Copyright 0 1992 by the Genetics Society of America
Tandem Repeats in Extrachromosomal Ribosomal
DNA
of
Dictyostelium discoideum, Resulting from Chromosomal Mutations
Robert
A. Cole and Keith
L.
Williams
School of Biological Sciences, Macquarie University, Sydney, New South Wales 2109, Australia Manuscript received June 1 7, 199 1
Accepted for publication December 24, 199 1
ABSTRACT
Extrachromosomal ribosomal DNA in the simple eukaryote Dictyostelium discoideum is readily separated from chromosomal DNA by orthogonal field electrophoresis (OFAGE), forming a promi- nent band in the 110-kb region of the gel. Here we show that mutations in at least two chromosomal genes give rise to a ladder of rDNA bands increasing in size up to about 300 kb. One of these mutations, the rrcA350 allele, which is recessive to wild type and maps to the centromere-proximal region of linkage group 11, has an unstable phenotype; spontaneous revertants, which no longer exhibit the rDNA ladder, have been recovered. Another mutation rrc-351, provisionally mapped to linkage group IV, is dominant to wild type. The rDNA ladder is caused by concatamerization of a 34-kb fragment in the nontranscribed central spacer region of the 88-kb linear rDNA palindrome. v Restriction enzyme analysis has revealed that each
two rDNA molecules.
T
H E cellular slime mold Dictyostelium discoideum is a simple amoeboid eukaryote that has been stud-ied as a model for cell differentiation (SPUDICH 1987). Cytological studies have revealed this organism has
7
chromosomes (ROBSON a n d WILLIAMS 1977; ZADA-
HAMES 1977), while contour-clamped homogenous electric field (CHEF) electrophoresis has indicated 6
chromosome-sized DNA molecules from 1 to 9 mb
(Cox et al. 1990), and genetic studies have clearly established 6 linkage groups (WELKER and WILLIAMS
1982). In addition there are 9 0 copies of an extra-
chromosomal 88 kb, linear ribosomal DNA palin-
drome (FIRTEL et al. 1976), which has two identical transcribed regions about 10-15 kb in length. These encode a 36s primary transcript which is processed to yield mature 26S, 1 7 s a n d
5.8s
rRNA. The distal ends of the molecule encode5s
rRNA (FRANKEL etal. 1977; COCKBURN, TAYLOR and FIRTEL 1978). The
remaining 70-75 kb is noncoding, and comprises central and terminal spacer regions (COCKBURN, NEW-
KIRK and FIRTEL 1976; COCKBURN, TAYLOR a n d FIR-
TEL 1978). Chromatin structure studies have revealed nuclease sensitivity in actively transcribed rRNA genes (NESS et al. 1983; PARISH, BANZ and NESS 1986). Nucleosome phasing occurs in the nontranscribed
central spacer region and in a more complex form in the terminal region (EDWARDS and FIRTEL 1984; PAR-
ISH, BANZ and NESS 1986). In the central nontran- scribed spacer region, specific nuclease hypersensitive zones have been mapped upstream of the transcrip- tional start of the rRNA genes (NESS, PARISH a n d
KOLLER 1986; BETTLER et al. 1988). Topoisomerase
I sites have been found in and around these zones
Genetics 1 3 0 757-769 (April, 1992)
concatamer is generated by crossovers between
leading to speculation that topoisomerase I binds in these regions to facilitate transcription (NESS, PARISH
and KOLLER 1986; BETTLER et al. 1988; NESS, KOLLER a n d THOMA 1988). Although the basic structure of
the linear rDNA palindrome is now well understood, its mode of replication and copy number regulation is still obscure.
In this paper we report the discovery of duplications
of a 34-kb spacer region of the rDNA which result in a prominent ladder of rDNA containing bands, rang- ing from about 1 10 to 300 kb in size on orthogonal
field electrophoresis (OFAGE) gels. This phenome- non occurs in many laboratory strains derived from
early genetic stocks of D. discoideum and is a result of
mutations at one of two chromosomal loci. A model is proposed to explain the amplification of this DNA.
