of - genetics · a genetic study of aggregation in the cellular slime mould dictyostelzum...

A GENETIC STUDY OF AGGREGATION IN THE CELLULAR SLIME MOULD DICTYOSTELZUM DZSCOZDEUM USING

COMPLEMENTATION ANALYSIS

KEITH L. WILLIAMS AND PETER C. NEWELL

Department of Biochemistry, Uniuersity of Oxford, South Parks Road, Oxford, O X I 3Ql7, Englmd

Manuscript received May 13: 1975 Revised copy received October 28, 1975

ABSTRACT

A series of aggregation-deficient (aggregateless) mutants were isolated in genetically marked haploid strains of the cellular slime mould Dictyostelium discoideum. Diploids were produced from pairs of such haploid mutants by a fusion system based on this organism’s parasexual cycle. The diploids were isolated from the haploids by using Complementation of non-allelic growth- temperature-sensitive mutations and selection at the restrictive temperature. Complementation between the aggregateless mutations was then assessed in 41 9 diploids so formed. The non-complementing aggregateless mutations fell into five complementation groups (agoA, B , C, D and E ) and a dominant aggregation class that allowed little or no aggregation when present in a diploid with any of the other mutations tested or the parental wild type. Complicating factors, including partial dominance, multiple mutations, and possible interallelic complementation, are discussed. Data on the linkage of the aggregateless mutations was obtained by using recessive drug resistance mutations on three linkage groups to segregate haploids from the diploids. Calculations from our results suggest a genetic complexity of about 50 genes that are specific and essential for aggregation.

HE most successful approach to studies of biochemical pathways and their control in prokaryotes has involved a combined biochemical and genetic

approach. It has been suggested by HOLLIDAY and PUGH (1975) that a necessary requirement for the solution of problems involved with differentiation in eukary- otes may similarly be a eukaryotic system which will allow such a combined approach. The cellular slime mould Dictyostelium would now seem to represent a system in which such an approach is feasible. I t is an organism that, while being eukaryotic and having a developmental program that proceeds (in the absence of cell division) to form spores and stalk cells, has many of the advantages that made the classical bacterial and bacteriophage systems tractable. The organism is, for example, easily cloned and the doubling time is short (3-4 hr when growing on bacteria or 10-12 hr in axenic medium). The developmental phase, moreover, requires only 24 hr and the resulting spores can be readily

Present address Department of Genetics, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra City, A C.T. Australia 2601

Geneti,s 82: 287-307 February, 1976

288 K. L. WILLIAMS AND P. C. NEWELL

stored. Since growth and development are not concurrent, mutations affecting specific developmental stages (and not affecting growth) can be easily isolated.

Until recently only the biochemical aspects of differentiation in D. discoideum had been analyzed since no workable genetic system had been available. Recent work in several laboratories, however, has made genetic analysis using the parasexual cycle a relatively straightforward procedure. Although D. discoideum is primarily a haploid organism, diploids can be isolated and they are relatively stable. The chromosomes can now be readily stained (BRODY and WILLIAMS 1974) and most of the seven mitotic linkage groups have been marked with pigmentation, spore shape, growth temperature sensitivity or drug resistance markers (KATZ and SUSSMAN 1972; WILLIAMS, KESSIN and NEWELL 1974a; KESSIN. WILLIAMS and NEWELL 1974). Moreover, on two chromosomes the gene order has been determined (WILLIAMS, KESSIN and NEWELL 1974a; GINGOLD and ASHWORTH 1974; KATZ and KAO 1974).

In this report we detail improved simple genetic techniques which have allowed us to study the process of aggregation in D. discoideum. We have used complementation testing to find the genetic relationship between a number of aggregation-deficient mutants with various phenotypic characteristics, and have attempted to assess the genetic complexity involved in the aggregation phase of development of this organism.

MATERIALS A N D METHODS

Media: SM agar (SUSSMAN 1966): 10 g glucose (Analytical reagent grade, Fisons), 10 g Bactopeptone (Difco), 1 g yeast extract (Oxoid), 1 g MgSO,, 2.2 g KH,PO,, 1 g K,HPO,, 15 g Oxoid agar No. 3, made up to 1 liter with distilled water and autoclaved at 15 lbs/sq. in. for 20 min; approximately 40 ml of SM agar was dispensed into each triple-vented plastic petri dish (Sterilin Ltd., Teddington, Middlesex, England),

SM agar containing inhibitors was prepared as described above except that methanol (Fisons AR grade, final conc. 2% v/v), filter-sterilized cycloheximide (Sigma, final cmc. 500 pg/ml), or filter-sterilized cobaltous chloride (May and Baker Lab. reagent grade, final conc. 300 pg/ml) was added after autoclaving and cooling to about 50”.

Bacterid strains: D. discoideum amoebae were grown on SM agar previously seeded with Aerobacter aerogenes (Strain 1033). This strain of A. aerogenes is unaffected by methanol (2% v/v) or cycloheximide (500pg/ml), but it is sensitive to cobaltous chloride (300 pg/ml). For growth o f D. discoideum on SM agar plates containing cobaltous chloride, a spontaneous cobalt- resistant strain of A. aerogenes was used.

Strains of D. discoideum: All strains used are derived from the NC4 isolate of D. discoideum (RAPER 1935); hence they are all mating type A, (ERDOS, RAPER and VOGEN 1973). For the complementation analysis suitably marked strains were constructed (see RESULTS). The origin of the strains is shown in Table 1, while details of the aggregateless strains derived from them arc‘ given in Table 2. Our nomenclature system is based on that used in bacterial genetics (DEMEREC et al. 1966). Several laboratories are commencing genetic studies on aggregateless mutants of D. discoideum. To avoid the possibility of different mutations being given the same name, the gene symbol for aggregateless mutations “ago” was chosen to specify our laboratory- i.e., “0” refers to Oxford.

Maintenance of stocks: Strains which formed spores were cloned and ‘mass’ plates were set up by inoculating A. aerogenes and about 104-105 spores or amoebae onto SM agar plates; spores were collected in horse serum (Oxoid), dried on to silica gel (6-18 mesh), and stored at 4”.

