Section A: Choice of Strains

Heterothallic and apogamic strains

Strains of P. polycephalum amoebae can be grouped into two
general types -heterothallic and apogamic - depending on their
ability to form plasmodia in clones. Heterothallic amoebae have
the advantage that they can usually be relied upon to proliferate
as amoebae, without producing plasmodia, even when cultured in
large quantities or in uncontrolled conditions. Heterothallic
strains cross readily, giving diploid plasmodia, when mixed with
amoebae of compatible genotype. Three mat loci, matA, matB and
matC, are important for the control of crossing (Youngman et al.,
1981; Kawano et al , 1987). To produce diploid, crossed
plasmodia, the two strains of amoebae must carry different
alleles of matA. Amoebae with the same matA alleles may fuse, but
the fusion cells do not develop into plasmodia. The matB locus
affects the efficiency of crossing: amoebae carrying different
matB alleles fuse much more frequently than amoebae carrying the
same alleles. The matC locus also affects the efficiency of
crossing but only at elevated pH; under other conditions, it has
little or no effect. Thus it is not essential to use strains
differing in matB or matC in order to obtain crossed plasmodia,
but plasmodia will form more rapidly and at higher frequency if
such strains are used. Since the three mat loci are unlinked and
multiallelic, strains carrying various combinations of alleles at
these loci can be easily constructed, and a wide choice is
available.

Apogamic amoebal strains have the advantage that they can produce
haploid plasmodia in clonal cultures; they are therefore ideal
for studies of gene expression since amoebae and plasmodia of
identical genotype can be obtained. Apogamic strains have not
been found in natural populations of P. polycephalum but have
been isolated in various laboratories. The most widely known
apogamic strain, CL (Colonia Leicester) carries a mutant allele
at (or close to) the matA locus, which is often denoted matAh.
This mutation allows the development of uninucleate, haploid
amoebae directly into multinucleate plasmodia (Bailey et al.
1987). Other apogamic strains have been isolated from various
heterothallic strains by screening amoebae after mutagenesis, and
have been designated gad mutants (Adler & Holt, 1977). The gad
mutation, which allows apogamic development, is usually located
very close to matA but the mating specificity of the original
heterothallic strain is often retained. The precise origin of CL
is unknown, but since the amoebae show matA2 mating specificity,
it is likely that this strain is descended from a mutant of a
matA2 heterothallic strain. For this reason, it is now usually
designated matA2 gadAh (Anderson, 1979). By choosing strains of
compatible mating types and by controlling culture conditions,
successful crosses between apogamic and heterothallic strains, or
even between apogamic strains, can be carried out (see Section
D).

Although apogamic (matAh or gad) strains are ideal for studies of
gene expression, they may sometimes be difficult to culture as
amoebae, because of their readiness to develop into plasmodia.
This difficulty has been avoided in several ways: by manipulation
of culture conditions and by genetic alteration of the strains.
Plasmodium formation can be prevented by maintaining cultures at
low cell density (see Sections B and C), but this is not a
satisfactory solution for some types of experimental work.
Fortunately, most apogamic strains are "heat-sensitive mutants":
they develop into plasmodia only at low temperature and can be
maintained in amoebal culture at high temperature (see Section
B). (In contrast, plasmodium formation by crossing is not
prevented by high temperature: see Section D). Apogamic strains
differ in their temperature-sensitivity, however, and some show
less tight control than others. Cultures of CL amoebae, for
example, eventually give rise to plasmodia even at high
temperature when incubated for long periods or allowed to reach
high cell density. 

One approach to the problem of culturing apogamic amoebae has
been to isolate strains carrying additional mutations that block
plasmodium development. The strain CLd was isolated from CL for
this reason; it carries a mutation npfC, tightly linked to matA,
which prevents apogamic plasmodium formation. This type of strain
has proved useful when a large number of amoebae or long
incubation of amoebal cultures has been necessary, for example
during mutagenesis and mutant isolation (see Burland, 1986).
Although this approach may seem to negate the original reason for
choosing an apogamic strain, the npfC mutation is not in fact the
obstacle it appears to be, since it readily reverts in
appropriate conditions, to give npfC+ amoebae which have regained
the ability to develop into haploid plasmodia. It must be
emphasised, however, that the use of such strains as CL and CLd
demands great care just because they do change so readily by
mutations (see Section B). Whatever strain is chosen for a
particular piece of work, one should never assume that it will
remain stable in its properties when cultured for long periods of
time.