MATERIALS AND METHODS
Growth of cells and preparation of chromosomal DNA
for pulsed field electrophoresis: All strains were stored desiccated on silica gel at 4". Spores were reactivated on SM plates previously spread with the bacteria Klebsiella aerogenes (SUSSMAN 1987). After incubation at 2 1 " cells were collected by harvesting approximately 10' amoebae from SM plates, then rotating (180 rpm) overnight in 100 ml starvation buffer (20 mM potassium phosphate, pH 6.4) to allow the amoebae to fully digest residual bacteria. Multi- cellular slugs were prepared according to BREEN, VARDY and WILLIAMS (1987). Spores were collected 1-2 days after sporulation by inverting and tapping SM plates containing fruiting bodies on the bench so the spores fell onto the lids. Amoebae were prepared for pulsed field electrophoresis as previously described (COLE and WILLIAMS 1988).
758 R. A. Cole and K. L. Williams
to the protocol of BERNARDS et al. (1986). Lambda/HindIII markers were supplied by Boehringer Mannheim.
OFAGE equipment and electrophoresis conditions: All gels were run in previously described orthogonal field elec- trode arrays (COLE and WILLIAMS 1988), under the follow- ing conditions: 20-sec pulse time, 15", 0.7% (w/v) agarose, 0.5 X T B E buffer (0.89 M Tris-borate, 0.89 M boric acid, 0.002 M EDTA, pH 8.3), 10 V/cm for 20 h. The bands were visualized and photographed under U V illumination (320 nm), after staining with ethidium bromide (0.8 pg/ml) for 1 hr and destaining for 10 min in water.
Band elution and purification: Bands were excised from OFAGE gels, placed in dialysis bags (6000-8000 M, cut off) and electrophoresed with OFAGE using 1 OO-sec pulse times for 3 hr at 10 V/cm. Prior to harvesting the DNA, the current was reversed for 3 min to remove bound DNA from the sides of the bags. Buffer containing the D N A was extracted with equal volumes of chloroform/phenol/isoamyl
alcohol, 25:24:1 (v/v/v), then precipitated twice with three volumes of 95% ethanol and 0.5 M (final concentration) ammonium acetate ( - Z O O , 45 min followed by centrifuga- tion 10,000 X g, 15 min, 4"). After washing the pellet in 70% ethanol and drying under vacuum, it was dissolved in 7 pl of T E buffer (10 mM Tris-HCI, 1 mM EDTA, pH 7.6) and stored at 4" until use.
DNA purification and electrophoresis: Amoebae were harvested from SM plates, while in vegetative or early aggregation phase. After shaking overnight at -5 X lofi cells/ml in starvation buffer, the cells were washed by two centrifugation steps (300 X g, 4 min). Final pellets (about 4
ml of packed cells) containing about 4 X log cells were lysed and DNA purified according to WELKER, HIRTH and WIL-
LIAMS (1 985). Restriction enzymes were supplied by Boeh-
ringer Mannheim and used according to their standard protocols.
DNA digests were electrophoresed through 0.8% (w/v) agarose gels, for 16 hr at 2.75 V/cm. After ethidium bro- mide staining for 1 hr the gels were destained in water for 30 min and photographed.
Probes: EcoRI fragment V ribosomal DNA was obtained from R. PARISH (Botany Department, La Trobe University, Melbourne, Australia) in a pBR322 plasmid vector. After digestion with EcoRI the 5.1-kb rDNA fragment was sepa- rated on an agarose gel, excised and gel purified with the BRESA (Adelaide, South Australia) Geneclean kit. "P-label- ing was carried out using the BRESATEC oligo-labeling kit. Other probes for "P-labeling were made by digestion of D.
discoideum genomic D N A (which included -90 copies of r D N A ) with the appropriate enzyme, separation of rDNA bands by agarose gel electrophoresis, and excision of the required band; the excised band was electrophoresed once more, purified and "'P-labeled as described.
Southern blotting and hybridizations: DNA from aga- rose gels was transferred to Zeta probe membrane (Bio- Rad) according to the protocol of REED and MAHN ( 1 985) with the addition of a depurination step (0.25 M HCI, 30
min) to facilitate the transfer of large DNA fragments. Hybridization was as previously described (COLE and WIL- LIAMS 1988), except 4% (w/v) sodium dodecyl sulfate (SDS) was used instead of 1% (w/v). The filters were washed 3
times (2 X SSC, 0.5% SDS; 2 X SSC, 0.1 % SDS; 0.1 X SSC, 0.1 % SDS), at room temperature for 15 min; finally the last wash was repeated at 65" for 30 min. Autoradiography was carried out on Fuji X-ray film at -80" for 1-5 days.
