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  Systematic cloning and functional analysis of the proteins encoded on human chromosome 21.

Warnatz, H.-J. (in preparation). Systematic cloning and functional analysis of the proteins encoded on human chromosome 21.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-7E0D-F Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0010-7E0E-D
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Warnatz, Hans-Jörg1, Author              
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1Human Chromosome 21 (Marie-Laure Yaspo), Dept. of Vertebrate Genomics (Head: Hans Lehrach), Max Planck Institute for Molecular Genetics, Max Planck Society, ou_1479652              

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Free keywords: Chromosom 21 Trisomie 21; Down-Syndrom; Subzelluläre Lokalisation; Protein-Protein-Interaktionen; Signaltransduktion
 Abstract: Understanding the complex interplay between proteins in physiological contexts poses a great challenge, since a striking number of human proteins still remain without solid functional annotation. To gain insight into the function of proteins encoded by human chromosome 21 (Hsa21) and to identify regulatory networks potentially relevant to Down syndrome (DS), 206 annotated open reading frames (ORFs) on Hsa21 were used as basis for amplification and cloning of ORFs and for collection of available full-length cDNA clones. This work resulted in the largest library of Hsa21 ORF clones available to date, covering 81% of the 206 annotated ORFs. A high success rate was reached in production of recombinant proteins (80%), ensuring appropriate downstream functional genomics analyses. For high-throughput determination of subcellular protein localizations, 89 constructs encoding N-terminally tagged proteins were introduced into HEK293T cells on transfected cell arrays. Localization information could be obtained for 52 out of the 89 Hsa21 proteins (58%). Of these, 28 subcellular localizations were described for the first time (published in Hu et al. 2006). All Hsa21 localization information has been entered into the public Gene Ontology (GO) database. For the identification of protein-protein interactions (PPIs), 53 constructs encoding non-autoactivating baits were screened against a prey set of 5,640 human proteins using a high-throughput mating array-based yeast two-hybrid (Y2H) system. Altogether, this mating array identified 56 new interactions for 24 different Hsa21 proteins. Twenty-three of the new interactions could be verified in an independent cotransformation Y2H set-up (56% of 41 tested). Cellular colocalization experiments in COS-1 cells and pull-down experiments confirmed five of six tested protein pairs (83%). All previously known protein interaction data for Hsa21 proteins were retrieved from available public sources. This resulted in a set of 684 new and known PPIs for 108 Hsa21 proteins, representing the largest Hsa21-centered interaction network to date. A proteome-wide interaction network was then generated by computational searches for indirect protein interactors in available data sets. Analysis of transcription factors showed a compelling inter-connection of thirteen Hsa21-encoded transcription factors. Detailed functional analysis of sub-networks involving NRIP1, UBE2G2, PCP4, MCM3AP and C21orf127 revealed novel functional properties of these Hsa21-encoded proteins. 35 Hsa21 proteins were found connected to nine signaling pathways, some of which have been previously implicated in the pathogenesis of DS, while others appear in this context for the first time. Further expansion of the Hsa21 interaction network will support the identification of new protein functions and regulatory networks. The data presented here shows that a Hsa21 interaction network is a useful resource for elucidating the biological contexts of new PPIs, providing an essential tool for the systems biology of Down syndrome.

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Language(s): eng - English
 Dates: 2009-02-18
 Publication Status: Not specified
 Pages: VIII, 196
 Publishing info: Berlin : Freie Universität Berlin
 Table of Contents: 1.1 Genetics, genomics and functional genomics . . . . . . 1
1.1.1 The origin of genetics . . . . . . . . . . . . . . . 1
1.1.2 The advent of genomics . . . . . . . . . . . . . . . 2
1.1.3 The field of functional genomics . . . . . . . . . . 3
1.1.4 Limitations and applications of functional genomics .5
1.2 Human chromosome 21 (Hsa21). . . . . . . . . . . . . . 6
1.2.1 Characteristics of Hsa21 . . . . . . . . . . . . . . 6
1.2.2 Gene content and gene functions . . . . . . . . . . .6
1.2.3 Diseases associated to Hsa21 . . . . . . . . . . . . 7
1.3 Trisomy 21 and Down syndrome (DS) . . . . . . . . . . 11
1.3.1 Incidence and impact . . . . . . . . . . . . . . . 11
