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BIOL 2416
Chapter 15: Gene Mapping
in Bacteria and Viruses
Gene mapping in bacteria by
• Conjugation
– Bacterial “sex” between donor (Hfr) and recipient (F-) cells
– Interrupted mating experiment determine gene order
• Transformation
– Donor DNA is isolated and added to recipient cells
– Donor DNA integrates by homologous recombination (Xover)
– Look for frequent co-transformation of close genes
• Transduction
– DNA transferred from donor cell to recipient cell by
transducing phages
– Can be generalized or specific transduction
– In generalized transduction, ANY random piece of DNA can
be transduced
– Look for high co-transduction frequency of selectable
markers to figure out which genes are close together.
Fig. 14.5a Transfer of genetic material during conjugation in E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 14.5b Transfer of genetic material during conjugation in E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 14.7 Interrupted-mating experiment
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 14.9 Interrupted-mating experiments with a variety of Hfr
strains, showing that
the E. coli linkage map is circular
100
minutes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 14.10 Transformation in Bacillus subtilis
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Fig. 14.11 Demonstration of determining gene order by
cotransformation
Done when conjugation or transduction is impossible.
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.
Frequent cotransformation
Indicates close gene proximity.
Temperate Phages such as Lambda may go through the lytic
or lysogenic cycle after infecting a bacterial host cell:
(involves packaging of viral DNA)
Fig. 14.13 Generalized transduction between strains of E. coli
Phage P1 can do
generalized transduction,
where ANY piece of
bacterial DNA can be
transferred to the next host
bacterium due to sloppy
packaging.
(Lambda phage can
only do specialized
transduction,
where only
certain sections of
host DNA can get
transferred.)
(sloppy
packaging
possible here)
Typical transduction mapping data
(looking for frequent cotransduction):
• Selected marker:
• Corresponding
unselected markers:
leu+ (does not require leu to grow)
50% = aziR (sodium azide resistant)
2% = thr+(does not require thr)
thr+
3% = leu+
0% = aziR
Gene map:
thr
leu azi
Mapping genes of bacteriophages
• By 2-, 3- or 4-gene crosses, involving
bacteria infected with phages of different
phenotypes
– Plaque = cleared area on a bacterial lawn where
the phage has lysed the bacterial host cells (so
indicates infected host cells, presence of phage)
• Distinguishable phage phenotypes may
include different plaque morphology and/or
host range
• Very similar strategy to 2-pont test crosses
seen before
Bacteriophage gene mapping, cont’d:
• Co-infect E.coli with two strains of T2 phage:
– T2-1 with genotype h+ r
• can lyse E.coli strain B, but not strain B/2 (h+),
• and forms large distinct plaques (r)
– T2-2 with genotype h r+
• can lyse both B and B/2 (h),
• and forms small, fuzzy plaques (r+)
• When phage DNA is packaged, can get parental combination:
– h+ r and h r+
• Or re-combination due to crossover between phage DNAs:
– h+ r+ and h r
• Frequency of recombinants = frequency of crossover = relative
genetic distance between viral genes
Complementation tests
• To determine how many genes are involved
in a set of mutations that produce a given
phenotype
• Used in many types of organisms
• E.g. two true-breeding mutant strains with
black bodies are mated > all F1 offspring
turns out to have wt grey bodies.
– First black parent had a mutation in the gene A
(aa BB genotype)
– Second black parent had a mutation in gene B
(AA bb genotype)
– The two mutations complemented (overcame) one
another (F1’s were all AaBb wt grey)