1. Microbial Genetics Genomic structure Replication of chromosomal DNA
Regulation of gene expression
Mutation, repair and recombination
Gene exchange in bacteria
Genetic engineering
微免所 何漣漪老師

2. Genomic structure
Eukaryotic microbes: fungi, yeasts Eukaryotic genome Chromosomal DNA Mitochondrial DNA Plasmids in yeast
Prokaryotic microbes: bacteria
Prokaryotic genome
Chromosomal DNA: double-stranded; circular; haploid.
Extrachromosomal genetic elements
Plasmids (autonomously self-
replicating)
Phages (bacterial viruses)
Transposons (DNA sequences that move within the same or between two DNA molecules)

3. Replication of chromosomal DNA
Replication of bacterial genome requires:
Replication origin
(oriC)
DNA polymerase
Primase
Helicase
Topoisomerase
Semiconservative
Bidirectional

4. Control of gene expression
A bacterial cell may regulate the expression of a certain set of its own genes in response to an environmental change by a variety of mechanisms.
Alternative s factors (e.g. sporulation)
Regulators (activators or repressors) activated or inactivated by an inducer or corepressor (e.g., cAMP in utilization of lactose; auto-inducers in quorum-sensing)
Attenuation

5. Regulation at transcriptional level (Example I)
Operon
Negative control
Repressor
Inducer
Operator
Lactose operon

6. Positive control
Activator
Inducer
Lactose operon

7. Regulation at transcriptional level (Example II)
Negative control
Repressor
Corepressor
Operator
Tryptophan operon

8. Attenuation
Transcription termination signal

9. Mutation Types of mutations may cause frameshift or null mutation
1. Base substitutions
Silent vs. neutral; missense vs. nonsense
2. Deletions
3. Insertions
4. Rearrangements: duplication, inversion, transposition
may cause frameshift or null mutation
Spontaneous mutations
Cuased by tautomeric shift of the nucleotides which lead to replication errors

10. Induced mutations Physical mutagens: e.g., UV irradiation
(heat, ionizing radiation)
Chemical mutagens
Base analog
Frameshift
intercalating agents
Base modification
Transposable elements

11. DNA Repair 1. Direct DNA repair (e.g., photoreactivation)
2. Excision repair
Base excision repair
Nucleotide excision repair
3. Mismatch repair
4. SOS response
5. Error-prone repair
Thymine-thymine dimer formed by UV radiation

12. Excision repair
Base excision repair
Nucleotide excision repair

13. Base excision repair

14. Nucleotide excision repair

15. SOS repair in bacteria
Inducible system used only when error-free mechanisms of repair cannot cope with damage
Insert random nucleotides in place of the damaged ones
Error-prone

16. Gene exchange in bacteria
Mediated by plasmids and phages
Plasmid
Extrachromosomal
Autonomously replicating
Circular or linear (rarely)
May encode drug resistance or toxins
Various copy numbers
Some are self-transmissible

17. Bacteriophage (bacterial viruse)
Structure and genetic materials of phages
Coat (Capsid)
Nucleic acid
Icosahedral tailess
Icosahedral tailed
Filamentous

18. Life cycle
Phage l as an example
Lytic phase
Lysogenic phase

19. Virulent phages: undergo only lytic cycle Temperate phages: undergo both lytic and lysogenic cycles Plaques: a hollow formed on a bacterial lawn resulting from infection of the bacterial cells by phages.

20. Mechanisms of gene transfer
Transformation: uptake of naked exogenous DNA by living cells.
Conjugation: mediated by self-transmissible plasmids.
Transduction: phage-mediated genetic recombination.

21. Transformation Artificial transformation Natural transformation
(conventional method and electroporation)
Natural transformation

22. Avery, MacLeod, and McCarty (1944)
Demonstration of
transformation
Avery, MacLeod, and McCarty (1944)

23. Conjugation mediated by self-transmissible plasmids
(e.g., F plasmid; R plasmids)

24. F plasmid --an episome F plasmid
F plasmid can integrate into bacterial chromosome to generate Hfr (high frequency of recombination) donors
Hfr strain
Excision of F plasmid can produce a recombinant F plasmid (F’) which contains a fragment of bacterial chromosomal DNA
F’ plasmid

25. phage-mediated genetic recombination
Transduction
phage-mediated genetic recombination
Generalized v.s. specialized transduction

26. Mechanism of Recombination
Homologous recombination Site-specific recombination
Transposition Illegitimate recombination
Intermolecular
Intramolecular
Double crossover
Homologous recombination

27. Importance of gene transfer to bacteria
Gene transfer provide a source of genetic variation in addition to mutation that alters the genotype of bacteria. The new genetic information acquired allows the bacteria to adapt to changing environmental conditions through the process of natural selection.
Drug resistance (R plasmids)
Pathogenicity (bacterial virulence)
Transposons greatly expand the opportunity for gene movement.

28. Transposons Mobile genetic elements May carry drug resistance genes
Sometimes insert into genes and inactivate them (insertional mutation)
Transposons

29. E Conjugational transposon

30. Spread of transposon throughout a bacterial population
Trans-Gram gene transfer

31. Cloning Cloning vectors plasmids phages Restriction enzymes Ligase
In vitro phage packaging

32. Library construction
Genomic library
cDNA library

33. Applications of genetic engineering
Construction of industrially important bacteria
Genetic engineering of plants and animals (transgenic pants or animals)
Production of useful proteins (e.g. insulin, interferon, etc.) in bacteria, yeasts, insect and mammalian cells
Recombinant protein (e.g. HBsAg) vaccines and DNA vaccines

35. Double-strand break repair
(postreplication repair)

36. End-joining (error-prone)
Translocation
Short deletion at the joining point