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Mechanisms of Genetic Exchange

Bacterial Conjugation
Mechanism of transferring genetic information from one bacterium to another, followed by recombination with the recipient bacteriums genetic material

Uptake of DNA from the surrounding medium and recombination into the recipient bacteriums genetic material

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

Lederberg and Tatum: More Work

Transfer is unidirectional
Some strains are always donors, some always recipients in an exchange

Strains designated F+ (fertility, donor) or F(recipient)

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)

Nearly always transferred to recipient cell during conjugation

Converting recipient to F+

Hfr Strains and Chromosome Mapping

Nitrogen mustard treatment of F+ strain to induce mutations (1950, 1953))
Mutant had recombination rate of 10-4 (vs. 10-7) Strains called Hfr for high-frequency recombination Unlike normal F+ strains, Hfr strains do not convert recipient cell to F+ Genes transferred at different rates
Some very commonly, some not at all something had changed

Hfr Strains and Chromosome Mapping

Wollman and Jacobs Interrupted Mating Technique
Allow mating (conjugation) to proceed for specified time and then transfer to blender
Sheer forces terminate transfer through pilus

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

Order of Transfer Same, First Gene and Direction Varies

First Prokaryotic Genetic Maps

Map units in minutes, not recombination frequency E. coli K12 map approximately 100 minutes total

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)

Discovery of rec Genes

Mutants isolated with diminished recombination ability
recA, recB, recC, and recD genes (at first) RecA protein involved in strand transfer reaction, integrating donor strand into recipient duplex (strand displacement) RecBCD complex cuts and unwinds strand from donor duplex

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
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

Cotransformation identifies linked genes

Transformation Process

Different Types of Bacteriophages

Lytic- infect the cell and force the replication of the viruses until the cell lyses (or splits the cell open) Lysogenic- infect the cell and integrates its genetic material into the bacterial DNA, remaining dormant until the cell shows signs of stress, when the phage becomes active and begins making copies of itself.

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)

Bacteriophage lambda can do either.

UV Induction



Lyctic cycle of bacteriophage T4

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.

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

Lysogenic or temperate phage Occurs when instead of replicating and lysing host cell, phage integrates its DNA into host chromosome

No new phage produced Integrated phage passed on to cell progeny Cell and progeny immune to further infection by similar phage

Genetic element that can either replicate independently or as part of the bacterial chromosome

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

DNA transfer by bacteriophage P22

Transduction Experiment
Prototrophs recovered 10-5 Filterable agent (FA)

Transduction Process

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

Intragenic exchange (Fine structure analysis of gene)

Seymour & Benzer - rII locus of bacteriophage T4 recombination= c.o in eukaroytes Occurs between DNA of individual bacteriophages during simultaneous infection of the host bacterium E.coli

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)

Mapping Within a Cistron

4x103 2 x ---------8x109 =2x0.5x10-6 =0.000001

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