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Transformation
Uptake of DNA from the surrounding medium and recombination into the recipient bacteriums genetic material
Transduction
Transfer of genetic material from one bacterium to another via bacteriophage
Bacterial Conjugation
Discovered by Lederberg and Tatum (1946)
Two auxotroph strains (one was met bio and the other thr leu thi) Culture together on complete medium Subculture on minimal medium
Some cells were prototrophs (10-7) and 2-3 independent gene mutations (genetic exchange &recombination) unlikely to create revertants One strain had provided genetic material to replace defective genes in the other
Bernard Davis
Demonstrated using a U-tube culture that contact between donor and recipient cells was necessary for the transfer of genetic material Now know transfer through F pilus
Bacterial Conjugation
F factor
Fertility conferred by a factor that could be lost and regained by a strain (from another F+ strain)
Mobile element
now known to be a plasmid (autonomous genetic element) 100 kbp in size Encodes 20 genes for genetic transfer (plus others)
Used antibiotic sensitive donor and resistant recipient Some genes always transferred sooner than others
Seemed to be a specific order Chromosome transferred linearly from a specific start point
Time Map
Times when individual genes first observed to have been transferred Time could vary depending upon Hfr strain
Order same Start point varied Minutes=map distance
Conversion of F+ to Hfr
F plasmid integrates into host chromosome Transfer always begins from one end of integrated F One strand of duplex peeled off and transferred through pilus Second strand synthesis and recombination occurs in recipieint
Hfr to F Conversion
Integrated F plasmid can excise
Often includes portion of host chromosome New plasmid called F Cell with F is partially diploid and called a merozygote (very useful for studying genetic regulation in bacterial systems)
Transformation
Foreign DNA enters the cell from the surrounding medium (Griffiths experiment) Two steps
Entry of foreign DNA into cell Replacement by donor DNA of resident DNA (but sometimes the donor DNA remains independent)
Transformation Process
Competence
A physiological state which allows the cell to take up foreign DNA into the cell
Natural competence requires specific receptors on the cell surface, energy and transport molecules dsDNA is taken up, one strand is degraded Surviving strand integrates into recipient chromosome, forming heteroduplex
Transformation Process
Lysis or Lysogeny
Lysis: Infection by phage produces many progeny and breaks open (lyses) the host bacterium Lysogeny: After infection, the phage DNA integrates into the host genome and resides there passively
No progeny No lysis of the host Can subsequently lyse (lysogeny)
UV Induction
Lysis
Lysogeny
Phage Genetics
TRANSDUCTION. There are two forms: Generalized Transduction: bacterial rather than phage DNA is packaged into a phage head. When another cell is infected, the bacterial DNA is injected and in a proportion of cases, may be incorporated into the chromosome by homologous recombination, replacing the existing genes. Frequency 105 - 108 per cell. More than one gene may be cotransduced - limit = packaging size = ~50kbp = ~1% of bacterial chromosome.
Phage Genetics
Specialized Transduction: Results from inaccurate excision of an integrated prophage; some phage DNA is lost and some bacterial genes are picked up and carried to the next host therefore phage are usually defective (noninfectious) and require replication-competent helper phage to replicate, depending on which phage genes are lost.
Bacteriophage
Viruses with bacterial hosts, phage for short Valuable models for genetic research T4 life cycle
Phage binds to host cell DNA injected into cell All host DNA replication, transcription stops Host chromosome degraded, phage DNA transcribed/replicated, phage proteins synthesized Phage particles assembled, host cell lysed to release progeny
Bacteriophage T4
T4 Life Cycle
Lysogeny
Lysogeny
Lysogenic or temperate phage Occurs when instead of replicating and lysing host cell, phage integrates its DNA into host chromosome
prophage
No new phage produced Integrated phage passed on to cell progeny Cell and progeny immune to further infection by similar phage
Episome
Genetic element that can either replicate independently or as part of the bacterial chromosome
Transduction
Zinder and Lederberg, 1952
Studying Salmonella typhimurium Recovered prototrophs from culture of two auxotrophs, but no F plasmid present U-tube experiment still allowed prototroph production when two auxotrophs remain separated
Filterable agent involved
Transduction Experiment
Prototrophs recovered 10-5 Filterable agent (FA)
Transduction Process
Transduction
Transduction mapping uses gene cotransfer frequency
Bacteriophage Genetics
Bacteriophage undergo genetic recombination Genetic maps can be constructed by mixed infection experiments
Simultaneous infection with two different phage mutants/strains (Seymour & Benzer, 1950s) h+r x hr+ gives some hr and h+r+ progeny Two loci intergenic recombination Recombination frequency= (h+r) + (hr+) / total plaquesX100 Detection of recombinants at 1 per 106 recombination=map distance between genes Negative interference
Complementation
Also discovered by Benzer studying rII locus of bacteriophage T4
rII mutants can lyse E. coli B but not E. coli K12(l) Simultaneous infection of K12 with certain pairs of rII mutants did produce plaques
Individual mutants fell into one of 2 groups
Pairs of mutations that produced plaques were said to complement each other (different complementation groups) Smallest unit of complementation called a cistron (equivalent to a gene today-smallest functional genetic unit)
Deletion Mapping
Mutants created with segments of the chromosome deleted Mutants that failed to complement a deletion mutant possessed a mutated locus (point) within the deletion
Preliminary mapping of mutants to a general location
Deletion Mapping
Benzers Significance
Combining the results from his studies, Benzer had defined an abstract unit (the gene) as a mutational and recombinational unit that was arranged in a specific order Now- nucleotides composing of DNA Experiment conducted before 1960sClassical examples of genetic experimentation