Densitometry scanning: Densitometry was performed with a LKB, 2202 Ultrascan Laser Densitometer on nega- tives of Polaroid, positive/negative 665 instant pack film. To obtain an accurate estimate of the amount of DNA in
2 3 4
1 2 3 4 5
5 6
1 2 3 4 5
FIGURE I.-Evidence for rDNA ladders in OFAGE gels of D.
discoideum DNA. A. T h e ethidium bromide staining pattern of the banding strain X22 (lane 1) is compared to wild-type strains AXSK (lane 2) NP20 (lane 3). WSJROB (lane 4), WS576 (lane 5 ) and O H I O (lane 6). T h e OFAGE gel fractioned DNA in the size range
0.1-1 mb. B, Different patterns ofbanding strains HU440, HU882. X 2 2 , M28 (lanes 1-4). and wild-type strains AXSK (lane .i). C , Gel
B probed with y2P-labeled cloned 5.l-kb RcoRl fragment V (from
D. discoideum rDNA, see Figure 7), excised from its pRR322 vecor: bands related to rDNA are numbered.
each band, each track was scanned five times. Band peaks were cut out, enlarged on a photocopier and weighed.
RESULTS
Strains with DNA concatamerization on OFAGE
gels: While using the technique of pulsed field elec-
trophoresis to examine chromosome rearrangements between strains of
D.
discoideum, it was noted that some strains with no known chromosomal re-arrangements gave different banding patterns com-
pared to the standard A X 3 K strain used in o u r origi-
nal study (COLE a n d WILLIAMS 1988). These strains exhibited an increase in the number and intensity of
bands in t h e 0.1-0.3 mb range. This was apparently
a laboratory-induced phenomenon in genetic stocks, which a r e mostly NC4-derived, as wild strains showed the same banding pattern as AXSK.
Figure 1A shows the OFAGE pattern of wild iso-
lates (WS380B, WS576,OH10, and NP20 and AXSK
rDNA Duplication in D. discoideum 759
exhibit only weak banding, have been designated “faint banders” and the banding strains “banders.” All four wild strains examined were faint banders; a fur- ther 30 NC4-derived strains were screened, 13 were faint banders while 17 were banders. T h e banding pattern in the faint bander strains results (with one exception, see below) from chromosomal fragmenta- tion which has been described previously (COLE and WILLIAMS 1988).
When banders were analyzed carefully, it became apparent that this phenomenon was not related to chromosome rearrangements (WELKER and WILLIAMS 1985) as many nontranslocated laboratory strains also exhibited banding patterns similar to those shown for X22 (Figure lA, lane 1). Figure 1B gives examples of four different banding types observed. T h e bands are numbered to emphasize the differences between pat- terns. T h e most prominent differences involved the intensity of staining between the first four bands in different strains. Strain M28 (lane 4) shows strong banding only in bands 1, 2 and 3. In contrast, HU440 (lane l), HU882 (lane 2) and X22 (lane 3) exhibit relatively strong banding extending to the unresolved DNA at the top of the gel. T h e bands in these strains appear equally spaced down the gel, except for band 6 (lanes 2 and 3) which is only slightly heavier than band 5. By increasing the pulse time we have found that the banding pattern of X22 and HU882 does not extend beyond 300 kb (data not shown). T h e unre- solved chromosomal DNA, at the top of the gel, is in excess of 1 mb (COLE and WILLIAMS 1988).
Position of the ribosomal DNA: COLE and WIL-
LIAMS (1988) identified the position of the 90 copies of extrachromosomal 88-kb ribosomal DNA in AX3K as being band 1 (Figure lB, lane 5), which stains intensely with ethidium bromide. It seemed likely that rDNA could also account for the strongly staining bands in the region 100-300 kb in the bander strains. T o investigate this possibility, bander strains were probed with the cloned EcoRI fragment V of
D.
dis-coideum ribosomal DNA (see Figure 7A). Figure 1C
shows that the strongly staining bands at the bottom of Figure 1B contain rDNA. It is interesting to note that a minor band in strain AX3K (Figure lB, lane 5, white dot), showed faint hybridization to this probe, indicating the presence of rDNA. Wild strains show a similar band (Figure l A , lanes 2, 3, 4 and 5). This band was not always apparent in faint bander strains (Figure lA, lane 6; Figure 3, lane 5).
