chromosome fragments in dictyostelium discoideum obtained from parasexual crosses between strains of...

CHROMOSOME FRAGMENTS IN DICTYOSTELIUM DZSCOZDEUM OBTAINED FROM PARASEXUAL CROSSES BETWEEN STRAINS

OF DIFFERENT GENETIC BACKGROUND

KEITH L. WILLIAMS+, GILLIAN E. ROBSON AND DENNIS L. WELKERt

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

Manuscript received November 11, 1979 Revised copy received January 28, 1980

ABSTRACT

The first aneuploid strains o€ Dictyostelium discoideum have been unam- biguously characterized, using cytological and genetic analysis. Three independently isolated, but genetically similar, fragment chromosomes have been observed in segregants from diploids formed between haploid strains derived from the NC4 and VI2 isolates of D. discoideum. Once generated, the fragment chromosomes, all of which have Vl2-derived centromeres, can be maintained in a NG4 genetic background. Genetic evidence is consistent with the view that all three fragment chromosomes studied encompass the region from the centromere to the whiA locus of linkage group I1 and terminate in the interval between whiA and acrA. From cytological studies, one of the fragment chromosomes consists of approximately half of linkage group 11.-We observed no deleterious effect: on viability or asexual fruiting-body formation in either haploid or diploid strains carrying an additional incomplete chromosome and hence are disomic or trisomic, respectively, for part of linkage group 11. The incomplete chromosome is lost at a frequency of 2 to 3% from disomic and trisomic strains, but surprisingly this loss is not increased in the presence of the haploidizing agent, benlate. A new locus (c lyA), whose phenotype is altered colony morphology, is assigned to the region of linkage group I1 encompassed by the fragment chromosome.

NEUPLOID strains have been used extensively for genetic studies in a A variety of organisms. For example, many markers have been mapped to linkage groups by studying gene dosage in aneuploid plants (CARLSON 1972; HART and LANGSTON 1977), fungi (MORTIMER and HAWTHORNE 1973) and insects (O'BRIEN and GETHMANN 1973). The ease of isolation of aneuploids and characteristics such as their stability and viability varies greatly in similar organisms [e.g., compare Aspergillus nidulans (=FER and UPSHALL 1973; =FER 1977) with Neurospora crassa (PERKINS and BARRY 1977)] and for different chromosomes within a single organism [e.g., A . nidulans (=FER and UPSHALL 1973)l. To extend genetic analysis in the cellular slime mold Dictyoste- Zium discoideum, we have been searching for aneuploid strains. It is established that diploids of D. discoideum haploidize via transient aneuploidy ( SINHA and

+ Present address M a x Plan& Institut fur Biochemie D-8033 Martmsried bel Munchen Fed. Rep. Germany.

Genetics 95: 289-304 June, 1980

290 K. L. WILLIAMS7 G . E. ROBSON A N D D. L. WELKER

ASHWORTH 1969; BRODY and WILLIAMS 1974), but there are no convincing reports of aneuploid strains. Although as yet we have found no strains containing an extra whole chromosome, we report here the isolation of strains with an extra chomosome that is apparently a centromere-attached fragment of linkage group 11.

MATERIALS AND METHODS

Media: SM agar and inhibitor containing SM agar was prepared exactly as described previously (WILLIAMS 1978; WILLIAMS and BARRAND 1978).

Strains: D. discoid&m was grown on a cobaltous-chloride-resistant strain of Klebsiella aerogenes, and strains used, which were derived from the NC4 or VI2 isolates, are described in Tables 1,2 and 3.

Maintenance of disomic and trisomic strains: Spores from “mass” plates were harvested and stored on silica gel at 4” (WILLIAMS and NEWELL 1976). For routine maintenance of the partially disomic and trisomic strains, amoebae or spores were suspended in Bonner’s salt solution (BONNER 1947), counted with a haemacytometer and known numbers plated to give single colonies on SM agar spread with K. uerogenes at 21 c I”. The strains were recloned each week. This method is a more rigorous way of obtaining single colonies than.the standard method for maintaining strains, which involves using a wire loop to streak a suspension of spores or amoebae onto a SM agar plate previously spread with K. aerogenes.

Parasexual diploid formiion: Standard techniques were used to form diploids (WILLIAMS and NEWELL 1976). In the case of diploids (e.g., DU707) formed between strains of opposite mating type, repetitive crossing was necessary, as such “escaped” diploids are formed only at a very low frequency (- 10-8) (ROBSON and WILLIAMS 1979).

