Section D: Genetic analysis

Mutants


It is difficult to lay down general rules for the isolation of
mutants, since widely differing approaches may be necessary to
isolate mutants with different characteristics. Most programmes
of mutant isolation in Physarum have been aimed at the
identification of mutations affecting the cell cycle or
plasmodium development, and these have yielded a number of
interesting mutants (Dee, 1982; Anderson et al, 1986, 1989;
Bailey et al, 1992; Solnica-Krezel et al, 1995). Other studies
have identified a few mutations that are useful as markers in
genetic analysis, for example mutations causing nutritional
requirements, drug resistance or plasmodial colour (Dee, 1982).

The first step in mutant isolation is obviously to decide upon
the phenotype required and the stage at which it should be
expressed. This decision is not necessarily straightforward. For
example, if the genes of interest code for essential cellular
functions, mutations leading to a loss of function may be lethal,
so that it is impossible to maintain the mutants for study. One
solution to this problem, of course, is to seek strains in which
expression of the lethal phenotype is conditional, and a good
example of the success of this approach is provided by the
isolation of temperature-sensitive cell cycle mutants in yeast.
It may be unnecessary to isolate such conditional mutants in
Physarum, however, if the expression of lethality is stage-
specific. For example, mutants carrying lethal mutations
affecting plasmodium development or plasmodial growth may be
readily maintained as amoebae. An alternative approach is to
isolate mutants in which there has been a change of gene function
rather than a loss. For example, knowing that the assembly of
Physarum tubulins was sensitive to benzimidazoles, Burland et al.
(1984) searched for and identified mutations of tubulin genes
which resulted in the expression of benzimidazole resistance.

Once the desired mutant phenotype has been chosen, each step of
the isolation procedure may be considered. It is often necessary
to use some form of mutagenic treatment to induce mutations at a
detectable frequency, and several methods, employing chemical
mutagens or UV-irradiation, have been published (see Chapters 7,
10 and 11 in Aldrich & Daniel, 1982, Vol. 2). It is important to
bear in mind the possibility that mutants obtained by such
treatments may sometimes carry multiple mutations or chromosomal
rearrangements, which may cause problems during subsequent
analysis. In practice, however, this does not appear to have been
a problem. If an efficient screening procedure is available, it
may be possible to dispense with any mutagenic treatment;  for
example, many apogamic (Gad) strains have been isolated as
spontaneous mutants, and are therefore unlikely to carry multiple
mutations.

Mutagenesis may be followed immediately by screening; for
example, amoebae may be mutagenised and then plated on a drug to
screen for resistant cells. Alternatively, it may be possible to
interpose an enrichment step which will specifically increase the
frequency of the desired mutants in the population to be
screened. For example, the frequency of rare npf mutant amoebae
lacking the ability to undergo plasmodium development can be
increased in a population of apogamic amoebae by allowing the
amoebae to form plasmodia, which are then separated from the
remainder of the amoebal population. Careful thought is required
when devising such enrichment procedures, since they may
sometimes favour only a subset of the mutants desired. This
appears to have happened with the enrichment for npf mutants; 
the vast majority of strains isolated using this procedure belong
to only two complementation groups (Anderson et al, 1989),
whereas isolations of npf mutants without the enrichment stage
have yielded mutants at many loci (Bailey et al, 1992; Solnica-
Krezel et al, 1995). Enrichment methods may also operate
efficiently to yield the wrong type of mutant.  For example, if
chemotactic response were used in an attempt to enrich for
mutants of altered motility, the method might be found to yield
mainly mutants with altered chemotactic response.

Mutagenesis, enrichment and screening need not all be carried out
at the same stage of the life cycle. For example, mutagenesis of
amoebae may be followed by screening of apogamically-derived
plasmodia. It may also be possible to screen for mutants at one
stage and then carry out more detailed analysis at another. For
example it might be convenient to isolate amoebal cell-cycle
mutants as amoebae but, provided the mutations were also
expressed at the plasmodial stage, it might then be desirable to
study the mutants more fully as plasmodia

Once mutant strains have been isolated, care must be exercised to
ensure that the strains can be maintained stably without loss or
change of the mutant characteristics. This is discussed in
Section B.

It is obvious that the foregoing considerations will all
influence the choice of suitable strains for mutant isolation.
For example, if screening for mutants is to be carried out in
plasmodia, it is essential to use an amoebal strain that forms
plasmodia at high frequency in individual colonies (e.g. CL,
LU610). It is important to remember that amoebae of the mutant
strains will be required for genetic analysis and it is therefore
desirable to retain amoebal cultures of all the screened clones. 
If the mutant plasmodia are able to sporulate, however, mutant
amoebae should be obtainable from spores.