In strains HU440, HU882 and X22 the bands heavier than 6, while remaining relatively strong in the ethidium bromide stained gel, are faint or absent in the autoradiogram (Figure IC). Prolonged expo- sure of the autoradiogram still failed to identify these bands (data not shown). There could be two possibil- ities to account for this observation. First, these bands
do not contain rDNA, but are a result of specific fragmentation of the much larger, unresolved chro- mosomal bands higher up the gel. Secondly, these bands may contain amplified rDNA, but EcoRI frag- ment V of the rDNA is greatly reduced. Although we have previously reported a weak, irregularly spaced series of bands in this region in AX3K, due to chro- mosomal breakage (COLE and WILLIAMS 1988), there was little correlation between the AX3K bands and the prominent ladder of bands reported here. Fur- thermore these unidentified bands occur at regular intervals continuing on the pattern shown with the bands 1, 2 and 3, which contain rDNA (Figure 1C). This suggested that they are more likely to be related to these ribosomal DNA containing bands. Strong evidence to support this conclusion is presented later.
Genetic basis of the enhanced OFAGE pattern:
After screening 34 genetically marked strains of
D.
discoideum it became apparent that the banding phe-
notype could not be explained as having resulted from a single event; there were at least two different genetic origins. These were most easily classified on the basis of the dominance of the effect. One class produced a banding pattern that was dominant to wild type. For example, Figure 2A shows that a parasexual cross between haploid faint banding strain NPl87 (WELKER and WILLIAMS 1982) and bander M28 resulted in a diploid (DU721) which was a bander. This dominant banding genotype (designated rrc-351, where rrc in- dicates ribosomal DNA recombination) has been traced to linkage group IV of strain M28, which contains the mutation bwnAl (KATZ and SUSSMAN 1972). For example, strain HU413 (WELKER and WIL-
LIAMS 1985) which contains bwnAl from M28, but has
linkage groups I, 111, VI and VI1 from other strains, retained the rrc-351 allele found in M28. Other crosses between banders containing linkage group IV of strain M28 (i.e., the bwnAl allele) with various combinations of other linkage groups produced dip- loids DU2748 (HL33 X M28), DU 1501 (HL1048 X
HU413), DU1172 (HL33 X HU413) that were also banders. T h e rrc-351 mutation is almost certainly the cause of the ladder in Figure 6, lanes 1 and 4 of
JENSEN et al. (1 989).
T h e second class exhibited banding that was reces- sive to wild type. This mutation (designated rrcA350)
was linked to the whiAl (white spore) allele of strain X22 and mapped to the proximal arm of linkage group I1 by analysis of a strain (HU329) that is disomic
for both the rrcA and whiA loci in this chromosomal region (WILLIAMS, ROBSON and WELKER 1980). Fig- ure 2B shows that strain HU329 (lane 4) is a faint bander, whereas strain HU440 (lane 3), a haploid segregant of HU329 which has lost the fragment of linkage group 11, exhibits the banding phenotype. T h e
760 R. A. Cole and K. L. Williams
LI
N
A
t-
1
2
3
B
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ure 2B, lane 2). This strain was derived from strain 'I312 and it has been thought to carry the same linkage group 11, since both strains carry the whiAI
(white spore) and tsgD12 (growth temperature sensi- tivity) alleles (WILLIAMS and NEWELL 1976). However, TS12 is wild type (faint bander) for the banding
phenotype (Figure 2B, lane 1). Although the evidence is inconclusive, it is probable that a rearrangement of linkage group I1 occurred when strain X22 was con- structed by haploidization of parasexual diploid DP4
( K . L. WILLIAMS, unpublished). Several strains de- rived from TS12 at a similar time that X22 was constructed show wild-type banding, so the event, which occurred in X22 leading to the banding phe- notype, was apparently specific to this strain. A series of strains derived subsequently from X22, which share its linkage group I1 (including HU440), all show the banding pattern (ie., carry t h e rrcA350 mutation). Therefore this phenotype is sufficiently stable to be observed in a range of genetic backgrounds.
The t l c A banding activity is found in all stages of
D. discoideum development: Although genetic analy-
sis of the banding phenotype has been straightfor- ward, this phenotype nevertheless is unstable. Two spontaneous revertants of strain X22 (one of which was named strain HU2551) have been obtained, which no longer show the banding pattern. Because we have no way of easily determining revertants, it
has not been possible to estimate the spontaneous reversion frequency. Both revertants retained the four genetic markers that identify strain X22 (round spores, white spores, growth temperature sensitivity and methanol resistance) and also the general fruiting morphology characteristic of X22. When we com- pared the banding phenotype of X22 and HU255 1 in
4
1
i
1
FIGURE 2."Ethidium hromide- stained OFAGE gels showing domi- nant and recessive mutations.
