Genetics

1. Genetics The Conversion of Information from Genetics to Protein

2. The Discovery of DNA and Its Role

3. The Turn of the Century • Optical Investigation • Chemical Analysis of the Nucleus • The Problem of Information • Griffith • Avery, MacCarty and McCloud • Chargaff

4. Watson and Crick

5. 5’  3’ Notation 5’ – ACTG – 3’ 3’ – TGAC – 5’

6. The Flow of Information

7. The Flow of Information Amino Acids Degradation tRNA Replication DNA mRNA Peptide Proteins Post Translational Processing Ribosome Transcription rRNA Transcription rProteins Translation

8. Replication

9. Chromosomal Level ORI Termination Point

10. Molecular Level

11. Practical Level • If given a replication problem you simply have to rely on Chargaff’s Rule to fill in the missing data. 5’ – ATGGTAGCTATGTAGTAGGAATT – 3’ 3’ – TACCATCGATACATCATCCTTAA – 5’ Remember that the second strand runs antiparallel to the original strand.

12. Transcription

13. Operon Structure Promoter Gene Sequences Terminator • Most Gene Sequences in Bacteria are arranged in this type of Promoter/Terminator system • Operons (grouped gene sequences under a single control) serve as a regulatory device. • Most Gene Regulation occurs at the level of Transcription.

14. Practical Level • Transcription occurs in a similar fashion to Replication, with the RNA Polymerase doing most of the duties. • The Hardest Part is telling the difference between coding and template strands. • Coding = the information • Template = the compliment of coding that is used to make the RNA strand

15. Practical Level 2 Note: The RNA is produced from the Template, but replicates the information on the Coding. Also note the use of “U” in place of “T.” Coding 5’ –CGCATGCCCGTGTGAGCGATAGT – 3’ Template 3’ –GCGTACGGGCACACTCGCTATCA – 5’ RNA 5’ –CGCAUGCCCGUGUGAGCGAUAGU – 3’

16. Translation

17. Translation Mechanism

18. Translation Mechanism 2

19. The Genetic Code

20. Practical • Always look for the first “Start” Codon from the 5’ end. • Separate the Codons • Translate the Codons till you reach a “Stop” Codon 5’ –CGCAUGCCCGUGUGAGCGAUAGU – 3’ 5’ –CGC AUG CCC GUG UGA GCG AUA GU – 3’ MET|PRO|VAL|STOP

21. Mutation

22. Mutations • Mutations are changes to the DNA of a cell. • Mutations occur either during Replication or between Replication cycles. • Mutations can be mistakes or changes caused by external sources. • A mutation that makes it through one replication cycle becomes permanent.

23. DNA Repair and Proofing • DNA Polymerase has a built in proofreading function. • The worst proofreading occurs in the viruses while the best is in the Eukarya. • Mistakes that make it through proofreading are often caught by the DNA repair systems

24. Types of Mutations • Structural • Indel– Bases are inserted or deleted from the sequence • Substitution – Bases are changed within the sequence • Functional • Missense– Changes the amino acid coded • Nonsense – Changes to a STOP codon • Frame-Shift – Changes the Reading Frame • Silent – No change to amino acid coded

25. Practical Level RNA 5’ – GCAUGCCAUGCAGAUGA – 3’ RNA 5’ – GC AUG CCA UGC AGA UGA – 3’ Protein Met-Pro-Cys-Arg-STOP RNA 5’ – GCAUGCCCUGCAGAUGA – 3’ RNA 5’ – GC AUG CCC UGC AGA UGA – 3’ Protein Met-Pro-Cys-Arg-STOP SUBSTITUTION / SILENT RNA 5’ – GCAUGUCAUGCAGAUGA – 3’ RNA 5’ – GC AUG UCA UGC AGA UGA – 3’ Protein Met-Ser-Cy-s-Arg-STOP SUBSTITUTION / MISSENSE

26. Practical Level 2 RNA 5’ – GCAUGCCAUGCAGAUGA – 3’ RNA 5’ – GC AUG CCA UGC AGA UGA – 3’ Protein Met-Pro-Cys-Arg-STOP RNA 5’ – GCAUGCCAUGAAGAUGA – 3’ RNA 5’ – GC AUG CCA UGA AGA UGA – 3’ Protein Met-Pro-STOP SUBSTITUTION / NONSENSE RNA 5’ – GC_UGCCAUGCAGAUGA – 3’ RNA 5’ – GCUGCC AUG CAG AUG A – 3’ Protein Met – Gln – Met INDEL / FRAME-SHIFT

27. Recombination

28. Horizontal Gene Transfer • Bacteria do not have sex, so they have evolved a different set of mechanisms to reshuffle genetic potential. • The ultimate source of genetic variation is mutation. • Horizontal Genetic Transferis transfer of genetic material within a generation.

