Post-transcriptional regulation of IgE [Elektronische Ressource] = Posttranskriptionelle Regulation von IgE / vorgelegt von Alexander Karnowski

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Post-transcriptional regulation of IgEPosttranskriptionelle Regulation von IgEInaugural-Dissertationzur Erlangung der Doktorwürdeder Fakultät der Biologieder Albert-Ludwigs-UniversitätFreiburg im Breisgauvorgelegt vonAlexander Karnowskiaus HamburgFreiburg im Breisgau, April 2002 Dekan der Fakultät: Prof. Dr. Hans Kleinig Promotionsvorsitzener: Prof. Dr. Samuel Rossel Betreuer der Arbeit: Dr. Marinus Lamers Referent der Arbeit: Prof. Dr. Gunther Neuhaus Koreferent: Dr. Hans U. Weltzien 3. Prüfer: Prof. Dr. Christoph F. Beck Tag der Verkündigung des Prüfungsergebnisses: 08.07.2002 Die vorliegende Arbeit wurde in der Zeit von November 1998 bis April 2002 in der Abteilung Entwicklung des Immunsystems von Prof. Dr. Thomas Boehm im Labor von Dr. Marinus Lamers am Max-Planck Institut für Immunbiologie in Freiburg im Breisgau durchgeführt Dedicated to Florienne LoderTable of contentsAbbreviations1 Introduction..............................................................................................................11.1 General Introduction...........................................................................................11.2 Short introduction to the immune system ............................................................21.2.1 Innate Immunity..........................................................................................21.2.
Publié le : jeudi 1 janvier 2004
Lecture(s) : 64
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Source : FREIDOK.UB.UNI-FREIBURG.DE/FREIDOK/VOLLTEXTE/2004/1175/PDF/DISSAK01.PDF
Nombre de pages : 130
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Post-transcriptional regulation of IgE
Posttranskriptionelle Regulation von IgE
Inaugural-Dissertation
zur Erlangung der Doktorwürde
der Fakultät der Biologie
der Albert-Ludwigs-Universität
Freiburg im Breisgau
vorgelegt von
Alexander Karnowski
aus Hamburg
Freiburg im Breisgau, April 2002
Dekan der Fakultät: Prof. Dr. Hans Kleinig


