Proc. Natl. Acad. Sci. USA Vol. 87, pp. 8247-8251, November 1990
Genetics
Electrophoretic karyotype for Dictyostelium discoideum (clamped homogeneous electric field electrophoresis/pulse-field gels/slime mold genetics)
EDWARD C. COX*, CATHY D. VOCKE, SONJA WALTER, KEQIN Y. GREGG, AND ERIC S. BAINt Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1003
Communicated by J. T. Bonner, August 1, 1990
ABSTRACT
This paper reports on the separation of the
Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.
The cellular slime mold Dictyostelium discoideum is widely used in the study of morphogenesis, cell-cell interaction, and signal transduction (for recent reviews, see refs. 1 and 2). Extensive genetic studies have helped to define the genes underlying these processes, and many genes have been mapped to well-studied linkage groups (reviewed in ref. 3). A considerable number have also been ordered by mitotic recombination (4, 5). Laboratory strains of D. discoideum are thought to have seven chromosomes (6, 7), although only six linkage groups have been identified (3). To aid in the correlation of linkage group with physical structure and to develop a rapid and accurate method for studying genetic fine structure in D. discoideum, we have examined the conditions necessary for separation of D. discoideum chromosomal-sized DNA molecules by pulsefield gel electrophoresis (8, 9). We report here on the number of electrophoretic species detected by clamped homogeneous electric field (CHEF) electrophoresis (10) and show, using a variety of molecular probes, that it is possible to correlate the genetic and physical map, thus enlarging the genetic possibilities in this organism.
MATERIALS AND METHODS Strains. D. discoideum AX3k and NC4 were received from R. Firtel (University of California, San Diego, CA) and J. T. Bonner (Princeton University, Princeton, NJ), respectively. Growth conditions. In most experiments, cells were propagated on 15-cm GYP plates (11) with Escherichia coli B/r as the food source. Strain AX3k was grown on E. coli B/r or axenically in HL5 medium (12). Incubation was at 21°C in a humidified growth chamber or at 21°C in a water bath on a rotary shaker. Cells were starved by plating well-washed cells at a density of approximately S x 104 amoebae per mm2 on 2% agar plates (20 g of agar per liter of distilled deionized water).
Cell Preparation. Cells were harvested at various stages of growth and development, washed several times at room temperature in phosphate buffer (13), and mixed at 390C with an equal volume of FMC InCert agarose. For most of the experiments reported here, cells were allowed to develop to the loose-mound stage before they were harvested. As we will discuss in Results, DNA prepared otherwise gives very poor resolution. The final cell concentration varied between 5 x 108 and 2 x 109 cells per ml. Aliquots (25 s.l) were pipetted into rectangular molds spaced to coincide with the wells of flat-bottom 96-well plates (Coming 25860). After cooling, the mold contents ("plugs") were pushed into the wells, which contained 100 ,ud of 0.5 M EDTA (pH 9.5). After incubation at room temperature for 1 hr, 100 tul of 0.5 M EDTA (pH 9.5) containing 2% sodium lauryl sarcosinate and 2 mg of proteinase K (BRL) per ml was added. The plates were sealed and incubated at 50°C for approximately 