Juliet Bailey

Summary

The two vegetative cell types of the acellular slime mould Physarurn polycephalurn - amoebae and plasmodia - differ greatly in cellular organisation and behaviour as a result of differences in gene expression. The development of uninucleate amoebae into multinucleate, syncytial plasmodia is under the control of the mating-type locus rnatA, which is a complex, multi-functional locus. A key period during plasmodium development is the extended cell cycle, which occurs in the developing uninucleate cell. During this long cell cycle, many of the changes in cellular organisation that accompany development into the multinucleate stage are initiated including, for example, alterations in microtubule organisation. Genes have been identified that show cell-type specific expression in either amoebae or plasmodia and many of these genes alter their pattern of expression during the extended cell cycle. With the introduction of a DNA transformation system for /? polycephalurn, it is now possible to investigate the functions of genes in the vegetative cell types and their roles in the cellular Accepted

7 July 1997 reorganisations accompanying development.

Introduction In developmental biology, much attention is being directed to unravelling the mechanisms underlying the differentiation of individual cells in multicellular organisms. Understanding these processes in a simpler system, such as the develop- ment of the slime mould Physarum polycephalum, may help to elucidate the mechanisms used by higher organisms.

In the life cycle of /? polycephalum, the two vegetative cell-types - uninucleate haploid amoebae and giant multi- nucleate syncytial plasmodia - are linked by a developmen- tal transition. These cell-types differ greatly in cellular behaviour, organisation and gene expression including, for example, differences in locomotion and mitosis. The initiation of development and the accompanying alterations in gene expression and cellular organisation are controlled by a single multiallelic mating-type locus matA. We are able to follow the alterations in cell behaviour and organisation in individual developing cells using a variety of techniques, including time-lapse cinematography and immunofluor- escence microscopy. The change from amoeba1 to plas- modial organisation and behaviour is first detectable in a single extended cell cycle that is a key period of develop- ment. Using a combination of classical genetic analysis and

mutagenesis, a range of developmental mutants with diverse phenotypes have been identified, many of which first show abnormalities during the extended cell cycle. We are also beginning to elucidate the genetic control of plas- modium development and to identify many of the genes involved in plasmodium formation. Cell-type-specific genes have been cloned and recent work has demonstrated that many of the changes in gene expression are initiated during the extended cell cycle. Molecular genetic techniques, in combination with the established cellular and classical genetic techniques, are now providing new information about the timing and control of alterations in cellular organi- sation and gene expression during development.

The life cycle Amoebae Amoebae are uninucleate, haploid cells with a diameter of 10-20 pm (Fig. 1). They move by amoeboid locomotion and feed by phagocytosis on fungal spores, bacteria and other micro-organisms. Mitosis is followed by cytokinesis and suc- cessive cell divisions give rise to a colony of genetically identical amoebae. In moist conditions, amoebae transform

Fig. 1. Events of the amoebal-plasmodia1 transition in apogamic development. M. mitosis. C, commitment; A. amoeba; F, flagellate; U. developing uninucleate cell undergoing closed mitosis at the end of the extended cell cycle: Bi, binucleate plasmodium in interphase. Bm. binucleate plasmodium in closed mitosis: Q. quadrinucleate plasmodium; P, macroplasmodium, S, sporangia. Bar, 10 pm except for P (15mm) and S (2mm). The coloured bars indicate the period of expression of amoeba-specific genes, e.g. proA (blue), plasmodium-specific genes, e.g frgp, prop (yellow) and genes regulated in development, e g. the red (orange). See text for more details.

into bi-flagellated cells (Fig. l ) , which swim, flagella fore- most, to dryer regions; flagellates cannot feed or undergo mitosis. When the flagellate settles onto a surface, the fla- gella are resorbed and the cell reverts to the amoeboid form. When amoebae encounter adverse conditions, such as starvation or cold, they secrete a resistant wall and trans- form into cysts. These dormant cells hatch to release amoe- bae when favourable conditions return.

