Microbial GeneticsPortland State University：微生物遺傳學波特蘭州立大學

Microbial Genetics WHY? terms Genotype refers to genetic makeup Phenotype refers to expression of that genetic makeup Heritable traits must be encoded in DNA Mutations occur randomlyMutants resistant to an antibioticKinds of mutants Auxotroph: loss of biosynthetic enzyme Cold- or temperature-sensitive: enzyme function is restricted to narrower temperature range Drug resistance or sensitivity: permeability, degradation, or site of action Morphology: change in capsule or other feature leads to change in colony Catabolic: loss of a degradative enzyme leads to, for example, inability to ferment a carbohydrate Virus-resistant: loss of surface feature that is a virus receptorResults of point mutationsFrame-shift mutationsReversions Same-site reversion: may be true revertant (same sequence) or just restore activity Second-site reversion Frame-shift Production of another enzyme that fulfills the functionMutagens Analogs for bases 5-bromo-uracil for thymine (5BU can pair with G as well as with A) 2-aminopurine for adenine (2AP can pair with C as well as with T) Intercalating agents (ethidium bromide) UV (260 nm) causes dimerization of adjacent thymines (photoreactivation) Ionizing radiation causes chemical changes to the DNA Generation of free radicals that can react with DNA SS or DS breaks in the DNA molecule Transposable genetic elementsRecombinationRecombinationGenetic exchange: transformationGenetic exchange: plasmid transfer Small, usually circular, independently replicating DNA molecules Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication Genes of replication control, timing initiation on plasmid (ori) Some plasmids integrate (F+, Hfr) Most are double-stranded About 1- 100kb Code for: R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins Degradation, tol, nah, Plasmid copy # Compatibility (inc) Avery ExperimentTransformation Competence: ability to be transformed Steps in transformation DNA binding and uptake (SS or DS, depending on species) Integration (recA) Competence may be induced by electroporation, CaMechanism of transformation Transduction Generalizedcan be carried out by either lytic or temperate phage Specializedrequires specific integrationGeneralized transductionSpecialized transductionSpecialized transductionPhage Conversion Lysogeny may cause other changes in the host cell Often the host acquires immunity to additional infection by that phage type There may be other changes that may be beneficial to the host Lysogenized Salmonella anatum acquires cell-surface changes Lysogenized Corynebacterium diphtherium acquires toxinGenetic exchange: conjugationConjugation: earlyCell-surface structureConjugation: middleConjugation: lateTransposons Rare events Mobile Genetic elements-”jumping genes” Carry a transposase, and flanked by inverted repeats 20bp to 100bp First discovered in maize/corn Phase variation (invertible elements, need invertase)Transposons Genetic Engineering-basics Basic steps in cloning Restriction-modification enzymes “shot gun” or PCR Ligase Recombination Plasmids (or phage)= vectors Expression vectors Selection of clones Looking for a clone with a specific gene Probes (DNA, RNA or antibody) Wave of the future: DNA chips or “microarrays”, BAC libraries, automated sequencing etcGenomics Bioinformatics Harvesting genes for biotech (Diversa) Recent Science article Comparing gene families Escherichia coli mapNext Tues NO quiz Chp 12, finish up where we left of Today