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Identification of New Genes in Human Polarized Intestinal Epithelial Cells Involved in Transport of Membrane Proteins

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Identification of New Genes in Human Polarized Intestinal Epithelial Cells Involved in Transport of Membrane Proteins 1 Department of Physiological Chemistry School of Veterinary Medicine Hannover Identification of new genes in human polarized intestinal epithelial cells involved in transport of membrane proteins THESIS submitted in partial fulfilment of the requirements for the degree PHILOSOPHICAL DOCTOR - Ph.D. - in the field of molecular and cell biology at the School of Veterinary Medicine Hannover by Dr. med. vet. Hiam Kamil Hameed Al-Bayati Khanakin, Iraq Hannover, Germany, 2002 2 Supervisor: Univ.-Prof. Dr. H. Y. Naim Advisory committee: Univ.-Prof. Dr. E. Töpfer-Petersen Univ.-Prof. Dr. E. E. Sterchi Oral examination: 3 June, 2002 3 To my husband M. AtaAlla and my daughter Juan 4 5 Contents 1. Introduction........................................................................................................................1 2. Review of the literature....................................................................................................3 2.1. Some aspects of polarized intestinal epithelial cells and sorting pathways ..............................................................................................3 2.2. Sucrase-isomaltase (SI) and colon adenocarcinoma cell line (Caco-2) as models for studies on differential expression of genes......................6 2.3. DAD-1, the defender against cell death ..................................................8 2.4. The cadherin family and its diversity ...................................................10 2.5. Homo sapiens transmembrane protein BRI..........................................13 2.6. Accompanying tools for isolation of differentially expressed genes .....15 3. Materials ............................................................................................................................20 3.1. Bacteria, plasmids and mammalian cell lines .......................................20 3.1.1. Host bacteria .......................................................................................................20 3.1.2. Plasmids...............................................................................................................20 3.1.3. Monoclonal antibodies (Abs) ............................................................................21 3.1.4. Mammalian cell lines..........................................................................................21 3.2. Enzymes.............................................................................................21 3.3. Chemicals and others .........................................................................21 3.4. Instruments ........................................................................................24 4. Methods .............................................................................................................................25 4.1. Construction of the subtracted cDNA library from polarized Caco-2 cells ...................................................................................................25 4.2. Cell lines, cultures ..............................................................................26 6 4.2.1. Colon adenocarcinoma cells (Caco-2)............................................................26 4.2.2. COS-1 cells..........................................................................................................26 4.2.3. Madin-Darby canine kidney cells (MDCK)......................................................26 4.3. Cultivation and storage of the cell lines ...............................................27 4.4. Subtraction hybridization ....................................................................28 4.4.1. Isolation of mRNA from Caco-2 cells...............................................................28 4.4.2. Subtraction of the differentiated cDNA............................................................30 4.4.2.1. Photobiotinlyation of the undifferentiated mRNA pool...........................30 4.4.2.2. Synthesis of first-strand cDNA from differentiated mRNA....................31 4.4.2.3. Subtraction hybridization............................................................................32 4.4.2.4. Synthesis of the double-strand cDNA......................................................33 4.4.2.5. Ligation..........................................................................................................33 4.4.2.6. Transformation of E. coli (DH10B)............................................................34 4.5. Arraying and storage of the library......................................................35 4.6. Colony hybridization based screening.................................................35 4.6.1. Arraying of the colonies onto nylon membranes ...........................................35 4.6.2. Preparation of the cDNA probes from cell cultures.......................................36 4.6.2.1. Isolation of the mRNA from Caco-2 cells ................................................36 4.6.2.2. Synthesis of the first-strand cDNA............................................................36 4.6.2.3. DNA probe labeling.....................................................................................37 4.6.2.4. Colony hybridization....................................................................................37 4.6.2.5. Post hybridization and stringency washing buffers................................37 4.7. Isolation plasmid DNA from E. coli.......................................................38 4.8. Digestions of cDNA clones..................................................................39 4.9. Sequencing of the recombinant plasmids ............................................40 4.10. Isolation of large-scale plasmid DNA from E. coli ...............................40 4.10.1. Plasmid DNA purification using the phenol-chloroform extraction method ...............................................................................................................40 4.10.2. Plasmid DNA purification using Qiagen method .........................................41 7 4 .11. Isolation of the insert DNA from plasmid DNA...................................41 4.11.1. Restriction digestion.........................................................................................41 4.11.2. Purification of DNA fragments from agarose gel.........................................42 4.12. Cloning of DNA fragments in mammalian expression vectors.............42 4.12.1. Cloning of pEGFP-N1 vector..........................................................................42 4.12.1.1. Preparation of pEGFP-N1 vector and ligation......................................43 4.12.1.2. Cloning of pcDNA3 vector .......................................................................44 4.13. Preparation of competent bacteria .....................................................44 4.13.1. Transformation of E. coli using heat shock method................................45 4.14. Polymerase chain reaction (PCR).......................................................46 4.15. Northern blot.....................................................................................46 4.15.1. Isolation of total RNA from cell cultures........................................................46 4.15.2. Northern blotting ...............................................................................................47 4.15.3. Assemble of a capillary blotting stack...........................................................48 4.15.4. Northern hybridization......................................................................................48 4.16. Screening of the human multiple tissues Northern blots* ...................49 4.17. Screening of human cancer profiling expression array.......................50 4.18. Transfection of COS-1 cells with DEAE-dextran method.....................51 4.19. Transfection, Biosynthetic labeling, and immunoprecipitation of transfected Caco-2 cell .....................................................................52 4.19.1. SDS-PAGE of proteins ....................................................................................55 5. Results...............................................................................................................................58 5.1. Subtraction hybridization ....................................................................58 5.2. Screening of the library by colony hybridization ..................................58 5.3. Insert size ...........................................................................................59 5.4. Database analysis ...............................................................................59 5.5. Protein location of the clones 78, 203, 432, and 530 in the cell..............61 8 5.6. Expression of the clones 432, 530, 78, and 203 in Caco-2 cells .............61 5.7. Expression of the clone 432 in human multiple tissue Northern blot (MTN)...................................................................................................63 5.8. Transcriptional analysis of the clone 432 in MDCK cells .......................64 5.9. Structural features of the protocadherin pcLKC...................................65 5.10. Expression of the novel cDNA clone 432 in human cancer profiling array ................................................................................................. 67 5.11. Effect of anti-sense recombinant clone 432 on the cell polarity...........69 5.12. Structural features of the novel gene encoded by cDNA clone 530......71 5.13. Expression of the clone
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