High Frequency Recombination During the Sexual Cycle of Dictyostelium discoideum

Corresponding author: David Francis, Department of Biological Sciences, University of Delaware, Newark, DE 19716, mkarcz{at}udel.edu (E-mail).

Communicating editor: S. L. ALLEN

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Analysis of Dictyostelium development and cell biology has suffered from the lack of an ordinary genetic system whereby genes can be arranged in new combinations. Genetic exchange between two long ignored strains, A₂Cyc^r and WS205 is here reexamined. Alleles which differ in size or restriction sites between these two strains were found for seven genes. Six of these are in two clusters on chromosome 2. Frequencies of recombinant progeny indicate that the genetic map of the two mating strains is colinear with the physical map recently worked out for the standard nonsexual strain, NC4. The rate of recombination is high, about 0.1% per kilobase in three different regions of chromosome 2. This value is comparable to rates found in yeast, and will permit fine dissection of the genome.

THE developmental phase of the haploid ameboid slime mold Dictyostelium discoideum has been subjected to intense scrutiny for the insights that it may offer into intercellular signaling and gene regulation during multicellular development. A tremendous hindrance has been the lack of a sexual system, such as has been invaluable in understanding developmental genetics of Drosophila. There is a sexual reaction between certain isolates of D. discoideum, but in general crosses between wild isolates yield only haploid progeny of parental phenotypes when the zygote-containing macrocyst hatches (FRANCIS and EISENBERG 1993 ). This lack of a meiotic sexual system was partly remedied by the development of a parasexual genetic technique, based on a selection of unstable diploid strains which result from fusion of haploid amebae from two sublines of the standard nonmating strain, NC4 (KATZ and SUSSMAN 1972 ). This permitted complementation studies and assignment of many genes to individual chromosomes (NEWELL 1982 ). More recently, genetic techniques based on transformation have been developed; these permit tagging mutagenesis (KUSPA and LOOMIS 1992 ), reporter gene experiments (NELLEN et al. 1984 ), and targeted mutagenesis (DELOZANNE and SPUDICH 1987). Similar advances in technology have not eliminated the usefulness of classical genetics for Drosophila, however, in such applications as counting the genes represented in a collection of mutants, in positional cloning of new mutations, and especially in generating doubly and triply mutated strains useful for understanding regulatory interactions between genes.

To be of maximal value for these purposes, the frequency of recombination (meaning the percent recombinant progeny per kilobase of chromosomal DNA) must be high. Here, we report crosses using a pair of long ignored D. discoideum strains (A₂Cyc^r and WS205). This pair is one of several where WALLACE and RAPER 1979 initially found recombination between two linked loci. Amazingly, nothing has been done with them since then. We describe recombination among a series of linked genes, with a frequency of about 0.1% per kiloase. This value lies in the range reported for Saccharomyces (OLIVER et al. 1992 ) and is sufficient to permit very detailed rearrangement of the genome.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Sequences of various genes of D. discoideum were obtained from the GenBank database. Primers for use in the polymerase chain reaction (PCR) were picked using Oligo software (National Bioscience, Plymouth, MN), and PCR reactions were performed according to SAMBROOK et al. 1989 , using nuclear DNA as template, isolated as described by FRANCIS and EISENBERG 1993 . Products of PCR reactions were separated on agarose gels and visualized with ethidium bromide. In a few cases, DNA extracted from a gel band was sequenced using an Applied Biosystems (Foster City, CA) Model 373A automated sequencing machine, so as to identify the cause of an observed size difference between two alleles. When no size difference was apparent in PCR products from the two mated strains, restriction fragment length polymorphisms (RFLPs) were sought by cutting the extracted DNA with a battery of ten restriction enzymes, the resulting fragments being separated on agarose gels.

Macrocysts were made by mixing amebae of strains A₂Cyc^r and WS205 (kindly provided by STEVE BARCLAY, University of Wisconsin, Madison, WI), and hatched as described in detail by FRANCIS and EISENBERG 1993 . Briefly, flooded petri dishes containing 2000–10,000 macrocysts were stored for three to six weeks; the macrocysts were washed exhaustively using a sieve, so as to remove stray amebae, spores, and contaminants; then they were spread thinly on nonnutrient agar plates. The plates were observed for germinating macrocysts from the fourth through the seventh day. A single clone derived from one of the amebae which emerged from a macrocyst was a "progeny." The frequency of germination observed was less than 1%.