A.
Dip- loid DU721 (lane I ) formed by the cross M2X (lane 2) X NI'I 87 (lane 3) is a bander. This indicates that the bander (M28) is dominant t o wild type(NPlX7). R, HU32I)(faint hand- ing. lane 4 ) is disomic for part of linkage group 11. When the fragment chromosome is lost (generating strain HU440), a bander phrnotype is oh- served (lane 3). Blnding strain X22 (lane 2) and faint hander T S I 2 (lane1 ) have been used to trace the origin of the banding phenotype in strain H U 4 4 0 (see text).
chromosomes
2
3
4
5
FIGURE 3,"Ethidium bromide-stained OFAGE gel comparing HU2551 (X22 handing revertant) lane I ; X 2 2 (from 19/1/1982 silica gel), lane 2; X 2 2 (from 19/1/1977 silica gel) lane 4; and wild strain AX3K (lane 5 ) . Lane 3: phage lambda ladder (50-kh incre- ments). T h e gel was run for 2 0 hr with 15-sec pulses. R;lnds are numbered according to Figure I R.
amoebae, the multicellular "slug" stage and spores, we obtained the same results (data not shown). There- fore, the banding phenotype does not alter with de- velopmental stage in the asexual cycle.
r D N A Duplication in D. discoideum 76 1
having the same size and number of bands, show markedly different staining intensities. For example, lane 2, has a strongly stained band 1 (wild-type rDNA), while there is only a faint band in the same position in lane 4. Nevertheless, the distinction between ban- der and faint bander phenotypes was always clear-cut. Lambda DNA oligomers have been used extensively 21s size markers for PFGE (SMITH et al. 1986). Previ- ously
D.
discoideum rDNA was found to have an ap- parent size of 150 kb when compared with the DNA of equine herpesvirus type 1 (COLE and WILLIAMS1988). JENSEN et al. (1989) have since reported that rDNA of AX3K runs at between 160-200 kb on transverse alternating field electrophoresis. Figure 3 shows that
D.
discoideum rDNA has an apparent size of about 1 10 kb when compared against lambda DNA oligomers. Restriction mapping has previously sized the rDNA at 88 kb (COCKRURN, TAYLOR and FIRTEL1978). Clearly there is a size discrepancy, probably due to the behavior of this palindromic DNA molecule under different electrophoresis conditions. However, despite these uncertainties, lambda DNA oligomers allow an estimate to be made of the size of rDNA containing bands in strain X22.
T h e two stocks of strain X22 shown in Figure 3 have ladders of bands. I f the distance between the leading edge of each band of both stocks of X22 is measured there is close agreement (data not shown). Thus, as with the lambda ladder (Figure 3, lane 3), there is a regular size increment. OFAGE gels do not separate uniformly along the gel and there is a greater distance between the 200- and 250-kb bands of lambda than the 150 and 200 kb. Similarly, there is a larger separation gap between bands 4 and 5 in both stocks of X22 (Figure 3, lanes 2 and 4), which occur in the same region of the gel. Note that a faint band between bands 3 and 4 (X22 lane 2, white dot) does not hybridize when probed with rDNA and therefore
is not part of the rDNA series (data not shown). T h e lambda ladder has 50-kb size increments. Therefore, by comparing the concatamers of X22 with lambda, bands 1-4 of X22 can be estimated to have a size increase of between 30 and 40 kb.
Comparison of the ribosomal restriction digest pattern of a bander and faint bander strain: T h e OFAGE gels indicated that the bander phenotype was associated with concatamerization of ribosomal DNA,
b u t it provided no clues as to the molecular nature of the changes. It was decided to examine t h e rDNA in order to understand the phenotype. Since genomic DNA from a bander strain contains a mixture of bands seen on the OFAGE gels, it was decided to excise individual bands from OFAGE gels, electroelute, pu- rify and digest them with restriction enzymes which produce known sized fragments. This made it possible to examine each concatamer to understand the nature
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FIGURE 4.-A, Composite gel showing EroRl restriction pattern of total DNA of the faint bander strains NP2. H U 2 5 5 l and bander strain X22. and bands I , 2 , 3 and 4 extracted from OFACE gels of strain X 2 2 . Bands were excised from OFAGE gels, electroeluted. phenol/chloroform extracted and ethanol precipitated twice. After digestion with EcoRI the DNA was electrophoresed on a standard agarose gel overnight. Lanes 1 ( N P 2 , a growth temperature sensi- tive mutant of strain AXSK), 2 (HU25.51) and 3 (X22) are controls showing genomic D N A digested with EcoRI. Lanes 4 , 5 . 6 and 7 are EcoRl digests of bands I , 2, 3 and 4 from OFAGE gels of strain X22. The Roman numerals refer to the EcoRl fragments which have been named previously (COCKBURN, NEWKIRK and FIRTEI.