Promotionsvorsitzener: Prof. Dr. Samuel Rossel


Betreuer der Arbeit: Dr. Marinus Lamers


Referent der Arbeit: Prof. Dr. Gunther Neuhaus


Koreferent: Dr. Hans U. Weltzien


3. Prüfer: Prof. Dr. Christoph F. Beck


Tag der Verkündigung des Prüfungsergebnisses: 08.07.2002





















Die vorliegende Arbeit wurde in der Zeit von November 1998 bis April 2002 in der
Abteilung Entwicklung des Immunsystems von Prof. Dr. Thomas Boehm im Labor von
Dr. Marinus Lamers am Max-Planck Institut für Immunbiologie in Freiburg im
Breisgau durchgeführt Dedicated to Florienne LoderTable of contents
Abbreviations
1 Introduction..............................................................................................................1
1.1 General Introduction...........................................................................................1
1.2 Short introduction to the immune system ............................................................2
1.2.1 Innate Immunity..........................................................................................2
1.2.2 Adaptive Immunity......................................................................................3
1.2.2.1 Immunoglobulins and B cells...............................................................3
1.2.2.2 Fc receptors ....................................................................................... 10
1.2.2.3 T cell receptor and T cells .................................................................. 11
1.3 The IgE Network .............................................................................................. 12
1.4 Project and aims................................................................................................ 15
2 Material and Methods............................................................................................ 17
2.1 Material ............................................................................................................ 17
2.1.1 Chemicals ................................................................................................. 17
2.1.2 Nucleic acid modifying enzymes............................................................... 17
2.1.3 Plasmids.................................................................................................... 17
2.1.4 Bacteria..................................................................................................... 17
2.1.5 Antibodies 17
2.1.6 Cell lines ................................................................................................... 18
2.1.7 Mouse Strains............................................................................................ 18
2.1.8 Standard Buffers and Media ...................................................................... 18
2.2 Methods 20
2.2.1 Working with DNA................................................................................... 20
2.2.1.1 Analytical purification of plasmid DNA (Alkaline Lysis Miniprep).. 20
2.2.1.2 Preparative purification of plasmid DNA (Alkaline Lysis Midiprep
and Maxiprep) ............................................................................................... 21
2.2.1.3 Purification of eukaryotic genomic DNA ........................................... 21
2.2.1.4 Measuring DNA concentration in solutions........................................ 22
2.2.1.5 Restriction digest of DNA.................................................................. 22
2.2.1.6 Gel electrophoresis of DNA using agarose gels.................................. 22
2.2.1.7 Dephosphorylation of DNA with calf intestine phosphatase (CIP)...... 23
2.2.1.8 Repairing 3' or 5' overhanging DNA ends to generate blunt ends........ 23
2.2.1.9 Ligation of DNA fragments ............................................................... 24
2.2.1.10 Polymerase chain reaction (PCR)..................................................... 25
2.2.1.11 Amplification of cloning inserts by polymerase chain reaction
(PCR) ........................................................................................................... 25
2.2.1.12 Phosphorylation of PCR fragments by T4 polynucleotide kinase...... 26
2.2.1.13 Isolation and purification of DNA fragments from agarose gels with
DEAE membranes......................................................................................... 27
2.2.1.14 Isolation and purification of large DNA fragments from agarose
gels................................................................................................................ 27
2.2.1.15 Making of chemical competent Escherichia coli (E. coli) ................. 28
2.2.1.16 Transformation of chemical competent E.coli .................................. 292.2.1.17 Rapid amplification of cDNA ends (RACE)..................................... 29
2.2.1.18 Southern Blot................................................................................... 31
2.2.1.18.1 Southern blotting onto a nylon membrane with an alkaline
buffer........................................................................................................ 31
2.2.1.18.2 Radioactive labelling of DNA probes....................................... 32
2.2.1.18.3 Hybridisation of DNA probes to a Southern Blot ...................... 33
2.2.1.19 Calculating molecular weight and copy numbers of plasmid DNA ... 34