29. Mechanisms • Conjugation – Exchange along a pilus • Transduction – Exchange through defective bacteriophage • Transformation – The uptake of naked DNA • Transposons – Interruption through “jumping” elements.

30. Biotechnology

31. Recombinant DNA (rDNA) • Recombinant DNA (rDNA) Technology is the insertion of a novel genetic sequence into a new host.

32. Basic rDNA Procedure • Isolation of gene of interest and suitable vector • Gene of interest is inserted in vitrointo vector • Modified vector is introduced into a host • Modified vector multiplies in host, making a line of clones. • These clones can be used for many purposes.

33. Tools of Biotechnology • Selection – a procedure where strains with a selective advantage of interest are favored in the environment and therefore become more numerous (contrast natural and artificial selection). • Mutation – Changes are made to selected DNA (either directed or random) to change the properties of the sequence. • Often these two techniques are used in concert.

34. Selection Example NA Plate NA Plate Select one colony and then replica plate on two different media NA Plate Plus Antibiotic

35. Restriction Enzymes • Useful tools in Molecular Biology to manipulate specific sequences of DNA • Evolutionarily are a type of viral defense mechanism • Serve as the Cut function in Molecular Biology • DNA Ligase (Replication) serves the Paste function

36. Vectors • Properties of Vectors (typically Plasmids) • Self-replication • Size capable of in vitro manipulation • Protection mechanism • Marker gene • Cross organism manipulation can be accomplished with shuttle vectors • Viral Nucleic Acids can also be used

37. Polymerase Chain Reaction

38. Inserting DNA • Various methods are used to insert foreign DNA into host cells, typically this is the step with the lowest efficiency. • Transformation • Electroporation • Protoplast Fusion • Ballistic Insertion • Microinjections

39. Choosing the Right Host • Which Host you use takes into consideration many factors, here are typical hosts. • Escherishia coli • Bacillus spp. (such as B. subtilisor B. megaterium) • Saccharomyces cerevisiae • Mammalian Cells • Plant Cells

40. Bioethics

41. Evolutionary Theory

42. Evolutionary Theory 101 • Core Tenets of Theory • All Populations possess Genetic Variation • Genetic Variation is passed Generationally • The Environment Selects the most adapted genetic variants. • Implication – Common descent means that there must be a common ancestor.

43. LUCA • LUCA – Last Universal Common Ancestor • Evidence for LUCA • Genetic Code • Conserved Information Mechanisms • Conserved Biochemical Pathways • Conserved Structural Details • What existed before LUCA? • Can we test the hypotheses experimentally?

44. Pathways Away from LUCA G+ Bacteria Proteobacteria ARCHAEA Viridiplantae Animals Spirochetes Protists Fungi Aquifex Bacteria LUCA Eukarya

45. Horizontal Gene Transfer and Endosymbiosis G+ Bacteria Proteobacteria ARCHAEA Viridiplantae Animals Spirochetes Protists Fungi Aquifex Chloroplasts Mitochondria Bacteria LUCA Eukarya

46. Taxonomy and Phylogeny

47. Taxonomy vs. Phylogeny • Taxonomy – A practical division of a complex group of items based on shared characteristics. • Phylogeny – A division of biological groups based on evolutionary understanding.

48. Classical Taxonomy • Organisms (not viruses) are classified by the classical Linnean System • Domain • Kingdom • Phylum • Class NOT ALL MAY BE DEFINED FOR • Order A PARTICULAR ORGANISM • Family • Genus • Species

49. What is a Species? • Since Prokaryotes do not have sex, it is impossible to apply the Biological Species Concept. • Microbiologists argue about how to define a species. • Functional Definition – A group of organisms that share a common evolutionary lineage, have conserved genetic sequences, similar biochemical markers and a shared environmental niche.

50. Identification of Organisms