1976) Lane 8: lambda/Hindlll size markers. The white circles represent the large EroRI fragment of plasmid Ddpl. The white squares represent a new rDNA KcoRI fragment (fragment I 1 light). B, EcoRl fragments I . I I , I I (light). I l l . 1V and V of r D N A bands 2 and S from 2 separate gels were scanned with a densitometer six times, averaged and adjusted for molarity. Band 1 w a s scanned five times from one gel. Fragment I l l , like fragment 11, did not change its staining intensity in a l l three hands and therefore was chosen as the standard which was divided into the other fragments. The figures in brackets are the closest molar ;tpproximations of each EroRI fragment in each band. The hyphen in fragment if, band 3 indicates that this fragment was too faint to scan with the tlensitom- eter.
of the progressive increase in rDNA size. Figure 4
shows bands 1, 2, 3 and 4 (lanes 4, 5 , 6 and 7) excised from an OFAGE run of X22 (e.g., Figure 3, lane 4)
762 R. A. Cole and K. L. Williams
tional 0.8% agarose gel. Because electroelution, ex- traction and precipitation gave poor recoveries of DNA and only small amounts of each concatamer were available, it was not possible to study bands larger that band 4 by this technique. Similarly, rDNA frag- ments VI1 and IX of an EcoRI digest of excised bands were too faint to study. However, genomic digests of wild-type strain NP2, X22 bander revertant HU2551, and X22 (Figure 4; lanes 1, 2 and 3), which reflect the total rDNA complement of each strain, revealed that fragments V and VI1 of X22 (lane 3) showed reduced staining compared to the same fragments of NP2 and HU2551 (lanes 1 and 2).
The genomic digests show that the revertant HU2551 (Figure 4, lane 2) has an almost identical restriction pattern to that found in wild-type strain NP2 (lane 1). This is not surprising since the rDNA of HU2551 runs at the same position as wild-type strains on OFAGE gels (Figure 3: HU2551, lane 1; AX3K, lane 5). T h e only difference was the appear- ance of a faint EcoRI fragment slightly below EcoRI fragment I1 in HU2551 (Figure 4; lane 2, white dot). This EcoRI fragment is also present in X22 (lane 3, white dot). X22 is' known to carry a plasmid Ddpl, which is 12.6 kb in size and has three EcoRI restriction sites, leading to a large EcoRI fragment of 10 kb (METZ et al. 1983; NOEGEL, METZ and WILLIAMS 1985). We probed a genomic digest of NP2 (which does not have this plasmid), HU2551 and X22 with Ddpl and found that the plasmid identifies this band in X22 and HU2551 and does not hybridize to NP2 DNA (data not shown). Therefore, the EcoRI band running slightly below EcoRI fragment I1 is the large EcoRI fragment of plasmid Ddp 1.
T h e genomic digest of X22 (Figure 4, lane 3) ex- hibited two further changes: it has an extra EcoRI band above EcoRI fragment 111 (white square) and different molarities of the known EcoRI fragments. In
particular, there was strong staining of EcoRI frag- ments I and IV and weak staining of fragments 11, 111, V and VII.
When rDNA band 1 (Figure 4, lane 4) from X22 was digested with EcoRI and electrophoresed, it dis- played the same restriction pattern as total genomic DNA of NP2 and HU2551 (Figure 4, lanes 1 and 2). This suggests that rDNA band 1 is the same as the wild-type rDNA molecule.
Band 2 (Figure 4, lane 5) is the first rDNA conca- tamer and it shows an extra EcoRI fragment running above EcoRI fragment I11 at about 8 kb in size (white square, hereafter designated IIL). This EcoRI frag- ment can also be seen in the EcoRI digest of rDNA bands 3 and 4 (Figure 4, lanes 6 and 7) and is the only additional EcoRI fragment observed in all three con- catamers. Otherwise the EcoRI fragment sizes were identical with those observed after digesting rDNA
band 1. However, there were distinct differences in staining intensities of the EcoRI fragments. These differences were more pronounced in the larger con- catamers. This could be most clearly seen in rDNA band 4 (Figure 4, lane 7), where the only EcoRI fragments prominent were I, and to a lesser extent I I L and IV, suggesting that these fragments may have increased copy numbers.