1.3.2 History and genetics . . . . . . . . . . . . . . . 11
1.3.3 Clinical characteristics . . . . . . . . . . . . . .13
1.3.4 Prenatal diagnosis and screening . . . . . . . . . 21
1.4 Molecular analysis of Down syndrome . . . . . . . . . 23
1.4.1 Defining a Down syndrome critical region (DSCR) . . 23
1.4.2 Mouse models for trisomy 21 . . . . . . . . . . . . 24
1.4.3 Dysregulation of transcript levels . . . . . . . . .26
1.4.4 Protein dosage imbalances . . . . . . . . . . . . . 28
1.4.5 Disturbed signal transduction in Down syndrome . . .29
1.5 Functional Genomics Analyses of Hsa21 Proteins. . . . 34
1.5.1 Cloning of open reading frames . . . . . . . . . . .34
1.5.2 Subcellular protein localization . . . . . . . . . 36
1.5.3 Protein-protein interactions . . . . . . . . . . . .40
1.5.4 Protein network analysis . . . . . . . . . . . . . .43
1.5.5 Connecting interaction data with signaling pathways 45
1.6 Objectives of this study . . . . . . . . . . . . . . .47
2 MATERIAL AND METHODS
2.1 Methods in molecular biology. . . . . . . . . . . . . 48
2.1.1 Culture of E. coli cells . . . . . . . . . . . . . 48
2.1.2 Transformation of E. coli . . . . . . . . . . . . . 49
2.1.3 Isolation of plasmid DNA from E. coli . . . . . . . 49
2.1.4 Agarose gel electrophoresis . . . . . . . . . . . . 50
2.1.5 Reverse transcription . . . . . . . . . . . . . . . 50
2.1.6 Polymerase chain reaction (PCR) . . . . . . . . . . 51
2.1.7 Cloning of PCR products . . . . . . . . . . . . . . 54
2.1.8 DNA sequencing . . . . . . . . . . . . . . . . . . .54
2.1.9 Subcloning into expression vectors . . . . . . . . 55
2.1.10 Quantification of nucleic acids . . . . . . . . . .56
2.2 Methods in protein biochemistry . . . . . . . . . . . 57
2.2.1 Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) . . . .........................................57
2.2.2 Coomassie staining of polyacrylamide gels . . . . . 57
2.2.3 Western blotting . . . . . . . . . . . . . . . . . .58
2.2.4 Immunodetection of proteins . . . . . . . . . . . . 58
2.2.5 Expression of recombinant proteins in E. coli . . . 59
2.2.6 Determination of protein solubility . . . . . . . . 60
2.2.7 Quantification of proteins . . . . . . . . . . . . 60
2.3 Methods in cell biology . . . . . . . . . . . . . . . 62
2.3.1 Transformation of yeast cells . . . . . . . . . . . 62
2.3.2 Autoactivation test . . . . . . . . . . . . . . . . 62
2.3.3 Yeast two-hybrid (Y2H) assays . . . . . . . . . . . 62
2.3.4 Culture of COS-1 cells . . . . . . . . . . . . . . .64
2.3.5 Transient transfection of COS-1 . . . . . . . . . . 65
2.3.6 Immunolocalization of proteins . . . . . . . . . . .66
2.4 Computational methods . . . . . . . . . . . . . . . . 68
2.4.1 ORF primer design . . . . . . . . . . . . . . . . . 68
2.4.2 Conversion of accession numbers to Entrez GeneIDs. .68
2.4.3 Retrieval of orthologous interaction data (interolog data). . . . . . .........................................68
2.4.4 Retrieval of known protein-protein interactions from
literature-curated databases . . . . . . . . . . . . . . .69
2.4.5 Retrieval of known protein-protein interactions from
previous large-scale Y2H screens . . . . . . . . . . . . .70
2.4.6 Retrieval of known protein-protein interactions from PubMed . . . .............................................70
2.4.7 Visualization of protein interaction networks . . . 71
2.4.8 Analysis of protein network topology . . . . . . . .72
2.4.9 Retrieval of Gene Ontology (GO) annotations . . . . 72
2.4.10 Retrieval of pathway connections through PPI data .72
3 RESULTS
3.1 Annotation of the proteins encoded on Hsa21 . . . . . 74
3.1.1 Annotation of open reading frames (ORFs) . . . . . .74
3.1.2 Retrieval of transcript sequences and
ORF coordinates ..........................................75
3.1.3 Primary structure of Hsa21 protein sequences . . . .76
3.1.4 Topology predictions for Hsa21 proteins . . . . . . 76
3.2 ORF cloning and protein expression . . . . . . . . . 79
3.2.1 Sources of ORF primers. . . . . . . . . . . . . . . 79
3.2.2 Amplification and cloning of Hsa21 ORFs . . . . . . 79
3.2.3 Subcloning of ORFs . . . . . . . . . . . . . . . . .84
3.2.4 Protein expression in bacterial cells . . . . . . . 85
3.2.5 Protein expression in yeast cells . . . . . . . . . 87
3.2.6 Protein expression in mammalian cells . . . . . . . 87
3.2.7 Comparison of protein expression in different cellular systems . . ..............................................88
3.3 Subcellular localization of Hsa21 proteins . . . . . 89
3.3.1 Cloning of mammalian expression constructs . . . . 89
3.3.2 Antibody selection for hexahistidine and cell organelles . . . . . . . .................................91
3.3.3 Determination of subcellular localizations on cell arrays . . . . . . . .....................................93
3.3.4 Protein translocation . . . . . . . . . . . . . . . 98