2.2.1.20 Quantitative real-time PCR .............................................................. 34
2.2.2 Working with RNA ................................................................................... 39
2.2.2.1 Purification of total RNA from cell culture and tissue ........................ 39
2.2.2.2 Measuring RNA concentration in solutions ........................................ 40
2.2.2.3 Removal of contaminating DNA by DNase I digests.......................... 40
2.2.2.4 First-Strand synthesis of cDNA.......................................................... 41
2.2.2.5 Northern Blot..................................................................................... 42
2.2.2.5.1 Electrophoresis of RNA denatured by glyoxal/DMSO treatment 42
2.2.2.5.2 Transfer of RNA from gel to membrane ..................................... 43
2.2.2.5.3 Non-formamide hybridisation of DNA probes to a
Northern Blot............................................................................................ 43
2.2.2.5.4 Removal of probes from Northern Blots 44
2.2.2.6 Improvement of the human mood by the synthesis and ingestion of
Apple Crumble .............................................................................................. 45
2.2.3 Cellular work 46
2.2.3.1 Stable transfection of eukaryotic cells through electroporation ........... 46
2.2.3.2 Isolating cells from mice.................................................................... 47
2.2.3.3 Lysis of erythrocytes.......................................................................... 47
2.2.3.4 Flowcytometric analysis of cells from cell culture or spleen............... 48
2.2.3.5 Dissociation of bound sIgE from FcεRII (CD23)................................ 49
2.2.3.6 Isolation of human peripheral blood mononuclear cells (PBMCs) ...... 49
2.2.3.7 Depletion of CD3+ cells by magnetic cell sorting (MACS) ................ 50
2.2.3.8 Cell culture ........................................................................................ 50
3 Results..................................................................................................................... 51
3.1 Expression of the murine ε HC locus ................................................................ 51
3.1.1 Expression analysis of ex vivo stimulated spleen cells by quantitative real-
time PCR ........................................................................................................... 51
3.1.2 Sequence and RACE analysis of the murine ε HC locus ............................ 55
3.1.2.1 Sequence analysis of the heavy chain isotypes ................................... 55
3.1.2.2 Determination of ex vivo poly(a) site usage by 3' RACE..................... 56
3.1.3 Regulation of alternative ε HC RNA processing ........................................ 59
3.1.3.1 Influence of the S-M intron regions on the mRNA expression pattern
of the ε HC gene............................................................................................ 59
3.1.3.1.1 The impact of regions in the S-M intron on the expression of m α
and m ε ...................................................................................................... 59
3.1.3.1.2 Construction of ε HC expression vectors .................................... 60
3.1.3.1.3 Quantitative real-time PCR analysis of ε HC intron deletion
expression vectors transfected cells........................................................... 62
3.1.3.2 Influence of the poly(a) signals on the mRNA expression pattern of
the ε HC gene............................................................................................... 63
3.1.3.2.1 Construction of ε HC expression vectors .................................... 633.1.3.2.2 Northern blot analysis of ε transfected cell lines ......................... 64
3.1.3.2.3 Study of ε HC mRNA profiles in ε transfected cell lines, by
quantitative real-time PCR ........................................................................ 68
3.1.3.2.4 mIgE surface expression in ε transfected K46 and J558L mb-1
cells .......................................................................................................... 70
3.2 Expression of the human ε HC locus................................................................. 74
3.2.1 Structure and comparison of the human µ HC gene and the ε HC gene...... 74
3.2.1 mRNA expression of the human ε HC and γ4 HC genes............................ 75
3.2.3 RT PCR analysis of ε HC mRNA in stimulated and non-stimulated human
B cells................................................................................................................ 75
3.2.4 Quantitative real-time PCR of ε HC and γ4 HC mRNAs 77
4 Discussion ............................................................................................................... 79
4.1 Introduction ...................................................................................................... 79
4.2 Ex vivo expression of IgE in mice and man ....................................................... 79
4.3 Alternative polyadenylation in ε mRNA expression, a common expression
regulator ................................................................................................................. 82
4.4 The impact of other genetic control elements on the alternative polyadenylation