Haploidizing diploid strains: Diploids or trisomic strains were routinely haploidized by plating low numbers of amoebae plus K. aerogenes on SM agar containing 20 ,ug/ml benlate (WILLIAMS and BARRAND 1978). In some cases, diploids that were heterozygous for a recessive resistance mutation were haploidized by plating - 2 x IO4 amoebae on SM agar containing the appropriate inhibitor (WILLIAMS, KESSIN and NEWELL 1974).

Chromosome studies: A rapid method was used to obtain amoebae arrested in metaphase (WELXER and WILLIAMS 1980). Briefly, amoebae were scraped from the edge of a clone on SM agar and suspended at between - 2 x 105 and 106Jml in 5 ml phosphate buffered saline pH 6.5 (DEERING et al. 1970) containing - IOS/ml E. coli B/r and 10 pg/ml thiabendazole (gift from Merck, Sharp and Dohme Research Labs., Ingleburn, N.S.W., Australia) and rotated at 150 rpm on an orbital shaker for 2.5 hr at 21 2 1 ’.

Amoebae were fixed and stained with Giemsa as described previously (ROBSON and WILLIAMS 1977). Slides were examined by bright-field light microscopy and photographs taken as described previously (WELKER and WILLIAMS 1980). The chromosome number of each strain was determined by examining at least 25 well-separated metaphases.

RESULTS

Partial disomic strains obtained from parasexual crosses between haploid strains derived from isolates NC4 and VI2 of D. discoideum In a previous report, we elucidated a vegetative incompatibility system in

D. discoideum in which diploids are only rarely formed (-IO-*) between strains of opposite mating type (ROBSON and WILLIAMS 1979). Such diploids were termed “escaped“ as they all had become homozygous for the mat& or mata2 allele at the mating-type locus. Several such diploids have now been formed between NC4 (matA2) -derived and VI2 (mata2) -derived strains; here we report

ANEUPLOID STRAINS OF D. discoideum 29 1

that, when they are induced to haploidize, these diploids occasionally produce chromosome breaks. The chromosome breakage is probably in a specific region of linkage group 11, and strains partially disomic for linkage group I1 are obtained. Partial disomic strains obtained from three “escaped” diploids are examined in detail here. One such “escaped” diploid, DU454, was subsequently shown to be a partial trisomic (it has 15 chromosomes), while the other two “escaped” diploids, DU707 and DP72, are normal 14-chromosome diploids. (1) Strain DU454, a partial trisomic for linkage group 11: Strain DU454 was

formed from a yellow-spored (whiA+) haploid strain (HU89) and a white- spored (whiA2) haploid strain (HU227, Table 1). As expected from the fact that whiA mutations are recessive to wild type, DU454 has yellow (wild-type) spores. However, the linkage group I1 genotype of haploid segregants from DU454 was either that of linkage group I1 of HU227 (whiA1, acrA1, tsgD12, sprB+, e.g., HU584) or a recombined linkage group I1 comprising whiA1, acrA+, tsgD+ and probably sprBP (e.g., HU440, Table 1). Hence, both linkage group I1 chromosomes in DU454 carry the mutant whiA1 allele. Yellow-spored “haploid’¶ segregants, carrying the whiA+ allele, were obtained from DU454, but these were unstable and spontaneously sectored white-spore strains. It seemed possible that these yellow-spored strains might be disomic for at least part of linkage group 11. This hypothesis was confirmed by chromosome studies, as approximately 95 % of metaphases of all yellow-spored “haploid” segregants examined from DU454 proved to have eight chromosomes, and white-spored segregants derived from them were all true haploids with at least 99% of metaphases having seven chromosomes. Details of four such partial disomic strains (HU317, HU329, HU336 and HU342) and haploids derived from three of them (HU584, HUM0 and HU588) are given in Figure 1 A through G and Table 1. Since no yellow-spored seven-chromosome haploids have been found from DU454, the chromosome containing the wild-type (yellow) allele at the whiA locus was probably incomplete.

From the above results, DU454 was predicted to be trisomic at the whiA locus (whiA1, whiA2, whiA+) and to have 15 chromosomes, despite the fact that it was formed from two normal seven-chromosome haploid strains, HU89 (Figure 2A) and HU227 (Figure 2B). This was confirmed cytologically (Figure 2C), and the presence of an additional fragment of linkage group I1 was inferred by its loss, revealed as the occurrence of white-spored (whiAl, whiAl) true diploids with 14 chromosomes (e.g., DU516 Figure 2D) at a frequency of about 1% in DU454 cultures.