AGGREGATION IN D. discoideum 289

c 9 E d

9 .- m

m c


TABLE 2

Aggregateless strains of D. discoideum

Strain Parent

NP37* XI NP38 X2 NP39 X2 NP40 X 2 NP41 X2 NP42 X 2 NP43 X2 NPM X2 NP45 X2 NP120 X36 NP122' X36 NP123 X36 NP124 X36 NP125 X36 NP127 X36 NPl28 X36 NP130 X36 NP134 X36 NP136 X36 NP142 X36 NP153 X36

Mutagen Gene Strain Parent Mutagen Gene

uv NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG NTH NTG NTG NTG NTG NTG

agoA3 ago-5 agoE6 ago-7 agoA8 ago-9 ago-I0 ago-I1 ago-I6 ago-55 agoA57 agoA58 ago-59 agoD6O ago-62 ago-63

ago-69 agoB71 agoC77 ago-91

ago-65

NP89 NP90 NP91 NP92 NP93 NP94 NP95 NP96 NP97 NP98 NP99 NPIOO NPlOl NP102 NP 104 NP105 NP 106 NP107 NP108 NP109 N P l l l NP112 NP113

x22 x22 x22 x22 x 2 2 x22 X22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22 x22

NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG NTG uv uv uv UV NTG NTG NTG NTG NTG NTG NTG NTG

ago-30 ago-31 ago-32 agoA33 ago-34 ago-35 agoB36 ago-37 ago-38 agoE39 agoD40 ago-41

agoC43 ago-45

ago-46 agoB47 ago-48 agoB49 ago-50 agoB52 agoC53 agoA54

ago-42

All aggregateless strains were isolated in this laboratory. * These strains may carry two aggregateless mutations.

Aggregateless strains were maintained by clonal passage at weekly intervals on A. aerogenes on SM agar at 22". Several different methods for storing aggregateless strains were investigated. For short-term storage (a few weeks) clonal plates, grown 4-7 days at 22", are stored at 7". In general this method is used for only 2-3 weeks as the viability of some strains declines rapidly; however s3me strains remain viable for at least 3 months.

Long-term storage of amoebae has been successful at either -70" or in liquid nitrogen, although systematic studies have not been made. Studies of storage of amoebae at -70" have been made by two other groups who have shown that storage is possible for at least a year (PROF. F. ROTHMAN, Brown University Providence, R.I.; DRS. J. LAINE, N. ROXBY, M. B. COUKELL, York University, Downsview, Ontario, Canada, personal communication and Can. J. Microbiol., manuscript in press). We have mainly used storage in liquid nitrogen which, although showing a lower percentage survival than at -70" initially, may allow longer storage. We have observed no loss in viability during one year of storage with the following method: Amsebae are scraped from the growing edge of 2 single clone (growing on a SM plate with A . aerogenes for about 5-7 days at 22") and mixed with approximately 1 ml of horse serum (Oxoid) which is 5% (v/v) with respect to dimethyl sulphoxide (S.L.R. Fisons). The concentration of amoebae is about 5 X 106/ml and bacteria (A . aerogenes) are not removed. The amoeba1 suspension is dispensed into 5 plastic straws (I" long) and each end is sealed with 'plasticine' modelling clay (Harbutt's Plasticine Ltd., Bath, England). The straws are coded at 0-5" for between % and 2 hr, then placed at approximately -20" for about 2 hr, then transferred to -70" for 2-3 hr, and finally transferred to liquid nitrogen. After 1-2 weeks, viability is checked by removing a straw and

AGGREGATION IN D. discoideum 29 1

thawing the amoeba1 suspension at room temperature, after which the amoebae are cloned on SM agar previously spread with A . aerogenes.

Finally, we have been able to store all aggregateless strains as spores on silica gel in the form of complementing diploids.

Isolation of aggregateless mutants: Strain X2 was mutagenized to about 13% survival with NTG (N-methyl-N-nitro-N-nitrosoguanidine, Ralph Emanuel Ltd., Wembley, England) as described previously (KESSIN, WILLIAMS and NEWELL 1974), and surviving amoebae were plated clonally on SM agar with A . aerogenes. Aggregateless mutant strains NP38-NP45 were isolated by inspection of the plates for colonies with aberrant aggregation. Bacterially grown amoebae of strains X36 and X22 were mutagenized to about 20% survival as described previously (KESSIN, WILLIAMS and NEWELL 1974) except that 0.2 mg/ml NTG was used instead of 1 mg/ml; aggregateless mutant strains NPl20-NP154 (from strain X36) and NP89-NP99, NP105-NP113 (from strain X22) were isolated from clonal platings. Aggregateless strains NP100-NP104 were isolated after UV treatment of strain X22 to 0.027% survival by exposure at 106 amoebae/ ml in salt solution (containing, per litre, NaCI, 0.6 g; KCl, 0.75 g; CaCI,, 0.3 g) for 3 min at 68 cm from a 50 W standard ultraviolet germicidal lamp (Hanovia Lamp Ltd., Slough, England).

Since the frequency of aggregateless mutants in non-mutagenized populations was low, and no division of amoebae was allowed after mutagenesis and prior to plating clonally on SM agar, all mutants are almost certainly of independent origin. Our definition of aggregateless mutants in this work encompassed those strains which grew normally (or only slightly slower or faster than the parent), and which showed either no aggregation at all or formed mounds of amoebae hut developed no further. The chosen end point for aggregation is clearly arbitrary, but the above definition was useful and unambiguous in most cases. To avoid bias we tried to isolate all aggregateless mutants arising in a few large mutagenesis experiments, rather than choosing small samples of aggregateless mutants in a number of experiments.

Complementation tests: Complementation tests involved the formation of diploids between two haploid aggregateless strains using a streamlined version of a method published previously (WILLIAMS. KESSIN and NEWELL 1974b). The tests were conducted in a sterile Falcon microtest I1 tissue culture plate (Cat. No. 3040) with lid (Cat. No. 3041). 'Fusion mixture' (0.1 ml) was added to each of the 96 wells. In initial experiments fusion mixture was water, but in most experiments it was 20mM CaCl,. The amoebae used in the complementation tests were obtained from clonal plates about 1 week old (i.e., last week's clonal plates were used for this week's experiments). A scrape of amoebae from near the growing edge of the clone was taken with a sterile toothpick and swirled into the appropriate well. A fresh sterile toothpick was used for each well. Each well contained approximately equal numbers of amoebae (similar blobs from a toothpick) of two strains to be fused; the final number of amoebae was usually between lo5 and 106. When all the wells had been inoculated with amoebae, the tissue culture plate was placed on an orbital shaker (150 cycles/min) at 22" for about 17 hr. After this incubation, the entire contents (0.1 ml) of each well were transferred with an Oxford sampler and sterile tip to a SM agar plate previously spread with A. aerogenes. The SM agar plates were incubated at 26.8 t 0.2" for 7 days, then transferred to 22". In most cases diploids were isolated from these plates at the time of transfer from 27" to 22", but sometimes they were obtained after several days at 22". Diploids were cloned at 22" before being analyzed further. Complementation was always analyzed on agar plates after growth on A. aerogenes, rather than on millipore filters. Diploids were assigned to a class between 1 and 5 only after inspection of aggregation and fruiting on both clonal and mass plating at 22".

Segregation of haploids, and diploids resulting from mitotic crossing ouer: Spores o r amoebae from a presumptive diploid clone were plated with A . aerogenes at 1-3 x 10*/plate onto two SM agar plates (one incubated at 22", the other at 26 8 " ) , and appropriate inhibitor-containing SM agar plates and incubated at 22". Resistant segregants from plates containing inhibitors were analyzed as described in the RESULTS.