Plasmodia Plasmodia are yellow, macroscopic syncytia with an intri- cate network of veins through which cytoplasmic streaming occurs (Fig. 1, P). Locomotion occurs as a result of this streaming, the direction of which alters every 60 seconds or so. These giant cells are easy to grow to a diameter of more than 30 cm and can contain in excess of 1O1O nuclei, which are moved around the cell during cytoplasmic streaming. The stages of the cell cycle, including DNA synthesis and mitosis, occur synchronously within all the nuclei of a plas- modium(’). Mitosis is not followed by cytokinesis and, as a result, both plasmodial mass and nuclear number double

every cell cycle. Under identical growth conditions, the cell cycles of amoebae and plasmodia are the same length. Genetically identical plasmodia fuse readily, leading to rapid increases in plasmodial mass. Plasmodia ingest bacteria, myxomycete amoebae and other microbes by phagocyto- sis, but are also capable of secreting extracellular enzymes to break down material which is then taken up by pinocyto- sis. They are unable to transform into flagellated cells but, in unfavourable conditions, transform into dormant resistant structures (sclerotia).

Genetic control of plasmodium development In all natural isolates of P polycephalum examined so far, sexual development is the norm; fusion of two haploid amoebae leads to the formation of a diploid plasmodium. Amoeba1 fusion and subsequent development of the fusion cell into a plasmodium are under the control of three unlinked mating-type genes. Multiple alleles of all three genes exist in natural population^(^-^). Two of these genes, matB and matC, affect the frequency with which pairs of

amoebae fuse; fusion occurs most frequently between amoebae carrying different alleles of these genes(5~6). Before amoebae are able to fuse, however, they have to become mating competent as the result of the action of an extracellular inducer secreted by amoebae during vegeta- tive growth@). Partial purification of the inducer showed that it has properties consistent with a high molecular weight gly- coprotein or gly~olipid(~). A more complete biochemical and functional characterisation of the inducer is needed to clarify its structure and mode of action.

Once amoebal fusion has taken place, subsequent devel- opment is under the control of the third mating-type gene, mafA; matB and matC have no effect on events that occur after amoebal When the matA alleles carried by the fusing amoebae are the same, all the subsequent events of development are blocked; the fusion cell either breaks down, or gives rise to a colony of diploid amoebae, but never develops into a plasmodium(g). If, however, the fusing amoe- bae carry different alleles of rnafA, plasmodium formation is initiated; nuclear fusion occurs in interphase shortly after amoebal fusion, giving rise to a diploid zygote. After a period of growth, the zygote undergoes mitosis without cytokinesis to become a binucleate plasmodium and then develops into a large plasmodium as a result of plasmodial growth and fusions(g). Plasmodia sporulate when starved in the light (Fig. 1, S); meiosis occurs in the spores, three of the four meiotic products disintegrate and the remaining spore is encased in a wall(1o). The spores are dispersed by wind, rain and animals and, in favourable conditions, each yields an amoeba or a flagellate, thus completing the life cycle.

Although development is usually sexual, mutant strains have been isolated in which haploid plasmodia form within clones of haploid amoebae” ,12). Genetic analysis showed that these apogamic strains carry dominant gadA mutations that map to matA (gackgreater asexual differentiation)(13). Development is temperature sensitive in all the apogamic strains currently used, with plasmodium formation inhibited at high temperatures (28-30°C), and permitted at low tem- peratures (21 -22°C)(14si5). At high temperatures, apogamic amoebae are able to undergo normal sexual development by mating with amoebae carrying a different matA allele, indicating that, although both map to matA, gadA function is distinct from mating-type ~pec i f i c i t y ( ’~~ ’~) . The matA locus is, thus, a complex genetic region with multiple functions and the master regulatory locus controlling plasmodium development .

