A rapid, small scale method for characterization
of plasmid insertions in the Dictyostelium genome Acknowledgements References
A rapid, small scale method for characterization of plasmid insertions
in the Dictyostelium genome
C. Barth, D. J. Fraser,
P. R. Fisher*
School of Microbiology, La Trobe University, Bundoora 3083, Melbourne,
Australia
Received January 7, 1998;Revised and Accepted May 13,
1998
ABSTRACT
A rapid, simple method for characterization of plasmid insertions
in the Dictyostelium discoideum genome was developed. It is based
on the capability of linear plasmid multimers in the insertions to recircularize
efficiently in Escherichia coli cells. This recombinational recircularization
of plasmid multimers provides a highly sensitive and reliable tool for
determining whether individual Dictyostelium transformants resulted
from restriction enzyme-mediated integration (REMI) or from recombinational
integration of plasmid (RIP). The method also reveals any rearrangements
in RIP insertions and provides an estimate of the vector copy number in
any particular transformant.
Insertional mutagenesis and gene tagging in Dictyostelium
discoideum are based on either restriction enzyme-mediated integration
(REMI) or recombinational integration of plasmid (RIP) into the genome
(1,2).
Within a single insertion site generated via RIP, the vector copy number
varies from a few to several hundred tandemly duplicated copies (2-4),
whereas successful REMI transformation results in the insertion of only
a single copy of the transforming vector (1,5).
Recently, we found that when Escherichia coli cells are transformed
with Dictyostelium genomic DNA (gDNA) containing tandemly duplicated
plasmid DNA, the plasmid multimers recircularize in vivo with high
efficiency and without deletions. The recircularization process involves
homologous recombination between the tandemly duplicated vector copies
present in such insertions, thereby regenerating exactly the circular monomeric
form of the original transformation vector (6).
Plasmid monomers, by contrast, recircularize inefficiently and inexactly
due to the lack of extended regions of homology that can direct the recombination.
Since REMI transformants contain only a single vector copy, E.coli
transformation with gDNA isolated from them is expected to be less efficient
than transformation with gDNA isolated from RIP transformants. However,
a significant proportion of presumed REMI transformants can contain plasmid
insertions that have arisen from RIP as a consequence of partial digestion
of plasmid DNA prior to transformation and/or preinsertional recircularization
events. This results in the formation of multicopy plasmid insertions (1,7).
As the efficient recovery of disrupted genes relies on the insertion of
only a single plasmid into an appropriate restriction site, putative REMI
mutants must be screened via a laborious process involving the preparation
and digestion of gDNA, gel electrophoresis and subsequent Southern blot
analysis. Here we report a rapid, small scale method for characterizing
plasmid insertions in the D.discoideum genome, that allows the efficient
identification of REMI transformants. It also allows the estimation of
the vector copy number present in the genomes of RIP transformants.
Relying on the high efficiency of E.coli electrotransformation,
we were able to simplify existing procedures for gDNA preparation (2,8)
in terms of time and labour, allowing the screening of large numbers of
Dictyostelium transformants. The transformants were cultured in
1.5 ml of HL-5 medium up to a density of 0.5-1.0 × 107
cells/ml. The cells were harvested by centrifugation (1-3 s, at 15 800
g in an Eppendorf centrifuge), washed once in water and then resuspended
in 300 µl TES buffer (10 mM Tris, pH 8.0; 1 mM EDTA; 0.1% SDS; 30
µg/ml RNase). After cell lysis by freezing (10 min, -70°C) and
thawing (on ice), 30 µg of Proteinase K was added and the lysate
was incubated for 1 h at 37°C. It was then extracted with phenol/chloroform/isoamylalcohol
(25:24:1 v/v/v) and ethanol precipitated. The recovered gDNA (always ~300
ng) was resuspended in 10 µl of water prior to electroporation (9)
into E.coli DH5[alpha] cells (estimated electrocompetence: 5 ×
107/µg pUC19 DNA).
Using this method we examined plasmid insertions in 43 Dictyostelium
RIP transformants and in 30 putative REMI transformants. All DH5[alpha]
transformations with gDNA of the RIP transformants resulted in high colony
numbers (15-300). In contrast to this, transformation with gDNA of 19 REMI
transformants failed to give any colonies. In a further 11 cases colony
numbers similar to those obtained in E.coli transformations with
gDNA of RIP transformants were obtained, suggesting that these presumptive
REMI transformants actually contained more than one plasmid copy per insertion.
