Section E: DNA Transformation



One of the most powerful developments of modern molecular biology
is the ability to affect gene function using DNA transformation.
In P. polycephalum, two separate advances were vital for the
development of a DNA transformation system. The first was the
isolation of amoebal strains able to grow in axenic medium (see
Section B; Dee et al., 1989). The use of axenic amoebae for
transformation provides large numbers of active haploid cells,
free of bacteria. The second was the isolation of promoters for
the Physarum  actin genes ardB and ardC (Burland et al., 1991,
1992); these genes are expressed constitutively at high levels
and thus provide ideal promoters for introduced genes. 

The optimisation of parameters for DNA transformation in P.
polycephalum utilised a reporter gene, (CAT - chloramphenicol
acetyl transferase), under the control of PardB or PardC (Burland
et al., 1992). DNA is transformed into axenic amoebae by
electroporation and expression of the CAT gene can be detected
easily. Since the vectors do not contain any Physarum replication
origin, reporter gene expression is transient and the plasmid
gradually disappears from the cells due to vector degradation and
dilution by cell multiplication. Using the CAT vectors,
electroporation and cell treatment parameters were optimised and
a standard protocol developed (Burland et al., 1992). A second
reporter gene system has now been developed which utilises the
firefly luciferase gene linked to PardB or PardC (Bailey et al.,
1994). This system is much more sensitive than the CAT-based
system and, since luciferase activity can be measured in a
luminometer, provides a quantitative measure of gene activity
under different conditions. The luciferase-based system is ideal
for analysis of regulatory elements, such as enhancers and
promoters (Bailey et al., 1994) as well as for routine
optimisation of transformation conditions.

Most DNA transformation experiments have been carried out using
the axenic strain LU352 although others have also been used
(Bailey et al., 1994). A wide range of axenic strains is
available, carrying many different combination of genes such as
matA, matB and fusA (see Section B). All these strains should be
suitable for use in DNA transformation but the electroporation
parameters must be optimised for each strain used. As with any
experiments using axenic strains, care should be taken to use
cells that have been maintained continuously in log phase growth.
It is also useful to re-optimise the electrical parameters every
10 - 15 subs as these alter slightly as amoebal growth rate
changes. If amoebae enter stationary phase they must need to be
allowed to "recover" for about 2 subcultures before being used
for DNA transformation. As mentioned in Section B, growth rates
in axenic medium increase as time in culture increases. Care
should be taken, however, to use cells that have been in culture
for less than about 60 subs. Cells grown for longer periods than
this have been found to lose normal cellular properties such as
ability to develop into plasmodia (see Section B). Such changes
make it difficult to detect phenotypes due to gene disruption. 

Techniques for stable DNA transformation have been developed
permitting experiments such as gene knockout and over-expression.
The selectable marker used is the hygromycin phosphotransferase
gene (hph) which confers resistance to hygromycin B; the levels
of spontaneous resistance to hygromycin B are extremely low in P.
polycephalum (Burland et al., 1993b). Integration of linearised
vectors carrying PardC-hph  occurs at one copy per genome and the
introduced DNA is stable through mitosis and meiosis even in the
absence of drug selection (Burland et al., 1993b). Most recently,
the first gene knockout was achieved in P. polycephalum (Burland
and Pallotta, 1995), that of the plasmodium-specific actin gene,
ardD. Homologous integration of the introduced DNA occurred in
about 5% of stable transformants leading to gene disruption,
although no phenotype was detectable. 

In the future, it will clearly be desirable to disrupt genes
expressed in amoebae. Using haploid amoebae it is not possible to
recover transformants in which genes essential for amoebal
survival have been disrupted. If diploid amoebae were used (see
Section D), it would theoretically be possible to use DNA
transformation to knock out one of the two copies of a gene. Such
transformant would  be allowed to form plasmodia and their
progeny analysed. If no progeny carrying a gene disruption can be
identified, the disrupted gene is essential for amoebal growth.