B Cell Diversity: Antigen Independent Mechanisms

Front 1

Name the gene segments that are involved in the formation of heavy and light chain variable regions.

Back 1

Heavy chain variable region - mew, epsilon, delta, gamma, alpha
Light chain variable regions - lambda and kappa

Front 2

Explain how the formation of Ig proteins from multiple gene segments contributes to the antibody repertoire

Back 2

Each Ig protein is produced from a unique combination of gene segments so that it can make the antibody repertoire have literally infinite specificities

Front 3

Describe the RSS feature of gene segments that ensures that the correct sequences are joined. ie what prevents a V segment from rearranging with another V segment

Back 3

the rss segment is when you have a heptamer and a nonamer seperated either by 12 sequences or 23 sequences. A 12 sequence can only recombine with a 23 sequence.

Front 4

Define junctional diversity and understand what causes it.

Back 4

When the segments are spliced together tDt adds new non genomic nucleotides to the splice junctions. that provides uniqueness and adds to the diversity of the Ig molecule

Front 5

What are the four antigen independent mechanisms that contribute to the development of the antibody repertoire?

Back 5

1) V and J combinations
2) V J and D combinations
3) junctional diversity
4) heavy chain and light chain combinations

Front 6

What is the purpose of the southern analysis of IgDNA from Liver DNA and B cell DNA?

Back 6

we want to see if we can see proof of DNA rearrangement

Front 7

How does the southern analysis of Liver and B cell DNA work? How do you do that experiment?

Back 7

1) you do a restriction enzyme digest of DNA from non lymphoid or B cell DNA population
2) seperate the DNA by gel electroporeisis and hybridize to membrane
3) probe for DNA complementary to heavy chain C region or heavy Chain V region C _H or V<sub>H<sub>

Front 8

What are the results of the experiment with Southern hybridization and what do they tell you?

Back 8

You get the same size fragment for the C region and V region in the B cell whereas you get different size fragments in the non lymphoid cell.
This tells you that DNA rearragement has occured because you get the same size fragment for both which means they are in the same area (?)

Front 9

what are the four organizational features of human Ig loci?

Back 9

1)one loci for heavy chains
2) one loci for kappa chains
3) one loci for lambda chains
4) multiple genes on each loci

Front 10

Where are the V regions of the Ig molecules located?

Back 10

at the 5' regions

Front 11

How are the v regions arranged?? (2 things)

Back 11

-300 bp
-families

Front 12

Which gene loci have multiple Cs and which gene loci have single Cs?

Back 12

kappa has a single c
heavy chain and lambda have multiple cs

Front 13

Where are the V, D and J, C segments located?

Back 13

All loci have the V, C and J segments
Only the heavy chain loci have the D segments

Front 14

What is the difference between the heavy chain variable region and the light chain variable region?

Back 14

-The heavy chain variable region loci has the V D and J genes
-The light chain variable region loci has the V and J genes

Front 15

What does V, D and J stand for?

Back 15

V = variable region
D = diversity region
J = joining region

Front 16

What is the 12-23 rule?

Back 16

an RSS consisting of a conserved heptamer and nonamer seperated by 12 bases can only recombine with a RSS consisting of a conserved heptamer and nonamer seperated by 23 bases

Front 17

What are the three stages of gene segement recombination?

Back 17

1)RAG binds the RSS at the heptamer on the two ends (heptamer of a V and heptamer of a J)
2) RAG cleaves the heptamer
producing a hairpin loop
3) ends are joined

Front 18

What is formed when the ends of the recombinant gene is ligated?

Back 18

1) signal joint of DNA that was eliminated
2) coding joint which is the V and J region connected

Front 19

What are the three steps of actually joining the recombination sequence?

Back 19

1) cleavage generates a palindromic sequence
2) cleaved ends can be modified before pairing
3) coding joint is formed with novel non genomic DNA

Front 20

What are the 8 steps of cleavage and recombination sequence in specifics?

Back 20

1) RAG binds to and cleaves double stranded DNA at RSS
2)creates a double stranded palindromic hair pin
3) hairpin is cleaved to yeild single stranded palindromic sequence (P sequence)
4) TdT adds nucleotides
5) single strands are paired
6) unpaired nucleotides are removed by exonuclease
7) gaps are filled by DNA synthesis and ligation
8)coding joint is formed with novel non genomic DNA

Front 21

What are the four antigen independent ways that we develop diversity?

Back 21

1) multiple v genes
2) V D J recombinations and V J recominbations
3) H and L combinations
4) junctional diversity

Front 22

How do you get VDJ next to C mew?

Back 22

differential RNA processing

Front 23

What combinations do you get to make IgM

Back 23

VDJ plus C mew

Front 24

What recominations do you get to make IgD

Back 24

VDJ plus V delta

Front 25

Are IgM and IgD expression antigen independent?

Back 25

yes