Biology Reference
In-Depth Information
Fig. 4
Random transposon mutagenesis of herpesvirus BACs and screening for the transposon
insertion site.
a
Transposon mutagenesis. A temperature-sensitive transposon donor plasmid (TnD)
is transformed into
E. coli
carrying the herpesvirus BAC. If a plasmid-selective transposon is used,
transposition leads to insertion of the transposon (
Tn
) into the BAC under antibiotic selection for
chloramphenicol (
Cm
) and ampicillin (
Amp
) at the permissive temperature (30°C). By following
selection with kanamycin (
Kn
) for the transposon and Cm for the BAC at a nonpermissive tempera-
ture (43°C), the donor plasmid gets lost and for transposon-inserted BACs can be selected.
b
Determination of the transposon insertion site. From individual
E. coli
clones carrying a viral BAC
with Tn insertion, DNA can be extracted and used for sequencing. Forward and reverse M13 primer
binding sites at both ends of the transposon allow sequencing from the Tn into the viral sequence,
thereby determining the exact nucleotide position of transposon insertion.
c
PCR screening of the
transposon mutant library contained in several 96-well plates. Eight rows (A-H) of 12 individual
E.
coli
clones (1-12) from each 96-well plate (
I
) are pooled together into eight single vials (
II
). These
eight vials are then pooled into one master vial (
III
) containing 96 different
E. coli
clones with
mutant BAC DNA. DNA extracted from individual master vials is used for the PCR screening reac-
tion. Here one specific primer binding up- or downstream to the viral gene of interest and the M13
forward (
M13-for
) and reverse (
M13-Rev
) primers binding to the ends of the transposon are used. A
clone with a Tn insertion within the gene of interest gives a positive PCR product generated by the
specific primer with one of the M13 primers. The master vial with the positive PCR product is
selected and the corresponding eight vials are tested using the same primers. After identification of
the corresponding positive vial, all 12 individual
E. coli
clones from this vial are tested and the clone
with the transposon insertion within the gene of interest is identified (
black circle
)
released under invasive conditions in host cells, leading to virus reconstitution
(Brune et al. 2001b). This procedure allows reconstitution of hundreds of random
mutant viruses, thereby setting up direct screens for specific phenotypes and selects
for nonessential genes because genomes in which Tns disable essential genes do
