Lecture 6 - Organization and Expression of Lymphocyte Receptor Genes

the diversity conundrum:
what can lymphocyte receptor genes make against any antigen?

antibodies

the diversity conundrum:
what can lymphocyte receptor genes synthesize?

novel antigens that do not occur in nature

the diversity conundrum:
what do humans have vast quantities of?
what was the hypothesis that was once thought of before it was proven incorrect?

- DNA
- thought there was enough DNA to account for diversity

the diversity conundrum:
where does the diversity in antigen recognition come from?
what does this allow for the production of?

- random genetic rearrangements
- more than 10^10 different antibodies

solving the conundrum:
what did the model for antibody production have to allow for? hint: there's three

- diversity in antibody specificities
- presence of variable region at N-terminus and constant region at C-terminus
- existence of different isotypes with same antigen specificity

solving the conundrum:
what two models were proposed?

- germ line model
- somatic hypermutation model

germ line theory:
what was the germ line theory?

genome contributed by germ line cells was sufficient for diversity

germ line theory:
why was this theory preferred? aka was it simple or complex?

due to its simplicity

germ line theory:
what does this theory not require?

no genetic mechanism to account for diversity

germ line theory:
how many antibodies exist in mouse models?
how many base pairs did each gene contain?

- at least 10^7 different antibodies exist
- each gene was 2000 bp

germ line theory:
how many nucleotides are required to produce antibodies?
how many nucleotides did the mouse genome contain?
was it enough?

- requires 10^10 nucleotides
- mouse genome 10^9 nucleotides
- not enough DNA

germ line theory:
knowing the above information, how many different antibodies can be produced?

at least 10^10 different antibodies

germ line theory:
what did the germ line theory demonstrate to be?

implausible

modified germ line theory:
what in the germ line DNA encode for immunoglobulin genes? hint: there's two

- few constant regions
- many variable regions

modified germ line theory:
what is a single variable region selected to pair with for light and heavy chain genes?

constant region

modified germ line theory:
it drastically reduces the genome required; how many aas (bp) are in the variable region?

110 aa (330 bp)

modified germ line theory:
what does this require?
where was this requirement previously only observed?

- requires somatic cell recombination
- recombination previously only observed during gametogenesis

somatic hypermutation theory:
this theory contains a small number of?

immunoglobin genes

somatic hypermutation theory:
why are a large number of antibodies produced?

due to hypermutability or hyper recombination

somatic hypermutation theory:
what did sequencing information reveal?

depth of problem

somatic hypermutation theory:
what were supporters of this theory not able to explain?

how hyper mutability was restricted to antigen binding site

somatic hypermutation theory:
what were germ line supporters not able to explain?

evolutionary mechanism for generating diversity in variable portion of heavy and light chains while preserving the constant regions

somatic hypermutation theory:
immunoreceptor genes have limited number of what that contributes to variable domain?

number of cassettes

somatic hypermutation theory:
what does recombination/genetic rearrangement confer?

high degree of variability

demonstrating non germline recombination:
what does recombination rearrange?
what happens to the fragments?
what do either case result in?

- dna
- may be moved or lost
- movement or loss of restriction enzyme sites

demonstrating non germline recombination:
what is possible to use to determine if somatic recombination occurs in B cells?

southern blot

demonstrating non germline recombination:
where is chromosomal dna isolated from? which one is immune cell and which one is non immune cell? what are they both digested with?

- b cells (immune cell)
- liver cells (non immune cell)
- digested with BamHI

demonstrating non germline recombination:
what would happen if recombination occurred in b cells?

fragmentation pattern would be different for different genomic digests

demonstrating non germline recombination:
what would happen if recombination would be absent?

fragmentation pattern would be the same

demonstrating non germline recombination:
what are b cells and liver cells probed with? hint: two types oligonucleotide

c region oligonucleotide and light chain oligonucleotide

demonstrating non germline recombination:
were the hybridization patterns for both different or the same?

different

demonstrating non germline recombination:
what did the c region probe produce?

one signal with liver cell dna

demonstrating non germline recombination:
what did the light chain probe produce?

multiple signals in liver cell dna

demonstrating non germline recombination:
what did both the c region probe and light chain probe produce? was the signal for both different or the same compared to the liver?

- single signal with b cell dna
- different

demonstrating non germline recombination:
where does substantive rearrangement occur in?

immune cells that do not occur in non immune cells

demonstrating non germline recombination:
what did probe production suggest that exists?

initially multiple variable genome segments exist

demonstrating non germline recombination:
what did probe production suggest separates variable genome segment from constant genome segment?

a great distance

demonstrating non germline recombination:
what did probe production suggest brings constant coding segment into proximity of variable coding segment?

recombination

final piece of the puzzle:
what did cloning and sequencing of genes reveal they were composed of? what are these things?