To confirm our studies on DU454, a trisomic strain, DU545, was constructed by parasexual fusion between HU329 and HU77 (Table 2). The results obtained were as expected. Strain DU545 had yellow spores and 15 chromosomes (Figure 2E) and segregated white-spored 14-chromosome diploids (e.g., DUllOl Table 2 and Figure 2F) at a relatively high frequency by loss of the fragment chromosome. All yellow-spored segregants of DU545 with small spores proved to have about 95 % 8-chromosome metaphases and to sector white-spored haploid strains (over 99% metaphases with 7 chromosomes) ; no yellow-spored ‘/-chromosome

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ANEUPLOID STRAINS OF D. discoideum 293 - - - - - . ..- ...

FIGURE 1.-Representative metaphases of disomic strains (n + 1 = 8) segregated from DU454: (A) HU317, (C) HU329 (E) HU342, (G) HU336 and haploid segregants (n = 7) from HU317, HU329 and HU342 respectively: (B) HU584, (D) HU440, (F) HU588. These cytological preparations of the disomic strains contained between 92% and 96% 8-chromosome metaphases. Of the remaining metaphases, between 3% and 7% were haploid (n = 7) and 1% had 9 chromosomes. Bacteria are present in most photographs. The bar represents 2 &m.

FIGURB 2.-Metaphases of haploid strains (n = 7) : (A) HU89, (B) HU227, trisomic strains (2n + 1 = 15): (C) DU454, (E) DU545, and diploid strains (2n = 14): (D) DU516, (F) DUllOl. A considerable number of the metaphases in the diploid and trisomic preparations had less than 14 or 15 chromosomes, respectively. The number observed depends on the number of generations since the strains were cloned (i.e., colony diameter). For example, when amoebae of colonies of approximately equal size (40 mm) of trisomic strain DU545 and diploid strain Dull01 were examined, the following distributions of metaphases were observed: DU545, 6% haploid (n = 7), 27% aneuploid (n + x = 8 + 13), 30% diploid (2n = 14), 33% trisomic (2n+ 1 = 15) and 3% trisomic or tetrasomic (2x14- 2 = 16); DUllOl, 5% haploid (n = 7), 60% aneuploid (n + x = 8-3 13), 33% diploid (2n = 14), 2% trisomic (2x1 + 1 = 15). Values similar to those presented for diploid DU1101 have been observed previously in other diploid strains (BRODY and WILLIAMS 1974). Bacteria are present in most photographs. The bar represents 2 pm.

294 K. L. WILLIAMS, G . E. ROBSON AND D. L. WELKER

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ANEUPLOID STRAINS OF D. discoideum 295

strains were observed. All partial disomic and trisomic strains exhibited normal development and asexual fruiting body formation; thus disomy or trisomy for part of linkage group I1 does not have a characteristic phenotype.

(2) Generation of the fragment chromosome in DU454 may involve mitotic crossing-over: DU454 has two unusual features: it contains both a recombined linkage group I1 and an incomplete additional copy of linkage group 11. We propose that this strain may have been formed as a result of chromosome breakage during mitotic crossing over and subsequent nondisjunction, as detailed in Figure 3. This hypothesis provides the simplest explanation for the unusual features of DU464, since the recombined linkage group I1 and fragment chromosome are generated as part of the same event. To explain just the recombined linkage group I1 by conventional mitotic crossing over requires two crossover events, one proximal and a second distal to the whiA locus.

( 3 ) “Escaped” diploids DU707 and DP72: “Escaped” diploids DU707 and DP72 (Table 3), plus several other “escaped” diploids, were shown to be normal 14-chromosome diploids and to have the expected genotypes on linkage group 11, since normal 7-chromosome haploids were obtained with either the NC4-derived linkage group I1 (whiAl, acrAl, tsgDl2, sprB+ ; phenotype: white spores, meth-

PARENTAL LINKAGE GROUPS I1 LINKAGE GROUP II OF DU454

whiAl a c r A l t s g D l 2 + wkiA1 a c r A l t sgD12 + 0- 0 . - U

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Mitotic crossover in the whiA-acrA interval

N o t recovered

FIGURE 3.-Possible mechanism, involving crossing over, for the generation of the fragment chromosome and the recombined linkage group I1 in DU454. The above mechanism involves a single crossover event and chromosome breakage. It is attractive because the observed recombined linkage group I1 and fragment chromosome are produced by the same event. If the recombined linkage group I1 and chromosome fragment were generated independently, DU454 would result from a most unusual series of events, since to generate just the recombined linkage group I1 by conventional mitotic crossing over requires two crossovers, one centromere-proximal to whiA and one between whiA and acrA.


