double edged sword:

double edged sword:pediatric leukemia and the biology of

adaptive immunity

Outline

pediatric leukemia and V(D)J Recombination

an in vitro assay that assesses aberrant V(D)J recombination in response to environmental exposure

mechanisms of genotoxin-induced recombination

A ROUGH AND RUDE IMMUNOLOGY PRIMER

Pediatric Leukemia

malignancies that arise from the uncontrolled proliferation of lymphoid or myeloid precursor cells

general types of pediatric leukemia include:

T- ALL & B-ALL

AML

PML

alterations of genomic DNA have been demonstrated to occur during fetal development.

Age specificity of pediatric leukemia

What is HPRT and why might we care?

X chromosome

Distribution of Aberrant V(D)J Recombinase Mediated Deletions and ALL in Children

Finette et al, 1996. Cancer Research

Incidence of ALL in Children and Young Adults

Over 50 publications linking V(D)J recombinase mediated genomic rearrangements with the development of lymphoid malignancies

% T

ota

l D

ele

tions

generating immune cell diversity

http://2012.igem.org/Team:Marburg_SYNMIKRO/Project

Normal

Abnormal

Cancer

Diversified Immune System

V(D)J Recombinase

consequences of a diverse immune system

Mechanism of V(D)J Recombination I

5’-CACAGTG-12/23-ACAAAAACC-3’

7-mer 9-mer

Recombination Signal Sequence (RSS)

Rag1

Rag2

I

II

DNA Damage & Repair

Mechanism of V(D)J Recombination II

Adapted from S.P. Jackson 2002 Carcinogenesis 25 (5):687

ArtemisXRCC4Ligase 4TdT

70 80

70 80

NHEJ proteins recognize RAG generated DSB and aid in the completion of V(D)J recombination

DNA-PK

DNA-PK

Ptrp

CAT

OOP

X

Rag1Rag2

OOP

OOPNHEJ

V(D)J Recombination of pGG51

AMP

CAT

Coding Joint

Ampr CamrPtrp

AMP

In vitro V(D)J recombination assay

Reh pre-B cells

X hours

pGG51

Amp/Cam colonies

Amp coloniesX 100 = % recombination frequency (Rf)

1 hour

amp amp/camamp amp/cam

Assay re-developed based on Gauss and Lieber 1993

48 hours

E. coli

Analysis of V(D)J signatures in rearranged plasmid substrates

pGG51

150 bp

5'...GCTTGG CTGCAGGTCGAC GGATCCCCGGGGATCAGCTTGGCGA...3'

Assessing DNArepair proteins

Gamma-radiation and Recombination frequency

Mutation Research/Fundamental and Molecular Mechanisms of MutagenesisVolume 686, Issues 1–2, 1 April 2010, Pages 30–38

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