Genetic analysis of a newly-isolated mutant

The genetic basis of a newly-isolated mutant strain can be
determined by analysis of an appropriate cross. For example, let
us suppose that a mutant of CL showing altered motility has been
isolated by screening plasmodia. Amoebae of the mutant strain
should be crossed with an appropriate wild-type strain to
generate a diploid plasmodium, which is then induced to undergo
sporulation. Since CL has matA2 mating specificity (see Section
A), an appropriate strain would have to carry a different matA
allele (e.g. matA1. To ensure efficient mating, the strain should
also carry a matB allele different from the matB1 allele carried
by CL (e.g. matB3). Spores of the crossed plasmodium should be
germinated to yield haploid amoebal progeny which are random
products of meiosis. If the newly-isolated mutant carried a
single nuclear gene mutation, the expected result is a 1:1
segregation of mutant : wild-type among the amoebal progeny.
Deviation from a 1:1 ratio may indicate that more than one
nuclear gene is involved, that the mutation is extrachromosomal,
or that there is differential viability of mutant and wild-type
spores;  further analysis is necessary to distinguish between
these possibilities.

It is important to realise that, for a variety of reasons, in
Physarum as in other organisms, crosses sometimes fail. Due to
genetic heterogeneity (see Section A), some combinations of
strains give rise to crossed plasmodia more readily than others,
and plasmodia may vary substantially in the vigour of their
growth, the readiness with which they can be induced to
sporulate, the viability of their spores, and in the frequency of
abnormal progeny. Even for the same combination of strains,
individual crosses may fail at some stage of the analysis. Much
of the following discussion will show that the causes of failure
can often be foreseen, and difficulties can be avoided by a
careful choice of strains and conditions.  It is always advisable
to replicate crosses, so that the failure of one or more
replicates will not delay progress. If checks are carried out at
each stage of the analysis, as explained below, it will be
possible to identify and reject any unsuccessful crosses without
undue wastage of effort.

When two amoebal strains are mixed, the plasmodia which arise may
not all be diploids of the desired genotype.  This is an obvious
risk when one or both of the amoebal strains are apogamic (as in
our example with a mutant derived from CL), since these may give
rise to haploid plasmodia by "selfing". Two precautions may be
taken to minimise the risk of selfing. First, since apogamic
development is heat-sensitive in most gad strains, the frequency
of selfing can usually be greatly reduced by carrying out crosses
at a temperature high enough to prevent  apogamic development
(e.g. 29-30¯C for CL). Crossing will not be inhibited at high
temperature. Second, the frequency of amoebal fusions can be
maximised by carrying out the cross under optimal conditions of
pH and ionic strength and by ensuring that the strains carry
different alleles of matB , and possibly also matC (Youngman et
al, 1981; Kawano et al, 1987). 

The measures described above will minimise the frequency of
selfing, but will not necessarily prevent it entirely.  Even
cultures of heterothallic amoebae may occasionally give rise to
plasmodia; these may result from gad mutations or they may be
"illegitimate" plasmodia resulting from "leaky" control of
development.  Some combinations of strains also yield significant
frequencies of haploid plasmodia through the poorly-understood
phenomenon of "stimulated selfing" (Anderson & Truitt, 1983).
Although the occasional formation of such plasmodia cannot be
prevented, it is possible to ensure that any selfed plasmodia can
be identified and rejected. This is usually achieved by using a
pair of amoebal strains which carry different alleles of fusA,
one of the plasmodial fusion loci.  The alleles fusA1 and fusA2
are codominant, and fusion occurs only between plasmodia carrying
identical fusA alleles. Thus crossed plasmodia will be unable to
fuse with selfed plasmodia (fusA1 or fusA2), and the three types
can be identified by their fusion behaviour with tester plasmodia
of known fusA genotypes. If selfing of one strain occurs at high
frequency, it will be helpful if this strain carries the whiA1
allele;  the other strain should carry the whiA+ allele. Since
whiA1 is recessive, crossed plasmodia will be yellow, and thus
readily distinguishable from white, selfed plasmodia formed by
the whiA1 strain. It will still be necessary to use the fusA
phenotypes of the yellow plasmodia to distinguish crossed
plasmodia from selfed plasmodia formed by the whiA+ strain.

