1 / 37

Regulation of gene expression

Regulation of gene expression. 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. Endolymph. Stria Vascularis.

casta
Download Presentation

Regulation of gene expression

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Regulation of gene expression

  2. Lecture planabout 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. Endolymph Stria Vascularis Tectorial membrane Outer hair cells Inner hair cells Spiral Limbus Supporting cells Basilar membrane Organ of Corti

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

  5. Differences in protein content • The human DNA consist of approximately 3x109 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. Italian Cuisine Italian Cuisine Cooking a cell Proteins DNA Proteins

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

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

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

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

  11. RNA pol. The Promoter nuclear factors TAAATA 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. Exon1 Exon1 GeneA GeneB Exon1 Exon1 GeneA GeneB selective gene expression

  15. RNA Pol. RNA Pol. selective gene expression Exon1 Exon1 GeneA GeneB Exon1 Exon1 GeneA GeneB

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

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

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

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

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

  21. T T T T C C C A A G G Probe hybridisation A-T C-G Probe

  22. T T T T T T C C C C C A A A A G G G G Probe hybridisation

  23. T T T T T T T T T T T T T T T T T C C C C C C C C C C C C A A A A A A A G G G G G G G G Probe hybridisation

  24. T T T T T T T T C C C C C C A A A A G G G G hybridisations • 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-PCRreverse transcriptase-polymerase chain reaction UGGGUACAAUGGGUACAAUGGGUAC Reverse trancriptase TGGGTACAATGGGTACAATGGGTAC ACCCATGTTACCCATGTTACCCATG

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

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

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

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

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

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

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

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

  34. 4th cycle

  35. Summary of gene expression detection techniques Technique Sensitivity Specificity Anatomical information Northern Blot Medium High Limited Adjustable RT-PCR Very High Very High Limited InSitu Hybridisation High High High

  36. 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.

More Related