Chromosome staining: Amoebae were fixed in methanol/acetic acid (3:1 v/v) and stained with Gurr's improved R66 Giemsa stain as described previously (BRODY and WILLIAMS 1974).

292 K. L. WILLIAMS A N D P. C. NEWELL

RESULTS

Complementation testing using parasexual genetics: The test: The complementation test described in MATERIALS AND METHODS used a very rapid and simple procedure for forming diploids from haploids. In particular no preparation of amoebae for experiments was needed since clonal plates used to maintain the strains were a source of amoebae for the test.

We found that some combinations of strains were more difficult to fuse to form diploids than others. This may not necessarily reflect differences in the frequency of formation of diploids; rather it may reflect differences in their ease of isolation or stability. Axenic strains derived either from AX2 or AX3 were more difficult to fuse than non-axenic strains. This was due at least in part to the fact that the wild-type (temperature-resistant) axenic strains (AX2, AX3) do not grow or fruit well on agar plates at the restrictive temperature (26.8"). In addition they contain mutations resulting in the production of small spore masses.

Because we wished to do biochemical studies (e.g., isolation of plasma mem- branes; GREEN and NEWELL 1974) on our aggregateless strains, we decided to use an axenic strain as one parent in our crosses. However to be able to use the complementation test described in MATERIALS AND METHODS it was necessary to use a non-axenic strain as the other parent. The non-axenic strain from which we constructed our parental strain X22 grew well and formed good fruiting bodies at the restrictive temperature, and spore masses were tall; all of these characteristics were almost completely dominant to the undesiriable features of the axenic strain (either XI, X2 or X36). Hence the diploids formed grew vigorously and produced tall spore masses on clonal platings at the restrictive temperature (if they sporulated).

For the success of the complementation test it was essential to have haploid growth-temperature-sensitive strains which were not at all leaky, since the numbers of amoebae plated at the restrictive temperature were only crudely estimated by the size of a blob on a toothpick. The axenic strains used (Xl , X2, X36) contained tsgA1, which is an excellent non-leaky growth-temperature- sensitive mutation (KESSIN, WILLIAMS and NEWELL 1974) , while the non-axenic strain (X22) was constructed with two leaky temperature-sensitive mutations (tsgD12, tsgE13: KATZ and SUSSMAN 1972; WILLIAMS, KESSIN and NEWELL 1974a) which together produced a very good growth-temperature-sensitive strain.

Using the strains described above, usually between 20 and 50 fusions yielded one o r more diploids in an experiment involving 96 fusions, although sometimes even greater success was achieved. In early experiments the amoebae were fused in water, but sometimes very poor fusion frequencies were obtained (10% or less of strains fused produced diploids). The major reason for the poor fusion was that in these cases the bacteria (which were not removed) overgrew and pre- vented fusion. Several treatments were successful in overcoming the problem of bacterial overgrowth. These included carrying out fusions in 20 mM CaCl,, 20 mM potassium phosphate pH 7.5, or chloramphenicol (50 pg/ml) . Eventually 20 mM CaC1, was chosen and this was used in most experiments.


Identification of diploids formed in the complementation test: Because of the large numbers of crosses involved in this work, we chose well marked strains which would allow us to distinguish diploids from haploids with little effort. The first criterion of diploidy was the ability to grow at the restrictive temperature, as a result of complementation between the different growth-temperature-sensitive mutations. Secondly, one parent contained the white spore marker (whi) , while the other parent was wild-type (yellow spores) at the whi locus but was mutant at the bwn locus, thereby producing brown pigment which not only

Paren ta l s : X22 (Haploid)

Phenotypes: Round, white spores .

Growth temp. s e n s i t i v e ,

non-axenic

Acr i f lav in /methanol

r e s i s t a n t .

Genotypes:

Linkage Group I + s p r A tsgE

I I whi acrA tsgD +

111

I V +

+ +

? +

X36 (Haploid)

E l l i p t i c a l , yellow spores .

Growth temp. s e n s i t i v e ,

axenic .

Forms brown pigment.

Cycloheximide and cobal tous

ch lo r ide r e s i s t a n t .

cycA + +

+ + + axeA

t s g A azeB

bwn

cobA

Diploid Phenotype: Oval, yellow spores. Grows a t r e s t r i c t i v e tempera-

t u r e . Non axenic . Forms no brown pigment.

S e n s i t i v e t o acr i f lav in /methanol , cycloheximide and

cobal tous ch lo r ide .

FIGURE 1:

FIGURE 1 .-Phenotypes and genotypes (including linkage information) of the parental haploid strains and the resulting diploids formed in the complementation tests on aggregateless mutants of D. discoideum. The order of genes on the chromosomes shown is in most cases unknown.

* Strains XI and X2 are genetically similar to X36 except that they are sensitive to both cycloheximide and cobaltous chloride.


colors spores but also diffuses into the SM agar. A complementing diploid was readily distinguished from either parent because the diploid had the phenotype yellow spores, no brown pigment, as both whi and bwn are recessive (KATZ and SUSSMAN 1972). Thirdly, use was made of spore shape mutations. One parent contained the mutant allele (round spores) at the sprA locus, while the other parent was wild-type (elliptical spores). Since the round spore mutation is incom- pletely dominant, the heterozygous diploid (.sprA/+, oval spores) can be distinguished from either homozygous diploid (sprA/sprA, round spores or +/+, elliptical spores). The size of the spores produced allows a reliable estimate of the ploidy, since diploid spores are readily distinguished from haploids under the microscope by their bigger size.

Fourthly, all diploids were plated on appropriate inhibitor-containing plates to determine whether they were heterozygous for the recessive resistance markers. All diploids constructed were heterozygous at the acrA locus (scored as methanol resistance; see WILLIAMS, KESSIN and NEWELL 1974a) , while diploids formed from mutants originally isolated from strain X36 were in addition heterozygous at the cycA locus (cycloheximide resistance at 500 pg/ml) and cobA locus. The acrA and cycA loci have been characterized previously (acrA: WILLIAMS, KESSIN and NEWELL 1974a; MOSSES, WILLIAMS and NEWELL 1975. cycA: KATZ and SUSSMAN 1972; WILLIAMS, KESSIN and NEWELL 1974a). The cobA locus. whose mutant phenotype is growth on bacteria in the presence of 300 pg/ml cobaltous chloride, is a new drug resistance locus unlinked to acrA and cycA and probably on an as-yet-uncharacterized linkage group. Full details of the characterization of this locus will be described elsewhere.

The characteristics of the parent strains and resulting diploids are summarized in Figure 1.

Finally if there was any doubt about the ploidy, or in cases where only limited analysis was possible (for example the diploid formed between strains NP43 and NP44) the chromosome number was determined by Giemsa staining.