The long cell cycle in development Time-lapse micro-cinematography is a valuable technique for analysing cell pedigrees and tracing the origins of cells which develop into plasmodia(9~14~i5~17~ia). The sequence of events deduced from filming can be correlated to changes in cellular organisation and gene expression using techniques such as immunofluorescence microscopy and northern blot-

ting (Fig. l)(15,1851g). Cinematographic studies on apogamic strains showed that a single amoeba develops into a haploid plasmodium without cell or nuclear fusion. The first indica- tion that an amoeba has entered the developmental path- way is when it fails to divide after a period of time equal to the length of an amoebal cell cycle. Instead, it continues to grow for a period about 2.3 times as long as the amoebal cell cycle (Fig. l)(i4xi5). During this extended cell cycle, the developing uninucleate cell becomes irreversibly committed to development, loses the ability to undergo the amoeba-fla- gellate transformation and gains plasmodial characteristics such as ability to ingest amoebae and to undergo plas- modial fusions. At the end of the long cell cycle, the develop- ing cell becomes binucleate by mitosis without cytokinesis and then grows into a larger plasmodium (Fig. 1).

Time-lapse cinematographic analyses of sexual plas- modium formation showed that the developing zygote also undergoes an extended cell cycle, indicating that this is a crucial period in the development of an amoeba into a p lasrn~d ium(~~ ’~) . The sequence and timing of events of sexual development are very similar to those of apogamic plasmodium formation. For example, loss of ability to transform into a flagellate occurs close to the time of com- mitment, although in this case commitment coincides with cell and nuclear fusion. Ability to ingest amoebae and to undergo plasmodial fusions are gained during the second half of the long cell cycle, as they are in apogamic develop- ment@). Thus, development in apogamic strains can be used as a model system for studying plasmodium develop- ment.

Differences between amoebae and plasmodia Gene expression in amoebae and plasmodia Comparisons of the proteins present in amoebae and plas- modia indicate that up to one quarter of abundant proteins show different levels of expression in the two cell types(20s2i). These differences are reflected at the level of gene expression; comparisons of cell-type specific cDNA libraries indicate that 5-10% of genes show amoeba- specific expression and a further 5-1 0% show plasmodium- specific e x p r e s s i ~ n ( ~ ~ > ~ ~ ) . More detailed analyses of some of these cell-type-specific cDNAs show that, in most cases, the alteration in the pattern of expression is initiated during the extended cell cycle, at about the same time as the devel- oping uninucleate cells become committed to plasmodium formation (Fig. 1)(23).

Microtubules in amoebae and plasmodia The microtubule cytoskeleton plays a vital role during nuclear division and in the maintenance of cell shape and polarity. Microtubules consist primarily of equal amounts of cx and p tubulin proteins and most organisms have multiple genes for both proteins. I? polycephalum contains four unlinked a-tubulin loci (altA to alto) and three unlinked

P-tubulin loci (betA to betC); some of these tubulin genes show cell-type specific expression(l ,24).

lmmunofluorescence and electron microscopy studies indicate that amoebae possess an intricate microtubule net- work similar to that of many animal cells; this network is re- organised during cellular transformations(25). In interphase amoebae, the microtubules radiate from a nucleus-associ- ated microtubule organising centre (MTOC), which consists of a pair of centrioles surrounded by amorphous material. At mitosis, the MTOC duplicates and forms the spindle poles, the nuclear envelope breaks down and an astral spindle forms; this open mitosis is followed by cy t~k ines is (~~) . When amoebae transform into flagellates, the centrioles form the basal bodies of the flagella, and the cytoplasmic micro- tubules give rise to the microtubules of the flagellate(26s27). Two-dimensional gel electrophoresis of amoebal proteins indicates that amoebae contain three tubulin isotypes (al, a3 and p1)(1328). The a3-isotype results from a post-transla- tional modification of a1 and is found in the flagellate and in the MTOC and spindle poles of amoebae, but cannot be detected in p l a s m ~ d i a ( ~ ~ ~ ~ ~ ~ ~ ~ ) .

