Chapter 7 - immunology

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3 sequence event in VDJ recombination

1- DH gene combine to JH 2- DHJH combine VH generate V domain 3- functional VL combines with funtional JLCL combinations

VDJ recombination series

1- RAG bind RSS, cleave DNA, unwind 2- other protein process hairpin loop that form after RAG react 3- Products of a recombined coding joint and a leftover signal joint that is later degraded

Nonamer conserved of

9- bp with sequense of 5'-ACAAACC-3'

combining of L and H chain can add

Final diversity of variable region

in VDJ recombination, J stand for ?

Joining segment

Role of Artemis generating diversity by adding P nucleotides

Once Artemis has been phosphorylated by DNA-PKcs, it opens the hairpin on the coding end joint.

Hairpin resolution results in

P-region nucleotide addition

Role of DNA-PKcs generating diversity by adding P nucleotides

Protein kinase that forms a complex with Ku70/80. It phosphorylates and activates Artemis, and recruits the ligation machinery VDJ recombination

what are 3 protein implicated in VDJ that are unique to lymphocytes?

RAG 1, Rag 2, and Terminal Dexynucleotidyl transferase (TdT)

What associated in gene segments joining?

RAG 1/2 recombinase

2 types of Rag

RAG1 & RAG2

High mobility group B proteins 1 and 2 (HMGB1/2)

Stabilize binding of RAG1/2 to recombination signal sequences (RSSs). Stabilize introduction of bend into 23 RSS DNA by RAG1/2

Where does TdT express and how it will express

TdT only happen at Lymphocytes and common in H chain. that can add up to 15-20 max nucleotides at cut end that create diversity

What is the segment for light chain?

V and J

What is the segment for heavy chain?

VDJ

List the 5 mechanisms that generate antibody diversity in naïve B cells

VDJ recombination, exonuclease trimming, P nucleotide addition, Non-templated nucleotide addition, combinatorial diversity

DNA ligation complex: DNA ligase IV, XRCC4, and XLF (Cernunnos)

XRCC4 maintains stability of DNA ligase IV and stimulates its catalytic activity. XRCC4 may also help to align DNA ends. DNA ligase IV is required for ligation of cut DNA ends, at both the coding and the signal joints.

how hairpin was introduce?

break near the tip of each hairpin in one of the DNA strands. Cutting of the hairpins generates sites for the addition of P-region nucleotides which adds to diversity.

Exonuclease trimming

common in 2 H chain V gene joints (V-D & D-J) than at L chain V-J joint. result in loss of coding nucleotide at joint

3 element in RSSs

conserved of "nonamer & heptamer", spacer 12 or 23 bp sequence, 12 bp RSSmust pair with 23bp RSS for recombination to occur (12/23rule)

TdT functions

develop only in lymphocytes. Adds nontemplated (N) nucleotides to V-D and D-J joints of Ig heavy chain and all joints of TCR chains in a template independent manner

What are the VDJ recombination?

directed by Recombination signal sequences (RSSs) that ensure each type of sequence is included in variable region gene

unique of RAG 1

express in all phases of cell cycle

hairpin formation

free 3'-OH on the cut DNA strand attacks the phosphodiester bond linking the opposite DNA strand to the SS, simultaneously producing a hairpin structure at the cut end of the CS & a flush, 5'-phosphorylated, double-strand break at the SS.

purpose of cutting hairpin

generates sites for addition of P-region nucleiotides

Why RAG1 are more more important

it form complex with RSS to stabilized biding with RAG2

Exonuclease trimming AKA

junctional flexibility

Non-templated N nucleotide addition are

mediated by TdT activity, adding in random nucleotides between joints

VDJ recombination are

of multiple gene segments

Function of hairpin structure

protects gene segment from being degraded, prevent them from being joined together immediately into functional gene

What doe RAG1/2 responsible for

recognizing and cutting DNA at immunoglobulin-encoding and the RSS

What is RAG?

recombination activating gene

both Rag 1/2 developed by

regulated in both T and B cells

what are 3 protein function in VDJ?

responsible for the generation of additional diversity in CDR 3 (complementary determining region) of the antibody heavy chain

combinatorial diversity are

same H chain can combine with different L chains, and vice-versa

unique of RAG 2

stable only in G0 or G1 phase cell

P nucleotide addition are

templated nucleotide addition between joints, resulting from assymetrical cleaving of hairpin structures

Role of TdT to generate diversity

the addition of N-nucleotides.

Exonuclease trimming are

trimming―sometimes occurs at joints, causing loss of nucleotides and changed reading frames

in VDJ recombination, V stand for?

variable region

define combinatorial diversiry

which heavy chains pair with which light chains

In human, what are the possible number of potential VDJ combination in Lambda light?

33 x 5= 165 possible VDJ combinations

In human, what are the possible number of potential VDJ combination in Kappa light?

41 x 5= 205 possible VDJ combinations

In human, what are the possible number of potential VDJ combination in heavy chain?

45 x 23 x 6= 6210 possible VDJ combinations

Heptamer conserved of

7- bp with sequence of 5'-CACAGTG-3'

who can form complex hairpins

DNA-dependent protein kinase (DNA-PK) and Artemis protein

in VDJ recombination, D stand for ?

Diversity segment


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