Complementation testing on 43 aggregateless mutants: Figure 2 shows the results of a series of complementation tests on 43 aggregateless mutants com- prising 20 strains containing tsgAl and 23 strains containing tsgD12 and tsgE13. (Aggregateless mutants are shown in Figure 3).

In approximately 50% of cases, diploids were isolated in two or more different experiments to confirm the complementation test, especially where non-complementation was suspected. At this stage approximately 91 % of possible diploids have been isolated (41 9 of a possible 460).

Some haploid aggregateless strains are clearly more difficult to fuse to form diploids than others. For example strains NP41 and NP92 formed diploids on all 5 different attempts, whereas a diploid has been isolated only once between strains NP42 and NP93 in 19 different experiments, and a diploid has not been isolated between strains NP43 and NP93 despite 19 attempts. The best approach for the difficult crosses seems to be to vary the incubation conditions used for fusion.

AGGREGATION IN D. discoideum

Strains containing tsg A1

295

r I l l l l l l l l l l l l l l l l l l I l l

FIGURE 2.-Complementation tests between aggregateless mutants of D. discoideum. Diploid strains were isolated a t the restrictive temperature from pairs of aggregateless haploid strains containing different growth-temperatwe-sensitive mutations. Diploids were assigned to one of 5 classes. class 1 representing wild-type aggregation and sporulation and class 5 representing the complete absence of aggregation and sporulation. The fire classes are explained fully in the HESULTS. A blank space indicates that ii diploid has not yet been isolated between the two haploid strains. All strains in this figure. except X22 and X36. are aggregateless mutants; strains X22 and X36 are parental (aggregating and sporulating) strainz. Five complementation groups are defined and these are indicated by different symbols: complementation group agoA, 0 ; agoB, 0 ; agoC,O; agoD,Q a g o E . 0 .

296 K. I,. WILLIAMS AND P. C. NEWELL ~ :'rXN-r" A 8 II"

FIGURE 3.-A (NP113), B (NP105), C (NP100): typical aggregateless mutants of D. discoideum used for the complementation analysis. The photographs were taken using transmitted light from a position looking vertically down onto clones of the mutants growing on agar plates. The growth ring where the amoebae are ingesting the bacterial associate Aerobacter uerogenes is moving toward the right-hand margin of each picture. Behind this ring the amoebae are attempting to aggregate. D (NP127) and E (NP94) show aggregateless strains with a distinctive colony morphology, while F shows the parental (aggregation competent) strain X36.

All aggregateless mutants so far isolated have proved to be capable of fusion with at least some of the other mutants tried. I t is interesting to note here, however, that some aggregateless mutants do not form macrocysts with a strain of opposite mating type. The macrocysts are thought to involve fusion of amoebae as part of the sexual cycle (ERDOS, RAPER and VOGEN 1973; CLARK, FRANCIS and EISENBERG 1973) but the relationship of the processes involved in such fusion to those involved in fusion during the parasexual cycle is as yet unexplored.

Each diploid formed in the complementation tests was assigned to one of five classes (Figure 2). Class 1 included diploids whose aggregation and sporulation


characteristics were indistinguishable from the diploid formed between the two wild-type strains (i.e. usually X22 X X36). Class 2 represents diploids whose aggregation and sporulation was less complete than the wild type, but only marginally so. Diploids in class 3 are those which displayed somewhat sparse aggregation and subsequent sporulation. Class 4 included diploids which produced essentially no aggregation and very few normal spore masses (less than 10 on a mass plate instead of about 5,000 as on a mass plate of class 1 diploids) or aberrant aggregation and the formation of a small number of tiny spore masses. Finally class 5 included diploids which did not aggregate further than one of the parent aggregateless strains and produced no spore masses on either clonal or mass platings.

A complementation test between two aggregateless mutations was scored posi- tive when diploids formed between the mutant strains fell into classes 1 or 2; this was observed in 85% of the tests that yielded diploids (357 of 419 genetically different diploids isolated). Classes 4 and 5 accounted for 43 diploids (1 1 %) .

Nineteen of the diploids in classes 4 and 5 involved crosses with strain NP89. In addition NP89 produced diploids of class 4 with two wild-type strains, including strain X36. We conclude that NP89 contains a mutation which is dominant to wild type. This is supported by the finding that the diploid formed between strains NP89 and X36 segregated both wild-type and aggregateless strains when haploids were selected from this diploid. The aggregateless mutation in strain NP89 (ago-30) is unlinked to the aggregateless mutations of at least 9 of the 19

TABLE 3

Complementation groups containing two or more aggregateless mutations

Complementation group

Mutant strain

Linkage Gene group

agoA

agoB

a g o c

agoD

ago E

NP3 7 NP41 NP92 NP113 NP122 NP123 NP95 NP136 NP108' NP111* NP136 NP102 NP112 NP142 NP99 NP 125 NP39 NP98

I

agoA3 agoA8 agoA33 I1 agoA54 agoA57 agoA58 agoB36 agoB47 agoB49 agoB52 agoB71 agoc43 agoC53 agoC77 agoD40 agoD60 agoE6 agoE39

~ ~~~ ~ ~~

* The non-complementation observed between NP108 and NP136 and N P l l l and NP136, may have a more complex origin.

298 K. L. WILLIAMS A N D P. C . NEWELL

aggregateless strains shown in Figure 2 with which strain NP89 formed a diploid (Table 4), since sporulating haploids were segregated from these 9 diploids (see Figure 4).

The aggregateless mutations found in 16 of the remaining 24 diploids assigned to classes 4 o r 5 can be readily assigned to five complementation groups (Table 3). Each of the 16 diploids which make up Table 3 segregated only aggregateless haploid and diploid strains on SM agar plates containing inhibitors (see Table 4 and Figure 4), and this result is consistent with the two aggregateless mutations in each diploid being in the same cistron.

Complexities of complementation analysis between aggregateless mutations: The above conclusions are relatively straightforward; most pairs of aggregateless mutations complement, but five different complementation groups are defined by non-complementation (class 4 or 5 ) in 16 diploids and one strain contains a dominant aggregateless mutation. There remain 8 diploids in class 4 or 5 and 19 diploids in class 3 that require more complex explanations.

i) Partial dominance: Many of the diploids formed in complementation tests involving strain NP107 aggregated and sporulated poorly. Hence five diploids were placed in class 4 or 5 , and a further six diploids were placed in class 3; the diploid formed with the wild-type strain X36 was also placed in class 3 (Figure

TABLE 4

Linkage between aggregateless mutations in class 4 and 5 diploids

Diploid Linkage Diploid Linkage

NP37/NP92 linked NP37/NP107 unlinked NP41/NP92 linked NP41/NP 107 unlinked NPl22/NP92 linked NP43/NP107 linked NP123/NP92 linked NP44/NP107 probably unlinked NP37/NP113 linked NP122/NP107 probably unlinked NP41 /NP1 13 linked NP 122/NP9 1 linked* NPlZ2/NP113 linked NP37/NP101 probably linked+ NP 123/NP113 linked NP37/NP104 probably linked+ NP95/NP136 linked NP37/NP89 unlinked NP106/NP136 linked NP39/NP89 unlinked NP108/NP136 linked NP41/NP89 unlinked NPlll/NP136 linked NP43/NP89 unlinked NP102/NP 142 linked NP44/NP89 unlinked NP112/NP142 linked NP45/NP89 unlinked NP39/NP125 linked NP128/NP89 unlinked NP39/NP98 linked NP130/NP89 unlinked

NP134/NP89 unlinked

Evidence for or against linkage was obtained as outlined in Figure 4. Ten class 4 or 5 diploids with strain NP89 as one parent (Figure 2) have not been examined in sufficient detail to determine linkage.

