BIOL 435 Ch. 6 Part Two (Organization+Expression of Lymphocyte Receptor Genes)

Question 1

recombination among various V gene segments

Answer 1

• generates a diverse repertorie of Ab combining sites

- 2.3x10^6

Question 2

heavy chain rearranged

Answer 2

• 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

Question 3

how does recombination of heavy chain increase diversity?

Answer 3

-each daughter cell has same heavy chain but a different light chain can be added
>4 possibilities

Question 4

recombination is directed by

Answer 4

-signal sequences

>RSS

Question 5

recombination signal sequences (RSS)

Answer 5

• 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

Question 6

12/23 rule

Answer 6

-spacing and arrangement dictates that a 12bp RSS must pair with a 23bp RSS for recombination to occur

Question 7

gene segments are joined by

Answer 7

-RAG1/2 recombinase

>both proteins are needed for recombination

Question 8

RAG

Answer 8

-recombination activating gene

Question 9

RAG1

Answer 9

• more important

- it forms a complex with RSSs stabilized by binding RAG2

Question 10

RAG1/2 complex

Answer 10

-responsible for recognizing and cutting DNA at the immuonglobin-encoding region and the RSS

Question 11

numerous other proteins are required for recombination

Answer 11

-including several that are not unique to lymphocytes

>non-homologous recombination proteins

Question 12

V(D)J recombination

Answer 12

• 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

Question 13

products from V(D)J recombination

Answer 13

• recombined coding joint
• leftover signal joint
• will later be degraded

Question 14

simple steps V(D)J H. chain recombination

Answer 14

1. Germline configuration
2. D to J recombination
3. V to DJ recombination
4. Transcription
5. Splicing: mRNA
6. Polypeptide chain

Question 15

simple steps V(D)J L. chain recombination

Answer 15

1. Germline DNA
2. V to J recombination
3. Transcription
4. Splicing
5. Polypeptide chain

Question 16

timeline for B-cells

Answer 16

1. Early pro-B cell
2. Late pro-B cell
3. Pre-B cell
4. Immature B-cell

Question 17

early pro-B cell

Answer 17

• H-chain gene rearrangement

- D-J rearrangements on both chromosomes

Question 18

late pro-B cell

Answer 18

-H-chain gene rearrangement
-V-DJ rearrangement on 1st chromosome
>if NOT good: V-DJ rearrangement on 2nd chromosome
»if NOT good: apoptosis

Question 19

pre-B cell

Answer 19

-L-chain gene rearrangement
-rearrange 1st L. chain gene on 1st chromosome
>go through all 4 if need be
»if none good: apoptosis

Question 20

immature B-cell

Answer 20

• rearrangement ceases

- cell expresses IgM lambda/kappa

Question 21

mice L. chain

Answer 21

-try kappa first

Question 22

length of helix 12/23

Answer 22

• related to turn
• 12=1 turn
• 23=2 turns

Question 23

unproductive rearrangements

Answer 23

-recombined V-segments where trimming caused loss of correct reading frame
>cannot encode Ab moleculesV0

Question 24

unproductive rearrangements

Answer 24

-recombined V-segments where trimming caused loss of correct reading frame
>cannot encode Ab molecules

Question 25

V(D)J recombination direction

Answer 25

- can occur between segments transcirbed in either same or opposite directions - as long as RSSs lined up, actual orientation of gene is irrelevant

Question 26

5 mechanisms generate diversity in naive B-cells

Answer 26

1. Inherit multiple V, D, and J gene segments 2. Recombine gene segments 3. Random assortment 4. Recombination inaccuracy 5. Somatic hypermutation

Question 27

recombine gene segments

Answer 27

-which gene segments are put together

Question 28

random assortment

Answer 28

-H/L chain combinational diversity (kappa or lambda)

Question 29

recombinational inaccuracy types

Answer 29

1. P-nucleotide addition 2. Exonuclease timing (mostly H-chain) 3. N-nucleotide addition (mostly H-chain)

Question 30

P-nucleotide addition

Answer 30

-templated nucleotides additions between joints, resulting from asymmetircal cleaving of hair pin strucutres

Question 31

exonuclease trimming

Answer 31

-sometimes occurs at junctions, losing nucleoties and chaning reading frames

Question 32

N-nucleotide addition

Answer 32

-mediated by Tdt actavity in (C heavy and beta chains) adding random nucleotides between joints

Question 33

chromatin alteration

Answer 33

- 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

Question 34

RAG1/2 binding affected by

Answer 34

-epigenetic modifications on histoines associated with target sequences

Question 35

histoine acetylation or methylation

Answer 35

-affects accessibility to enzymatic activity in chromatin

Question 36

rosette-containing chromatin regions

Answer 36

- cotain accesible gene loci | - recombination events are topologically limited to genes located here

Question 37

rosette containing D, J, C heavy regions

Answer 37

-defines scope of RAG activity in earliest pre-pro-B cell precursor

Question 38

D-J heavy recombination enables

Answer 38

-loop structure alteration for V-D heavy recombination in later pro-B cell stage

Question 39

allelic exclusion

Answer 39

-ensures that each B-cell synthesizes only one heavy and one light chain

Question 40

heavy chains are

Answer 40

-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

Question 41

if SLC will pair with H-chain

Answer 41

-machinery starts up again | >L-chain recombination takes place

Question 42

nonproductive arrangements

Answer 42

-lead to programmed cell death (apoptosis) during development

Question 43

receptor editing

Answer 43

-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

Question 44

mature B-cells express both

Answer 44

- IgM and IgD antibodies | * mRNA splicing mechanism is the cause

Question 45

spacer RNA sequence

Answer 45

-between VDJ and C regions -mRNA splicing removes the intervening spacer >leaves VDJC mRNA ready to be translated

Question 46

primary transcripts for IgM heavy chains

Answer 46

-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

Question 47

mRNA splicing also controls

Answer 47

- whether the cell produces membrane bound or secreted IgM | * always membrane bound first than differentiated