In contrast to the situation in amoebae, the microtubules in interphase plasmodia have no specific orientation relative to the veins or nuclei, and radiate from cytoplasmic, not nuclear, foci(32.33). The nuclear envelope remains intact throughout the closed plasmodial mitosis and the anastral spindles are nucleated by intranuclear organising centres, distinct from the interphase Two-dimensional gel electrophoresis of plasmodial proteins shows that plas- modia express al, a2, P1 and P2 tubulin isotypes(1.28). The P2-isotype is found in the plasmodia but is not present in a m ~ e b a e ( ~ ~ % ~ ~ ) .

lmmunofluorescence studies have provided valuable information about the alteration from amoebal to plasmodial microtubule organisation that occurs during develop- ment(36s37). The changes are initiated during the extended cell cycle, although they take several cell cycles to complete and there is variation in timing between individual cells. For example, the P2-tubulin isotype is usually first detectable around the time of commitment, after loss of ability to trans- form into a flagellate, although some developing uninucleate cells can form P2-tubulin-positive flagella(36). The a3-tubulin isotype normally disappears at the same time as the P2-iso- type appears, but some quadrinucleate plasmodia still con- tain a 3 - t u b ~ l i d ~ ~ ) . Since there is no strict correlation between changes in cellular organisation and alterations in tubulin isotype usage, it has been concluded that loss of a3- tubulin and accumulation of P2-tubulin are not sufficient to bring about the reorganisation of microtubules during devel- opment(36'37).

One group of proteins that could play a key role in regulat- ing the changes in microtubule organisation are the micro- tubule associated proteins, or MAPs. Very few studies have looked at MAPs in F! polycephalum to ascertain which fami- lies are present, and whether these show cell-type specific

expression. Albertini et identified six proteins from amoebae which had some properties of MAPs. One of these proteins, a 125 kDa protein, appeared to be present in both amoebae and plasmodia, but it was not clear whether the proteins in the two cell types were encoded by the same or different genes. Further studies of F! polycephalum MAPs might help to determine whether alterations in MAP expression bring about the rearrangement of the micro- tubule organisation associated with development.

The actin-based cytoskeleton in amoebae and plasmodia The actin-based microfilament network found in all cells is vital for locomotion and cell division. As might be expected given the very different morphologies of amoebae and plas- modia, the actin organisation of these two cell types is very different. In amoebae, there is a layer of actin just beneath the cell membrane with a higher concentration of actin in the pseud~pod ia(~~) ; actin also becomes concentrated in the cytokinetic furrow of mitotic cells. In flagellates, an actin-rich backbone runs along the dorsal axis of the cell from anterior to p o ~ t e r i o r ( ~ ~ 5 ~ ~ ) . The actin rearrangements that occur dur- ing the amoeba-flagellate and flagellate-amoeba transfor- mations have been well c h a r a c t e r i ~ e d ( ~ ~ ~ ~ ~ ) . Like amoebae, plasmodia possess an actin layer just below the cell mem- brane, but they also have a more complex fibrillar microfila- ment network. This latter system forms a three-dimensional network in areas without veins. Close to the veins, actin cables are found parallel to the veins and also encircling them as helically twisted bundled4'). The contraction of these actin networks provides the propulsive force for cyto- plasmic streaming and plasmodial locomotion. Little is known, however, about the reorganisation of the microfila- ment network during development and how this relates to the observed changes in locomotion and behaviour.

F! polycephalum, like most eukaryotes, possesses a fam- ily of actin genes; five genes have been identified, desig- nated ardA to ardE(42)43). The ardD gene is expressed pri- marily in spherulating plasmodia, while the time of expression of ardE is unknown(42). The remaining three actin genes, ardA, ardB and ardC, are expressed at high levels at all stages of the life cycle(44). There are some differ- ences in the coding sequences of these three actin genes but they give rise to identical proteins(44). Thus, the differ- ences in actin organisation between amoebae and plas- modia cannot be due to changes in actin gene expression.