	RESULTS AND DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS AND DISCUSSION LITERATURE CITED

Marker genes:
The mating pair A₂Cyc^r and WS205 is one of several pairs described by WALLACE and RAPER 1979 as yielding recombinant progeny. These two strains were searched for PCR-RFLP primers based on sequences known from the standard NC4 -derived strains of D. discoideum. All primer pairs yielded PCR products from both A₂Cyc^r and WS205 nuclear DNA as template, implying that these strains are very similar in sequence to NC4.

Some PCR products from these two strains were obviously different in size and directly usable as genetic markers; in others, RFLP differences were found in the PCR products from the two strains. Of 15 genes examined, seven were found which showed an allelic difference (Table 1).

Recombination of linked genes:
Three crosses were made. In one of these an axenic substrain of A₂Cyc^r (IR1; made by ROBERT INSALL, University College, London) was used. A total of 107 progeny were examined for alleles of the seven marker loci. Genetic distances were calculated from the data. It was found that pspD, cotA, pspB, and sasA are in a cluster, in the order given. CotA and PspB are very close, with only four recombinants found (Table 2). This pair is flanked by pspD and sasA, each about 22 map units from cotA (Table 2). At the other end of chromosome 2 are dscA and carA (LOOMIS et al. 1995 ), separated by 38 map units (Figure 1). This value is just significantly different from the 50 map units expected if these genes were not linked by a chi-square test. Neither dscA, carA or glpD (on chromosome 5) show significant linkage to any gene in the pspD-sasA cluster (Table 2).

View larger version (6K): In this window In a new window Download PPT slide   Figure 1. —Map of chromosome 2 of Dictyostelium discoideum, summarizing genetic distances found in the current study and physical distances reported for a clone of strain NC4 by LOOMIS et al. 1995 . "% rec" indicates percent recombination.

The observed frequencies of recombinants are consistent with the arrangement of genes on the physical map of LOOMIS et al. 1995 and KUSPA and LOOMIS 1996 , except for sasA. No attempt has yet been made to locate sasA on the physical map (W. F. LOOMIS, personal communication). The data allow calibration of genetic map units with physical map units at three locations, all indicating a frequency of about 0.1% recombination per kilobase of DNA (Table 2). It is anticipated that this value will be different for different locations in the genome, as observed in yeast (OLIVER et al. 1992 ), for example.

Parental types:
One worry from previous work (FRANCIS and EISENBERG 1993 ) was that parental types would be heavily overrepresented in the progeny. In that study, it was found that no genetic exchange occurred in crosses between a large variety of wild isolates of D. discoideum. Normal appearing sexual structures (macrocysts) were formed, and these hatched well, but all haploid progeny were genetically identical to one parent or the other. If all seven loci recombine with the frequencies shown in Table 2, then the expected frequency of progeny that are identical to one parent or the other is (0.62)(0.50)(0.78)(0.96)(0.81)(0.5) = 0.09, or nine progeny in the 101 that were examined for all seven traits. This is not significantly different from the ten such progeny that were actually found, so an excessive number of parental types did not occur.

Germination rate:
0.1–0.5% of macrocysts germinated in the 4-day-period of observation (see MATERIALS AND METHODS). A concern is that this small sample of progeny was biased, and that certain genotypes occurred only in macrocysts that did not germinate in this period. This would result in underrepresentation of some classes of progeny. For example, if the P1 allele of carA was linked to a gene which delayed germination, then the P1P1 and P1P2 classes of progeny would be underrepresented in the carA-discA line of Table 2. Careful search of the data has detected no significant imbalances of this type. This result suggests that the low frequency of germination currently obtainable will not seriously interfere with genetic mapping, although it is of considerable nuisance value.

Conclusion:
The results confirm the finding of WALLACE and RAPER 1979 that genetic exchange occurs between strains A₂Cyc^r and WS205 of D. discoideum. The genetic markers employed were naturally occurring allelic differences between these two strains. However, any mutation generated by transformation with cloned material can be mapped as well. Conventional transformation protocols should be immediately feasible with the axenic IR1 substrain of A₂Cyc^r, and it should be possible to map and recombine mutations caused by insertions of transformed material. The rate of recombination can be as high as 0.1% per kb. This value means that the sexual system of D. discoideum is well-suited to the purpose of generating strains carrying multiple mutations.

	ACKNOWLEDGMENTS

I am grateful to STEVEN BARCLAY of the University of Wisconsin for strains A₂Cyc^r and WS205, to ROBERT INSALL of University College, London, for strain IR1 and to LING TAO, VINAY HARPALANI, TOM RUTKOWSKI, CHEN YA, ANDY DINSMORE, CHUN XIAO and PETER RINALDI for help in the search for allelic differences. This work was supported by grants from the University of Delaware.

Manuscript received June 26, 1997; Accepted for publication December 24, 1997.

	LITERATURE CITED

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