8.5 mutant selection

DNA mediated transformation

"naked" DNA is taken up from the environment by a bacterial cell

homologous recombination

-if a chromosomal fragment is transferred, then it must become integrated into a replicon to be maintained in a population. -can only happen if the donor DNA is similar in nucleotide sequence to a region in the recipients cells genome.

genes can be trf from donor to a recipient by 3- different mechanism

1. DNA mediated transformation 2. transduction 3. conjugation

mutations are rare events. 2 methods are used to isolate mutants:

1. direct 2. indirect

conjugation

DNA is transferred during cell to cell contact

Transduction

DNA is transferred from one bacterial cell to another by a bacteriophage (a virus that infects bacteria)

vertical gene transfer

trf of DNA from parent organism during reproduction

ames test

-a quick, cheap tests that was developed to screen for possible carcinogens. -uses histidine req. auxotroph to measure the mutagenicity. 1st chemical to be screened is added to a culture on glucose-salt agar plate 2nd the agar medium lacks histidine so, auxotrophs cannot grow, whereas prototrophs can. 3rd mutations will occur in the prototrophic phenotype

replicon

-following gene trf, recipient cells must replicate the DNA to pass it on to daughter cells. This can only happen if the DNA is a replicon, meaning it has an origin of replication. -plasmids and chromosomes are replicons but fragments of chromosomal DNA are not

recombinants

have properties of each of the original strains. -horizontal gene transfer can be studied only if donor and recipient cells are genetically different, which is why mutants are useful. these diff. make it possible to determine if the recipients have indeed acquired new characteristics. -

when 2 bacterial strains that are his-/trp- (req. his and trp for growth) and leu-/thr- (req. leu and thr for growth) are mixed together and plated on minimal media any colonies that form are likely due to one strain acquiring genes from another strain is called:

horizontal gene transfer

in the Ames test, it is expected that a mutagen would __?__ the reversion rate of a histidine requiring auxotroph of Salmonella?

increase

is used to isolate an auxotroph from a prototrophic parent strain, because no selective medium exists to inhibit the parent is called:

indirect selection

in replica plating, colonies that grow on the master plate but cannot grow on the glucose-salts plates are?

auxotrophs

carcinogens

cancer-generating -most carcinogens are mutagens

chemicals that cause cancer are called ___?___ and most of them are ___?___

carcinogens; mutagens

selecting penicillin resistant mutants by plating them on a medium containing penicillin is an example of:

direct selection

penicillinase

enzyme added to destroy penicillin

penicillin enrichment

is sometimes used before replica plating to increase the proportion of auxotrophs in broth culture. -kills only growing cells (aka prototrophs ) -auxotrophs cannot multiply in the glucose-salt medium so, they are not killed by the penicillin

replica plating

is a clever method for indirect selection of auxotrophic mutants , devised by joshua and esther lederberg in 1950. 1.a plate of bacterial colonies is pressed onto the surface of a sterile velvet -colonies imprint on velvet 2.cells adhering to the velvet are transferred to the sterile media, and incubated -one nutrient agar -one glucose and salt agar =resulting in exact replicas of the original plate. 3.auxotrophic mutants form colonies on the nutrient agar but not on the glucose-salt agar.

indirect selection

is used to isolate an auxotrophic mutant from an prototrophic parent strain. -this method is more difficult cause any medium that supports the growth of the mutant also allows the growth of the parent.

penicillin enrichment is a technique:

is which mutant cells are incubated in glucose-salt broth with penicillin before plating on nutrient agar in order to increase the ratio of auxotrophs to prototrophs

direct selection

mutants that can grow under conditions in which the parent cells cannot are usually easy to isolate by direct selection. -method used: cells are inoculated onto a agar medium that supports the growth of the mutant but does not allow the parent to grow.

DNA in the environment that is not contained within a cell or virus is called :

naked DNA pg 217

plating bacteria in duplicate patterns on both a nutrient agar plate and a glucose-salt agar plate is an example of :

replica plating

the Ames test measures the effect of potential carcinogens on the __?___ rate of a histidine-requiring Salmonella auxotroph

reversion

horizontal gene transfer

trf of DNA from a source other than the parent organism.