FU Berlin Digitale Dissertation
Ingo Marenholz : Molecular Genetic Characterization of the Epidermal Differentiation Complex (EDC) on Human Chromosome 1 Molekulargenetische Charakterisierung des Epidermalen Differenzierungskomplexes (EDC) auf Chromosom 1 des Menschen
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Abstract In recent years, three structurally related gene families which are important for the formation of a functional epidermis were localized to the long arm of human chromosome 1. They encode structural as well as regulatory proteins which cooperate during epidermal differentiation. Coordinated expression and the close linkage of the genes in chromosomal region 1q21 lead to the definition of a new gene complex, the epidermal differentiation complex (EDC). Two resources essential for the characterization of the EDC were generated within this study. To examine the genomic organization of the EDC, a contig of 24 yeast artificial chromosomes (YACs) covering 6 Mb of region 1q21 was assembled. A total of 43 genes, 20 newly generated hybridization markers, and seven polymorphic sequence-tagged site (STS)-markers were mapped within the contig. Except for two smaller DNA segments all of these loci were at least doubly linked. To achieve a higher resolution, a SalI restriction map of the YAC contig was generated and, to verify the mapping results, a genomic restriction map covering approximately 4,5 Mb of region 1q21. Using two YACs flanking the EDC as probes for dual colour fluorescence in situ hybridization (FISH) the chromosomal orientation of the gene complex was determined. The positioning of seven STS-markers yielded an integrated map of region 1q21 as a valuable tool for linkage analyses. For the transcriptional analysis of genes involved in epidermal differentiation a gridded cDNA library synthesized from differentiating keratinocytes was constructed. This library was unique in terms of its size (10 filters carrying 18432 doubly spotted clones each) and the included transcripts which even covered late stages of differentiation and enabled rapid and systematic investigations of the whole keratinocyte transcriptome. In addition, a database for the management of the cDNA library was developed. Furthermore, a gene identification method using YACs as probes to screen a cDNA library was improved. The subtractive hybridization of a gridded cDNA library with non overlapping YACs resolved nearly all problems with non specific hybridization previously described; approximately 90% of the subtractively selected cDNA clones could be mapped within the EDC. The cDNA sequences represented 40 different transcripts originating from 33 genes. Of these 21 genes were newly assigned to the EDC: ADAR1, ANXA9, HAX1, LAMRL6, PIAS3, PIP5K1A, PSMB4, PSMD4, PSMD8L, RBM8, and TPM3, as well as ten previously uncharacterized genes (NICE1 to NICE10), one of which was homologous to NOTCH2. Four additional genes, CHRNB2, RPTN, S100A11, and TDRKH, were localized within the EDC independently of the subtractive hybridization. The established gene families of the EDC were extended by two new members, RPTN and S100A11, and a polymorphic transcript of the SPRR3 gene was identified. Expression and sequence analyses of NICE1 mapping as well within the core region of the EDC suggested the existence of a new, differentiation-specifically expressed gene family. The recognition of the corresponding genes in the mouse genome verified an orthologous linkage group on mouse chromosome 3. Likewise, paralogous regions on human chromosomes 1, 6, 9, and 19 were confirmed. Moreover, four of the newly assigned EDC genes have homologues on the long arm of chromosome 15, indicating a fifth paralogous region; a further step in the elucidation of the evolution of chromosomes. As a result of this study the EDC has been extended from 2 to 6 Mb, now including 52 genes which are almost completely expressed in keratinocytes and therefore potentially contribute to the formation of the epidermis. Because the transcriptional analysis was not performed exhaustively, the number of genes should still increase. Moreover, the extension of the EDC on chromosome 1 is still unknown, making further investigations of this remarkable region of the human genome desirable.