* I t is likely that linkage between aggregateless mutations in this case represents linkage between ago-32 (strain NP91) and a second aggregateless mutation in strain NP122 (i.e., not agoA57), since ago-32 is unlinked to ago& and agoA8 and is not located on linkage group 11.

+Probable linkage does not involve ago& in strain NP37, since neither ago-42 (strain NP101) nor ago-45 (strain NP104) is located on linkage group 11.

AGGREGATION IN D. discoideum

FIGURE 4.

Aggregateless haploid Aggregateless haploid

299

Induce cell fusion

1 Select diploids at 27' and test f o r ability to aggregate

k Aggregateless diploids:

1 Segregate on cycloheximi , methano

Wild-type diploids:

and cobalt plates

Observation 1:

Aggregateless and wild type haploids on at least one type of

inhibitor containing plate.

Explanation:

Dominant aggregateless

mutation unlinked to

I

4 Recessive, unlinkcd

aggregateless mutations.

recessive aggregateless

mutation

Observation 2:

Only aggregateless haploids observed on all three types of

inhibitor containing plates.

4 Recessive, linked, non-allelic

.1 Explanation:

Recessive allelic mutations

or aggregateless mutations.

Dominant aggregateless

mutation linked to recessive

aggregateless mutation.

FIGURE 4.-Complementation tests with D. discoideum aggregateless mutants. The figure illustrates the fusion of haploids to form diploids, the resegregation of haploids on inhibitor- containing agar plates and the system for obtaining information on dominance, complementation, and linkage. To avoid complications only the haploids that segregated on inhibitor plates are shown, although diploids resulting from mitotic crossing over are also observed.


2). The simplest explanation of these results is that the aggregateless mutation in strain NP107 (ago-48) is partially dominant. In agreement with this explanation it seems likely that class 4 or 5 diploids formed between strain NP107 and strains NP37, NP41 and NP122 probably do not represent non-complementation between the aggregateless mutations, since there is convincing evidence that the aggregateless mutations of strain NP107 and strains NP37 and NP41 are unlinked (Table 4). Moreover both sporulating and aggregateless haploid segregants were obtained from the diploids NP107/NP37 and NP107/NP41 when amoebae were plated on SM agar plates containing methanol. In addition strains NP43 and NPM (which form class 4 and 5 diploids, respectively, with NP107) were shown to complement each other. This was established by fusing strains NP43 and NP44 as described previously (WILLIAMS, KESSIN and NEWELL 1974b) and plating amoebae at low numbers (104/plate) with A. aerogenes at the permissive temperature. Selection of diploids at the restrictive temperature was not possible as both strains have the same growth-temperature-sensitive mutation. An aggregating and sporulating diploid was obtained, however, from this cross by non-selective methods, indicating that NP43 and NP44 have non-allelic aggregateless mutations. It is concluded that the complementation pattern of diploids involving strain NP107 is complex but can reasonably be accounted for on the basis of its aggregateless mutation (ago-48) being partially dominant.

ii) Multiple mutations: The class 4 diploid formed between strains NP91 and NP122 may reflect the fact that strain NP122 contains a second aggregateless mutation (in addition to agoA57) which does not complement the aggregateless mutation (ago-32) of strain NP91. Likewise the class 4 diploids NP37/NP101 and NP37/NP1 04 (Figure 2) may reflect non-complementation between second and even third aggregateless mutations (in addition to agoA3) in strain NP37 and the aggregateless mutation in strain NPlOl and NP104. We have no information as to whether or not the aggregateless mutations of strains NPlOl and NP104 are allelic. The suggestion that strains NP37 and "122 may contain more than one aggregateless mutation is not surprising in view of the known multiple mutations caused by NTG in other systems. Indeed NTG seems to be highly mutagenic in D. discoideum since in the series of aggregation-deficient strains isolated for this study we have observed three cases where one of the outside markers was mutated. Firstly, out of 35 aggregation-deficient strains (NP120-NP154) isolated in strain X36 (which produces brown pigment) one strain was found (NP153) that no longer produced the brown pigment. We have shown that this is due to the presence of a suppressor mutation which is unlinked to both the aggregation deficiency mutation (ago-91) and the brown pigment locus (bwn) . A detailed study of this suppressor mutation will be presented elsewhere. Secondly, out of 19 aggregation-deficient mutant strains (NP89-NP113, Table 2) that were isolated in the round-spored parent strain X22, one strain (NP90) is genetically wild-type for spore shape (i.e., if it sporulated it would produce elliptical spores). Whether this is a reversion of sprA or a suppressor mutation has not yet been established. Thirdly, strain NP38, whose parent strain X2 is methanol-sensitive, shows resistance to methanol (2%) and is probably a mutation at the acrA locus.


iii) Possible leakiness of aggregateless mutations: Most of the strains used in this work are completely non-leaky on agar plates at the permissive temperature (22"), although a few strains can be induced to aggregate and sporulate by lowering the temperature to 7" for a short period. However some strains, notably NP95, NP109 and NP111, produce some sparse aggregation and sporulation after abnormally prolonged incubation. This presented no problems here except for strain NP109 where six diploids were scored as class 3 (Figure 2). It is possible that in the case of one or more of the class 3 diploids the two aggregateless mutations were non-complementing, but leakiness of the aggregateless mutation in strain NP109 allowed better aggregation and sporulation than might otherwise be expected.

iv) Interaction between aggregateless mutations: There is some evidence of a negative interaction between some aggregateless mutations. For example, strain NP107 produced a class 3, two class 4 and one class 5 diploids with different members of complementation group agoA (Figure 2) despite the fact that strain NP107 is almost certainly wild-type at the agoA locus (Table 4 ) . Likewise strain NP98 produced a class 1, a class 2 and two class 3 diploids with different members of complementation group agoA (Figure 2) suggesting some interaction despite the fact that NP98 is wild-type at the agoA locus.

v) Interallelic complementation: The complementation tests are a very sensitive system for demonstrating interallelic complementation between aggregateless mutations in the same cistron. Aggregation can be observed over a prolonged period, and any fruiting bodies formed can be easily spotted against the flat back- ground of non-aggregating cells even if the fruiting bodies are sparsely spaced and are very small.