24 hr. The plugs were stored at 4°C. Plugs prepared in this way give reproducible electrophoretic patterns for more than 1 year. Electrophoresis. For running gels, 0.8% "electrophoresis grade" agarose (BRL) was dissolved in running buffer (27 mM Tris base/27 mM boric acid/0.75 mM EDTA, pH 8.5). Agarose plugs were sealed in the wells of the running gel with 0.8% FMC InCert agarose dissolved in running buffer. Electrophoresis was for 240 hr at 10°C in running buffer with a CHEF apparatus of the dimensions described by Chu, Volrath, and Davis (10). The starting voltage and amperage were 1.85 V/cm and 50 mA. The pulse time was increased linearly from 2000 to 9600 sec during the run, and the voltage was decreased from 1.85 V/cm to 1.48 V/cm at a pulse time of 7000 sec. Molecular Probes. With the exception of mitochondrial DNA (mtDNA), rRNA-encoding DNA (rDNA), and the phage Agt11 inserts, protein-coding sequences were cut and purified from vectors carrying cDNA clones (Table 1). The tRNAval probe was the one unique 250-base-pair (bp) HindIII-BamHI subfragment described in ref. 14. The ribosomal probe was homologous to the 5.8S, 17S, and 26S RNA (fragment VII of ref. 35). The mitochondrial probe was the entire plasmid containing a mtDNA insert. Probes C1 to C17 were isolated from a Agtll cDNA library prepared from cells starved for 8 hr. Inserts were amplified from single Agtll plaques by the polymerase chain reaction with Agtll forward and reverse primers (New England Biolabs). Southern Blots. Agarose gels were stained with ethidium bromide and photographed. The DNA was depurinated in acid, nicked in alkali, and transferred to nylon membranes as described by Church and Gilbert (36). After the DNA was cross-linked to the nylon by exposure to a high-intensity Abbreviations: CHEF, clamped homogeneous electric field; rDNA, rRNA-encoding DNA; tRNAVal, tRNAvaIGUUB3s3; Gf,8 guanine nucleotide-binding regulatory protein ,32 subunit; CARl, cyclic AMP receptor 1; CP1 and CP2, cysteine proteinases 1 and 2; PDE, phosphodiesterase; PsA, glycoprotein PsA. *To whom reprint requests should be addressed. tPresent address: University of California San Francisco School of Medicine, San Francisco, CA 94143-0724.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8247
8248
Genetics: Cox et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
Table 1. DNA probes used in this study Probe Linkage group Name Abbreviation Ref. No. Ref. a-Actinin 15 I 16 I Glycoprotein PsA PsA 17 18 Actin 15 19 I, II 20 II Discoidin I 21 22 II 23 A* Cyclic AMP receptor 1 CAR1 II 24 G protein ,82 subunit A* GP3 tRNAvaJGUUB353 25 III or VI tRNAval 14 IV PDE 26 A* Phosphodiesterase Myosin heavy chain 27, 28 IV 29 Severin VII 30 31 Contact site A 32 VII Bt CP1 33 VII A* Cysteine proteinase I Ct CP2 34 Cysteine proteinase 2 Ct Ct C1.,C17 Ct 35 rDNA fragment VII Ct mtDNA D§ G protein, guanine nucleotide-binding regulatory protein. *A, D. Welker, personal communication. tB, E. Wallraff, personal communication. tC, this work. §D, S. Alexander, personal communication.
ultraviolet light, the membranes were blocked and probed (36). 32P-labeled probes were prepared by nick-translation (37). Probing was at 60°C followed by washing at 65°C under the Church and Gilbert conditions. Membranes were then exposed to x-ray film at -20°C before development.
