|
Epicentre Forum 3 (4)
||
John Jakubczak, Brian Paul, Sankar Adhya, Glenn Merlino, and Susan
Garges Laboratory of Molecular Biology, National Cancer Institute,
Bethesda, MD 20892
Mice transgenic for bacteriophage lambda have proven invaluable for
assaying changes in mutation frequency caused by chemical exposure or
introduction of genetic changes into the mice.1 These
mice have multiple copies of bacteriophage lambda inserted in a head-to-tail
array in the genome. To assay changes in mutation frequency, total mouse
DNA is isolated from the target organ and the lambda DNA is packaged in
vitro using high efficiency phage packaging extracts. Escherichia
coli is then infected with the packaged lambda to see if a mutation
has occurred in the DNA. If the mutation frequency has increased in lambda
DNA from experimental animals or tissue compared with lambda DNA from
control animals or tissue, then it is inferred that mutation frequency
increased in that particular mouse or organ of that mouse.
We have recently developed a new direct selection method for mutation
detection of bacteriophage lambda in mice that presents distinct advantages
over previous systems.2 It is inexpensive,
requires no specialized media or equipment, and can be used with any
of the mice transgenic for bacteriophage lambda that are currently available.
Moreover, sequence changes resulting in mutation can be easily characterized
because of the small size of the mutable target gene. Our method is as
efficient for detecting both spontaneous and induced mutations in
vivo as the lacI-based screening method.3 Additionally,
as the selection is based on a target gene that is expressed and has
its effects before DNA replication occurs, false positives from E.
coli-derived mutations are unlikely.
The selection method identifies changes in the bacteriophage lambda cII gene.
When lambda infects a wild type E. coli strain, the phage either
lysogenizes or lyses the cell, depending on the level of cII protein.
The phage cII protein activates transcription of the lambda cI repressor
and Integrase genes, both of which are required for establishing lysogeny.
Lysogeny occurs at a much greater frequency in E. coli that have
mutations in the hflA and the hflB genes.4 These
genes normally encode proteases that degrade the cII protein. In hfl- strains
there is little degradation of cII, therefore the levels of cII are extremely
high. As a result, the level of transcription of cI repressor
and Integrase genes is high, and thus virtually 100% of the incoming
phage become lysogenized. As all of the phage lysogenize, there is no
lysis and consequently there are no plaques. However, if there is a mutation
in the cII gene, no cI repressor or Integrase proteins
are made, and therefore plaques are formed even on an hfl- strain.
We have taken advantage of this genetic system to develop a method for
mutation detection in mice transgenic for bacteriophage lambda.2 This
method is a positive selection for occurrence of mutation in the cII gene.
We select for mutations in the cII gene of lambda by observing
plaque formation on an hfl- strain. The selection scheme
is diagrammed in Figure 1. The selection is further biased for cII mutants
by doing the selection at low temperature. We have found that at low
temperature, lambda that is cI- (even [delta]cI)
does not form plaques on an hfl- strain, despite the
absence of the cI repressor protein.2 Table
1 shows that only [lambda] cII- mutants will form plaques
on the hfl- strain. In spite of its small size, the cII gene
appears highly mutable and can be sequenced easily.
 |
Figure 1. Selection for mutant plaques on an E. coli hfl- host. In
the example, a mouse transgenic for bacteriophage lambda has been
treated with a chemical to test for chemical mutagenesis. DNA is
extracted from the tissue of interest and used as substrate in an in
vitro packaging reaction. The lambda DNA from the mouse is packaged
into infective particles which are then used to infect E.coli.
Only if mutations have occurred in the lambda cII gene will plaques
appear on the hfl- host. A hfl+ host
is used to titer the number of phage packaged. |
|
|
[lambda] cII+ |
[lambda] cII- |
|
hfl+
|
+
|
+
|
|
hfl-
|
-
|
+
|
|
Our protocol using Epicentre MutaPlax™ Packaging Extract with
the cII selection strains is shown in Table 2. To characterize
the mutations in the phages, the mutant plaques should be purified on E.coli strain
G1225. The cII gene from the bacteriophage can be amplified and
the DNA sequenced using standard techniques.2
|
Table 2. MutaPlax cII-Select DNA Packaging Protocol.