- gene segments
- coding regions separated by non coding dna

final piece of the puzzle:
what gene segments do kappa and lambda light chains have?

v, j, and c gene segments

final piece of the puzzle:
what gene segments do heavy chain genes?

v, d, j, and c gene segments

final piece of the puzzle:
in kappa, lambda, and heavy chains, what does the c segment code for?
what is each v region preceded by? what is this required for?

- c segment codes for constant region
- preceded by a signal or leader peptide
- required for secretion

final piece of the puzzle:
when is leader peptide cleaved? what is this not part of?

- upon entry into er
- not part of mature antibody

kappa-chain multigene family:
what did nucleotide sequence code for? hint: what segment?
what is it preceded by?

- the first 97 aa of antibody - variable segment (v)
- preceded by leader peptide

kappa-chain multigene family:
what is the coding sequence for the remaining 13 aa separated by? hint: what segment?

- kilobases of dna - joining segment (j)

kappa-chain multigene family:
what is required to place v and j segments in proximity to one another?

recombination event

kappa-chain multigene family:
how many v segments do humans have? what about j segments? c segments?

- 41 v segments
- 4 j segments
- single C segment

lambda chain multigene family:
how many v segments do humans have? how many j segments? c segments?

- 33 v segments
- 4 j segments
- 4-5 c segments

lambda chain multigene family:
what is the term for when not all of the segments are functional?

pseudogenes

lambda chain multigene family:
recombination is directional, meaning v1 can recombine with what c segments? what c segments can it not recombine with?

- c3 or c1
- not c2 or c4

lambda chain multigene family:
what c segments can v2 and v3 recombine with? what does it recombine with more frequently?

- any c segments
- more frequently with c2 and c4

heavy chain multigene family:
how many v segments does it have? what about j segments?

- 45 v segments
- 6 j segments

heavy chain multigene family:
what are the v segments proceeded by?

leader peptide

heavy chain multigene family:
what gene segments do heavy chains have that light chains don't have? how many do they have?

- diversity segments (d)
- 23

heavy chain multigene family:
where are d segments located?

between v and j segments

heavy chain multigene family:
what is required to assemble heavy chain variable regions?

two recombination events

heavy chain multigene family:
heavy chains have multiple c segments, what are their greek letters? what does it dictate? hint: there's two and it revolves around antibody

- mu, delta, gamma, epsilon, alpha
- dictates both the isotype and function of antibody

multiple germ line/combinatorial v(d)j joining and association of light and heavy chains:
how many possibilities can occur with the combination of Vh, D, and Jh segments?

6610 possibilities

multiple germ line/combinatorial v(d)j joining and association of light and heavy chains:
how many possibilities can occur with the combination of Vk and Jk segments?

205 possibilities

multiple germ line/combinatorial v(d)j joining and association of light and heavy chains:
how many possibilities can occur with the combination of Vl and Jl segments?

165 possibilities

multiple germ line/combinatorial v(d)j joining and association of light and heavy chains:
what can any assembled heavy chain combine with?

any assembled light chain

how do b cells know where to recombine:
what did dna sequencing lead to the identification of?
what are the two types? hint: think of base pairs
what does this denote?

- recombination signal sequences (rsss)
- 12 or 23 base pairs
- denotes spacer between consensus sequences

how do b cells know where to recombine:
what consensus sequences is this: CACAGTG

heptamer consensus sequence

how do b cells know where to recombine:
what consensus sequence is this: ACAAAAACC

nonamer consensus sequence

how do b cells know where to recombine:
what is the heptamer consensus sequence adjacent to?

cassette

how do b cells know where to recombine:
how much does the 12 bp rss span?

1 turn of double helix (1 turn rss)

how do b cells know where to recombine:
how much does 23 bp rss span?

2 turns of double helix (2 turn rss)

how do b cells know where to recombine:
what can 1 turn rss only recombine with?

2 turn rss (1 turn 2 turn joining rule)

how do b cells know where to recombine:
what rss does Vk have? what rss does Jk have?

Vk \= 1 turn rss
Jk \= 2 turn rss

how do b cells know where to recombine:
what rss does Vl have? what rss does Jl have?

Vl \= 2 turn rss
Jl \= 1 turn rss

how do b cells know where to recombine:
what rss do Vh and Jh have? what rss does Dh have?

Vh and Jh \= 2 turn rss
Dh \= flanked by 1 turn rss

how do b cells know where to recombine:
what do recombination signal sequences ensure?

proper recombination events

generating light chain diversity:
this process is the same for lambda and kappa
dna is first transcribed into rna, what does rna contain?

