BIOL 435 Ch. 6 Part Two (Organization+Expression of Lymphocyte Receptor Genes) Flashcards
recombination among various V gene segments
- generates a diverse repertorie of Ab combining sites
- 2.3x10^6
heavy chain rearranged
- first
- if functional, it will be expressed on Pre-B cells with a surrogate light chain (gamma5 and VPreB)
- Pre-B cell undergoes several rounds of replication
- light chain rearrangement then begins in each daughter cell
how does recombination of heavy chain increase diversity?
-each daughter cell has same heavy chain but a different light chain can be added
>4 possibilities
recombination is directed by
-signal sequences
>RSS
recombination signal sequences (RSS)
- flank each Ab gene segment
- each has a conserved nonamer (9) and heptamer (7) sequence
- in between the nonamer/heptamer lies either a 12 or 23 bp spacer sequence
12/23 rule
-spacing and arrangement dictates that a 12bp RSS must pair with a 23bp RSS for recombination to occur
gene segments are joined by
-RAG1/2 recombinase
>both proteins are needed for recombination
RAG
-recombination activating gene
RAG1
- more important
- it forms a complex with RSSs stabilized by binding RAG2
RAG1/2 complex
-responsible for recognizing and cutting DNA at the immuonglobin-encoding region and the RSS
numerous other proteins are required for recombination
-including several that are not unique to lymphocytes
>non-homologous recombination proteins
V(D)J recombination
- occurs in a series of well-defined steps
1. RAG proteins bind to RSSs and cleave the DNA
2. Other proteins process the hairpin loops that form after RAG reacts
3. Products that will later be degraded
products from V(D)J recombination
- recombined coding joint
- leftover signal joint
- will later be degraded
simple steps V(D)J H. chain recombination
- Germline configuration
- D to J recombination
- V to DJ recombination
- Transcription
- Splicing: mRNA
- Polypeptide chain
simple steps V(D)J L. chain recombination
- Germline DNA
- V to J recombination
- Transcription
- Splicing
- Polypeptide chain
timeline for B-cells
- Early pro-B cell
- Late pro-B cell
- Pre-B cell
- Immature B-cell
early pro-B cell
- H-chain gene rearrangement
- D-J rearrangements on both chromosomes
late pro-B cell
-H-chain gene rearrangement
-V-DJ rearrangement on 1st chromosome
>if NOT good: V-DJ rearrangement on 2nd chromosome
»if NOT good: apoptosis
pre-B cell
-L-chain gene rearrangement
-rearrange 1st L. chain gene on 1st chromosome
>go through all 4 if need be
»if none good: apoptosis
immature B-cell
- rearrangement ceases
- cell expresses IgM lambda/kappa
mice L. chain
-try kappa first
length of helix 12/23
- related to turn
- 12=1 turn
- 23=2 turns
unproductive rearrangements
-recombined V-segments where trimming caused loss of correct reading frame
>cannot encode Ab moleculesV0
unproductive rearrangements
-recombined V-segments where trimming caused loss of correct reading frame
>cannot encode Ab molecules
V(D)J recombination direction
- can occur between segments transcirbed in either same or opposite directions
- as long as RSSs lined up, actual orientation of gene is irrelevant
5 mechanisms generate diversity in naive B-cells
- Inherit multiple V, D, and J gene segments
- Recombine gene segments
- Random assortment
- Recombination inaccuracy
- Somatic hypermutation
recombine gene segments
-which gene segments are put together
random assortment
-H/L chain combinational diversity (kappa or lambda)
recombinational inaccuracy types
- P-nucleotide addition
- Exonuclease timing (mostly H-chain)
- N-nucleotide addition (mostly H-chain)
P-nucleotide addition
-templated nucleotides additions between joints, resulting from asymmetircal cleaving of hair pin strucutres
exonuclease trimming
-sometimes occurs at junctions, losing nucleoties and chaning reading frames
N-nucleotide addition
-mediated by Tdt actavity in (C heavy and beta chains) adding random nucleotides between joints
chromatin alteration
- involved in regulation of V(D)J gene recombination
- catalytic activity of RAG1/2 binding occurs in extraordinary complex nuclear environment
- since V, D, J gene segments are spread out, higher order chromatin structure must play a role
RAG1/2 binding affected by
-epigenetic modifications on histoines associated with target sequences
histoine acetylation or methylation
-affects accessibility to enzymatic activity in chromatin
rosette-containing chromatin regions
- cotain accesible gene loci
- recombination events are topologically limited to genes located here
rosette containing D, J, C heavy regions
-defines scope of RAG activity in earliest pre-pro-B cell precursor
D-J heavy recombination enables
-loop structure alteration for V-D heavy recombination in later pro-B cell stage
allelic exclusion
-ensures that each B-cell synthesizes only one heavy and one light chain
heavy chains are
-recombined and expressed first
>expression of a functional H-chain shuts down recombination machinery temporarily
>paired with a surrogate L-chain (SLC) to from a pre-BCR
if SLC will pair with H-chain
-machinery starts up again
>L-chain recombination takes place
nonproductive arrangements
-lead to programmed cell death (apoptosis) during development
receptor editing
-of potentially auto reactive receptors occurs in L-chains
-functional antibody in an immature B-cell may bind to self-Ag
>recombination machinery can be turned back on to edit and salvage rearrangement or at least inactivate it
mature B-cells express both
- IgM and IgD antibodies
* mRNA splicing mechanism is the cause
spacer RNA sequence
-between VDJ and C regions
-mRNA splicing removes the intervening spacer
>leaves VDJC mRNA ready to be translated
primary transcripts for IgM heavy chains
-may result from RNA polymerase transcribing through both IgM and IgD constant regions
>dependin on which C segment becomes polyadenylated, IgM or IgD could end up being produced
mRNA splicing also controls
- whether the cell produces membrane bound or secreted IgM
* always membrane bound first than differentiated
