2. AP Biology 2007-2008 Control of Eukaryotic Genes – Chapter 19

3. The BIG Questions… How are genes turned on & off in eukaryotes? How do cells with the same genes differentiate to perform completely different, specialized functions?

4. Evolution of gene regulation Prokaryotes single-celled evolved to grow & divide rapidly must respond quickly to changes in external environment exploit transient resources Gene regulation turn genes on & off rapidly flexibility & reversibility adjust levels of enzymes for synthesis & digestion

5. Evolution of gene regulation Eukaryotes multicellular evolved to maintain constant internal conditions while facing changing external conditions homeostasis regulate body as a whole growth & development long term processes specialization turn on & off large number of genes must coordinate the body as a whole rather than serve the needs of individual cells

6. Points of control The control of gene expression can occur at any step in the pathway from gene to functional protein 1.packing/unpacking DNA 2.transcription 3.mRNA processing 4.mRNA transport 5.translation 6.protein processing 7.protein degradation

7. How do you fit all that DNA into nucleus? DNA coiling & folding double helix nucleosomes chromatin fiber looped domains chromosome from DNA double helix to condensed chromosome 1. DNA packing

8. Nucleosomes “Beads on a string” 1 st level of DNA packing histone proteins 8 protein molecules positively charged amino acids bind tightly to negatively charged DNA DNA packing movie 8 histone molecules

9. DNA packing as gene control Degree of packing of DNA regulates transcription tightly wrapped around histones no transcription genes turned off  heterochromatin darker DNA (H) = tightly packed  euchromatin lighter DNA (E) = loosely packed H E

10. DNA methylation Methylation of DNA blocks transcription factors no transcription  genes turned off attachment of methyl groups (–CH 3 ) to cytosine C = cytosine nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body

11. Histone acetylation Acetylation of histones unwinds DNA  loosely wrapped around histones enables transcription genes turned on  attachment of acetyl groups (–COCH 3 ) to histones conformational change in histone proteins transcription factors have easier access to genes

12. 2. Transcription initiation Control regions on DNA promoter nearby control sequence on DNA binding of RNA polymerase & transcription factors “base” rate of transcription enhancer distant control sequences on DNA binding of activator proteins “enhanced” rate (high level) of transcription

13. Model for Enhancer action Enhancer DNA sequences distant control sequences Activator proteins bind to enhancer sequence & stimulates transcription Silencer proteins bind to enhancer sequence & block gene transcription Turning on Gene movie

14. Transcription complex Enhancer Activator Coactivator RNA polymerase II A B F E H TFIID Core promoter and initiation complex Activator Proteins regulatory proteins bind to DNA at distant enhancer sites increase the rate of transcription Coding region T A Enhancer Sites regulatory sites on DNA distant from gene Initiation Complex at Promoter Site binding site of RNA polymerase

15. 3. Post-transcriptional control Alternative RNA splicing variable processing of exons creates a family of proteins

16. 4. Regulation of mRNA degradation Life span of mRNA determines amount of protein synthesis mRNA can last from hours to weeks RNA processing movie

17. RNA interference Small interfering RNAs (siRNA) short segments of RNA (21-28 bases) bind to mRNA create sections of double-stranded mRNA “death” tag for mRNA triggers degradation of mRNA cause gene “silencing” post-transcriptional control turns off gene = no protein produced NEW! siRNA

18. Action of siRNA siRNA double-stranded miRNA + siRNA mRNA degraded functionally turns gene off Hot…Hot new topic in biology mRNA for translation breakdown enzyme (RISC) dicer enzyme

19. RNA interference 1990s | 2006 Andrew Fire Stanford Craig Mello U Mass “for their discovery of RNA interference — gene silencing by double-stranded RNA”

20. 5. Control of translation Block initiation of translation stage regulatory proteins attach to 5' end of mRNA prevent attachment of ribosomal subunits & initiator tRNA block translation of mRNA to protein Control of translation movie

21. 6-7. Protein processing & degradation Protein processing folding, cleaving, adding sugar groups, targeting for transport Protein degradation ubiquitin tagging proteasome degradation Protein processing movie

