Biology 216 - Lect 4 (Genetics)
Objective: genetics as it pertains to med bact
I. Review replication, transcription, translation if unfamiliar 
	Necessary for discussion of antibiotic MOA (rif, str, tet, clin); 
	Topoisomerases - Quinolones TopoII
   Overview - circular x-some, ori, bidirectional, topoII (gyrase), 
	RNA pol (single one) at -35 and -10 (promoter) + sigma,
	concensus sequence, tRNA and rRNA stable, translation,
	 ribosome (L&S proteins), charged tRNA, 
	rbs (Shine Dalgarno), EFGvsII, release factors. 
II. Mutation - alteration in DNA base sequence
A. Basic types: Frameshift (from deletion or insertion)
	Single base change (transversion, transition) - less severe.
B. Causes - Spontaneous (due to infidelity during replication or repair) 
	- 10(-7) - 10(-12)
	Induced - physical (uv) or chemical mutagens, Tn,
		 site specific mutagenesis
C. Effect - Maladaptive, cryptic, adaptive (increase likelihood of survival) 
	ex. SmR - combined Ab therapy
D. Ames test (Bruce Ames - UCB) - based upon carcinogen = mutagen
	Library of his- Salmonella -> growth w/o his?
E. Isogenic mutants to test virulence factors
III. Gene transfer in bacteria
A. Transformation - if chromosome must recombine; if plasmid autonomous
	Used by Avery, MacLeod, McCarty to 
		demonstrate DNA is genetic (S. pn)
	Natural in some Gm+; Must make Gm- competent
 	Used in cloning after in vitro manipulations.
B. Conjugation - Requires pilus
	Mapping in E. coli with F+, Hfr, and F' (fine structure)
	Similar mapping done in Ps (toxA, LasB, Hly)
      1. R-factor plasmids (resistance factor plasmids) - modified F-factors
	a. RTF - Resistance transfer factor = pilus
	b. Antibiotic resistance
	c. Other - Resistance often Tn
	   Often multiple resistances - Shigella
	   Promiscuous - often cross genus (Ps -> E.coli -> Legionella)
	   Antibiotic in animal feed (R-factor transferred to human pathogen)
C. Transduction - Lytic (general) vs lysogenic (specialized)
	Ex. lambda-gal; diphtheria-tox; botulism-tox; cholera toxin 
		(recently discovered); O157 verotoxin
IV. Molecular cloning - Know basics since used in med bact research (Fig 5.12)
A. Principles - passenger, vector (plasmid/phage) -> microbe -> product
B. Applications - Products, Vaccines (hepB, S. typhi w/ LT,
	 rotavirus, Shigella surface, A-B+ V. chol)
   	Research on Ag Shift - Borrelia, Trypanosomes, 
   	Research on bugs which cannot grow in vitro 
	- Treponema pallidum, M. leprae
 	Production of DNA probes 
	(ex. probe to TB rRNA, GenProbe and Chlamydia/GC)
   	PCR - principles, use to amplify (bacterial and host mRNA),
V. Sequencing entire organisms
A. Why? New drugs, vaccines
B. Pathogens sequenced:
1995 - Haemophilus influenzae, Mycoplasma genitalium
1996 - Mycoplasma pneumoniae
1997 - Helicobacter pylori
1998 - Borrelia burgdorferi, Mycobacterium tuberculosis, Treponema pallidum,
	Rickettsia prowazekii, Chlamydia trachomatis
1999 - Chlamydia pneumoniae
2000 - Campylobacter jejuni, Neisseria meningitidis, Vibrio cholerae, 
	Ureaplasma, Pseudomonas aeruginosa
2001 - Mycobacterium leprae, Pasteurella multocida, E. coli O157:H7
VI. End of Review portion of course -
	Use Baron and Dictionary to fill in gaps