1. Regulation of gene expression

2. Lecture plan about 40 min to be given during digestion of plasmids Concepts of DNA genes etc Structure of a gene Details of transcription Transcription factors Environment and transcription factors Examples in the ear

3. Organ of Corti

4. Cell diversity Outer hair cells Inner hair cells Supporting cells Different shapes Different function Different protein content Nuclei DNA: same in all cells of an organism (with a few exceptions) How can cells that have the same DNA express different proteins?

5. Differences in protein content The human DNA consist of approximately 3x10 9 bases and contain about 100,000 genes. Any one cells may contain 5-10,000 different proteins. Within a cell a protein may be present in about 10 copies while another protein may be present in 100,000 copies (even if there is only one DNA sequence for each of these proteins). Within the same cell some proteins are present throughout the life of the cell, while others are present for only very limited periods. How can all this be possible?

6. Cooking a cell Italian Cuisine Italian Cuisine DNA Proteins

7. Regulation of Gene Expression The cell can select: –which genes to express –when to express them –how much to express them DNA Promoter Exons Introns Gene

8. Transcription hRNA (heteronuclear) GUACUGCAUUAGCGAUGCAUACGAUGCAUGCAAAGGCAUAC RNA polymerase ACGTCTAGTACTGCATTAGCGATGCATACGATGCATGCAAAGGCATAC TGCAGATCATGACGTAATCGCTACGTATGCTACGTACGTTTCCGTATG GUAC nuclear factors RNA Exon 1Intron 1 DNA gene ACGTCTAGTACTGCATTAGCGATGCATACGATGCATGCAAAGGCATAC TGCAGATCATGACGTAATCGCTACGTATGCTACGTACGTTTCCGTATG Exon 2Exon 3Intron 2 mRNA (messenger) splicing GUACUGCAUUCAUACGGGCAUACAAAAAAAAAAAA polyA tail

9. Alternative splicing hRNA GUAGAUGAUUAGCGAUGCAUACGAUGCAUGCAAAGGCUAAC GUAGAUGAUUCAUACGGGCUAAC GUAGAUGAUUGGCUAAC Met Ile His Thr Gly StopMet Ile Gly Stop

10. The Promoter RNA pol. nuclear factorselongation factors complex RNA polymerase binding site DNA exon1 TAAATA Responsive elements

11. The Promoter TAAATA nuclear factors RNA pol. DNA RNA

12. The Promoter RNA DNA

13. Nuclear Factors proteins that bind to specific DNA sequences and that influence gene expression also known as: transcription factors regulatory factors the DNA sequences that they bind to are known as:transcription element regulatory elements regulatory consensus sequences the regulation of expression and activity of nuclear factors determines which proteins will be expressed in the cell

14. selective gene expression GeneAGeneB Exon1 GeneAGeneB Exon1

15. selective gene expression GeneAGeneB Exon1 GeneAGeneB Exon1 RNA Pol. RNA Pol.

16. Signal transduction to the nucleus adenylate cyclase cAMP PKA CREB P P CREB = cAMP responsive element binding protein nucleus G RNA

17. GeneAGeneB Exon1 GeneAGeneB Exon1 Gene expression RNA Pol. P CREB Signals can induce gene expression in specific cells

18. Time scale of gene expression Birth Death Time Gene A Gene B Gene C

19. Gene gene interaction Precursor Cell type A Precursor Cell Type B Time Differentiated Cell type A Differentiated Cell type B

20. Gene expression assays Protein levels Western Blot Immunocytochemstry Northern Blots PCR InSitu Hybridization RNA levels ACGTA UGCAU ACGTA

21. Probe hybridisation AT C TT CG A T C G Probe A-T C-G

22. Probe hybridisation AT C TT CG A T C G A T C G A T C G

23. A T C TT C G A T C TT C G A T C TT C G T C G A C TT C G T AA TT CC G T G A T C G

24. hybridisations A T C G A T C TT C G A T C G A T C TT C G Northern Blot RNA extraction gel electrophoresis blot to nitrocellulose hybridise with probe wash unbound probe visualize probe In situ hybridisation fix tissue Cut thin sections (10-50 um) hybridise with probe wash unbound probe visualize probe

25. RT-PCR reverse transcriptase-polymerase chain reaction TGGGTACAATGGGTACAATGGGTAC UGGGUACAAUGGGUACAAUGGGUAC ACCCATGTTACCCATGTTACCCATG Reverse trancriptase

26. RT-PCR TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 94°C Denaturation 100- 75- 50- 25- 0- -25-

27. RT-PCR TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 55-60°C TACCC TACAA Annealing 100- 75- 50- 25- 0- -25-

28. RT-PCR TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 72°C TACAATGGGTACAATGGGTAC Elongation ( Taq DNA polymerase) ACCCATGTTACCCATGTTACCC 100- 75- 50- 25- 0- -25-

29. 100- 75- 50- 25- 0- -25- RT-PCR-2nd cycle TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 94°C TACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCC

30. RT-PCR (2nd cycle) TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 55-60°C TACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCC TACCC TACAA TACCC TACAA 100- 75- 50- 25- 0- -25-

31. RT-PCR (2nd cycle) TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 72°C TACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCC ATGTTACCCATGTTACCC TACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCC TACAATGGGTACAATGGG 100- 75- 50- 25- 0- -25-

32. RT-PCR 3rd cycle TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG 94°C TACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCC ATGTTACCCATGTTACCC ACCCATGTTACCCATGTTACCC TACAATGGGTACAATGGG TACAATGGGTACAATGGGTAC 100- 75- 50- 25- 0- -25-

33. RT-PCR 3rd cycle TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG TACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCC ATGTTACCCATGTTACCC ACCCATGTTACCCATGTTACCC TACAATGGGTACAATGGG TACCC TACAA TACCC TACAA TACAATGGGTACAATGGGTAC TACCC 55-60°C 100- 75- 50- 25- 0- -25-

35. 4th cycle

36. Summary of gene expression detection techniques TechniqueSensitivitySpecificityAnatomical information Northern BlotMediumHighLimited Adjustable RT-PCRVery HighVery HighLimited InSitu HybridisationHighHighHigh

37. Study of gene expression the regulation of expression and activity of nuclear factors determines which proteins will be expressed in the cell nuclear factors can be modulated by external and/or internal signals the expression of one gene often affects the expression of other genes, both in the same cell and in other cells. the characterisation of the ”signal to gene” and “gene to gene talk” is one of the key focus of molecular biology.