One group of proteins that could be responsible for the differences in actin organisation between amoebae and plasmodia are the actin-binding proteins. F! polycephalum contains representatives of many classes of actin-binding proteins, including those which bind to actin monomers or filaments, and those which sever actin filaments(41). The genes coding for several of these proteins have been identi- fied and their pattern of expression has been determined. With the exception of the calcium binding myosin light chain(45) and the myosin-like protein, m l ~ A ( ~ ~ ) , all those

examined so far show cell-type-specific expression; i.e. they are expressed in amoebae but not plasmodia, or vice versa@). In the case of the ~ r o f i l i n ( ~ ~ ) , myosin heavy

18 kDa myosin light chain(50) and fragrnin(’9151) proteins, gene families exist, one member of which is expressed in amoebae and the other in plasmodia. Little is known, however, about any differences in function between the members of these gene families. Do, for example, the amoebal and plasmodia1 profilins differ in their actin binding affinity, as has been shown for two isoforms of bovine brain ~rofi l in(~*)? Any differences in activity between members of a family of actin-binding proteins might be related to the dif- ferent functions of the proteins within the two vegetative cell- types, reflecting the observed differences in microfilament organ isat ion.

Analyses of cell behaviour during development indicate that alterations in actin-requiring processes, such as loco- motion and cytokinesis, begin during the extended cell cycle; this is the same stage at which changes in micro- tubule organisation and tubulin gene expression are known to be initiated(36s37s53). For example, the transformation of an amoeba into a flagellate involves the reorganisation of the actin cytoskeleton; the ability to undergo this transformation is lost around the time of c ~ m m i t m e n t ( ~ , ’ ~ . ~ ~ ) . Similarly, cytokinesis does not occur following the mitosis at the end of the extended cell c y ~ l e ( ~ $ ~ ~ ) , suggesting that the processes of mitosis and cytokinesis have become uncoupled by this stage, as they are in mature plasmodia. Detailed examina- tion of the expression patterns of genes coding for cell-type specific actin-binding proteins shows a correlation between the timing of the changes in gene expression and the alter- ations in actin organisation. For example, the plasmodium- specific fragmin (frgP)(’g) and profilin mRNAs (J. Bailey, unpublished observations) are first detectable during the extended cell cycle and gradually increase in abun- dance during development, being maximally abundant in plasmodia (Fig. 1). As the prop mRNA increases in abun- dance, the amount of mRNA from the amoebal profilin gene (proA; Fig. 1) decreases until no expression can be detected in plasmodia(47) (J. Bailey, unpublished observations). Thus, changes in the expression of genes coding for actin-binding proteins coincide with alterations in cell organisation and behaviour. It still remains to be determined, however, if the changes in gene expression cause the alterations in actin organisation.

The role of mafA in development Apogamic strains have proved valuable tools for studying plasmodium development in F? polycephalum since both amoebae and plasmodia are haploid. The genomes of apogamic amoebae and plasmodia are identical and con- tain the same single copy of each gene, facilitating analysis of gene expression during development(23). This character- istic of apogamic genomes has also aided studies of muta-

tions affecting development since, for all strains carrying such mutations, including recessive ones, the phenotype is expressed. The developmental mutants isolated so far have been termed npf mutants (no plasmodium formation). Some of these map to loci other than mafA and show a wide variety of terminal phenotype~( ’~3~~) . The majority of npf mutants isolated so far, however, carry mutations that are genetically inseparable from mafA and which block the initiation of development(16). These fall into two complemen- tation groups, designated npfB and npfC(16). Since the npfB and npfC mutations blocked apogamic development, it seemed possible that they resulted from reversion of gadA to gad+; genetic analysis indicated that this was not the case(16). The simplest interpretation for these results is that the npf6 and npfC mutations define two further functions of the mafA locus. Thus, including mating-type specificity and gadA function, there may be as many as four functions map- ping to this complex locus(16).

The genetic and cellular evidence indicate that the matA locus controls the initiation of development. Thus, under- standing the mechanism of action of mafA is an important area for future work and any model for the action of this locus must explain the following: Firstly, the role of inducer in the acquisition of mating competence. Secondly, the initiation of heterothallic development by the presence, in the same cell, of any two of the many mafA alleles. Thirdly, the ability of gadA mutations to allow haploid, apogamic development and the temperature sensitivity of this type of development. Fourthly, the mechanism by which the npf6 and npfC mutations prevent apogamic development. Fifthly, the mechanisms by which the mafA locus controls the acti- vation of plasmodium-specific genes and the repression of amoeba-specific ones. For convenience, we tend to repre- sent the four functions of matA as mapping to separate genes within a single complex, as seen in the A mating-type factor of Coprinus c i n e r e ~ s ( ~ ~ ) . Alternative proposals can be formulated, however, where these functions all result from the product of a single gene. Several models have been put forward to explain the mechanism of action of mafA, but until this locus is cloned and sequenced in a variety of normal and mutant strains, these must remain s p e ~ u l a t i v e ( ~ ~ ~ ~ ~ ~ ~ ) .

