Exploring Microbial Genetics: Fundamentals and Applications

1. Microbial Genetics

2. What is genetics? • Study of inheritance and traits expressed in the genetic material of the organism. • GENOTYPE: genetic “make-up” of the organism • PHENOTYPE: expression of the genotype - the organism and its traits.

3. Genome • Entire genetic compliment of the organism. • DNA - all prokaryotes and eukaryotes • DNA or RNA - viruses

4. Structure of DNA • Until 1953 the exact structure of DNA was unknown. • James Watson and Francis Crick discovered the structure of DNA. • In 1962, Crick, Watson, and another researcher named Wilkins were awarded the Nobel prize of this work.

5. Watson and Crick with the model of DNA

6. The Watson-Crick model • The model proposes the following: • DNA molecule is a double helix • The “backbone” of the helix is composed of the sugar, deoxyribose, alternating with a phosphate group • Four nitrogenous bases form the interior “ladder” of the molecule and carry the genetic code for life

7. Nitrogen Bases in DNA • Adenine, thymine, guanine, cytosine • The bases ALWAYS pairs as follows: • Adenine-Thymine • Guanine-Cytosine • Base pairs are held together by hydrogen bonds

8. Base Pairing

10. Key Terminology • 1. REPLICATION – new copy of DNA being made • 2. TRANSCRIPTION – gene being copied from DNA sequence into messenger RNA • 3. TRANSLATION – mRNA read and protein produced

11. Central Dogma of Molecular Biology

12. DNA Replication(the secret is in the base pairing) • 3’ A-T-G-G-C-T-G-T-C-G-G-C-T 5’ • 5’ T-A-C-C-G-A-C-A-G-C-C-G-A 3’ • The strands run ANTI-PARALLEL • DNA Replication is said to be SEMI-CONSERVATIVE • http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120076/bio22.swf::Meselson and Stahl Experiment

13. Semi-conservative replication

14. DNA Replication

15. Structure of deoxyribose • The carbons are numbered 1’ to 5’ • The phosphate is linked to the sugar between the 3’ and 5’ carbons • Sugar – phosphate backbone

16. 5’ and 3’ ends of DNA • The 5’ end has the free phosphate (P) • The 3’ end is the free OH end • The strands are anti-parallel

17. Direction of replication • DNA replication proceeds 5’→ 3’ • A free 3’ end is needed to add another nucleotide

18. DNA Replication • 1. The molecule “unzips” to expose free ends. One is the 3’ end the other is the 5’ end • 2. DNA synthesis can now begin • 3. Synthesis on the free 3’ end is CONTINUOUS, this is called the LEADING STRAND • 4. Synthesis on the free 5’ end is DISCONTINUOUS, this is the LAGGING STRAND • 5. The enzyme that is responsible for DNA synthesis on BOTH strands is DNA polymerase BUT this enzyme requires a free 3’ end at which it can add new bases!

19. DNA Replication(continued) • 6. Synthesis of the lagging strand is in short segments of DNA, about 1000 bases • 7. These short segments are called OKAZAKI FRAGMENTS • 8. The enzyme DNA ligase seals the gaps between these fragments to produce a gaps free discontinuous strand

21. Directions of replication

22. RNA compared to DNA

23. Types of RNA • 1. Messenger RNA (mRNA) • 2. Ribosomal RNA (rRNA) • 3. Transfer RNA (tRNA)

24. Transcription • 1. This is the process of making a copy of a gene (sequence of DNA that codes for a protein or functional product) • 2. The enzyme responsible for this process is RNA POLYMERASE • 3. Copies the gene is a 5’→ 3’ direction • 4. Gene transcription begins at a site called the PROMOTER and ends at another site called the TERMINATOR

25. Transcription(making mRNA)

26. Example of transcription and translation • 3’ TACAGAGTACGAACT 5’ (antisense) this is copied • 5’ ATGTCTCATGCTTGA 3’ (sense)    • 5’ AUG|UCU|CAU|GCU|UGA 3’ mRNA (gene copy) • 3 bases = codon = amino acid • Met-ser-his-ala-stop Protein • 3’strand of DNA is the TEMPLATE or ANTISENSE strand THE STRAND THAT IS COPIED! • 5’ strand of DNA is the INFORMATIONAL or SENSE strand

27. Transcription (s

28. Another example of transcription • 3’ GGGGGGGGGGGGGGG 5’ anti-sense • 5’ CCCCCCCCCCCCCCC 3’ sense Transcription (anti-sense) 5’ CCCCCCCCCCCCCCC 3’ Translation Pro-pro-pro-pro-pro-pro- Remember-copy the 3’ strand and by the rules of base paring you get the sense strand sequence!

29. Genetic code

30. Protein Synthesis(Translation)

31. Translation

32. Translation

33. Translation

34. Translation

35. Transfer RNA

36. Exons and Introns • 1. These are terms unique to eukaryotic cells • 2. EXONS are information regions in DNA that must be expressed • 3. INTRONS are non-coding regions in DNA that are not expressed

37. RNA processing in eukaryotic cells

38. Control of Gene Expression • 1. Control is at the level of TRANSCRIPTION • 2. Genes that are NOT needed are NOT expressed, i.e., the gene is not transcribed • 3. Many genes are always expressed because the cell always needs the gene product • 4. Such genes are CONSTITUTIVE

39. Genetic Control • 1. REPRESSION – inhibition of gene expression • 2. INDUCTION – switching on gene expression

40. Model of Inducible Gene System • Lactose operon • OPERON is defined as a set of operator and promoter sites and the genes that they control • Described in E. coli by Francois Jacob and Jacques Monod • Genetic ON/OFF switch

41. Operon

42. Off and On of the lac operon

43. Tryptophan operon(repressible operon)

44. Mutations • Change in the base sequence of DNA • May or may not have an effect on the organism • The potential magnitude of the change depends on the gene affected

45. TACTTCAAACCGATT AUGAAGUUUGGCUAA Met-lys-phe-gly-stop TACTTCAAATCGATT AUGAAGUUUAGCUAA Met-lys-phe-ser-stop MISSENSE MUTATION BASE SUBSTITUTION

46. TACTTCAAACCGATT AUGAAGUUUGGCUAA Met-lys-phe-gly-stop TACATCAAACCGATT AUGUAGUUUGGCUAA Met-STOP NONSENSE MUTATION BASE SUBSTITUTION

47. TACTTCAAACCGATT AUGAAGUUUGGCUAA Met-lys-phe-gly-stop TACTTCAACCGATT AUGAAGUUGGCUAA…. Met-lys-leu-ala…. FRAMESHIFT MUTATION BASE DELETION

48. Bacterial SEX!!! • 1. Transformation • 2. Conjugation • 3. Transduction

49. Transformation(Griffith 1928)

50. Transformation