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anol resistant, temperature sensitive for growth, elliptical/oval spores) or the Vl2-derived linkage group I1 (whiA+, acrA+, isgD+, sprB2; phenotype: yellow spores, methanol sensitive, not temperature sensitive, thin elliptical spores). This showed that the Vl2-derived linkage group I1 was not lethal in a NC4 genetic background.

Partial disomic strains were readily isolated from “escaped” diploids. Details of two such strains, HU572 and X42. which segregated from DU707 and DP72, and a true haploid strain, HU571, obtained from HU572 are given in Table 3. The partial disomic strains were readily recognized as yellow-spored, methanol- resistant “haploids,” which in turn segregated white-spored true haploids. In all cases examined, the partial disomic strains had eight chromosomes in approximately 95% of metaphases (Figure 4A, X42; Figure 4B, HU572) while at least 99% of haploid metaphases had seven chromosomes. A partial trisomic strain (DU938) was constructed by crossing HU572 (seven chromosomes + the linkage group I1 fragment) with HM3 [a haploid, n = 7, white-spored (whiA900) strain derived from V12M2 by J. H. TRENT and R. R. KAY; see ROBSON and WILLIAMS 19791.

These 14-chromosome “escaped” diploids have not provided new information concerning the mechanism of the generation of the fragment chromosome, but they have shown that formation of this fragment is a general property of diploid strains carrying a NC4 linkage group I1 and a VI2 linkage group 11. The frequency of fragment chromosome formation can be quite high; e.g., one of 16 yellow-spored “haploid” segregants of DP72 obtained on 20 pg/ml benlate was a partial disomic strain. Likewise, partial disomic strains are often encountered when these diploids are segregated on methanol (WILLIAMS, KESSIN and NEWELL 1974).

So far, we have no evidence for partial disomy of other linkage groups from these diploids, nor have we encountered partial disomy at linkage group I1 from diploids formed between two NWderived or two V12-derived strains. Screening of a considerable number of strains, using newly developed, rapid chromosome staining techniques (WELKER and WILLIAMS 1980), has also failed to uncover any aneuploid or partial aneuploid strains other than those associated with the NC4/V12 linkage group I1 effect.

U A B C FIGURE 4.--Representative metaphases of independently derived disomic strains (n + 1 = 8)

(A) X42 and (B) HU572 (C) Late prophase of strain X42 in which the presumptive fragment chromosome (arrow) appears to be paired with the intact linkage group I1 chromosome. Bacteria are present in most photographs. The bar marker represents 2 pm.

298 K. L. WILLIAMS, G. E. ROBSON AND D. L. WELKER

Size of the fragment chromosomes, genetic evidence The genetic markers used to characterize linkage group I1 in this study are

whiA, acrA, tsgD and spB, and the gene order is centromere, whiA, acrA (tsgD/ sprB), with tsgD and sprB not yet ordered (MOSSES et al. 1975). All three fragments examined here (from DU454, X42 and HU572) carry a centromere, presumably from linkage group 11, and the wild-type allele at the whiA locus. On the other hand, all three fragment chromosomes must carry the mutant allele at acrA if they extend to the acrA locus because strains HU317 (Table l ) , X42 (Table 3) and HU572 (Table 3) are all methanol resistant; the phenotype of acrA mutations is recessive resistance to methanol. All three fragment chromosomes must also carry the wild-type allele at the sprB locus if they extend to that locus, since none of the three strains HU317 (Table l) , X42 (Table 3) and HU572 (Table 3) express the sprB2 phenotype, which is dominant to wild type. Thus, all three fragment chromosomes examined carry the Vl2-derived genotype from the centromere to the whiA-acrA interval; if they extend to and beyond the acrA locus, they carry the NC4-genotype for ncrA and distal markers (e.g., sprB+). The simplest interpretation of these results is that the fragment chromosomes terminate in the whiA-acrA interval (Figure 3 ) .

Size of the fragment chromosomes, cytological evidence We sought cytological confirmation of our proposal that the additional chro-

mosome represents an incomplete linkage group 11. The fortuitious pairing of the presumptive fragment chromosome and its corresponding linkage group I1 in a metaphase cell of X42 (Figure 4C) allowed an estimate to be made of the length of the fragment chromosome in this strain; the fragment encompassed about 50 % of linkage group 11.