In many systems, gene products which control the activa- tion of other genes do so by binding to specific conserved sequences within the promoters of specific genes; i.e. they are transcription factors. During the development of the fruit fly Drosophila melanogasfer, for example, the anterior-pos- terior axis of the developing larva is defined through the action of the bicoid protein(57). This protein regulates the transcription of the genes whose promoters it binds to, and these genes in turn regulate the activity of another set of genes. Similarly, the products of the MAT locus in Saccha- romyces cerevisiae are regulatory proteins that activate and repress gene expression to determine cell type(58). A role as a transcription factor, or as part of a transcription factor com- plex, can be postulated for rnafA.

If matA functions as a transcription factor, does it directly activate all the plasmodium-specific genes and switch off all the amoeba-specific genes? Or, does it stand at the head of a cascade of gene action? In many of the npf mutants carry- ing mafA-unlinked mutations, developmental abnormalities are first detectable in the second half of the long cell ~ y c l e ( ~ ~ 5 ~ ~ ) . Cytological analyses of development in some strains carrying two matA-unlinked npf mutations show that the developing cells have defects characteristic of both single mutants, indicating that the mutations act in separate, independent pathways(i8). In other double-mutant combina- tions, the developing cells show the phenotype of only one mutant, suggesting that the mutations are in the same path- way(18). These analyses support the idea that matA initiates a cascade of gene action involving other transcription fac- tors and structural genes rather than acting directly on all the genes involved in development.

The lack of a strict correlation between changes in gene expression and changes in cellular organisation for some of the tubulin genes(36z37) suggests that these genes are not directly responsible for the observed alterations in microtubule organisation. They are, therefore, most likely to be downstream members of the gene cascade, some steps removed from matA. Genes that are directly acti- vated by mafA might be important for the regulation of development and would form a link between matA and the downstream genes. These intermediate genes should have a different pattern of expression from either amoeba- specific or plasmodium-specific genes, with their peak levels of expression during development (primarily in the long cell cycle) when much of the cellular re-organisation is initiated (Fig. l ) , and lower or no expression at other times. Studies to identify genes in this new category (termed red genes for regulated in development) are under way(59). The results of such studies should help to explain how the changes in gene expression are regulated and co-ordinated so that plasmodium development pro- ceeds smoothly, as well as shedding light onto the mecha- nism of action of matA.

DNA transformation One of the most important advances in the study of /? poly- cephalum biology over recent years has been the develop- ment of a DNA transformation system. The isolation of amoebal strains able to grow in axenic liquid culture(60) and the cloning of the ardB and ardC actin gene promoterd6’) made this advance possible. Using either the chlorampheni- col acetyltransferase gene(61) or the firefly luciferase gene(62) as reporter genes under the control of the actin pro- moters, conditions for DNA transformation have been opti- mised and the effects of various enhancers and other DNA elements investigated. Stable transformations have been carried out using the hygromycin phosphotransferase gene as a selectable drug-resistance marker(63) and transforma-

tion techniques are now routine in many Physarum labora- tories.