RESULTS We knew from previous experiments with Polysphondylium pallidum that long pulse times and correspondingly long run times would be required to separate chromosome-sized D. discoideum DNA molecules. In both species, we found that DNA from vegetative cells could be made to enter 0.8% gels, but that it did so as a single diffuse band, somewhat like the results reported by Cole and Williams (38). The reasons for this are not known, but by using starved cells, D. discoideum AX3k and NC4 DNA could be resolved into seven major bands as shown in Fig. 1. With the exceptions noted below, individual probes hybridized to unique bands, to DNA rea 2
D. discoideum AX3k
b
4*ii
1
maining in the sample plugs, and to fragmented chromosomal DNA running near the mtDNA and rDNA bands. NC4 band number 1 was labeled by all probes, although, because of the various exposure times used to produce the Southern blots for Figs. 1 and 2, this is only apparent for the tRNA, contact site A, and actin probes. This result suggests that this region of the NC4 gel contains sequences from the entire genome. The largest band in AX3k does not exhibit this behavior. Finally, the results with AX3k were the same whether or not the cells were propagated on E. coli, and the electrophoretic patterns in both strains have proven to be highly reproducible from preparation to preparation over an 18-month period. The majority of the chromosomal-sized DNA molecules hybridized to our probes unambiguously and can be assigned to known linkage groups (Table 2). There is good agreement between the published linkage map and the electrophoretic karyotype outlined here (compare Table 1 with Table 2). The correspondence is especially good for AX3k. In four casesactin, tRNAv1, CP2, and probe C5-there are copies of closely related or identical sequences on one or more additional chromosomes. We found that the GP (see Table 1) and PDE probes hybridized to NC4 bands 4 and 5, respectively, whereas they hybridized to bands 1 and 3 in AX3k. Representative data illustrating this point for GP are shown in Fig. 2, where the cyclic AMP receptor CAR1, GP, and discoidin I probes have been hybridized to AX3k and NC4. Although CAR1 and discoidin both clearly mapped to band 1 in AX3k (linkage group II) and band 6 in NC4 (also linkage group II), GP hybridized to band 4 of NC4. Perhaps most interestingly, known linkage group II markers hybridized to the largest band in AX3k and to the smallest in NC4. The NC4 result was unexpected since, as we point out in Discussion, linkage group II appears by genetic criteria to be the largest D. discoideum chromosome. The two fastest AX3k species, bands 6 and 7, were not always well resolved and coded for rDNA and mtDNA sequences, respectively, along with variable amounts of degraded chromosomal DNA (Fig. 1). In NC4, rDNA and mtDNA sequences had approximately the same electrophoretic mobility. The intensities of bands 6 and 7 varied from preparation to preparation and were independent of whether or not AX3k was grown axenically. The reasons for this are not understood. D. discoideum NC4 4m:
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Genetics
Electrophoretic karyotype for Dictyostelium discoideum (clamped homogeneous electric field electrophoresis/pulse-field gels/slime mold genetics)
EDWARD C. COX*, CATHY D. VOCKE, SONJA WALTER, KEQIN Y. GREGG, AND ERIC S. BAINt Department of Molecular Biology, Princeton University, Princeton, NJ 08544-1003
Communicated by J. T. Bonner, August 1, 1990
ABSTRACT
This paper reports on the separation of the
Dictyostelium discoideum chromosomes by pulse-field electrophoresis and the correlation of the electrophoretic pattern with linkage groups established by classical genetic methods. In two commonly used laboratory strains, five chromosome-sized DNA molecules have been identified. Although the majority of the molecular probes used in this study can be unambiguously assigned to established linkage groups, the electrophoretic karyotype differs between the closely related strains AX3k and NC4, suggesting that chromosomal fragmentation may have occurred during their maintenance and growth. The largest chromosome identified in this study is approximately 9 million base pairs. To achieve resolution with molecules of this size, programmed voltage gradients were used in addition to programmed pulse times.
The cellular slime mold Dictyostelium discoideum is widely used in the study of morphogenesis, cell-cell interaction, and signal transduction (for recent reviews, see refs. 1 and 2). Extensive genetic studies have helped to define the genes underlying these processes, and many genes have been mapped to well-studied linkage groups (reviewed in ref. 3). A considerable number have also been ordered by mitotic recombination (4, 5). Laboratory strains of D. discoideum are thought to have seven chromosomes (6, 7), although only six linkage groups have been identified (3). To aid in the correlation of linkage group with physical structure and to develop a rapid and accurate method for studying genetic fine structure in D. discoideum, we have examined the conditions necessary for separation of D. discoideum chromosomal-sized DNA molecules by pulsefield gel electrophoresis (8, 9). We report here on the number of electrophoretic species detected by clamped homogeneous electric field (CHEF) electrophoresis (10) and show, using a variety of molecular probes, that it is possible to correlate the genetic and physical map, thus enlarging the genetic possibilities in this organism.