Mapping a mutation that aflects colony morphology using the fragment chromosome A mutation affecting colony morphology whose phenotype is a wide band of

unaggregated amoebae at the edge of the colony (Figure 5B) , which we named cZyA351, has been identified on the intact chromosome proximal to acrA in the region encompassed by the fragment chromosome. Two of the disomic strains, HU329 and HU342, have a narrow band of unaggregated amoebae to the edge of their colonies (Figure 5A), while in their haploid segregants the mutant phenotype (wide band of unaggregated amobeae) is unmasked. Strain HU329 and its haploid segregant HU440 are shown in Figure 5A and 5B, respectively. Haploids spread more rapidly, which explains why disomic strains such as HU329 are overtaken by haploids if regular clonal isolations are not made. The cZyA351 mutation is expressed in the parent HU227 and in various other strains, since it resides on the chromosome carrying the whiAl allele. This chromosome can be traced to strain TSl2 (KATZ and SUSSMAN 1972), which also has a wide band of unaggregated amoebae. The phenotype is suppressible since some strains carrying clyA351 (eg., X22, WILLIAMS and NEWELL 1976) do not have a wide unaggregated edge to their colonies. The colony morphology phenotype of cZyA351 is


B FIGURE 5.-Colony morphology of (A) disomic strain HU329 (narrow band of unaggregated

amoebae) and (B) strain IKJ440, a spontaneous haploid segregant of I-IU329 that has a wide band of unaggregated amoebae. Between 10 and 20 amoebae of each strain were mixed with a 0.1 ml suspension of K . nerop~nes and spread on a 9cm SM agar plate. After the liquid had dried, the plates were incubated at 21.5 zk 1 O for &x days and then photographed.

not associated with the change in genome balance caused by the fragment chromosome, since HU317 and its haploid segregants (e.g., HU584) have a similar colony morphology.

Characterizalion of the stability and effect of benlate on a fragment chromosome.. The loss of the fragment chromosome, assayed mostly by the formation of

white-spored colonies. and the effect of the haploidizing agent, benlate, have been examined in several genetic backgrounds. The results are all similar to those presented in detail in Table 4 for the disomic strain HU329 and the trisomic strain DU545. Both strains showed greater than 80% viability on clonal platings on SM agar, as did haploid (HU440) and diploid (DUllOI) segregants from them. Both the disomic strain (HU329) and the trisomic strain (DU545) lost the fragment chromosome at a similar frequency; about 3% of colonies obtained from clonal plating of amoebae from colonies -20 mm diameter were haploid or diploid, respectively (Table 4). A similar frequency of haploid mitotic figures (IO of 220 = 5%) was observed in cytological preparations from the same freshly cloned colonies of disomic strain HU329. When the disomic and trisomic strains were not cloned regularly, the percentage of haploid and diploid amoebae respectively was greatly increased. Surprisingly, 20 pg/ml benlate, which causes essentially 100% haploidization of diploid strains (WILLIAMS and BARRAND 1978), did not affect the disomic strains (e.g., HU329) ; i.e., the fragment chromosome did not appear to be “recognized” as a chromosome (Table 4) . The plating efficiency of HU329 remained high in the presence of 20 pg/ml or 35 pg/ml benlate, and fewer than 5% haploids were obtained from clonal platings of HU329 on such benlate-containing SM agar plates. DU545 behaved like a normal diploid


TABLE 4

Viability of strains carrying the fragment chromosome (disomic or trisomic for part of linkage group 11) and the effect of the haploidizing agent benlate

Treatment

Disomic strain HU239(1 SM agar + K. aerogenes + benlate (20 pg/ml) + benlate (35 pg/ml)

