Diversification of the immunoglobulin genes [Elektronische Ressource] : analysis of the molecular mechanisms in the chicken B cell line DT40 / Ulrike B. Schötz

Technische Universität München
Lehrstuhl für Experimentelle Genetik



Diversification of the immunoglobulin genes: analysis of the molecular
mechanisms in the chicken B cell line DT40

Ulrike B. Schötz




Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades
eines

Doktors der Naturwissenschaften
genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. E. Grill

Prüfer der Dissertation:
1. apl. Prof. Dr. J. Adamski
2. Univ.-Prof. Dr. A. Gierl
3. Univ.-Prof. Dr. M.J. Atkinson



Die Dissertation wurde am 13.5.2009 bei der Technischen Universität München eingereicht und durch die
Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 02.09.2009
angenommen.









































Für Achim
Für meine Eltern


If we falter in resolve
Just because the task is hard,
No accomplishment can follow:
It is the world´s way.
- Emperor Meiji -

Cut, if need be, through thick briars,
Knots of brambles, tangled thorns,
For the path that´s yours to follow
Must be trodden to the end.
- Empress Shōken -
Table of contents
ZUSAMMENFASSUNG ..................................................................................................................................... 1

SUMMARY.......................................................................................................................................................... 3

1 INTRODUCTION........................................................................................................................................5
1.1 THE INNATE AND ADAPTIVE IMMUNE SYSTEMS ACT TOGETHER FOR A COMPLETE IMMUNE RESPONSE ............ 5
1.2 B AND T CELLS RECOGNIZE FOREIGN PATHOGEN SUBSTANCES BY THEIR HIGHLY VARIABLE RECEPTORS ......... 7
1.3 COMPOSITION OF A B CELL ANTIBODY MOLECULE ..................................................................................... 7
1.4 DIVERSIFICATION IS GAINED BY SEVERAL DISTINCT PROCESSES.................................................................... 8
1.4.1 V(D)J recombination is adding diversity to the gene loci by gene rearrangement ............................................ 8
1.4.2 Hypermutation increases specificity of the antibody after encounter of antigen ............................................. 10
1.4.3 Gene Conversion needs pseudogene templates to diversify the antibody genes............................................... 11
1.4.4 Class Switch Recombination changes the effector functions of the antibody molecule..................................... 12
1.5 DIVERSIFICATION IS REGULATED BY THE B CELL SPECIFIC ENZYME ACTIVATION-INDUCED CYTIDINE
DEAMINASE (AID).............................................................................................................................................. 12
1.5.1 AID has high homology to the RNA-editing enzyme APOBEC-1............................................................... 13
1.5.2 Regulation of AID is necessary to avoid genomic instability....................................................................... 14
1.6 URACIL DNA GLYCOSYLASE (UNG) IS INVOLVED IN THE PROCESSING OF THE AID-INDUCED DNA LESIONS15
1.7 THE THREE DIVERSIFICATION PROCESSES INVOLVE DIFFERENT FACTORS OF DNA REPAIR........................... 15
1.7.1 Error-prone repair in HM ends up with a diversified antibody gene after replication..................................... 16
1.7.2 Repair pathways that lead to GCV and CSR .......................................................................................... 17
1.8 SPECIFICITIES OF AID-DEPENDENT DIVERSIFICATION PROCESSES ............................................................. 18
1.8.1 Mutation frequency in hypermutating cells is strongly enhanced ................................................................ 18
1.8.2 The role of primary Ig sequences – AID can also target other sequences ...................................................... 19
1.8.3 Strand bias......... 19
1.8.4 Strong transcription is a prerequisite for AID-mediated diversification processes.......................................... 19
1.8.5 Chromatin modifications seem to play a minor role in AID targeting ......................................................... 21
1.9 TRANS-ACTING FACTORS AND CIS-ACTING ELEMENTS IN IG GENE DIVERSIFICATION................................... 21
1.9.1 AID is expressed only in B cells ............................................................................................................. 22
1.9.2 AID recruitment needs additional factors ............................................................................................... 22
1.9.3 The role of Ig enhancers and matrix attachment regions (MARs) is not completely clarified.......................... 23
1.9.4 E-box motifs seem to be important for HM............................................................................................. 24
1.9.5 E2A transcription factors bind to E-box motifs........................................................................................ 24
1.10 DT40 CELLS AS MODEL SYSTEM............................................................................................................... 25
1.11 EFFECT OF E2A KNOCKOUT ON HM ....................................................................................................... 26
1.12 AIMS.................................................................................................................................................... 29




i TABLE OF CONTENTS


2 MATERIALS................................................................................................................................................31
2.1 BACTERIAL STRAIN.31
2.2 BUFFERS AND SOLUTIONS....................................................................................................................... 31
2.3 CELL CULTURE....... 33
2.4 CELL LINES............. 33
2.5 CHEMICALS............ 34
2.6 CONSUMABLES....... 35
2.7 DNA SIZE MARKER.35
2.8 ENZYMES AND DNTPS............................................................................................................................ 35
2.9 EXPERIMENTAL KITS.............................................................................................................................. 36
2.10 IMMUNO-STAINING ANTIBODIES & ANTI-ANTIBODIES .............................................................................. 36
2.11 INSTRUMENTS ....................................................................................................................................... 36
2.12 MEDIA.................... 37
2.13 NUCLEOTIDE SEQUENCES....................................................................................................................... 38
2.14 OLIGONUCLEOTIDES ............................................................................................................................. 38
2.15 PLASMIDS............... 38
2.16 SOFTWARE.......... 38

3 METHODS...................................................................................................................................................39
3.1 VECTOR DESIGN..... 39
3.1.1 Databases........... 39
3.1.2 E2A complementation vector................................................................................................................ 40
3.1.3 IgL targeting vectors for cis-element study............................................................................................... 40
3.2 MOLECULAR BIOLOGY ........................................................................................................................... 44
3.2.1 Culture of E.coli... 44
3.2.2 DNA ligation...... 44
3.2.3 Transformation... 45
3.2.4 E.coli DH5 α competent cell preparation ................................................................................................ 45
3.2.5 PCR amplification .............................................................................................................................. 46
3.2.6 Analysis of DNA by electrophoresis ....................................................................................................... 46
3.2.7 PCR purification & Gel purification of DNA.......................................................................................... 47
3.2.8 DNA Purification by phenol/chloroform extraction................................................................................. 47
3.2.9 Ethanol precipitation of DNA .............................................................................................................. 47
3.2.10 Topo Cloning.....................................................................................