L peptide, vj, and c coding sequences

generating light chain diversity:
what are l and c coding sequences still separated from?

vj coding sequence

generating light chain diversity:
what makes the l and c coding sequences join vj coding sequences?

mrna splicing

generating light chain diversity:
when mature mrna is translated, where is the protein secreted? what happens to the leader peptide?

- secreted into er
- leader peptide is cleaved

generating heavy chain diversity:
first, recombination occurs to join what segments?

join d and j segments

generating heavy chain diversity:
second recombination occurs to join what segments?

v and d segments

generating heavy chain diversity:
what does the transcript contain?

l, vdj, and two coding sequences (mu and delta)

generating heavy chain diversity:
what joins l to vdj? what does this determine?

- mrna splicing
- determines what isotype of antibody will be produced

generating heavy chain diversity:
when mature mrna is translated, where is the protein secreted? what happens to the signal peptide?

- secreted into er
- signal peptide is cleaved

generating heavy chain diversity:
what do heavy and light chains assemble into in the er?

antibodies

initiation of v(d)j recombination:
what is v(d)j recombinase encoded by?

recombination activating gene 1 and 2 (rag1 and rag2)

initiation of v(d)j recombination:
what does RAG1/2 form? where is it only expressed in?

- forms tetramer, two molecules of each
- only expressed in lymphocytes

initiation of v(d)j recombination:
- what does rag1 contain that is required for dna cleavage? what are the names?
- what does the central region bind? hint: consensus sequence
- where is the heptamer binding motif? what does it also contain?

- 3 catalytic amino acids (D600/D708/E962)
- nonamer consensus sequence
- core region; contains RAG2 binding motif

initiation of v(d)j recombination:
- what does RAG1 contain? what are the names?
- what assists with DNA binding and binds H3?
- what does H3 interaction inhibit? what does it recruit?
- when H3 is ubiquitinated and removed, what does it promote?

- two zinc fingers: ZnA and ZnB
- zna assists with DNA binding
- inhibits cleavage; recruits ubiquitin ligase
- when removed, it promotes cleavage

initiation of v(d)j recombination:
where is the core region of RAG2? what is it essential for?

- at amino terminus
- essential for dna cleavage

initiation of v(d)j recombination:
where is the plant homeodomain (PHD) motive?
what does it promote? what does it bind?
what does binding enhance?

- downstream of core region
- promotes dna binding
- binds H3K4me3 modified histone
- binding enhances dna cleavage activity

initiation of v(d)j recombination:
what does carboxy terminal amino acid t490 regulate?
what is t490 phosphorylated by? hint: think of cell cycle
what does it promote? what does it ensure?

- regulates activity
- cdk in s, g2, and m phases of cell cycle
- promotes degradation
- ensures v(d)j recombination does not occur in dividing cells

other factors involved in v(d)j recombination:
where is the terminal deoxynucleotidyl transferase (TdT) uniquely expressed in?
tdt uses template independent polymerase to add what to v(d)j junctions?
what does it randomize? what does it add? what does it increase?

- expressed in lymphocytes
- adds non templated nucleotides
- randomizes reading frame, adds new codons, increases variability

other factors involved in v(d)j recombination:
these other machinery are not unique to lymphocytes aka they are expressed in many cells
- what does high mobility group B proteins 1 and 2 (HMGB1/2) assist?
- what does the non homologous end joining pathway (NHEJ) complete?
- what are both of these expressed in? hint: two

- assist RAG1/2 with rss binding
- completes v(d)j recombination
- both expressed in either germ line recombination or error prone dna repair

v(d)j recombination:
it starts with rag1/2 binding to what? what does it require?

- 12/23 rss
- requires hmgb1/2

v(d)j recombination:
what does binding of 12 rss cause? what does it create?
note to self: this can occur vice versa

- causes conformational change
- creates 23 rss binding site

v(d)j recombination:
what does intervening dna form?

loop

v(d)j recombination:
what does rag1 catalyze at 3' end of v and j segments? what does it create?

- ssdna nick
- creates 3'-OH

v(d)j recombination:
what does the 3'-OH perform? what does this create for both v and j segments? what does it require? hint: requires three things

- a nucleophilic attack on 5' phosphate of opposite strand
- creates hairpin
- RAG1/2, HMGB1/2, and Ataxia Telangiectasia Mutated (ATM) serine/threonine kinase

v(d)j recombination:
what happens to the intervening region of dna? what are the hairpin structures formed called?

- it is lost
- coding end

v(d)j recombination:
what does the nhej dna ligase IV protein catalyze?

ligation that forms the hairpins

v(d)j recombination:
how are the hairpins opened? this is catalyzed by what? hint: it is an nhej nuclease

- ssdna nick
- catalyzed by artemis

v(d)j recombination:
the ssdna nick is random meaning that it may create three different types, what are they?

5' overhang
3' overhang
blunt end