We have assumed that interallelic complementation explained the very limited aggregation and sporulation of those diploids in class 4 whose aggregateless mutations were assigned to complementation groups agoB, Cy D and E (Table 3 ) . If as is thought likely, ago-48 (in strain NP107) is not a member of complementation group agoA, no interallelic complementation has been observed between any of the strains containing mutations in agoA (Table 3 ) . This result would be consistent with the gene product of agoA being a protein with only a single subunit.

It remains possible that substantial interallelic complementation between aggregateless mutations in the same cistron may result in diploids scored as class 3, although we have no evidence for this suggestion at present.

Fine structure analysis of particular aggregateless loci: If there is recombination within the aggregateless cistron in a non-complementing diploid containing two aggregateless mutations in trans, the result may be the formation of a diploid which aggregates and sporulates normally (class 1 ) . Such diploids have been observed in complementation groups agoA (from diploids between NP37/NP113; NP122/NPl13; NP123/NP113) and agoB (from the diploid NP106/NP136).

Analysis of intragenic recombinants allows the fine structure mapping of particular aggregateless loci. This is most easily studied in complementation group agoA where there is no interallelic complementation, so that any diploid spores formed almost certainly result from intragenic recombination. On clonal platings

302 K. L. WILLIAMS AND P. C . NEWELL

of aggregateless diploids containing two different agoA mutations in trans occa- sionally a sporulating sector arises that proves to be a class 1 diploid on clonal isolation. This contrasts strongly with the very rare spore masses observed in class 4 diploids which on clonal isolation produce only class 4 diploid colonies.

The frequency with which diploids with two agoA mutations sector such class 1 diploid colonies varies depending on the two agoA mutations used. For example, the aggregateless diploid containing agoA54 and agoA57 in trans produces occasional class 1 intragenic recombinant diploids, whereas none have been observed on several clonal platings of the diploid containing agoA8 and agoA54 in trans. Experiments are in progress to order several agoA mutations and to construct a map of the agoA locus.

Assignment of aggregateless mutations to linkage groups: Although the experiments reported here were not designed primarily to test linkage of different aggregateless mutations, information was gained concerning linkage when haploids were segregated from diploids on SM agar plates containing cycloheximide, methanol or cobalt (Figure 4). Hence some aggregateless mutations have been provisionally assigned to linkage groups. A few examples are given here.

Linkage group Z: The following results are consistent with agoB72 (in strain NP136) being located on linkage group I proximal to cycA or on the other chromosome arm. The mutations agoB71 and cycAl were arranged in cis on linkage group I in 25 platings of different diploids of the genotype agoB7I cycA2 +/+ 4- sprA. The order centromere-cycA-sprA has been previously established ( KATZ and SUSSMAN 1972; WILLIAMS, KESSIN and NEWELL 1974a). When these diploids were plated onto SM agar containing cycloheximide, aggregateless haploid strains were observed in all 25 platings, while cycloheximide-resistant diploid colonies that sporulated were also observed in 9 platings. These sporulating diploids presumably arose a5 a result of mitotic crossing over distal to agoB7l but proximal to cycAl . In addition, haploid segregants which sporulated were obtained by segregating heterozygous diploids of the genotype shown above on methanol- or cobalt-containing plates. All had round spores and were sensitive to cycloheximide, indicating the presence of the chromosome bearing the wild-type allele at both the cycA and agoB loci and the mutant allele at the sprA locus.

Linkage group ZZ: The locus agoA has been located on linkage group I1 proximal to the white spore locus (whi) or on the other chromosome arm, by analysis of diploids containing either agoA33 (from strain NP92) or agoA54 (from strain NP113), by analysis of a number of diploids of the genotype agoA whi acrA tsgD/+ + + +. The map order is centromere-whi-acrA-tsgD (MOSSES, WILLIAMS and NEWELL 1975). All haploid strains that segregated from such diploids on methanol-containing plates (i.e., containing acrA) were aggregateless, while both white-spored and yellow-spored diploid strains were obtained. These sporulating diploids presumably arose from mitotic crossovers distal to agoA but proximal to acrA, and either proximal or distal to whi.

Another aggregateless mutation ago-41 (in strain NPIOO) is probably located on linkage group I1 proximal to whi or on the other arm of linkage group 11, since when diploids of the genotype ago-41 whi acrA tsgD/+ 4- + 4- were segre-


gated on methanol in ten different experiments, all haploids were aggregateless but on four occasions yellow-spored diploids were observed, while on one occasion a single white-spored diploid clone was observed.

In many of the diploids, the two aggregateless mutations were unlinked, since they sectored haploid strains which sporulated (Figure 4). By analyzing linkage relationships between different aggregateless mutations, it is clear that aggregateless mutations are spread widely over the seven linkage groups. Experiments are in progress to assign each of the aggregateless mutations to a specific linkage group.

Morphological characteristics of the aggregateless strains: We find that a number of mutant strains with a similar type of morphology fall into different complementation groups. One such type (Figure 3A) shows no sign of aggregation and amoebae remain as a flat lawn when starved. In general members of groups agoA, D and E fall into this category. A second type forms loose mounds of variable size with no normal streaming pattern. These mounds invariably fall apart after prolonged incubation. Members belonging to this type include those in group agoB.

While the same mutant morphology may be found in different complementation groups. the morphology within a group is generally similar with minor variations. For example, in group agoA strains NP37, 113,122 and 123 all show no sign of aggregation, while strains NP41 and NP92 can sometimes show loose shallow mounds o r ripples which later degenerate into a flat lawn of cells.

The vast majority of our aggregateless mutants have a morphology similar to that shown in Figure 3A or 3B; hence most aggregateless mutants in different complementation groups cannot be distinguished on morphological grounds. However, we have observed a few very distinctive morphological types which may represent specific complementation groups. Strain NP94 forms relatively tight, stable mounds of amoebae (Figure 3E). The aggregateless mutation ago-35 in this strain complements all 39 other aggregateless mutations tested (Figure 2 and additional experiments) and also differs morphologically from all of them. Similarly the aggregateless mutation ago-62 in strain NP127 (which shows extensive streaming patterns, Figure 3D) complements 20 other aggregateless mutations (Figure 2), none of which have the phenotype of NP127.