Trisomic strain DU546(I SM agar f K. aerogenes + benlate (20pg/ml)$ + benlate (35 ,fig/ml)s

~~~ ~ ~

Number off Percentage

Viability* Haploids Diploids Disomicst Trisomics Haploids/ Diploids/ (%) (n=7) ( 2 1 ~ 1 4 ) (n-!-l=8) (2nfl=15) Disomics Trisomics

82 7 NA 194 NA 3/97 NA 80 2 NA 97 NA 2/98 NA 73 3 NA 126 NA 2/98 NA

83 0 Ill 0 33 o/o 3/97 40 22 0 8 1 -71,’-26 O/-3

7 6 0 4 0 -60/--4.0 O/O

* Viability determined as the percentage of colonies that grew after plating a known number of amoebae, determined by haemacytometer counts, from single disomic or trisomic clones (diameter 15-30 mm) growing on SM agar + K. aerogenes.

1. Haploids and disomics were distinguished from diploids and trisomics on the basis of their smaller spore size. Haploids and diploids had white spores, while disomics and trisomics had yellow spores.

3 Thirty-six of the yellow-spored colonies from HU329 and the I 2 yellow-spored colonies from DU545 were toothpicked on SM agar + K. aerogems to confirm that they sectored white; all 48 colonies examined did sector white-spored colonies.

S; Bias against disomic strains in favour of haploid strains here, since the colonies were scored after picking to SM agar, which favors haploids overtaking the disomic strain (see text).

11 A white-spored colony with large spores; confirmed to be a diploid (2n = 14) by counting chromosomes.

(1 The genotypes of HU329 and DU546 are given in Table 2. NA = Not applicable.

strain on benlate-containing SM agar. There was a marked decrease in the plating efficiency [similar to that reported in diploids by WILLIAMS and BARRAND (1978)l and essentially 100% reduction in ploidy from 15 to seven or eight chromosomes (Table 4). Given that there was some bias against the detection of 8-chromosome strains (see legend to Table 4), the segregation of haploid to disomic strains was not greatly different from 1:l; i.e., again the benlate did not cause loss of the fragment chromosome; it clearly did so for the intact chromosomes, since diploids or trisomics were only rarely obtained on SM agar containing benlate. The fragment chromosome can be maintained in a NC4 genetic background

All three fragment chromosomes discussed in this paper can be traced to the Vl2-derived parent. Essentially all D. discoideum genetics has been done in the NC4 genetic background; therefore, we asked whether a fragment chromosome could be maintained in this genetic background. Strains with eight chromosomes were segregated from DU545 and two independently isolated, 8-chromosome strains (HU580 and HU581) carrying six NC4-derived chromosomes and the fragment chromosome are shown in Table 2. Since linkage group V is currently unmarked ( RATNER and NEWELL 1978; WILLIAMS and WELKER unpublished), we cannot determine whether this chromosome is NC4- or Vl2-derived. The


fragment chromosome in strains HU580 and HU581 had a stability similar to that reported for HU329 in Table 4. Results of studies on other 8-chromosome strains obtained from DU454, or carrying the fragment chromosomes found in X42 or HU572, suggest strongly that, once generated, fragment chromosomes are similarly stable in a genetic background of strain NC4, in a mixture of NC4 and VI2 chromosomes or in a Vl2 genetic background, although the studies on strains with the V12 genetic background are less complete. The effect is unre- lated to mating type, as strains of either mating type can carry the fragment chromosome (Tables 2 and 3).

DISCUSSION

The partial aneuploid strains described here were discovered because they are heterozygous at the wZriA locus on linkage group 11; hence they are readily observed as apparently haploid (on the basis of spore size) yellow-spored colonies that sector white-spored true haploids. Cytological studies confirmed that the yellow-spored colonies had eight chromosomes (n = 7). While this case is the first unambiguous demonstration of aneuploid strains in the cellular slime moulds, there are other reports of aneuploid metaphases and aneuploid strains. There is little doubt that aneuploid amoebae do exist during the haploidization of diploid strains in the parasexual cycle (SINHA and ASHWORTH 1969; BRODY and WILLIAMS 1974) and when amoebae are treated with antimitotic compounds (WELKER and WILLIAMS 1980). Under these conditions, the viability is low and no aneuploid strains were obtained from cultures that contained 50 % aneuploid metaphases (BRODY and WILLIAMS 1974). We propose that it may not be possible in D. discoideum to isolate strains disomic or trisomic for whole chromosomes because of genome inbalance resulting in both mitotic instability and inviability. The fact that previously described putative aneuploids carrying both mating- type alleles, which were obtained from germinating macrocysts, were resolved into strains of one or other mating type within three clonings (D. discoideum, WALLACE and RAPER 1979; D giganteum, ERDOS, RAPER and VOGEN 1975), could be consistent with a mixture of amoebae rather than aneuploidy. Aneuploidy was suggested in another cellular slime mold species, Polysphondylium violaceum, by WARREN, WARREN and Cox (1976) to explain segregation of colonies of different morphology from clones with close to haploid spore size. This report requires substantiation using chromosome studies because in unpublished experiments we have observed similar sectoring of colonies of different morphology from D. discoideum in which both the parent and sectored colonies had seven chromosomes.