Plating Bacteria Preparation
- The day before performing the packaging reactions, inoculate
5 ml of LB broth with a single colony of E. coli hfl+ strain
G1217 and another of the hfl- strain G1225.
- The day the packaging reactions are performed, centrifuge
the overnight cultures of G1217 and G1225 and resuspend the pelleted
cells in 10 mM MgSO4, adjusting the optical density
(at 600 nm) to 0.5. Place the cells on ice until infection.
Packaging Reactions
- Thaw the appropriate number of MutaPlax Packaging Extracts.
Remove half of each packaging extract to another tube; keep on
ice for later use.
- Add the DNA to the packaging extract using a wide-bore tip.
Mix slightly (stirring or pipetting up and down once). Continue
with the other tubes, and hold at room temperature until all
are ready.
- Incubate at 30°C for 90 minutes.
- At the end of the incubation, add the remaining 25 µl
of packaging extract, and mix the packaging reactions by gently
pipetting up and down about five times with a wide-bore tip,
avoiding the introduction of any bubbles.
- Incubate at 30°C for an additional 90 minutes.
- Stop the reaction by adding 0.6 ml of phage dilution buffer
to the packaging reactions. Vortex mix for 10 seconds at medium
speed. Keep the tubes on ice until ready to infect. We have found
that the titer of the packaged phage drops fairly rapidly, so
infection should be carried out as soon as possible. The addition
of chloroform to the packaging extract does not prevent the drop
in titer.
Plating the Packaged Phage
- For each DNA to be tested, you will need 2 plates for assaying
recovery of packaged phage on G1217, and 10 plates for mutant
detection on G1225. For G1217, 0.2 ml of the cells are mixed
with 3 µl of the packaged phage by pipetting up and down
a few times. For G1225, 0.2 ml of the cells are mixed with 50 µl
of the packaged phage, as above.
- Incubate the tubes for 30 minutes at room temperature to allow
for adsorption.
- Add 2.5 ml of melted top agar to the tubes and pour these tubes
onto TB plates. Incubate at room temperature (no higher than
25°C). Plaques will be visible in 24-48 hours.
- Count the plaques on the G1217 plates and obtain the average.
Divide by 3 to determine the number of packaged pfu (plaque forming
units) per µl. Total the plaques appearing on the G1225
plates.
To determine the mutation frequency:
(total plaques on the G1225 plates)
(G1217 pfu/µl X 50µl/plate X # of G1225 plates)
|
We have described a new positive selection system for measurement of
mutation frequency in mice transgenic for bacteriophage lambda. We have
successfully used it to study the effect of chemical exposure and introduction
of an oncogene on mutation frequency and found it compares favorably
with other mutation screening systems.2 This
assay has a number of advantages. First, because our assay is based on
selection of mutants (i.e., appearance of a plaque), only those phage
containing a mutation in the gene cII are recovered. Some alternative
screening methods require large numbers of plaques at low plating densities
using chromogenic substrates, which is expensive and laborious. Second,
this assay can be used with any lambda-based mouse mutagenesis system,
including Big Blue3 and Muta Mouse.5 Third,
the small size (294 bp) of the cII gene allows easy sequencing
to determine the nature of the mutation. Finally, false positives derived
from mutations occurring after phage infection of E.coli are unlikely
to be seen with this assay, as commitment to the lytic cycle takes place
before DNA replication.4
- Mirsalis, J.C. et al. (1995) Annu. Rev. Pharmacol. Toxicol. 35,
145.
- Jakubczak, J.L. et al. (1996) Proc. Natl. Acad. Sci. USA 93,
9073.
- Kohler, S.W. et al. (1991) Proc. Natl. Acad. Sci. USA 88,
7958.
- Herskowitz, I. and Hagen, D. (1980) Annu. Rev. Genet. 14,
399.
- Gossen, H.A. et al. (1989) Proc. Natl. Acad. Sci. USA 86,
7971.
|
