Genetics Flashcards
Direct selection
Mutants can grow
Observe after simple plating on specific medium
Antibiotic resistance
Ability to use specific nutrient (ex lactose)
Ability to synthesize nutrient (separate slide)
Morphology (LPS, pili, flagellin -> colony shape)
Types of mutations
Silent (redundant code or conservative/similar amino acid)
Point -> missense or nonsense
- transition = purine->purine, etc
- transversion = purine -> pyrimidine, etc
Deletion, insertion, frameshift
Reversion - restores phenotype
- true - reverse of original mutation
- suppressor - restores phenotype but not genotype
Nutrition selection
Prototroph - can synthesize necessary metabolites
- grows on minimal medium (C, N, P only)
Auxotroph - missing enzyme, requires supplementation (ex Arg auxotroph)
Can assess complementation (order of enzymes)
ex Missing enzyme #3 -> can make earlier precursors -> diffuse to bordering areas that are missing earlier enzymes -> robust growth
Can select with penicillin (kills ONLY growing cells = prototrophs)
Minimal medium + penicillin -> prototrophs killed -> grow autotrophs on enriched medium
Selection of antibiotic resistance
Can prove spontaneous generation by replica plating
-> same colonies appear on non-selective and antibiotic medium
(Darwinism > Lamarkism)
Can also isolate colonies by direct plating onto antibiotic medium
Conditional mutants
Only mutant under certain circumstances
Temperature sensitive
- “permissive” low temp - protein folds correctly
- non-permissive high temp -> abnormal folding -> loss of fx
Can include mutations in essential genes (no growth at high temp)
Types of recombination
Homologous (normal repair mechanism, high frequency, conserved)
Site-specific - small region of homology + recombinase
- ex lysogenic lambda bacteriophage
Illegitimate - no homology, random, low frequency
- ex transposition
Population genetic study
Most bacteria cannot be cultured
- require symbiosis
- fastidious conditions
Study via genetics - ex “small subunit RNA” - 16S
- generic probe PCR -> array or sequencing
Whole genome sequencing
Only identification for intracellular and difficult to culture
Short genome! 4-5 kB with 4k-5k genes
- selected for speed of replication = no junk
Bacteriophage
Virus that infects bacteria
Either RNA or DNA, variety of structures
Specific for organism (capsid binding)
Either lytic or lysogenic or temperate (either)
Grow on active bacterial plate -> plaques
Morphology, size help identify
Virulent phage
aka lytic
Adsorption -> introduction -> transcription -> replication of DNA, translation of capsid proteins -> packaging -> lysis and release
-> clear plaques (kills cells)
Not binary fission (duh)
Lambda phage
Temperate - either lytic or lysogenic
Turbid plaques - some lysis but not all
Lysogenic phase:
Circularizes -> site specific recombination -> prophage
(represses lytic genes -> replication in genome)
Host cell stress -> excises -> lytic phase
Complementation
Multiple genes in same biosynthetic pathway -> same phenotype
Co-infect to test number of different genes
- > rescue phenotype = different genes/groups
- > absent phenotype = same gene/group
Restriction enzymes
Bacterial protection -> cleave foreign DNA
Palindromic sequences
Modify their own DNA to resist cleavage (EcoR1 methylase)
Also used for lab recombination
Transposons
Mobile genetic elements
Integrated into multiple areas
- nonhomologous (illegitimate)
- requires insertion sequence element - ex transposase enzyme
Can also carry antibiotic resistance, toxins
May replicate during transfer -> spread via plasmids
May also disrupt host genes
Transformation
Uptake of naked DNA -> integration to homologous chromosome -> autonomous plasmid Wide range of bacteria - can increase "competence" via electroporation, CaCl2
Generalized transduction
Random uptake of host DNA into phage
-> homologous recombination with recipient
(does not rely on integration of phage DNA)
Can select for integration of antibiotic or metabolic markers
Ex - distance measured by co-transduction of genes
Specialized transduction
Messy excision of integrated phage DNA ->
uptake of nearby DNA -> recipient cells -> homologous integration
(Also carry phage DNA, can become permanent phage genome)
Must be a temperate (integrated) phage (ex lambda)
Can carry toxins (ex Shigella)
Plasmid structure
Circular, supercoiled, double strand
Small to very large (400 kb)
Extrachromosomal (can separate with centrifuge)
Origin of replication - autonomous
Control copy number via encoded repressor mechanisms
(same repressor mechanism -> can’t grow in same cell = “incompatibility group)
Plasmids and resistance
“R” plasmid = type of F with antibiotic resistance
Rtf = resistance transfer factor
- R plasmid with IS sequences -> insert into other Rtf’s -> multiple resistance
Selection only if antibiotic exposure (otherwise slows replication)
Can duplicate -> highly resistant if shortened course
Can transfer beween species
Conjugation
Sexual cell-cell transfer
F+ donor -> pilus/bridge, transfer genes (tra), origin of transfer
Hfr = high-frequency transfer
Includes insertion sequences -> integration into host genome -> can carry host genes after excision (F’ plasmid)
Mapping - constant origin of transfer and directionality
- interrupt transfer -> map sequence of phenotypes