of the ε HC locus.................................................................................................... 85
4.5 The mIgE expression in B cell lines transfected with different ε HC expression
constructs ............................................................................................................... 90
4.6 A new model for murine and human IgE regulation ......................................... 92
4.7 Therapeutic aspects of the regulation of IgE production .................................... 94
4.8 Evolutionary aspects of the IgE regulation ........................................................ 96
5 Summary .............................................................................................................. 102
6 Literature references............................................................................................ 104
Acknowledgements.................................................................................................. 119
Curriculum Vitae .................................................................................................... 120Abbreviations
APC antigen presenting cell
α alpha heavy chain
Ab antibody
Amp ampicillin
AREs AU-rich elements
BCR B cell receptor
bp base pair
BSA bovine serum albumin
CBC cap binding complex
CD cluster of differentiation
cDNA complementary DNA
CF cleavage factor
CH exon encoding constant domain from immunoglobulin heavy chain
Ci Curie
CIP calf intestine phosphatase
CPSF cleavage and polyadenylation specificity factor
CstF cleavage stimulation factor
CTD carboxy terminal domain
D diversity segment
δ delta heavy chain
dATP deoxy adenosine triphosphate
DC dendritic cell
dCTP deoxy cytidine triphosphate
DEAE diethylaminoethyl
DEPC diethylpyrocarbonate
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DNase deoxyribonuclease
dNTP deoxyribonucleoside triphosphate
dpm disintegration per minute
dsRNA double stranded RNA
DTT dithiothreitol
ε epsilon heavy chain
E-CF eosinophil-chemotactical-factor
EDTA ethylenediaminetetraacetic acid
eIF-2a eukaryotic initiation factor 2a
ER endoplasmatic reticulum
ES embryonic stem cells
EST expressed sequence tag
FACS fluorescence activated cell sorter
Fc invariant constant region of immunoglobulins
FceRI high affinity receptor of IgE
FceRII low affinity receptor of IgE
FCS fetal calf serum
FITC fluoro-iso-thiocyanate
g gram
γ gamma heavy chain
GAPDH glyceraldehyde-3-phosphate dehydrogenaseGPT glutamic-pyruvic transaminase
HC heavy chain
hnRNP heterogeneous nuclear ribonucleoprotein
IFN- γ interferon gamma
I intron
Ig immunoglobulin
IgA immunoglobulin A
IgD immunoglobulin D
IgE immunoglobulin E
IgG immunoglobulin G
IgM immunoglobulin M
IL interleukin
J joining segment
kb kilo base pair
kDa kilo Dalton
KO knock out
L ligand
l litre
LPS lipopolysacharides
M exon encoding membrane / transmembrane domain from
immunoglobulin heavy chain
M molar
µ mu heavy chain
MW molecular weight
mAb monoclonal antibody
MACS magnetic cell sorting
mg milligram
µg microgram
MHC I major histocompatibility complex class I
MHC II major histocompatibility complex class II
mIg membrane-bound immunoglobulin
mIgA membrane-bound IgA
mIgD membrane-bound IgD
mIgE membrane-bound IgE
mIgG membrane-bound IgG
mIgM membrane-bound IgM
µl microlitre
ml millilitre
mLIFR murine leukemia inhibitory factor receptor
mM millimolar
M-MLV RT moloney murine leukemia virus reverse transcriptase
mRNA messenger RNA
NF- κB nuclear factor κB
ng nanogram
NK natural killer
NO nitric oxide
OD optical density
PAB poly(A) binding protein
PAF platelet activating factor
PAP poly(A) polymerase
PBC peripheral blood cellsPBMC peripheral blood mononuclear cell
PBS phosphate buffered saline
PCR polymerase chain reaction
PDGF platelet-derived growth factor
PE phycoerythrine
PGE prostaglandin 22
PIPES 1,4-piperazinediethanesulfonic acid
poly(A) poly adenylic acid residues
poly(a) polyadenylation
RACE Rapid amplification of cDNA ends
RNA ribonucleic acid
RNase ribonuclease
rpm revolution per minute
RT reverse transcriptase
RT room temperature
S switch region
S α-M α intron intron which is located between the exon coding for the third
constant domain and the exon coding for the transmembrane and
cytoplasmic domain of α heavy chain
SDS sodium dodecyl sulfate
sec second
S ε-M ε intron intron which is located between the exon coding for the fourthtransmembrane
domain of ε heavy chain
sIg secreted immunoglobulin
sIgA secreted IgA
sIgE secreted IgE
sIgG secreted IgG
sIgM secreted IgM
snRNP small ribonucleoprotein particle
SOB SOB medium [Hanahan's broth]
SOC SOC medium
sPLA -R secreted PLA receptor2 2
SSC sodium chloride sodium citric acid
STAT signal transducer and activator of transcription
TB terrific broth
TBE Tris borate/EDTA electrophoresis buffer
TCR T cell receptor
TE Tris ethylenediaminetetraacetic acid
TF II D transcription factor II D
Th naive T helper cell type 00
Th type 1 T helper cell1
Th type 2 T helper cell2
Tlr Toll-like receptor
TNF tumour necrosis factor
TNP trinitrophenol
Tris Tris(hydroxymethyl)aminomethane
U unit of enzyme or hormone activity
υ upsilon heavy chain
UTR untranslated region
UV ultravioletV variable segment
V Volt
VDJ variable region
VEGF vascular endothelial growth factor
vol volume

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