DISCUSSION

We show in this paper that the process of aggregation in D. discoideum is amenable to analysis using parasexual genetic techniques. The diploids formed from our genetically marked haploid strains are sufficiently stable to allow their cultivation and inspection for possible complementation of aggregateless mutations. In the absence of auxotrophic markers in this organism we have employed recessive drug resistance mutations to enable us to select for known linkage groups. We have found that with the aid of such selective markers on three linkage groups (to select haploid clones for heterozygous diploids) we have been able to demonstrate whether or not given aggregateless mutations are linked. It


is clear that aggregateless mutations are spread widely on the chromosomes and represent a number of different complementation groups. Five complementation groups have been defined by patterns of non-complementation and they are reported here as named groups (agoA, B, C, D and E ) , agoB being on linkage group I and agoA on linkage group 11.

A potentially important outcome of the complementation experiments is the finding of the dominant aggregateless mutation ago-30 in strain NP89. This mutation is dominant over the wild type in diploids but is not dominant at the extracellular level; i.e., wild-type amoebae aggregate and form normal spore masses in the presence of equal numbers of amoebae of strain NP89. A dominance mechanism simply involving an extracurricular diffusible inhibitor is thereby ruled out. We suggest that the mutated gene may code for some regula- tory macromolecule that controls expression of genes necessary for aggregation. The mutation may therefore be somewhat allalogous to the super repressor (is) mutation found in bacteria (JACOB and MONOD 1961), although other possibilities must be considered.

The results of the complementation tests detailed in Figure2 can be used to estimate the genetic complexity of aggregation in D. discoideum. In a similar study of the genetic complexity of aggregation in the slime mould Polysphondylium uiolaceum (WARREN, WARREN and Cox 1975), an estimate of approximately 50 complementation units was made. This estimate was based on the use of a statistical test known as the maximum likelihood estimator, which is described in detail in J. WARREN’S Ph.D. thesis (Princeton University, 1974). An important assumption of the maximum likelihood estimator is that all genes required for aggregation are equally mutable, and that in the process of isolating the aggregateless mutants there was no differential selection for or against a strain carrying a particular aggregateless mutation. This assumption does not hold for the study of WARREN, WARREN and Cox (1975), and it seems that in this study strains carrying mutations in complementation groups agoA and agoB were observed at a frequency higher than would be expected at random. WARREN, WARREN and Cox (1975) excluded the ‘hot spots’ from their calculations. If the same process is applied here (by excluding agoA and agoR) , we obtain an estimate similar to that of WARREN, WARREN and Cox (1975). Hence we conclude that in D. discoideum there are of the order of 50 aggregation-specific genes in which mutations prevent aggregation. I t is not possible to provide a more accurate estimate at this stage because not all crosses have been performed, and it may be argued that there are still some ambiguities in the interpretation of some complementation tests. It is of interest to note that from genetic data the number of operons thought to be involved in the sporulation sequence in Bacillus subtilis has recently been estimated to be about 40 (PIGGOT 1973; HRANUELI, PIGGOT and MANDELSTAM 19 74).

Our estimate of the order of 50 aggregation-specific genes is at least an order of magnitude lower than the estimate of FIRTEL (1972) based on single-copy DNA/RNA hybridization studies. FIRTEL (1972, Figure 4) estimates that 1000 new transcripts are produced over the period 0-6 hours and 4000 new transcripts


are produced over the entire aggregation period of 0-12 hours in D. discoideum. A similar disparity between estimates of gene numbers based on genetic analysis and single-copy DNAJRNA hybridization studies has also been observed in Drosophila melansgaster (JUDD, SHEN and KAUFMAN 1972; HOCHMAN 1971 ; TURNER and LAIRD 1973) and possibly suggests that a gene defined by complementation tests may contain approximately ten times more RNA transcript than that actually translated into protein (see BISHOP 1974 for a discussion of this point). This suggestion is contradicted in D. discoideum by the finding that primary RNA transcripts are only about ZOO/, longer than messenger RNA (LODISH et al. 1974).

There are a number of possible reasons for the discrepancy between the results cf FIRTEL (1972) and ourselves, but only two which can be tested experimentally will be mentioned. If there are between 1000 and 4000 newly transcribed genes during aggregation (excluding those involved with “housekeeping” functions) and only about 50 (approximately 5 % ) prevent aggregation, the remaining new transcripts may code for m l l genes for aggregation; for example they may code for proteins required later in development that are involved with stalk and spore differentiation. If this suggestion is correct one would predict that complementation analysis of developmental mutants affected after aggregation would reveal a large number of different genes (at least 1000). Alternatively the remaining 95% of newly transcribed genes may code for proteins involved with ‘Lfine tuning” of aggregation. Hence one may propose that the coarse control of aggregation (on/off) involves a small number of genes, while large numbers are required for “fine tuning”. This suggestion can also be tested experimentally using complementation analysis of mutants in which aggregation occurs but is aberrant. We are collecting mutants with various alterations in aggregation, ranging from strains which form small closely spaced fruiting bodies to those which produce large widely spaced fruiting bodies. I t is important to establish whether these mutants are leaky variants at the complementation groups defined in this study, or whether they represent additional (fine tuning) loci, and if so how many such loci there are.

There is a considerable amount of biochemical information concerning aggregation in D. discoideum. It may be asked whether the genetic complexity estimated here is consistent with the biochemical information. Known compon- ents specific for aggregation in D. discoideum include chemotactic and cell contact systems. The chemotatic system involves the ability to secrete pulses of cyclic AMP, to respond to these pulses (which involves binding the cyclic AMP) and to relay cyclic AMP in response to receiving it. A phosphodiesterase system (believed to be involved with lowering the cyclic AMP concentration) is also present, as is an inhibitor of phosphodiesterase (BONNER et al. 1969; GERISCH 1968; MALCHOW and GERISCH 1974; ALCANTARA and MONK 1974; CHANG 1968; RIEDEL et al. 1972. For reviews see NEWELL 1971 and 1975; GERISCH, MALCHOW and HESS 1974). Less is known about cell contact, but it is known that there are antigenic cell surface sites specific for aggregation (BEUG, KATZ and GERISCH 1973). We consider that from such studies the number of gene products known


to be involved with aggregation is not inconsistent with there being approximately 50 genes that are specific and essential for the aggregation process.

We gratefully acknowledge the financial support of the Science Research Council (UK) . We also thank MR. JOHN HUGHES for technical assistance, DR. R. H. KESSIN for isolating mutants NP37-NP45 during his stay in Oxford, DR. B. COUKELL and PROFESSOR F. ROTHMAN for details of their methods for storing aggregateless strains a t -70" and DR. J. WARREN for preprints of her papers and Ph.D. thesis.

LITERATURE CITED

Signal propagation during aggregation in the cellular slime

Dynamics of antigen membrane sites relating to

ALCANTARA, F. and M. MONK, 1974 mould Dictyostelium discoideum. J. Gen. Microbiol. 85 : 321-334.

BEUG, H., F. E. KATZ and G. GERISCH, 1973 cell aggregation in Dictyostelium discoideum. J. Cell Biol. 56: 647-658.

