FU Berlin Digitale Dissertation
Georg Wilhelm Otto : The zebrafish genome: Strategies and applications for physical and radiation hybrid mapping Das Zebrafischgenom: Strategien und Anwendungen für die physikalische und radiation hybrid Kartierung
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Abstract The zebrafish has been shown to be a powerful system for the genetic, molecular and embryological study of vertebrate development. In spite of a growing number of available genomic resources (genetic maps, radiation hybrid maps, genomic libraries, cDNA libraries) the cloning of genes disrupted in mutants is still difficult and time-consuming. The availability of physical maps of the genome anchored to the genetic and radiation hybrid maps can facilitate the identification and cloning of mutated genes and is crucial as a framework for sequencing the genome. Gene catalogues containing expression and mapping data of transcripts also help to identify candidate genes for mutations and hence have the potential to enable the cloning of the affected genes. The subject of this thesis is the establishment and application of physical and radiation hybrid mapping techniques in the zebrafish genome. The work can be divided in three parts: In the first part, interspersed repetitive sequence (IRS)-PCR was adapted and optimised for the zebrafish system, especially for physical and radiation hybrid mapping. Different repetitive elements, primer annealing sites and PCR conditions were tested. A new zebrafish repetitive element was identified and characterised. Libraries of IRS-PCR products derived from 5 different zebrafish strains were constructed and 27,000 clones were picked. The libraries were normalised and clustered by oligonucleotide fingerprinting, suggesting 11,000 different IRS-PCR products in the library. This method also identified IRS products with +/- polymorphism among different zebrafish strains. IRS-PCR products were further characterised by sequencing. The average frequency of single nucleotide polymorphisms (SNPs) between IRS-PCR products of different strains tested was found to be p = 0.013. Thus IRS-PCR products can serve as a reduced representation subset of the zebrafish genome for SNP mapping and detection. Radiation hybrid mapping of IRS-PCR products was established. Part two describes the construction of a physical framework map of zebrafish linkage group 20. A mapping procedure was set up by hybridising STS-derived oligonucleotides on a PAC library, and by hybridising IRS-products to YAC and PAC pools. So far, 344 STS derived oligonucleotide probes and 284 IRS probes have been used. A total of 3416 PACs were identified as positive, and are currently being restriction fingerprinted for integration in the Tuebingen restriction fingerprinting map and for selection of tiling paths for sequencing. A subset of the hybridisation results was used for contig construction. This consisted of 388 probes, hitting 2007 clones, representing a marker density of 1 per 205 kb. The contig assembly resulted in 249 contigs, of which 19% were anchored by more than one probe. These data agree with theoretical predictions. Thus, an STS-based framework map of zebrafish chromosome 20 has been constructed, which is anchored to genetic and radiation hybrid maps, and integrated in the restriction fingerprint map. This is the first map of its kind of a zebrafish chromosome. In the third part of this work zebrafish ESTs with homologies to human disease genes or with localised expression patterns were mapped on the radiation hybrid map. So far we mapped > 120 clones. The aim of this work is to identify candidate genes for zebrafish mutations and to refine the map of human:zebrafish syntenic relations; synteny data itself are used to determine if sequences homologous between zebrafish and human are true orthologues.