelotides (poly-A-tails) are added to polyadenylation sites at 3’ of Ck
- mRNA exists the nucelus to bind ribosomes in the rough ER - translation
(4) L sequence pulls growing polypeptide into lumen of ER and is then cleaved off
(5) processing and glycosylation of the protein
if there wass a mutation in the RAG genes (recombinase enzymes that act on the variable regions of Ig) would Igs be made? would B-cells be made?
niether would be made
the heavy chain locus is found on what chromosome?
14
describe formation of the heavy chain
one D segment joins with a J segment, then a V segment joins with the D-J segment
describe the process of heavy chain rearrangement (6 steps)
(1) first rearrangement - somatic recombination: D-J joining (regulated by RAG)
(2) second rearrangement - somatic recombination: V-DJ joining (regulated by RAG)
(3) transcription and rna processing (splicing)
(4) mulitple adeinin nucelotides (poly-A-tails) are added to polyadenylation sites at 3’ of Cu (mu)
- mRNA exists the nucelus to bind ribosomes in the rough ER - translatio
(5) L sequence pulls growing polypeptide into lumen of ER and is then cleaved of
(6) processing and glycosylation of the protein
each unique heavy chain associates with a light chain to create a unique Ig, true or false?
true
the variable regions are the:
exons
how many variable heavy chain regions have we idenitifed?
100-200
how many diversity (D) heavy chain regions have we identified?
50
how many joining heavy chain regions have we identified?
6
recap: how many constant gene segments are there in the kappa light chain ?
1
recap: what is unique about the J lambda 4 gene?
it is a pseudogene, or a non-functional gene
recap: what gives rise to antibody specificity (peritope)?
the V-DJ (heavy) and the V-J (light) regions
how do we make different classes of antibodies with the same antigenic specificity?
class switch recombination - type of gene rearrangement where the constant gene regions in the Ig heavy chain switch from being one class to another (e.g. go from IgM to IgG)
where does class switch recombination occur?
btwn the switch sites which are located upstream of the CH regions
can switch rearrangement occur with IgD? why or why not?
no - has no switch site
with what Igs can IgM do switch rearrangement with?
IgM –> IgG, IgA
this way only - can’t go from IgA to IgM
can switch rearrangement occur from IgM to IgE?
yes - but have to go from IgM to IgG then from IgG to IgE
how many possible heavy and light chain associations are there?
2.64 million
how many possible combinations of VJ rearrangements are there for k light chains?
200
how many possible combinations of VJ rearrangements are there for λ light chains?
100
how many heavy chain V segments are there?
51
how many heavy chain D segements are there?
27
how many J segments are there?
6
how many possible heavy chain V regions are there?
8262
recap: what primarily allows for Ig diversity?
gene rearrangement
what are recombination singal sequences?
conserved sequences of noncoding DNA that function as signals for the recombination process in heavy and light chain rearrangements
where are recombination singal sequences found in terms of where recombination takes place?
directly adjacent to the point at which recombination takes place
recombination singal sequences are recognized by ____ during what process?
RAG1 and RAG2 during VDJ recombination
what is RAG1? what is its role?
- enzyme encoded by recombination activating gene 1
- plays important role in the rearrangement and recombination of antibody and t-cell receptor molecules
what is RAG2? what is its role?
- enzyme encoded by recombination activating gene 2
- plays important role in the rearrangement and recombination of antibody and t-cell receptor molecules
where are recombination singal sequences found on the V segment?
3’ end
where are recombination singal sequences found on the J segment?
5’ end
where are recombination singal sequences found on the D segment?
both 3’ and 5’ ends
what do all recombination singal sequences contain? (2)
- a conserved palindromic heptamer
- a conserved AT-rish nonamer sequence
- the two are separated by either 12 or 23 non-conserved bps (if there’s 1 or 2 turns of the DNA helix)
what does the one-turn/two-turn joining rule state?
that signal sequences with a one turn spacer can only join with a sequence that has a two turn spacer (and vice versa)
what happens when a one-turn and a two-turn sequence join?
the resulting gene sequence forms a loop, then the RAG enzyme cuts the excess gene segment
are the 12/23 bp spacers conserved when one-turn and two-turn sequences join?
no
are the palindromic and AT-rich signal sequences conserved when one-turn and two-turn sequences join?
yes - they form circular dna that gets degraded
what is the result of the looping that occurs due to the palindromic and AT-rich sequences?
the merging of VJ segments
rearrangement can be productive or nonproductive, true or false? why?
true
the amino acid sequence variation generated by gene rearrangement in the coding joint has been shown to fall within:
the third hypervariable region (CRD3) in immunoglobulin heavy and light chain DNA
CRD3 plays a key role in:
the recognition of unique epitopes
what is productive rearrangement?
the joining of VDJ segments in phase to produce VJ or a VDJ unit that can be translated in its entirety
what does one productive rearrangement mean?
one allele is enough to make the immunoglobulin
what is nonproductive rearrangement?
rearrangement in which gene segments are joined out of phase so that the triplet-reading frame for translation is not preserved (i.e. there is a stop codon)
what happens if there is a nonproductive rearrangement?
the immunoglobulin chain is not made
the site where polyadenylation occurs directs:
whether the antibody produced will be membrane bound or secreted
the constant regions of IgM and IgD are processed at the same time, true or false? why?
true - they are the first immunoglobulins produced
where do immature developing B-cells favour polyadenylation? what is the result?
at poly-A site 2 and 4 to produce membrane bound antibodies
where do plasma cells favour polyadenylation? what is the result?
at poly-A site 1 and 3 to produce secreted antibodies
describe the process of class switching
ds dna breaks are generated at the switch sites, removing unwanted portions of the CH and the remaining segments are re-joined by non-homologous end-joining to produce a different antibody isotype
does antigen specificity remain the same following class switching? why or why not?
yes - its dictated by the VH region which stays the same
what does the CH change (due to class switching) modify in terms of Ig function?
the effector molecules the antibody can interact with
what are the means of Ig diversity that we covered?
(1) junctional flexibility (VDJ)
(2) combinatorial addition of light/heavy chains
(3) nucelotide addition (P-region/N-region)
(4) somatic hypermutation
where is the thrid hypervariable loop of Igs located?
at the joint btwn the V and J gene segment
in the heavy chain, CRD3 is partially encoded by the:
D gene
in both heavy and ligth chains, the diversity of CRD3 is significantly increased by:
the addition or deletion of nucleotides
when does nucleotide addition occur?
during the rearrangement of gene segments in the initial development of B-cells
describe how the addition of N-nucleotides adds diversity to the antigen binding site
- these are nontemplate-encoded
- these are added by TdT to single stranded ends of coding dna after hairpin cleavage
describe how the addition of P-nucleotides adds diversity to the antigen binding site
- these make up palindromic sequences
- these are added to the ends of gene segments (in the antigen binding site)
are somatic mutations random?
yes
where do somatic mutations occur?
in germinal centers where b-cells proliferate ONLY
what do somatic mutations target?
rearranged V regions (CRDs) located within the DNA requence influencing the VJ/VDJ rearrangement process
why are Ig somatic mutations reffered to as somatic hypermutations?
bc they have a frequency 100,000 times higher than the spontaneous rate in other genes
what are 2 processes that somatic mutations are associated with?
(1) class switching
(2) affinity maturation
what triggers class switching?
antigen stimulation
what is affinity maturation?
increase in the average affinity of an antibody for an antigen during the course of an immune response or in subsequent exposures to an antigen (resulting in the lock and key fit)
when does combinatorial diversity manifest?
when 2 separate units of genetic information combine
