Gene Technology

1. Fundamental Features of Eukaryotic Genes Fundamental Features of Eukaryotic Genes Gene Technology Processing

2. Cotranscritional processing of eukaryotic mRNA: -> Modification of 5’ end (Cap) -> Modification of 3’ end (poly A) -> Splicing (removal of introns) Gene Technology

3. Gene Technology Eukaryotic transcription products (from RNA polymerase II) are processed triphosphate 7-methylguanylate end Polyadenylation of 3’ end Capping 5’ end

4. Poly(A) tail -> only added to mRNA transcribed by RNA polymerase II Gene Technology

5. The Intron/Exon concept Gene Technology

6. The Intron/Exon concept Gene Technology Organization of the β-globin (human β subunit of hemoglobin) gene

7. Splicing -> cutting out of introns Gene Technology 20-50 bp Splicing enhancers – silencers -> regulate alternative splicing Human: ~25.000 genes coding for ~100.000 proteins !!!

8. Splicing -> cutting out of introns Gene Technology

9. Splicing is done by a RNA and protein complex -> spliceosome Small nuclear RNAs in spliceosomes catalyse the splicing -> small nuclear ribonucleoprotein particles (snRNPs) Gene Technology

10. Spliceosome assembly The catalytic center of the spliceosome

11. Gene Technology Self-splicing A rRNA precursor of Tetrahymena (protozoan) splices itself in the presence of guanosine (G) as co-factor The L19 RNA is a intron that is catalytical active This TappingMode scan of the protozoan, Tetrahymena, shows its cilia-covered body and mouth structures. The sample was dried onto a glass slide and scanned; no other preparation was required. 50 micron scan courtesy C. Mosher and E. Henderson, BioForce Laboratory and Iowa State University.

12. Gene Technology Self-splicing mechanism

13. Gene Technology

14. Gene Technology

15. mRNA Processing Gene Technology Splicing factors -> recognize splice sites Polyadenylation factors -> recognize poly(A) sites

16. Gene Technology Alternative Splicing -> different proteins

17. Alternative Splicing -> different proteins Gene Technology

18. Alternative Splicing + Alternative Polyadenylation-> different proteins Gene Technology

19. Alternative Splicing + Alternative Polyadenylation-> different proteins Gene Technology Cell type specific adenylation !!!

20. Alternative Splicing +Alternative Polyadenylation-> different proteins Gene Technology Cell type specific modification of the mRNA of the α-tropomyosin gene

21. Alternative Splicing +Alternative Polyadenylation-> different proteins Process of sex determination in Drosophila: -> splicing event directs development of embryo Gene Technology

22. Errors in splicing -> cause disease Anemia: defect synthesis of hemoglobin Mutations affecting splice sites cause around 15% of all genetic diseases Gene Technology Creates a new splice site Disruption of splice site in β-globin gene -> β-Thalassemia

23. Introns have been aquired and lost during evolution Gene Technology • Archaea -> have introns (self-splicing) • Bacteria -> have no introns (except: Cyanobacteria -> involved in development of plants) • Eukaryotes -> have introns • -> during evolution introns are aquired or lost -> gene families !!! • -> insulin or tubulin

24. Introns have been aquired and lost during evolution Gene Technology

25. Exons often define protein domains -> exon shuffling -> protein evolution Introns -> facilitate genetic variation By recombination of exons (domains) -> exon shuffling -> New protein have been designed by nature by putting protein domain blocks together Gene Technology Low density lipoprotein receptor Result from known proteins -> all proteins are made up of only 1000-7000 exons !!!

26. Introns can have function Gene Technology Tumor repressor genes : different promoter, but share 2 exons -> Genes can also be found within introns of other genes

27. Protein families arise from gene duplication Duplication allows one copy to undergo mutations without selection pressure -> accumulations without selection -> genetic drift Genetic drift leads to new function of protein (enzyme acting on different substrate) -> genetic diversity Gene Technology β-globins: -> Evolved from same gene -> different subunits have different physiological properties -> expressed at different times during development

28. Protein families arise from gene duplication Unequal cross-over between similar sequences (nonhomologous) -> chromosome with duplication Mutations can be corrected by gene conversion -> break of DNA of normal allele -> base pairing of mutant allele with normal allele -> way of eliminating deleterious mutations Gene Technology

29. Protein families arise from gene duplication Gene duplication can also lead to pseudogenes -> nonfunctional genes Gene Technology

30. RNA Editing -> posttranscriptional Addition or substraction of nucleotides -> correct reading frame Gene Technology

31. RNA Editing Guide RNA (gRNA) acts as template to add nucleotides to pre-mRNA -> not coded by DNA Gene Technology

32. RNA Editing Gene Technology Editing by diamination C -> U Apolipoprotein B: Plasma lipoprotein RNA editing -> introduction of STOP codon -> cell type specific protein

33. Different levels of gene expression control Gene Technology