Comparative analyses of tumour related genes in dogs as model systems for human cancer [Elektronische Ressource] / vorgelegt von Hugo Istvan Murua Escobar

Hugo Istvan Murua Escobar

Sujets

• Gesundheit

Informations

• Publié par: universitat_bremen
• Publié le: 01 janvier 2004
• Nombre de lectures: 24
• Langue: Deutsch
• Poids de l'ouvrage: 26 Mo

Extrait

Centre for Human Genetics Bremen
University of Bremen





“COMPARATIVE ANALYSES OF TUMOUR RELATED GENES IN
DOGS AS MODEL SYSTEM FOR HUMAN CANCER”

Dissertation

zur Erlangung des Grades eines Doktors der Naturwissenschaften
- Dr. rer. nat. -

Dem Promotionsausschuß Dr. rer. nat.
Fachbereich Biologie/Chemie Universität Bremen

vorgelegt von
Hugo Istvan Murua Escobar






1. Gutachter: Prof. Dr. Jörn Bullerdiek
2. Gutachter: Prof. Dr. Ingo Nolte

Bremen, Mittwoch, 29. September 2004 Hiermit erkläre ich, Hugo Istvan Murua Escobar, geboren am 28.05.1973, daß für das
Verfassen der vorliegenden Dissertation “Characterisation of the canine counterparts
of the human tumour relevant HMGA and HMGB Protein family genes and further
potential tumour relevant canine genes and evaluation as molecular targets for
therapeutic approaches using the dog as model system” folgende drei Aussagen
zutreffen:

1. Ich habe die Arbeit ohne unerlaubte fremde Hilfe angefertigt.
2. Ich habe keine anderen als die von mir angegebenen Quellen und Hilfsmittel
benutzt.
3. Ich habe die den benutzten Werken wörtlich oder inhaltlich entnommenen Stellen
als solche kenntlich gemacht


Bremen, Montag, 27. September 2004


Hugo I. Murua Escobar




"You're guaranteed to miss 100 percent of the shots you never take."
Wayne Gretzky



Contents
1 Introduction ........................................................................................................................ 8

2 Material and Methods....................................................................................................... 13
2.1 Tissues ...................................................................................................................... 13
2.2 Cell Culture and Cell Lines...................................................................................... 13
2.3 DNA Isolation........................................................................................................... 13
2.3.1 Plasmid DNA Isolation..................................................................................... 13
2.3.2 BAC DNA Isolation .......................................................................................... 14
2.3.3 Genomic DNA Isolation ................................................................................... 14
2.4 PCR .......................................................................................................................... 14
2.5 Gelelectrophoresis ................................................................................................... 14
2.6 Cloning and Sequencing of DNA Fragments ........................................................... 15
2.7 DNA Restriction Endonuclease Digestion ............................................................... 15
2.8 In Silico Analysis...................................................................................................... 15
2.9 RNA and mRNA Purification.................................................................................... 15
2.9.1 Total RNA Purification Using TRIzol LS ......................................................... 15
2.9.2 Total RNA Purification Using RNeasy............................................................. 16
2.9.3 mRNA Purification Using Oligotex.................................................................. 16
2.10 cDNA Synthesis ........................................................................................................ 16
2.10.1 Synthesis Using M-MLV RT ............................................................................. 16
TM2.10.2 Synthesis Using Superscript ......................................................................... 17
2.11 DNase Treatment 17
2.12 RNA Gelelectrophoresis and Northern Blotting ...................................................... 17
2.13 DNA / cDNA Probes................................................................................................. 17
2.14 Radioactive Probe Labelling and Purification ........................................................ 17
2.15 Hybridisations .......................................................................................................... 18
2.15.1 BAC Screening 18
2.15.2 Northern Blots.................................................................................................. 18

3 Results .............................................................................................................................. 19
3.1 Canine Gene Characterisations (Chronological Order) ......................................... 19
3.1.1 The Canine HMGB1......................................................................................... 19
3.1.2 The Canine LHCGR 20
3.1.3 The Canine HMGA1 21
3.1.4 The Canine ZNF331 22
3.1.5 The Canine CCND1 23
3.2 Canine Point Mutation Screening............................................................................ 23
3.2.1 Canine ras Gene Hotspot Mutation Screening ................................................ 23
3.3 Gene Mapping Studies (Chronological Order)........................................................ 24
3.3.1 ERBB-2............................................................................................................. 24
3.3.2 HMGA1 ............................................................................................................ 24
3.3.3 HMGB1 24
3.3.4 LHCGR 24
3.3.5 ZNF331 24
3.3.6 KRAS2 .............................................................................................................. 24
3.3.7 NRAS ................................................................................................................ 24
3.3.8 AKT3................................................................................................................. 24
3.3.9 FASTK 24
3.3.10 CCND1 ............................................................................................................. 24 3.4 Further Publications ................................................................................................ 25
3.4.1 The HOPE-Technique ...................................................................................... 25
3.4.2 Human ZNF331 Expression in Follicular Thyroid Adenomas. ....................... 26
3.4.3 Identification of a Gene Rearranged by 2p21 Aberrations in Thyroid
Adenomas. ........................................................................................................ 27

4 Discussion ........................................................................................................................ 28

5 Summary .......................................................................................................................... 38

6 References 40

7 Publications in Reverse Chronological Order.................................................................. 57

8 Danksagung...................................................................................................................... 80 Abbreviations

aa Amino acid
AGE Advanced glycation end products
AKT3 Protein kinase b, gamma gene
bp Base pair
CCND1 Cyclin D1 gene
cDNA Complementary DNA
CDS Coding sequence
CFA Canis familiaris
C-terminal Carboxy terminal
Da Dalton
dATP 2’-Deoxyadenosine 5’-triphosphate
dCTP 2'-Deoxycytidine 5'-triphosphate
DNA Deoxyribonucleic acid
DNase Deoxyribonuclease
dNTP Deoxyribonucleotide triphosphates
EDTA Ethylenediaminetetraacetic acid
EST Expressed sequence tags
FISH Fluorescence in situ hybridisation
GAPDH Glyceraldehyd-3-phosphat-dehydrogenase
h Hour
ERBB-2 Human epidermal growth factor receptor 2
FASTK FAS-activated serin/threonin kinase
HMG High mobility group
HMGA High mobility group protein A
HMGA1a High mobility group protein A1 Isoform a
HMGA1b High mobility group protein A1 Isoform b
HMGB1 High mobility group protein B1
HOPE Hepes-glutamic acid buffer mediated Organic solvent Protection Effect
KRAS2 v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog
kb Kilo base pair
kDa Kilo Dalton
LB Luria Bertani
LHCGR luteinizing hormone/choriogonadotropin receptor
M Molar
MAP Mitogen-activated protein
MAPK Mitogen-activated protein kinase
min Minute
ml Millilitre
mM Millimolar
M-MLV Moloney murine leukemia virus
µl micro litre
NaAc Sodium acetate
NRAS Neuroblastoma RAS viral (v-ras) oncogene homolog
ORF Open reading frame
PCR Polymerase chain reaction
RAGE Receptor for advanced glycation end products
RT Reverse Transcriptase
RT-PCR Reverse-Transcription-PCR s Second
SDS Sodium dodecyl sulfat
SSC Standard saline citrate
THADA Thyroid adenoma associated
U Unit
UTR Untranslated region
V Volt
ZNF331 Zinc finger protein 331


Manufacturers

Ambion Ambion, Cambridge, United Kingdom
Amersham Biosciences Amersham Biosciences, Buckinghamshir