BISHOP, J. O., 1974 The gene numbers game. Cell. 2: 81-85. BONNER, J. T., D. S. BARKLEY, E. M. HALL, T. M. KONIJN, J. W. MASON, G. O'KEEFE and P. B.

WOLFE, 1969 Acrasin, acrasinase and the sensitivity to acrasin in Dictyostelium discoideum. Devel. Biol. 20: 72-87.

BRODY, T. and K. L. WILLIAMS, 1974 Cytological analysis of the parasexual cycle in Dictyo- stelium discoideum. J. Gen. Microbiol82 : 371-383.

CHANG, Y. Y., 1968 Cyclic 3'5'-Adenosine monophosphate phosphodiesterase produced by the slime mould Dictyostelium discoideum. Science 161 : 57-59.

CLARK, M. A., D. FRANCIS and R. EISENRERG, 1973 Mating types in cellular slime moulds. Biochem. Biophys. Res. Commun. 52: 672-678.

DEMEREC, M., E. A. ADELBERG, A. J. CLARK and P. E. HARTMAN, 1966 A proposal for a uniform nomenclature in bacterial genetics. Genetics 54.: 61-76.

ERDOS, G. W., K. B. RAPER and L. K. VOGEN, 1973 Mating types and macrocyst formation in Dictyostelium discoideum. Proc. Natl. Acad. Sci. US. 70 : 1828-1830.

FIRTEL, R. A., 1972 Changes in the expression of single-copy DNA during development of the cellular slime mould Dictyostelium discoideum. J. Mol. Biol. 66: 363-377.

GERISCH, G., 1968 Cell aggregation and differentiation in Dictyostelium. Current Topics in Devel. Biol. 3: 157-197.

GERISCH, G., D. MALCHOW and G. HESS, 1974 Cell communication and cyclic-AMP regulation during aggregation of the slime mould, Dictyostelium discoideum. Mosbacher Colloguium der Gesellshaft f i i r Biologic Chemie. Edited by L. JAENICKE. 25: 279-298.

Evidence for mitotic crossing over during the parasexual cycle of the cellular slime mo~ild Dictyostelium discoideum. J. Gen. Microbid. 84: 70-78.

GREEN, A. A. and P. C. NEWELL, 1974 The isolation and subfractionation of plasma membrane from the cellular slime mould Dictyostelium discoideum. Biochem. J. 140 : 313-322.

HOCHMAN, B., 1971 Analysis of chromosome 4 in Drosophila melanogaster. 11: Ethyl methane sulfonate induced lethals. Genetics 67: 235-252.

HOLLIDAY, R. and J. E. PUGH, 1975 DNA modification mechanism and gene activity during development. Science 187: 226-232.

HRANUELI, D., P. J. PICCOT and J. MANDELSTAM, 1974 Statistical estimate of the total number of operons specific for Bacillus subtilis sporulation. J. Bacteriol. 119: 684-690.

JACOB, F. and J. MONOD, 1961 Quant. Biol. 26: 193-211.

GINGOLD, E. B. and J. M. ASHWORTH, 1974

On the regulation of gene activity. Cold Spring Harbor Symp.


The anatomy and function of a segment of the X chromosome of Drosophila melanogaster. Genetics 71 : 139-156.

Evidence for mitotic recombination in the cellular slime mould Dictyostelium discoideum. Proc. Natl. Acad. Sci. US. 71 : 4025-4026.

Parasexual recombination in Dictyostelium discoideum. Selection of stable heterozygotes and stable haploid segregants. Proc. Natl. Acad. Sci. U.S. 69: 495-49s.

Linkage analysis in Dictyostelium discoideum using temperature-sensitive growth mutants selected with bromodeoxyuridine. J. Bacteriol. 119: 776-783.

Synthesis of messenger RNA and chromosome structure in the cellular slime mould. Proc. Natl. Acad. Sci. U.S. 71 :

Short-term binding and hydrolysis of cyclic 3’5’-adenosine monophosphate by aggregating Dictyostelium cells. Proc. Natl. Acad. Sci. U.S. 71 : 2423- 2427.

The use of mitotic crossing-over for genetic analysis in Dictostelium discoideum: mapping of linkage group 11. J. Gen. Microbiol. 90 : 247-253.

NEWELL, P. C., 1971 The development of the cellular slime mould Dictyostelium discoideum. A model system for the study of cellular differentiation. Essays in Biochemistry 7: 87-126. _- , Cellular communication during aggregation of the slime mould Dictyostelium. In: Microbiology. Edited by M. DWORKIN and L. SHAPIRO. Am. Soc. for Microbiol. publica- tion. (In Press.)

PIGGOT, P. J., 1973 Mapping of asporogenous mutations of Bacillus subtilis. A minimal estimate of the number 3f sporulation operons. J. Bacteriol. 114: 1241-1253.

RAPER, K. B., 1935 Dictyostelium discoideum, a new species of slime mould from decaying forest leaves. J. Agr. Res. 50: 135-147.

RIEDEL, V., D. MALCHOW, G. GERISCH and B. NAGELE, 1972 Cyclic AMP phosphodiesterase interaction with its inhibitor of the slime mould Dictyostelium discoideum. Biochem. Biophys. Res. Commun. 46: 279-284.

Biochemical and genetic methods in the study of cellular slime mould development. pp. 397-410. In: Methods in Cell Physiology. Vol. 2. Edited by D. PRESCOTT. Academic Press, New York.

TURNER, S. H. and C. D. LAIRD, 1973 Diversity of RNA sequences in Drosophila melanogaster. Biochem. Genet. 10: 263-274.

WARREN, A. J., W. D. WARREN and E. C. Cox, 1975 Genetic complexity of aggregation in the cellular slime mould Polysphondylium violaceum. Proc. Natl. Acad. Sci. US. 72 : 1041- 1042.

Parasexual genetics in Dictyostelium disco‘deum: mitotic analysis of acriflavin resistance and growth in axenic medium. J. Gen. Microbiol. 84: 59-69. --, 197413 Genetics of growth in axenic medium of the cellular slime mould Dictyostelium discoideum. Nature 274 : 142-143.

Corresponding editor: D. R. STADLER

JUDD, B. H., M. W. SHEN and T. C. KAUFMAN, 1972

KATZ, E. R. and V. KAO, 1974

KATZ, E. R. and M. SUSSMAN, 1972

KESSIN, R. H., K. L. WILLIAMS and P. C. NEWELL, 1974

LODISH, H. F., A. JACOBSON, R. FIRTEL, T. ALTON and J. TUCHMAN, 1974

51 03-51 08.

MALCHOW, D. and G. GERISCH, 1974

MOSSES, D., K. L. WILLIAMS and P. C. NEWELL, 1975

1975

SUSSMAN, M., 1966

WILLIAMS, K. L., R. H. KESSIN and P. C. NEWELL, 1974a

of - genetics · a genetic study of aggregation in the cellular slime mould dictyostelzum...

Documents