22. Ubiquitin “Death tag” mark unwanted proteins with a label 76 amino acid polypeptide, ubiquitin labeled proteins are broken down rapidly in "waste disposers" proteasomes 1980s | 2004 Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside

23. Proteasome Protein-degrading “machine” cell’s waste disposer breaks down any proteins into 7-9 amino acid fragments cellular recycling play Nobel animation

24. initiation of transcription 1 mRNA splicing 2 mRNA protection 3 initiation of translation 6 mRNA processing 5 1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription - mRNA processing - splicing - 5’ cap & poly-A tail - breakdown by siRNA 5. translation - block start of translation 6 & 7. post-translation - protein processing - protein degradation 7 protein processing & degradation 4 4 Gene Regulation

25. DNA Technology Chapter 20

26. Quick Definitions: Genetic engineering – the direct manipulation of genes for practical purposes. Biotechnology – manipulation of organisms or their components to perform practical tasks or provide useful products. We will now look at many different techniques that assist us in these two fields:

27. GOAL: To make recombinant DNA and put it to use.

28. Producing Recombinant DNA Gene cloning – getting well-defined, gene- sized pieces of DNA and making multiple copies that we can put to use. This is a multi-step process. Restriction enzymes – Discovered in bacteria in 1960’s They cut foreign DNA at a restriction site (specific recognition sequence). Produces restriction fragments.

29. Restriction Enzymes Restriction Site Sticky Ends

30. How do we insert our gene? Cloning vectors – DNA molecules that carry foreign DNA into a cell and replicate. i.e. viruses, plasmids How do you tell if it is successful? Grow plates on antibiotic impregnated plates Nucleic acid hybridization – Figure 20.4 Figure 20.3

31. Nucleic Acid Hybridization 1.Transfer cells to filter. 2.Treat cells to denature DNA on filter (chemical or heat) 3.Add radioactive / fluorescent probe to filter. 4.Take a picture (autoradiography). 5.Return to original culture and select those colonies that showed radioactivity / fluorescence.

32. Genomic Libraries When a gene is cloned, not just the desired gene is inserted into the bacteria cells. There may be many other genes of interest, so you can save these bacteria with the recombinant genes for later experiments in a genomic library. cDNA library – this is made from the mRNA present = the genes being transcribed. You produce the complimentary DNA from the mRNA so you only get the coding sequences.

33. PCR Polymerase Chain Reaction

34. Analysis of cloned DNA NOW WHAT? We have all this DNA, how do we make sense out of it? Gel electrophoresis

35. Southern Blot – More specific E. M. Southern – 1975 Steps: 1. Take DNA from source, splice. 2. Load fragments into gel and separate by electrophoresis. 3. Blotting – takes DNA sample from gel and transfers to paper. 4. Hybridize – use radioactive probe and develop film. 5. Study bands and the differences that may exist.

36. More techniques… RFLP’s – restriction fragment length polymorphisms – differences that exist in the non-coding sequences of genes. In-Situ Hybridization – allows you to locate specific gene on a chromosome in a genome. Much like labeling your DNA with a probe Southern blotting.

37. Mapping Entire Genomes Baker’s yeast 6,000 Fruit fly 13,000 Roundworm 19,000 Mustard 25,000 Human 35,000

38. Human Genome Project Officially began in 1990 – international effort to map the entire human genome, determining every nucleotide sequence. Types of Mapping: Genetic (linkage) Mapping – mapping the genome with genetic markers as reference points Physical Mapping – determine the distance between markers – cut DNA into manageable fragments DNA Sequencing – determine the sequence of these fragments.

39. What did we learn? Only about 3% of our DNA is protein-coding. The number of human genes is far smaller than previously thought The absolute number of human proteins remains unknown but far exceeds the number of genes. Very few genes (~1%) are “uniquely human” – almost all are seen in other organisms. On average, the DNA sequence of one person differs from another once every 700 nucleotides.

40. Applications of DNA Technology Medicine: Diagnosis of disease Human gene therapy Pharmaceutical products

41. Applications of DNA Technology Forensics DNA Fingerprinting Agricultural Transgenic organisms Transgenic plants Environmental Bioremediation Nitrogen fixation “power houses”