MATERIALS AND METHODS Strains. D. discoideum AX3k and NC4 were received from R. Firtel (University of California, San Diego, CA) and J. T. Bonner (Princeton University, Princeton, NJ), respectively. Growth conditions. In most experiments, cells were propagated on 15-cm GYP plates (11) with Escherichia coli B/r as the food source. Strain AX3k was grown on E. coli B/r or axenically in HL5 medium (12). Incubation was at 21°C in a humidified growth chamber or at 21°C in a water bath on a rotary shaker. Cells were starved by plating well-washed cells at a density of approximately S x 104 amoebae per mm2 on 2% agar plates (20 g of agar per liter of distilled deionized water).
Cell Preparation. Cells were harvested at various stages of growth and development, washed several times at room temperature in phosphate buffer (13), and mixed at 390C with an equal volume of FMC InCert agarose. For most of the experiments reported here, cells were allowed to develop to the loose-mound stage before they were harvested. As we will discuss in Results, DNA prepared otherwise gives very poor resolution. The final cell concentration varied between 5 x 108 and 2 x 109 cells per ml. Aliquots (25 s.l) were pipetted into rectangular molds spaced to coincide with the wells of flat-bottom 96-well plates (Coming 25860). After cooling, the mold contents ("plugs") were pushed into the wells, which contained 100 ,ud of 0.5 M EDTA (pH 9.5). After incubation at room temperature for 1 hr, 100 tul of 0.5 M EDTA (pH 9.5) containing 2% sodium lauryl sarcosinate and 2 mg of proteinase K (BRL) per ml was added. The plates were sealed and incubated at 50°C for approximately 24 hr. The plugs were stored at 4°C. Plugs prepared in this way give reproducible electrophoretic patterns for more than 1 year. Electrophoresis. For running gels, 0.8% "electrophoresis grade" agarose (BRL) was dissolved in running buffer (27 mM Tris base/27 mM boric acid/0.75 mM EDTA, pH 8.5). Agarose plugs were sealed in the wells of the running gel with 0.8% FMC InCert agarose dissolved in running buffer. Electrophoresis was for 240 hr at 10°C in running buffer with a CHEF apparatus of the dimensions described by Chu, Volrath, and Davis (10). The starting voltage and amperage were 1.85 V/cm and 50 mA. The pulse time was increased linearly from 2000 to 9600 sec during the run, and the voltage was decreased from 1.85 V/cm to 1.48 V/cm at a pulse time of 7000 sec. Molecular Probes. With the exception of mitochondrial DNA (mtDNA), rRNA-encoding DNA (rDNA), and the phage Agt11 inserts, protein-coding sequences were cut and purified from vectors carrying cDNA clones (Table 1). The tRNAval probe was the one unique 250-base-pair (bp) HindIII-BamHI subfragment described in ref. 14. The ribosomal probe was homologous to the 5.8S, 17S, and 26S RNA (fragment VII of ref. 35). The mitochondrial probe was the entire plasmid containing a mtDNA insert. Probes C1 to C17 were isolated from a Agtll cDNA library prepared from cells starved for 8 hr. Inserts were amplified from single Agtll plaques by the polymerase chain reaction with Agtll forward and reverse primers (New England Biolabs). Southern Blots. Agarose gels were stained with ethidium bromide and photographed. The DNA was depurinated in acid, nicked in alkali, and transferred to nylon membranes as described by Church and Gilbert (36). After the DNA was cross-linked to the nylon by exposure to a high-intensity Abbreviations: CHEF, clamped homogeneous electric field; rDNA, rRNA-encoding DNA; tRNAVal, tRNAvaIGUUB3s3; Gf,8 guanine nucleotide-binding regulatory protein ,32 subunit; CARl, cyclic AMP receptor 1; CP1 and CP2, cysteine proteinases 1 and 2; PDE, phosphodiesterase; PsA, glycoprotein PsA. *To whom reprint requests should be addressed. tPresent address: University of California San Francisco School of Medicine, San Francisco, CA 94143-0724.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 8247
8248
Genetics: Cox et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
Table 1. DNA probes used in this study Probe Linkage group Name Abbreviation Ref. No. Ref. a-Actinin 15 I 16 I Glycoprotein PsA PsA 17 18 Actin 15 19 I, II 20 II Discoidin I 21 22 II 23 A* Cyclic AMP receptor 1 CAR1 II 24 G protein ,82 subunit A* GP3 tRNAvaJGUUB353 25 III or VI tRNAval 14 IV PDE 26 A* Phosphodiesterase Myosin heavy chain 27, 28 IV 29 Severin VII 30 31 Contact site A 32 VII Bt CP1 33 VII A* Cysteine proteinase I Ct CP2 34 Cysteine proteinase 2 Ct Ct C1.,C17 Ct 35 rDNA fragment VII Ct mtDNA D§ G protein, guanine nucleotide-binding regulatory protein. *A, D. Welker, personal communication. tB, E. Wallraff, personal communication. tC, this work. §D, S. Alexander, personal communication.