3.3.5 Comparison of N- and C-terminal fusion tags . . . .100
3.3.6 Comparison with computational predictions . . . . .101
3.4 Identification of new Hsa21 protein-protein interactions . . . . .................................................104
3.4.1 Yeast two-hybrid constructs for interaction screening . . . . . . . . . .......................................104
3.4.2 Mating array yeast two-hybrid (Y2H) screen . . . . 106
3.4.3 Interaction confirmation by cotransformation Y2H . 108
3.4.4 Confirmation by cellular colocalization assays . . 109
3.4.5 Confirmation by pull-down assays . . . . . . . . . 111
3.5 Data collection for known protein interactions . . . 114
3.5.1 Retrieval of interaction data for Hsa21 orthologous proteins . . . . ........................................114
3.5.2 Retrieval of previously known Hsa21 interactions . 116
3.5.3 Consolidation and overlap of interaction data sets.118
3.6 Assembly and analysis of Hsa21 interaction networks .119
3.6.1 Computational generation of a Hsa21 interaction network . . . . . .......................................119
3.6.2 Functional analysis of interaction subnetworks . . 121
3.6.3 Prediction of new protein functions from interaction networks . . . ..........................................123
3.6.4 Retrieval of pathways with involvement of Hsa21 proteins . . . . . ......................................132
4 DISCUSSION
4.1 On the annotation of genes and ORFs on Hsa21 . . . . 135
4.1.1 Protein-coding versus non-protein-coding genes . . 135
4.1.2 On the quality of the gene annotations used in this study . . . . . . .......................................136
4.1.3 On the quality of the ORF annotations used in this study . . . . . . .......................................136
4.2 ORF cloning as resource for functional genomics . . .137
4.2.1 Approaches in functional genomics and proteomics . 137
4.2.2 Hsa21 ORF cloning for clone-based proteomics . . . 138
4.3 New subcellular localizations for Hsa21 proteins . . 139
4.3.1 Different small fusion tags resulted in similar localizations . . . . . .................................139
4.3.2 Success rate of subcellular
localization experiments ................................140
4.3.3 Observed localizations and potential effects of epitope tagging. . ......................................140
4.3.4 Known and new localizations in different compartments . . . . . . . ...........................................141
4.3.5 Protein translocation . . . . . . . . . . . . . . 147
4.3.6 Comparison with computational predictions . . . . .148
4.4 New protein interactions for Hsa21 proteins . . . . .149
4.4.1 On the identification of new PPIs by mating array-based Y2H screening . . . . . . . . . . . . . . . .149
4.4.2 On the verification of protein-protein interactions151
4.5 On current protein-protein interaction data sets . . 152
4.5.1 Retrieval of PPI data from databases and large Y2H screens . . . ...........................................152
4.5.2 Retrieval of PPI data from literature records . . .153
4.5.3 Characteristics of proteins without PPI information153
4.6 On the analysis of protein interaction networks . . 154
4.6.1 Higher-order protein networks . . . . . . . . . . 154
4.6.2 Mapping protein interactions to cellular processes 155
4.7 Interaction networks and signaling pathways . . . . .156
4.7.1 Previously known pathway connections of
Hsa21 proteins . . . . . ................................157
4.7.2 Newly identified pathway connections of Hsa21 proteins . . . . . . .............................................161
4.8 Conclusion and outlook . . . . . . . . . . . . . . . 163
5 BIBLIOGRAPHY . . . . . . . . . . . . . . . . . . . . . 165
6 APPENDIX
6.1 Sources of cloned ORFs . . . . . . . . . . . . . . . 180
6.1.1 ORFs cloned from public cDNA clones . . . . . . . .180
6.1.2 ORFs cloned from cDNA . . . . . . . . . . . . . . 182
6.1.3 ORFs cloned from genomic DNA . . . . . . . . . . . 183
6.1.4 ORFs obtained from public Gateway clones . . . . . 184
6.2 List of primer sequences. . . . . . . . . . . . . . 185
6.2.1 Primer for ORF cloning . . . . . . . . . . . . . . 185
6.2.2 Primer for C-terminal epitope tagging . . . . . . 188
6.2.3 Primer for insert verification and sequencing . . .189
6.3 Bacterial and fungal strains and mammalian cell lines189
6.3.1 Escherichia coli strains . . . . . . . . . . . . . 189
6.3.2 Saccharomyces cerevisiae strains . . . . . . . . . 190
6.3.3 Cercopithecus aethiops cell line . . . . . . . . . 190
6.3.4 Homo sapiens cell line . . . . . . . . . . . . . . 191
Publikationen . . . . . . . . . . . . . . . . . . . . . .192
Curriculum vitae . . . . . . . . . . . . . . . . . . . . 193
Erklärung . . . . . . . . . . . . . . . . . . . . . . . 195
Danksagungen . . . . . . . . . . . . . . . . . . . . . . 196
 Rev. Type: -
 Identifiers: eDoc: 456391
 Degree: PhD

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