The DNA transformation system will play a vital role in studies of genes required for the development of a normal plasmodium. Once a gene of interest has been cloned, it is now possible to use homologous gene replacement to investigate gene function(G5). For functional investigation of genes that are expressed only during development or only in plasmodia, apogamic strains are particularly useful. Gene replacement can be carried out in the amoebae, which will then carry an alteration in the single copy of the targeted gene, but should be unaffected by the disruption since they do not express the gene. Once gene disruption is achieved in the apogamic amoebae, development can be induced and the effects of the gene disruption on development or plasmodia1 morphology can be investigated. The pheno- types of the strains carrying the disruption can be examined using a wide variety of techniques, including time-lapse cin- ematography and immunofluorescence microscopy, as has already been done for strains carrying matA-unlinked npf mutation^('^,'^). The first gene disruption was recently car- ried out in P polycephalum by Burland and P a l l ~ t t a ( ~ ~ ) , who replaced the wild-type copy of the spherulation-specific ardD actin gene with an altered copy; unfortunately no phenotype was detected in the disruptants. The efficacy of this approach has been proved, however, and disruption of more genes is sure to follow, including the redgenes and the plasmodium-specific genes, such as propand frgP(47,19).

A different approach is needed for the identification of genes that have not been cloned, such as matA and the matA-unlinked npf genes. Here, cloning by complementa- tion would be the method of choice for identifying the wild- type copy of the gene of interest. Random fragments of DNA from suitable strains would be placed into the same vector as the selectable marker and transformed into amoebae. In the case of the npfmutants, the strains used would be ones which could develop into phenotypically normal apogamic plasmodia when ‘rescued’ by the introduced DNA. Drug- resistant colonies able to develop normally would be identi- fied and classical and molecular genetic techniques used to confirm that the introduced DNA did carry the wild-type copy of the gene. For the mafA locus, amoebae that are heterozy- gous for matA (i.e. carry two different copies of the matA locus in the same amoebal cell) readily form plasmodia in colonies(66), unlike normal heterothallic amoebae. Thus, the introduction into heterothallic amoebae of one mating-type, of DNA carrying a different copy of this locus, should give rise to drug-resistant matA-heterothallic amoebae, which readily develop into plasmodia. In both the above cases, the hph gene could be used as a tag to recover the introduced DNA. Unlike the gene disruption technique described above, these approaches utilise random integration of the introduced DNA into the genome; this occurs at higher fre- quency than homologous i n t e g r a t i ~ d ~ ~ . ~ ~ ) . Cloning by com- plementation does, however, require higher levels of stable

transformation than are currently achievable and so must await further advances in DNA transformation technology for I? polycephalum.

Conclusions The development of amoebae into plasmodia is a useful system in which to investigate alterations in cellular organi- sation since we can study single cells and the changes that occur within them. The differences in cellular organisation between these two vegetative cell-types have been well studied and an increasing number of differentially expressed genes are being identified. Many of these genes code for cytoskeletal proteins, including tubulins and actin- binding proteins, making this a useful system in which to analyse the functions of such proteins.

The extended cell cycle in the developing uninucleate cell is a crucial period in development, during which many of the changes in cellular organisation and gene expression are initiated. The key locus controlling development, matA, probably activates a cascade of gene action but its structure and mode of action are unknown at present. Important future lines of investigation include cloning the matA locus and characterisation of its role in initiating development and controlling expression of the cell-type-specific genes. Another key area is understanding the roles of the redgenes in development and how their expression is related to matA action. Additional lines of investigation will include further studies of the functions of the cell-type-specific actin-bind- ing proteins in organising the cytoskeleton in amoebae and plasmodia and during development.

Apogamic strains are sure to play a vital role in future investigations since they develop without change in DNA or gene content and so are ideal for studies of the genes required for the development and maintenance of plas- modia. With the introduction of a DNA transformation sys- tem, it is now possible to study genes important for plas- modium development at many levels - cellular, molecular and genetic - in order to investigate their roles in the cell- types in which they are expressed.

Acknowledgements I would like to thank Jennifer Dee, Roger Anderson, Lyn- nette Cook, Peter Meacock and Jan Gettemans for their helpful comments on the manuscript and Kamlesh Chan- darana for help with the illustrations. I am grateful to The Wellcome Trust for financial support (grants 034879 and 042524) and to my colleagues in the Physarum field who have been generous with their time and ideas over the past few years.

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Juliet Bailey is at the Department of Genetics, University of Leicester, University Road, Leicester LEI 7RH, UK (E-mail: jab9 @ le.ac. uk).