The fragment chromosomes described here in D. discoideum, which comprise about 50% of the intact linkage group I1 chromosome and approximately 10% of the total genome, have little, if any, effect on the genome balance in D. discoideum, as both haploid and diploid strains carrying the fragment show normal morphogenesis and have high viability. Their mating capacity in the sexual cycle is probably unimpaired (i.e., disomic strains form macrocysts when paired with

302 K. L. WILLIAMS, G. E. ROBSON AND D. L. WELKER

haploids of opposite mating type). However, the fragment chromosomes do not behave normally at mitosis since they are mitotically unstable and treatment with benlate, which is an extremely effective haploidizing agent, does not result in their loss. It has been shown that benlate binds to tubulin and affects the mitotic spindle in A . nidulans (DAVIDSE and FLACH 1977), and it probably has a similar action in D. discoideun (WELKER and WILLIAMS 1980). Thus, it seems possible that the fragment chromosomes do not bind to the mitotic spindle in the normal way.

The mechanism for the generation of the fragment chromosome has not been conclusively elucidated here, although breakage in the whiA-acrA interval associated with mitotic crossing over is consistent with all current information and is the simplest explanation. If this interpretation is correct, the “fragment” chromosomes may not have a telomere in the classic sense since the acquisition of a telomere would involve an additional chromosome break ( CAVALIER-SMITH 1974). The reason for a high level of chromosome breakage associated with putative mitotic recombination in this region (Figure 3) remains obscure. Since the fragment chromosomes have been found only in segregants from diploids carrying linkage group I1 of both the NG% and V12 isolates, some chromosomal rearrangement between linkage group I1 of these isolates may be involved. While the genetic events involved are as yet unclear, it is apparent that similar events have been observed in a variety of organisms. Possible fragment chromosomes were obtained in N . crassa in meiotic studies as unusual pseudo-wild-type ascospores (THRELKELD 1962). Chromosome loss has been associated with mitotic exchange in S . cerevisiae, and there is preliminary evidence for chromosome breakage (formation of telomeric chromosomes from metacentrics) in that work (CAMPBELL and FOGEL 1977). Finally, there is convincing evidence for non- random chromosome breaks in human lymphocyte cultures ( WELCH and LEE 1975; BEATTY-DESANA, HOGGARD and COOLEDGE 1975; HECHT et al. 1975). The system outlined here in L). discoideum may be useful for probing the genetic features of these unusual observations, since the fragment chromosomes may be recognized both visually, using heterozygosity at the whiA locus, and cytologically.

A fragment chromosome was used to locate a mutation that affects colony morphology, cZyA, to linkage group I1 in this study. In principle, it should be possible to locate other mutations involved in complex morphological phenotypes, such as cell patterning, using partial disomic strains and their haploid segregants, since only the region covered by the fragment chromosome is examined. Also, it should be possible to commence mapping using gene dosage (CARL- SON 1972; MORTIMER and HAWTHORNE 1973; O’BRIEN and GETHMANN 1973), although for this work additional fragment chromosomes are needed. One approach to obtaining strains disomic for other chromosome fragments may be to examine segregants from diploids formed between other pairs of wild isolates in the hope that other linkage groups may carry the factors (e.g., rearrangements) that lead to generation of fragment chromosomes. Several such diploids have already been constructed (ROBSON and WILLIAMS 1979).


We thank J. A. PATEMAN and M. J. D. WHITE and one of the referees for helpful comments. D. L. WELKER acknowledges support by Public Health Service Fellowship 1 F32 CA06162-01 from the National Cancer Institute. We thank AILSA CHAMBERS for technical assistance.

LITERATURE CITED

BEATTY-DESANA, J. W., M. J. HOGGARD and J. W. COOLEDGE, 1975 Non-random occurrence of

BONNER, J. T., 1947 Evidence for the formation of cell aggregates by chemotaxis in the develop-

BRODY, T. and K. L. WILLIAMS, 1974 Cytological analysis of the parasexual cycle in Dictyo-

CAMPBELL, D. A. and S. FOGEL, 1977 Association of chromosome loss with centromere-adjacent