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title 0 Title page, table of contents 1 Introduction 1 1.1 Zebrafish as a model organism for studying vertebrate development 2 1.2 Mapping and sequencing of complex genomes 8 1.2.1 Genetic linkage mapping 9 1.2.2 Radiation hybrid mapping 12 1.2.3 Physical mapping 14 1.2.4 Sequencing strategies for complex genomes 17 1.3 High throughput characterisation of the zebrafish transcriptome by gene catalogues and whole mount in situ screens 19 1.4 Cloning of genes identified in mutants 20 1.5 Genome mapping by interspersed repetitive sequence (IRS)-PCR. 21 1.6 Repetitive elements in the zebrafish genome 23 1.7 Physical mapping of zebrafish chromosome 20 26 2 Objective 30 3 Materials and Methods 31 3.1 Materials 31 3.1.1 Laboratory equipment 31 3.1.2 Chemicals and enzymes 32 3.1.3 Oligonucleotides 33 3.1.4 Kits 34 3.1.5 Other materials 34 3.1.6 Nucleic acids 35 3.1.7 E. coli strains, cell lines and zebrafish strains 35 3.1.8 Libraries 36 3.1.9 Buffers and Solutions 36 3.1.10 Culture Media 38 3.2 Methods 39 3.2.1 Isolation of genomic DNA from tissue or adult fish 39 3.2.2 Plasmid preparation 40 3.2.3 Lysis of yeast cells 40 3.2.4 DNA precipitation 41 3.2.5 Restriction digest 41 3.2.6 Dephosphorylation of DNA 5'-ends 41 3.2.7 Ligation of restriction fragments 41 3.2.8 Preparation of E. coli cells for electroporation 42 3.2.9 Transformation 42 3.2.10 Blue/white selection 42 3.2.11 Picking and handling of libraries 42 3.2.12 Spotting of bacterial colonies onto nylon membranes 43 3.2.13 Spotting of DNA onto nylon membranes 43 3.2.14 Capillary blotting of DNA onto nylon membranes ("Southern Blotting") 44 3.2.15 Labelling by random hexamer priming 44 3.2.16 Hybridisation of DNA probes labelled by random priming. 45 3.2.17 Labelling of oligonucleotides by polynucleotide kinase 45 3.2.18 Labelling of "overgo" probes 45 3.2.19 Hybridisation of oligonucleotides 46 3.2.20 Generation of amplified restriction fragments 46 3.2.21 Addition of T-overhangs in cloning vectors 47 3.2.22 Cloning of PCR products using the pAMP10 system 48 3.2.23 IRS-PCR 48 3.2.24 Amplification of plasmid inserts 49 3.2.25 DNA Sequencing 49 3.2.26 Radiation hybrid mapping of ESTs 49 3.2.27 Computational tools and bioinformatics 49 4 Results 51 4.1 IRS-PCR based methods for mapping the zebrafish genome 51 4.1.1 Analysis of the repetitive Mermaid/DANA element. 51 4.1.2 Identification of a new interspersed repetitive element in the zebrafish genome 59 4.1.3 Tests of IRS-PCR Primers 64 4.1.4 Construction of an IRS marker library 66 4.1.5 Characterisation and normalisation of the IRS marker library by oligonucleotide fingerprinting. 67 4.1.6 Characterisation of IRS-PCR products by sequence analysis 73 4.1.7 Radiation hybrid mapping of IRS-PCR products 76 4.1.8 High-density arrays of IRS products of pooled large-insert clones on nylon membranes. 78 4.1.9 Detection of Single Nucleotide polymorphisms in orthologous IRS-PCR products of different zebrafish strains. 83 4.2 Physical mapping of zebrafish linkage group 20 86 4.2.1 Mapping of PACs by hybridisation of oligonucleotides. 87 4.2.2 Mapping of PACs and YACs by hybridisation of IRS-PCR products 90 4.2.3 Assembly of the physical framework map of zebrafish linkage group 20 91 4.3 Radiation hybrid mapping of expressed sequence tags 94 4.4 Genetic mapping using amplified fragment length polymorphism (AFLP) 102 5 Discussion 105 5.1 IRS-based methods for mapping the zebrafish genome 105 5.1.1 Identification and testing of anchor sites for IRS-PCR 105 5.1.2 Size distribution of IRS-PCR products 106 5.1.3 Complexity of the IRS-PCR amplicon 107 5.1.4 Repeat and SNP content 107 5.1.5 IRS-PCR pools for physical mapping 107 5.1.6 Characterisation of a new interspersed repeat 108 5.2 Physical mapping of the zebrafish linkage group 20 109 5.2.1 Mapping strategy 110 5.2.2 Mapping results 110 5.2.3 Significance of the map 112 5.3 Radiation hybrid mapping 113 5.3.1 Mapping of ESTs with specific expression patterns during embryogenesis 113 5.3.2 ESTs homologous to human disease genes 114 5.3.3 Mapping and determination of conserved synteny 115 5.4 Conclusion and outlook 116 6 Summary 119 6.1 Abstract 119 6.2 Zusammenfassung 121 7 Appendix 123 7.1 STS markers used to generate oligonucleotide probes 123 7.2 Oligonucleotide probes for hybridisation on PAC filters 129 7.3 Primers for radiation hybrid mapping 134 7.4 Abbreviations 137 7.5 IUPAC-code of wobble nucleotides 139 7.7 Publications 8 Literature 140

More Information:
Online available:	http://www.diss.fu-berlin.de/2002/168/indexe.html
Language of PhDThesis:	english
Keywords:	zebrafish genome mapping EST STS SNP
DNB-Sachgruppe:	32 Biologie
Date of disputation:	29-May-2002
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Professor Dr. Hans Lehrach
Second Referee:	Professor Dr. Volker Erdmann
Contact (Author):	georg.otto@ tuebingen.mpg.de
Contact (Advisor):	lehrach@molgen.mpg.de
Date created:	26-Aug-2002
Date available:	16-Sep-2002

 

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