Mechanism of Generalized Transduction
Any piece of DNA can be transferred
lysis
Strain A
Can only get a headful of DNA (90 kb in P22).
Recipient engineered cell is called a transductant
HOW CAN ONE USE
TRANSDUCTION TO MAP
GENES IN BACTERIA???
A headful of DNA is about 2 minutes of bacterial
chromosome. Therefore, can only get cotransduction
if the genes are very close.
Provides a method for fine scale mapping of genes.
If two genes are close together, they are co-transduced
in the same viral particle.
Strain B
Linkage mapping of bacterial genes by transduction
Linkage mapping of bacterial genes by transduction
• One phage can only transduce a single small
fragment of bacterial DNA.
• The closer two genetic markers are, the higher
the cotransduction frequency!!!!!
A
Done as a selection and then a screen.
Can select for any marker first- unlike conujugation.
B
C
Markers
Cotransduced
A
A
A+B
B
B+C
B+C
B+C
A
B
Linkage Analysis
by Transduction
C
phage infection
Donor:
leu+ thr+ arg+
Cotransduction Frequency: B-C > A-B > A-C
A-B=
A+ B+
X 100 = % cotransduction
A+
• The denominator is the marker
that was selected.
Recipient:
leu - thr - arg -
High transduction frequency = close genes. Opposite of
map units or minutes, where the smaller the map units or
minutes, the close two genes.
Donor:
leu+ thr+ arg+
What is the order of the leu thr and arg genes????
Recipient:
leu - thr - arg -
Selection
Transducing lysate
Centrifuge bacteria to bottom.
Phage stay in the lysate.
select transductants
Donor:
leu+ thr+ arg+
Recipient:
leu - thr - arg -
Exp # 2: MM + leu + arg
Exp # 1: Minimal media + thr + arg
Replica plate
Replica plate
MM + arg
# thr+leu+ = 2%
Screen
MM + thr
# leu+arg+ = 50 %
2 Possible
maps
thr
leu arg
MM + arg
# leu+thr+ = 2 %
or
thr
arg leu
2 Possible
maps
thr
Final Map
leu arg
MM + leu
# thr+arg+ = 0%
or
thr
arg leu
KanR lys+ arg+ X KanS lys- arg-
Donor Recipient
Example = Cross is A+ B+ C+ X A- B- C-
donor
recipient
What is the order of A B and C genes?
Expt #1 Transductants
A+ = 60
A+B+ = 30
A+C+ = 0
Cells are selected on a master plate with Kan Lysine and Arginine,
Then replica plated on the following plates.
C
A
B
A
B+ = 60
B+C+ = 20
B+A+ = 30
C
Master
A
Expt #2 B
B
C
What are the cotransduction frequencies?
A-B? =
B-C? =
A-C? = During generalized transduction, all markers in the genome are
transferred at an equal rate, because the phage can package any
piece of DNA. Later, a strain was discovered which transduced the lac genes
from one strain to another at a much higher frequency.
What might this be?
SPECIALIZED TRANSDUCTION
KanR arg+ lys+ KanR arg+ lys- KanR arg lys+
KanR arg lys
Replica 1 KanR arg+
lys+
Replica 2 KanR arg+ lys+ KanR arg+ lys- Replica 3 KanR arg+ lys+
KanR arg- lys+
Cotrans freq Kan arg = Cotrans freq Kan lys = Temperate Phage can lysogenize bacteria
Just like F
prime
plasmids,
sometimes
when the
phage comes
back out, it
rips out
some
bacteria
genes.
Excision can be induced, occasionally
leading to specialized transduction phage
λ prophage
bacterial lysogen
Alternative λ
developmental
pathways
Phage will lay
dormant, until
some signal,
like UV light,
signals it to
jump out and
go lytic.
lac+
λ
Cell carrying prophage
is called a lysogen
lac+
λ
lac+
THE
END
progeny phage
Don’t worry about
the details of
regulation in book.