We confirmed this by Southern blot analysis of gDNAs of 10 RIP and 10 putative
REMI transformants, the latter including five whose transformation into
E.coli resulted in high colony numbers (Fig. 1).
The gDNA was digested with BamHI, separated by pulsed field gel
electrophoresis (PFGE) and blotted onto nylon membrane prior to screening
for plasmid DNA. BamHI cuts once in the Dictyostelium transformation
vector, releasing a plasmid fragment of identical size to the original
transformation vector only if more than one vector copy is present in the
insertion. In BamHI-digested gDNA of the REMI transformants whose
DNA transformation did not yield any E.coli colonies, a band similar
in size to the linearized transformation vector was not detected, excluding
a multicopy arrangement of the vector in these transformants (Fig. 1,
lanes 1-5). In contrast to this, BamHI digestion of gDNA of the
five putative REMI transformants whose gDNA transformed E.coli with
high efficiency, released a band identical in size to the linearized transformation
vector (Fig. 1,
lanes 6-10). This indicates that the plasmid insertions of these transformants
did not contain single plasmid molecules as desired, but instead contained
multiple, tandemly duplicated plasmid copies. Similar results were obtained
with BamHI-digested gDNA of all RIP transformants (Fig. 1,
lanes 11-20). It is probable that those transformants isolated by the REMI
method which are found to contain plasmid multimers arise by recircularization
in Dictyostelium and subsequent RIP rather than by REMI (7).
Thus, transformation of E.coli with Dictyostelium gDNA is
a reliable tool for distinguishing multicopy from single copy plasmid insertions
and thereby allows efficient screening for real REMI transformants.
Figure1.
Composition of plasmid insertions in the genome of Dictyostelium
transformants. The transformation vector used in all experiments was pPROF160,
containing a neomycin and a blasticidin resistance cassette under the control
of Actin15 promoters. BamHI-digested gDNA of REMI and RIP transformants
was separated by PFGE (10 h, 4 s switching time, 200 V, 14°C), blotted
according to standard protocols and screened for plasmid DNA using a DIG-labeled
probe (Boehringer Mannheim, Germany). Lanes 1-5, gDNA of REMI transformants
that did not yield any E.coli colonies; lanes 6-10, gDNA of REMI
transformants that transformed E.coli efficiently; lanes 11-20,
gDNA of RIP transformants. HindIII-digested DNA size standards ([lambda])
and the size of the linearized transformation vector (shown by the arrow;
6.7 kb) are indicated.
The method also allows rapid characterization of the multimeric insertions
found in RIP transformants. Recombinational recircular-ization faithfully
reveals the existence of rearrangements or deletions within such insertions
because of the exactness of recombinational recircularization in E.coli
(6). Estimation of
the vector copy number per Dictyostelium genome is also readily
performed because of the correlation we observed between the vector copy
number in the Dictyostelium genome and the number of E.coli
transformants obtained after electrotransformation with Dictyostelium
gDNA (Fig. 2A).
It is therefore possible to estimate the vector copy number in any Dictyostelium
transformant simply by comparing the relative number of the colonies obtained
after electroporating E.coli with similar amounts of gDNA of this
transformant and a strain containing a known vector copy number in its
genome. We confirmed the usefulness of this method by transforming E.coli
with gDNA of 25 Dictyostelium transformants, one (HPF261) known
to contain 35 vector copies (6)
and the others containing an unknown copy number. Comparison of the colony
numbers obtained led to copy number estimates that agreed well with the
estimates based on Southern blot analysis (Fig. 2B).
Thus, the method presented here allows a rapid screening for REMI transformants
or, in overexpression studies, for RIP transformants containing large insertions
without rearrangements.
Figure2.
(A) Correlation between vector copy number per Dictyostelium
genome and E.coli colony numbers obtained. DH5[alpha] cells were
transformed with Dictyostelium gDNA (HPF274, 100 vector copies per
genome) after serial dilution with gDNA of an untransformed wildtype strain
(AX2). A total of 1 µg of gDNA was used in all cases except those
transformations indicated by an arrow, in which 2 µg of HPF274 gDNA
was used to simulate a transformation involving 200 copies per genome.
The regression line was fitted by the least squares method. (B)
Comparison of vector copy number per Dictyostelium genome as estimated
from Southern blots (stippled bars) and the electroporation method (solid
bars). Data is shown for 24 individual transformants arranged in ascending
order according to the Southern blot estimates. Transformants 1-5 had confirmed,
single copy REMI insertions and did not yield any E.coli colonies.