CARLSON, P. S., 1972 Locating genetic loci with aneuploids. Molec. Gen. Genet. 114: 273-280. CAVALIER-SMITH, T., 1974 Palindromic base sequences and replication of eukaryote chromosome

ends. Nature 250: 467470. DAVIDSE, L. C. and W. FLACH, 1977 Differential binding of methyl benzimidazol-2-yl carbamate

to fungal tubulin as a mechanism of resistance to this antimitotic agent in mutant strains of Aspergillus nidulans. J. Cell Biol. 72: 174-193.

DEERING, R. A., M. S. SMITH, B. K. THOMPSON and A. C. ADOLF, 1970 Gamma-ray-resistant and sensitive strains of slime mould (Dictyostelium diswideum). Radiation Res. 43: 711-728.

ERDOS, G. W., K. B. RAPER and L. K. VOGEN, 1975 Sexuality in the cellular slime mold Dictyo- stelium discoideum. Proc. Natl. Acad. Sci. U.S. 72: 970-973.

HART, G. E. and P. J. LANGSTON, 1977 Chromosomal location and evolution of isozyme structural genes in hexaploid wheat. Heredity 39: 263-277.

HECHT, F., B. K. MCCAW, D. PEAKMAN and A. ROBINSON, 1975 Non-random occurrence of 7-14 translocations in human lymphocyte cultures. Nature 255: 243-244.

KHFER, E., 1977 Meiotic and mitotic recombination in Aspergillus and its chromosomal aberra- tions. Advan. Genet. 19: 33-131.

KAFER, E. and A. UPSHALL, 1973 The phenotypes of the eight disomics and trisomics of Asper- gillus nidulans. J. Heredity 64: 35-38.

KATZ, E. R. and M. SUSSMAN, 1972 Parasexual recombination in Dictyostelium discoideum: Selection of stable heterozygotes and stable haploid segregants. Proc. Natl. Acad. Sci. U.S. 69: 4.95498.

Genetic mapping in Saccharomyces. IV. Mapping of temperature-sensitive genes and use of disomic strains in localizing genes. Genetics. 74: 33-54.

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

7-14 translocations in human lymphocyte cultures. Nature 255 : 242-%3.

ment of the slime mold Dictyostelium diswidaum. J. Exp. Zool. 106: 1-26.

stelium discoideum. J. Gen. Microbiol. 82: 371-383.

mitotic recombination in a yeast disomic haploid. Genetics 85: 573685.

MORTIMER, R. K. and D. C. HAWTHORNE, 1973

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

247-259. NEWELL, P. C., 1978 O'BRIEN, S. J. and R. C. GETHMANN, 1973 Segmental aneuploidy as a probe for structural genes

PERRINS, D. D. and E. G. BARRY, 1977 The cytogenetics of Neurospora. Advan. Genet. 19:

RATNER, D. I. and P. C. NEWELL, 1978 Linkage analysis in Dictyostelium discoideum using multiply marked tester strains: establishment of linkage group VI1 and the reassessment of earlier linkage data. J. Gen. Microbiol. 109 : 225-236.

Genetics of the cellular slime molds. Ann. Rev. Genet. 12: 69-93.

in Drosophila: mitochondrial membrane enzymes. Genetics 75: 155-167.

133-285.

3 04 K. L. WILLIAMS, G. E. ROBSON A N D D. L. W E L K E R

ROBSON, G. E. and K. L. WILLIAMS, 1977 The mitotic chromosomes of the cellular slime mold Dictyostelium discoideum: a karyotype based on Giemsa banding. J. Gen. Microbiol. 99: 191-200. --, 1979 Vegetative incompatibility and the mating-type locus in the cellular slime mold Dictyoszelium discoideum. Genetics 93: 861-875.

SINHA, U. and J. M. ASHWORTH, 1969 Evidence for the existence of elements of a parasexual cycle in the cellular slime mould Dictyostelium discoideum. Proc. Roy. Soc. B. 173: 531-540.

THRELKELD, S. F. H., 1962 Some asci with nonidentical sister spores from a cross in Neurosporu crassa. Genetics 47: 1187-1198.

WALLACE, M. A. and K. B. RAPER, 1979 Genetic exchanges in the macrocysts of Dictyostelium discoideum. J. Gen. Microbiol. 113 : 327-337.

WARREN, A. J., W. D. WARREN and E. C. Cox, 1976 Genetic and morphological study of aggregation in the cellular slime mold Polysphondylium violaceum Genetics 83: 2547.

WELCH, J. P. and C. L. Y. LEE, 1975 Non-random occurrence of 7-14 translocations in human lymphocyte cultures. Nature 255: 241-242.

WELKER, D. L. and K. L. WILLIAMS, 1980 Mitotic arrest and chromosome doubling using thiabendazole, cambendazole, nocodazole and benlate in the slime mould Dictyostelium discoi- hum. J. Gen. Microbiol. 116: 397-407.

Characterization of dominant resistance to cobalt chloride in Dictyo- stelium discoideum and its use in parasexual genetic analysis. Genetics 90: 37-47.

Parasexual genetics in the cellular slime mould Dic- tyostelium discoideum: haploidisation of diploid strains using benlate. FEMS Microbiol. Letters 4: 155-159.

Parasexual genetics in Dictyostelium discoideum: mitotic analysis of acriflavin resistance and growth in axenic medium. J. Gen. Microbiol. 84: 59-69.

A genetic study of aggregation in the cellular slime mould Dictyostelium discoideum using complementation analysis. Genetics 82 : 287-307.

Corersponding editor: D. SCHLESSINCER

WILLIAMS, K. L., 1978

WILLIAMS, K. L. and P. BARRAND, 1978

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

WILLIAMS, K. L. and P. C. NEWELL, 1976

chromosome fragments in dictyostelium discoideum obtained from parasexual crosses between strains of...

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