Axenic amoebal strains

Laboratory strains of P. polycephalum originated from isolates of
plasmodia, sclerotia or spores, collected in several different
natural habitats. Amoebae derived from these isolates were
cultured on live or formalin-killed bacteria but, unlike the
plasmodia, they were not cultured successfully in synthetic
axenic media. Attempts to culture CLd amoebae in liquid, axenic
conditions resulted eventually in a genetically altered strain of
amoebae, designated CLd-AXE, which grew well in various synthetic
media, including one of the semi-defined media used for plasmodia
(McCullough et al, 1978). Unfortunately, CLd-AXE differed from
CLd in a number of other ways also and it could not be used to
study apogamic development in axenic conditions. By crossing CLd-
AXE with various laboratory strains, a range of new amoebal
strains able to grow in axenic media ('Axe' strains) were
constructed (Dee et al, 1989). The genetic basis of the ability
to grow in axenic media is not yet understood, but it is likely
that mutant alleles at only one or two loci (axe loci) are
responsible. The axe alleles are inherited by a large proportion
of progeny (up to 50%) in crosses between Axe and non-Axe
strains, and it is therefore not difficult to construct new Axe
strains. Both heterothallic and apogamic Axe strains are
available, and they carry various combinations of alleles at
other genetic loci.

Axe amoebal strains are obviously useful for any work involving
large-scale culture of amoebae, such as biochemical studies or
DNA transformation (see Section E). Axe amoebae can also grow on
bacterial lawns and can be manipulated by the methods formerly
developed for genetic analysis of non-Axe amoebal strains.
Plasmodia derived from Axe amoebae are not obviously different in
their properties from other plasmodia. Some of the Axe strains
have been more thoroughly studied than others and are therefore
specially recommended for particular types of research. For the
isolation of mutants and studies of gene expression in amoebae
and plasmodia, a strain (LU352) has been selected which resembles
CLd in having the genotype matA2  gadAh npfC5 . This strain is
also known to undergo the amoeba-flagellate transformation in
axenic conditions (see Section C). Reversion of the npfC5
mutation occurs frequently, as it does in CLd, giving rise to
npfC+ amoebae which are capable of apogamic development into
haploid plasmodia.  An apogamic clone of amoebae, resulting from
reversion in LU352, and designated LU610 has been used for
studies of the amoebal-plasmodial transition in axenic conditions
(see Section C). LU610 amoebae can be assumed to differ from
LU352 amoebae at the npfC locus only. Different revertants,
however, may result from mutations at different sites in npfC and
may consequently differ in their properties. LU610 is a suitable
strain for studies of gene expression in amoebae and plasmodia,
but LU352 amoebae might be preferable for certain types of work,
since, like CLd amoebae, they can be cultured in large quantities
without giving rise to plasmodia (see Section B).

Although LU352 and LU610 are comparable to CLd and CL
respectively in some of their properties, it must be emphasized
that they may differ from the latter strains at many genetic
loci. LU352 was isolated as a progeny clone from a cross between
CLd-AXE and a non-Axe strain and is a recombinant clone, know to
carry alleles derived from both parents. With the exception of
CLd-AXE, which arose from CLd, all other Axe strains have been
derived from crosses and are therefore genetically diverse; this
must be borne in mind if experiments are to be done on a range of
strains.

Genetic heterogeneity among strains

It may be useful to distinguish between several different types
of genetic heterogeneity found among laboratory strains of P.
polycephalum.

Firstly, natural polymorphism occurs at several loci; that is,
different alleles at these loci are all present at high frequency
in natural populations. Some polymorphism has been retained in
laboratory strains because it has proved useful in controlling
genetic manipulations; at the three mat loci already discussed,
for example, and at some fus loci, which control fusion between
plasmodia (see Section D).

Secondly, mutants of various classes have been isolated in
laboratories: some of these have been picked because they affect
particular processes in the organism, such as plasmodium
development or the cell cycle;  others were isolated because they
provided useful markers for genetic analysis, affecting drug
resistance or colour, for example. As with other organisms, such
mutations presumably occur in natural populations also but at low
frequency because they confer some disadvantage on the organism.
Such mutants may be lost as the result of selection in favour of
revertants, unless care is taken to maintain them. 

Thirdly, it is very probable that P. polycephalum, in common with
other out-breeding organisms, is naturally heterogeneous at many
other loci, not yet recognized by genetic analysis, due to the
occurrence of mutations in natural populations. Although
deleterious mutations affecting the amoebal stage may be
eliminated by selection, recessive mutations in genes that are
not expressed in amoebae may accumulate, even if they have
deleterious effects on plasmodia, since most plasmodia in natural
habitats are diploid and heterozygous. Such mutations may be
expressed in laboratory conditions when plasmodia are produced by
back-crossing or selfing amoebal strains. Polymorphism at various
loci, causing subtle variations in cellular properties or growth
rate of plasmodia or amoebae may also be present in natural
isolates. Since laboratory strains of P. polycephalum are derived
from more than one natural isolate, further variations may be
expected to result from genetic differences between isolates.