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 Titel I Inhaltsverzeichnis III Danksagung VII Symbole und Abkürzungen IX I EINLEITUNG 1 1 Die Haut - Aufbau und Funktion 1 2 Die Differenzierung der Epidermis 2 3 Molekularbiologie der Keratinozyten 4 4 Die Bedeutung der Region 1q21 für die Epidermisdifferenzierung 9 5 Der Epidermale Differenzierungskomplex (EDC) 12 6 EDC-assoziierte Krankheiten 12 7 Genomanalyse 15 8 Genetische Kartierung 16 9 Physikalische Kartierung 17 10 Methoden zur Identifizierung neuer Gene 20 11 Das Humangenom-Projekt 22 12 Ziele der Arbeit 25 II MATERIAL & METHODEN 27 1 Geräte 27 2 Arbeitsmaterial & Hilfsmittel 27 3 Reagenzien 28 4 Enzyme 29 5 Reagenziensets 29 6 Nucleinsäuren 30 6.1 Vektoren 30 6.2 DNA-Sonden 30 6.3 Oligonucleotid-Primer 31 6.4 Molekulargewichtsstandards 31 7 DNA-Bibliotheken 32 8 Lösungen 32 8.1 Stammlösungen 32 8.2 Puffer 33 8.3 Nährmedien 35 9 Zellen und ihre Aufzucht 35 9.1 Zellkultur 35 9.2 Hefekultur 36 9.3 Bakterienkultur 36 10 DNA-Isolierung 36 10.1 Genomische DNA des Menschen (hoch- bzw. niedermolekular) 36 10.2 DNA aus Hefezellen (hoch- bzw. niedermolekular) 37 10.3 YAC-DNA (Elektroelution) 38 10.4 Plasmid-DNA 39 10.5 Plasmidinserts (Elution mit Glas-beads) 39 10.6 Konzentrationsbestimmung von Nucleinsäuren 40 11 Restriktionsanalyse 40 11.1 Restriktion von DNA in Lösung 40 11.2 Restriktion von DNA in Agarose 40 12 DNA-Klonierung 41 12.1 Ligation 41 12.2 Herstellung kompetenter Zellen 42 12.3 Transformation 42 13 Gelelektrophorese 43 13.1 Polyacrylamidgelelektrophorese (PAGE) 43 13.2 Agarosegelelektrophorese 43 13.3 Gelelektrophorese im rotierenden Feld (ROFE) 43 13.4 DNA-Nachweis mit Ethidiumbromid 44 14 Southern-Blotting 44 15 Radioaktive Hybridisierung 45 15.1 DNA-Sonden 45 15.2 Markierung der Sonde 45 15.3 Hybridisierung 46 15.4 Autoradiographie 47 16 Polymerasekettenreaktion (PCR) 48 17 DNA-Sequenzierung (Didesoxy-Verfahren) 49 17.1 Sequenzierungsreaktion 49 17.2 Denaturierende Polyacrylamidgelelektrophorese 50 17.3 Detektion und Fehlerkorrektur 50 18 Computergestützte Sequenzanalysen 51 19 Herstellung einer feingerasterten (gridded) cDNA-Bibliothek 51 19.1 Aufziehen der Klone 52 19.2 Automatisches Auflesen der Klone (picking) 54 19.3 Vervielfältigung der cDNA-Bibliothek (replicating) 54 19.4 Automatisches Aufbringen der Klone auf Membranen (spotting) 55 19.5 Verarbeiten der Filter (processing) 56 19.6 Verwalten der cDNA-Bibliothek 56 III ERGEBNISSE 58 1 Erstellung eines YAC-Contigs der Region 1q21 58 1.1 Auswahl der YAC-Klone 58 1.2 YAC-Größenbestimmung 61 1.3 Charakterisierung instabiler YACs 61 1.4 Zusammensetzen eines Contigs des EDC 62 1.5 Erweiterung des Contigs durch die Integration genetischer Marker 63 1.6 Herstellung und Kartierung neuer Sonden zur Erhöhung der Markerdichte 66 1.7 Identifizierung rearrangierter YACs 67 1.8 Chromosomale Orientierung des EDC 67 1.9 Diskussion 69 2 Identifizierung neuer EDC-Gene durch subtraktive Hybridisierung 70 2.1 Eine direkte Methode zur Identifizierung neuer Gene 71 2.2 Hybridisierung der feingerasterten cDNA-Bibliothek mit einem YAC 71 2.3 Auswertung der Hybridisierung 72 2.4 Hybridisierung der feingerasterten cDNA-Bibliothek mit einem zweiten YAC 75 2.5 Subtraktive Auswertung 75 2.6 Hybridisierungsergebnisse zweier weiterer YACs aus der centromeren Region 78 2.7 Mögliche Fehlerquellen und ihre Vermeidung 78 3 Kartierung der neuen EDC-Gene 79 3.1 YAC-Restriktionskarte 80 3.2 Genomische Restriktionskarte 85 3.3 Fusion der physikalischen Karten 88 3.4 Integration der genetischen Marker 88 4 Charakterisierung der cDNA-Sequenzen 90 4.1 Ermittlung der cDNA-Sequenzen 91 4.2 cDNA-Sequenzen bekannter EDC-Gene 91 4.3 cDNA-Sequenzen anderer bekannter Proteine 94 4.4 cDNA-Sequenzen unbekannter Funktion 94 IV DISKUSSION 102 1 Die Ressourcen 102 1.1 Das YAC-Contig des EDC 102 1.2 Die feingerasterte Keratinozyten-cDNA-Bibliothek 104 2 Die Hybridisierungsmethode 106 3 Die integrierte Karte der Region 1q21 108 4 Die neuen EDC-Gene 109 5 Der Genkomplex 116 5.1 EDC oder "CDC"? 116 5.2 Kontrolle der Genexpression 117 5.3 Orthologie im Mausgenom - der EDC der Maus 119 5.4 Paralogie im menschlichen Genom 121 6 Ausblicke 124 Va ZUSAMMENFASSUNG 126 Vb SUMMARY 128 Literaturverzeichnis 130 Publikationsliste 149

More Information:
Online available:	http://www.diss.fu-berlin.de/2002/195/indexe.html
Language of PhDThesis:	german
Keywords:	epidermal, differentiation, complex, EDC, 1q21, chromosome 1
DNB-Sachgruppe:	32 Biologie
Date of disputation:	02-Oct-2002
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Prof. Dr. Andreas Ziegler
Second Referee:	Prof. Dr. Volker A. Erdmann
Contact (Author):	ingo.marenholz@kispi.unizh.ch
Contact (Advisor):	andreas.ziegler@charite.de
Date created:	18-Sep-2002
Date available:	25-Sep-2002

 

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