1. BIOL 2416 Chapter 15: Gene Mapping in Bacteria and Viruses

2. 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.

3. Fig. 14.5a Transfer of genetic material during conjugation in E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

4. Fig. 14.5b Transfer of genetic material during conjugation in E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

5. Fig. 14.7 Interrupted-mating experiment
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

6. Fig 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.

7. Fig. 14.10 Transformation in Bacillus subtilis
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

8. Fig. 14.11 Demonstration of determining gene order by cotransformation
Done when conjugation or transduction is impossible.
Frequent cotransformation
Indicates close gene proximity.
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

9. Temperate Phages such as Lambda may go through the lytic
or lysogenic cycle after infecting a bacterial host cell:
(involves packaging of viral DNA)

10. 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)

11. Typical transduction mapping data (looking for frequent cotransduction):
Selected marker:
leu+ (does not require leu to grow)
thr+
Corresponding unselected markers:
50% = aziR (sodium azide resistant)
2% = thr+(does not require thr)
3% = leu+
0% = aziR
thr leu azi
Gene map:

12. 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

13. 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

15. 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)