Fourthly, natural isolates may differ from one another by
chromosomal rearrangements, such as translocations, or
inversions. These are known to be common in populations of other
organisms but would not be easily detected in P. polycephalum
because it is not possible at present to study the chromosomes
either by cytological methods or by genetic mapping. Difficulties
in analysing some crosses between strains derived from different
isolates has suggested that such variation does occur. The
obvious solution to this problem would seem to be the use of
strains from one isolate only, but this is unfortunately not
feasible for certain genetic manipulations which depend on the
effects of alleles derived from different isolates; for example,
alleles of matB, necessary for the construction of diploid
amoebae (see Section D).

As far as possible, the 'hidden' heterogeneity among laboratory
strains has been reduced by programmes of inbreeding, and all
strains now used for genetic analysis are at least partially
isogenic (Dee, 1982). Since heterogeneity at certain loci, for
example matB, was intentionally retained, however, it is possible
that strains may also remain heterogeneous for genetic regions
closely linked to such loci, particularly if the original
strains, derived from different isolates, differed in chromosomal
rearrangements.

On balance, heterogeneity among laboratory strains of P.
polycephalum has proved an asset rather than an obstacle, and it
does not seriously interfere with genetic analysis. It has been
dealt with at some length, however, because serious errors of
experimental design or interpretation could occur if possible
heterogeneity is ignored. Methods for detecting and avoiding the
problems that strain differences may cause during genetic
analysis will be discussed in detail in Section D, but one other
type of problem is indicated here. Let us suppose, for example,
that a researcher wishes to compare the cell-surface components
of plasmodia carrying different alleles of fusA, a locus that
affects plasmodial fusion. There is no problem in producing
haploid plasmodia carrying different alleles of fusA and isogenic
for all other known loci; it is impossible, however, to ensure
that the plasmodia are isogenic at all loci other than fusA.
Since mutations of naturally-polymorphic loci such as fusA have
never been observed in the laboratory, it is not possible to
overcome the problem by isolating mutants from a single clone. To
conduct such an experiment, therefore, it is necessary to base
the comparison on samples of progeny clones segregating for fusA
alleles, isolated from a single cross. If correlations are found
between particular surface components and particular alleles of
fusA, one may be able to deduce that fusA, or the genetic region
near to it, is responsible for the differences observed. With
some understanding of the genetics of P. polycephalum, such
experiments would not be difficult to arrange and the greater the
range of strains and progeny tested, the firmer will the
conclusions become.

Finding the strain you need

It is not always possible, of course, to choose a new strain for
one's next experiment; many research workers feel obliged to
continue using a strain with known disadvantages because they
have invested so much time and effort into previous work and they
fear that a different strain may give different results.
Sometimes, however, experiments on several different strains can
strengthen an interpretation; or it may be discovered that some
previous results were misleading due to the use of a strain with
an aberrant genetic constitution.

If you are in the fortunate position of embarking on a new
project, and particularly if you are considering genetic
approaches at any stage in the project, it is worth making a very
careful choice of the strain or strains to be used. If you are
contemplating the isolation of mutants, for example, and the
study of their expression in amoebae and plasmodia, you may want
to use a strain capable of apogamic development and amoebal
growth in axenic medium. Before you leap into action, however, it
is important to consider whether you might need to analyse your
mutants by crossing them with a compatible strain carrying
particular genetic markers, whether you might want to carry out a
particular selective technique, depending on drug resistance for
example, and whether you may want to compare your own mutants
with others already isolated by some other research worker in a
strain of different genetic background. We hope that this booklet
will help you to base your choice on consideration of all the
important factors and to anticipate and avoid difficulties before
they arise.

It is, of course, impossible to list all the strains that are
available, since every cross generates a large number of progeny
clones, carrying different combinations of known mutant alleles
or marker alleles, and many such strains are stored in
laboratories engaged in Physarum genetics. We have described in
detail a few strains that are particularly useful for certain
kinds of work. If you do not find the strain you require in this
booklet, it may still be available in someone's freezer; if you
know what to ask for, we may be able to find it. Alternatively,
it may be that, after studying this booklet, you will feel
competent to make your own ideal strain;  we hope that at least
some readers will be inspired to reach this point.