Even when plasmodia with the appropriate hybrid fusA phenotype
have been identified, the subsequent analysis of progeny from
spores may indicate that the plasmodium did not result from a
successful cross. For example, it is sometimes found that most or
all of a set of progeny are identical to one parent strain for
all tested markers. Several lines of evidence suggest that some
hybrid plasmodia may initially be heterokaryons, containing
diploid, crossed nuclei together with haploid, unfused nuclei of
one parental type. While such plasmodia will probably give
satisfactory results if sporulation occurs soon after plasmodium
formation, the proportion of haploid nuclei will increase rapidly
over subsequent subcultures (see Section B). Two measures will
reduce the risk of problems of this type. First, plasmodia should
be cultured for as short a time as possible before sporulation.
Second, before isolating a large number of progeny for detailed
analysis, a sample of progeny should always be tested for
segregation and recombination of unlinked marker genes such as
matA and matB. Where one parent in a cross is an apogamic strain,
as in our example with the mutant isolated from CL, half the
progeny are expected to form plasmodia in tests of selfing.  If
the mutant has been crossed with a whiA1 or leuA1 strain,
recombination can be tested by scoring the selfed plasmodia for
these markers. Since matA and whiA are unlinked, 50% of the
selfed plasmodia are expected to be white if the progeny are
showing free recombination. If the mutant had been isolated in a
matA2 gadAh npfC5 strain, such as CLd, half the progeny from a
cross with a heterothallic strain will inherit matA2 gadAh npfC5. 
Haploid plasmodia can therefore be derived from selfing tests of
these progeny clones as a result of reversions of the npfC5
mutation. If free recombination of unlinked markers is not
obtained, further analysis of the spore batch should not be
carried out.

Other indications of problems with particular crosses may be
obtained by observing the morphology of the colonies that form
when spores germinate. It is normal to find a small proportion of
colonies (<1%) in which plasmodia appear when the colony is very
small, even at 29-30¯C;  such colonies are often diploids,
heterozygous for matA (Adler & Holt, 1975). It is also normal to
find occasional colonies with an abnormal, "fuzzy" morphology;
such colonies may be aneuploids. If diploid or aneuploid colonies
appear at high frequencies, however, further analysis of the
progeny should not be carried out. Some combinations of strains
appear particularly prone to high frequencies of abnormal
progeny;  in the event of such difficulties it may be advisable
to check that both parent amoebal strains are haploid (Section
B).

Most of the colonies that form following spore germination appear
to be clonal. A small proportion are mixed colonies, however,
possibly derived from the germination of two or more adjacent
spores. It is therefore usually essential to establish pure
clones of progeny amoebae before any analysis is carried out.

Strain construction and linkage analysis

It will sometimes be necessary to construct a new strain carrying
a particular combination of markers for a particular research
project. For example, it may be desirable to study, under axenic
conditions, a developmental mutant which was isolated in a non-
axenic amoebal strain. If the mutant is crossed with amoebae of
an axenic strain, it should be possible to identify recombinant
progeny amoebae which are able to grow axenically and express the
mutation. A range of heterothallic Axe strains is available for
this purpose, which carry various alleles of matA, matB and fusA.

A further reason for carrying out crosses is to study the linkage
relationships of genes. Such analyses may provide confirmation
that a mutation is chromosomally located and give important
evidence for or against allelism of independently isolated
mutations. Where a set of mutants with similar phenotypes have
been independently isolated, it will be necessary to cross them
with one another in order to determine the number of loci
involved. However, if the mutants have been isolated in the same
strain, they cannot be directly crossed with one another, and
preliminary crosses with other strains will be necessary to
produce recombinants carrying the mutant alleles with appropriate
matA, matB and fusA alleles. These strains can be used for both
linkage analysis and complementation tests.

It is important to realise that a detailed genetic map showing
all linkage groups is not available for Physarum;  since the
haploid chromosome complement is approximately 40 (Mohberg,
1977), and since relatively few markers are available, there has
been little point in expending effort on the construction of such
a map. Molecular techniques, such as analysis of restriction
fragment length polymorphisms (RFLPs) or pulsed-field gel-
electrophoresis, may ultimately provide the route to a linkage
map. Linkage analysis based upon RFLPs has already been of value
in the correlation of benzimidazole-resistance loci with -
tubulin genes (Burland, 1986).

Dominance and complementation tests

Where more than one mutant has been isolated with a similar
phenotype, tests of complementation may be used to determine
whether the mutations affect the same function. For mutations
expressed in plasmodia, these tests can be carried out by
crossing mutant amoebal strains carrying appropriate combinations
of matA, matB and fusA (see previous section). Alternatively,
haploid mutant plasmodia may be fused to generate a heterokaryon;
this will only be possible if the plasmodia are of identical
fusion phenotype. For mutations expressed only at the amoebal
stage, it is necessary to construct diploid, heterozygous amoebae
for complementation tests. Such amoebae can be isolated by
setting up amoebal mixtures in which the strains carry different
alleles of matB (and usually also matC) but the same allele of
matA.  The amoebae in such mixtures are able to fuse efficiently
to form fusion cells, which do not develop into plasmodia because
they are homoallelic for matA. In some of these fusion cells,
nuclear fusion occurs to generate diploid cells which proliferate
as amoebae. Other markers can be incorporated into the strains to
facilitate the identification of these diploids (Anderson &
Youngman, 1985; Anderson et al, 1989).

Methods similar to those used for complementation analysis can be
used to determine whether a mutation is recessive or dominant to
wild type.  For example, diploid amoebae heterozygous for axe
genes were unable to grow in axenic medium, showing that at least
one of the axe mutations is recessive (Dee et al., 1989).