ultraviolet light, the membranes were blocked and probed (36). 32P-labeled probes were prepared by nick-translation (37). Probing was at 60°C followed by washing at 65°C under the Church and Gilbert conditions. Membranes were then exposed to x-ray film at -20°C before development.
RESULTS We knew from previous experiments with Polysphondylium pallidum that long pulse times and correspondingly long run times would be required to separate chromosome-sized D. discoideum DNA molecules. In both species, we found that DNA from vegetative cells could be made to enter 0.8% gels, but that it did so as a single diffuse band, somewhat like the results reported by Cole and Williams (38). The reasons for this are not known, but by using starved cells, D. discoideum AX3k and NC4 DNA could be resolved into seven major bands as shown in Fig. 1. With the exceptions noted below, individual probes hybridized to unique bands, to DNA rea 2
D. discoideum AX3k
b
4*ii
1
maining in the sample plugs, and to fragmented chromosomal DNA running near the mtDNA and rDNA bands. NC4 band number 1 was labeled by all probes, although, because of the various exposure times used to produce the Southern blots for Figs. 1 and 2, this is only apparent for the tRNA, contact site A, and actin probes. This result suggests that this region of the NC4 gel contains sequences from the entire genome. The largest band in AX3k does not exhibit this behavior. Finally, the results with AX3k were the same whether or not the cells were propagated on E. coli, and the electrophoretic patterns in both strains have proven to be highly reproducible from preparation to preparation over an 18-month period. The majority of the chromosomal-sized DNA molecules hybridized to our probes unambiguously and can be assigned to known linkage groups (Table 2). There is good agreement between the published linkage map and the electrophoretic karyotype outlined here (compare Table 1 with Table 2). The correspondence is especially good for AX3k. In four casesactin, tRNAv1, CP2, and probe C5-there are copies of closely related or identical sequences on one or more additional chromosomes. We found that the GP (see Table 1) and PDE probes hybridized to NC4 bands 4 and 5, respectively, whereas they hybridized to bands 1 and 3 in AX3k. Representative data illustrating this point for GP are shown in Fig. 2, where the cyclic AMP receptor CAR1, GP, and discoidin I probes have been hybridized to AX3k and NC4. Although CAR1 and discoidin both clearly mapped to band 1 in AX3k (linkage group II) and band 6 in NC4 (also linkage group II), GP hybridized to band 4 of NC4. Perhaps most interestingly, known linkage group II markers hybridized to the largest band in AX3k and to the smallest in NC4. The NC4 result was unexpected since, as we point out in Discussion, linkage group II appears by genetic criteria to be the largest D. discoideum chromosome. The two fastest AX3k species, bands 6 and 7, were not always well resolved and coded for rDNA and mtDNA sequences, respectively, along with variable amounts of degraded chromosomal DNA (Fig. 1). In NC4, rDNA and mtDNA sequences had approximately the same electrophoretic mobility. The intensities of bands 6 and 7 varied from preparation to preparation and were independent of whether or not AX3k was grown axenically. The reasons for this are not understood. D. discoideum NC4 4m:
w
2 3
164b
A
-w' ,?f
I"W
f,
.ii Am-
3
uK
Am
is.i-
5
,I..
WIN
I 3.5 I
}
6r
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