Quantitation of rDNA in different bands: T o quantitate the above observations, lanes 4, 5 and 6 (rDNA bands 1,
2
and 3) in Figure 4 were scanned with a laser densitometer. Figure 4B is a summary of the results. In wild-type rDNA each EcoRI fragment is present at 2 mol/molecule, except for fragment IX (4 mol/molecule) and fragment I, which spans the hinge of the palindrome and is therefore present at only 1 mol/molecule. After each EcoRI fragment was scanned it was adjusted to the correct molarity, then divided by fragment 111 (which gave consistent molar- ities in all three bands) to give a clearer indication of the relationship between the EcoRI fragments. The densitometer scan for rDNA band 1 (Figure 4B, row 1) showed that EcoRI fragments 11, 111, IV and V were present in approximately equimolar proportions, but EcoRI fragment 1 was recovered at a lower than expected amount (0.3 1 instead of 0.5; Figure 4B, row 1). A similar trend was observed with the scans of EcoRI fragment I from both rDNA bands 2 and 3. Therefore the most accurate way of interpreting these data is comparing across lanes rather than along them.A
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rDNA Duplication in D. discoideum 763
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FIGURE 5.-EcoRI digestion and probing of N P 2 (lane l ) , H U 2 5 5 1 (lane 2) and X 2 2 (lane 3) with gel purified EcoKI and f'vull rDNA f r q p e n t s to establish the identity of EcoRI fragment I 1 (light). A and E, ethidium bromide stained agarose gel of EcoRl digestcd genomic DNA. B. C and D probing of gel A (with removal of label between each probing); F, probing of gel E. .Y'P-l,abeled probes were prepared froln digests ofgenomic DNA; B, fragment IlL. EcoRl digest from X22; C, fragment 11, EcoRl digest from HU2.5.51: D. 7.1-kb Pv~ll-l:'roRI fragment from HU2551; F. 4.5-kb Puull fragment from HU2551 which encompasses part of the EcoRl fragments I 1 and V (see text). The squares indicate the position of fragment IIL. The position of the Puull site within EcoRl fragment 11 is indicated in Figure 7.
determined by comparison with lambda ladders (Fig- ure 3).
Identification of fragment I I L After establishing which fragments were involved in the concatameri- zation process, it became important to identify the new fragment IIL. Genomic DNA from NP2, HU2551 and X22 was digested with EcoRI, electro- phoresed overnight, then ethidium bromide stained (Figure 5A and E). Radioactive EcoRI fragment IIL
was prepared by gel purification of the fragment from
a genomic EcoRI digest of X22 and "P-labeling. Fig- ure 5 B (lane 3) shows EcoRI fragment IIL (black square) hybridized to EcoRI fragment I1 in all strains and to itself in strain X22. This clearly indicates that fragment IIL has sequences in common with fragment
11 and no other fragments. However, the hybridiza- tion of fragment IIL was also stronger to itself than to fragment 11. This is surprising, as Figure 5A (white square) shows that there is only about half the amount of D N A in fragment IIL compared to fragment I 1 (Figure 5A, lane 3). This suggested that only a small
part of fragment I 1 contained fragment IIL sequences. T o confirm this result the membrane was stripped and re-probed with EcoRI fragment I 1 (Figure 5C). While fragment I 1 hybridized back to itself strongly there was only weak hybridization to fragment IIL
(Figure 5C, black square). I t was reasoned that if fragment IIL contained only a segment of fragment
11, this region could be identified, by probing with subfragments of EcoRI fragment 11. B E ~ L E R et al.
(1988) have extensively restriction mapped the rDNA region around EcoRI fragment I1 and V. A 7.1-kb
EcoRI-PuuII fragment defines the region of fragment
I1 that adjoins fragment IV (see Figure 7). Genomic DNA from HU255 1 was digested with EcoRI, electro- phoresed, EcoRI fragment I1 electroeluted, purified and digested with PuuII. After a second electropho- resis step the appropriate 7.1-kb fragment was gel excised, purified and radioactively labelled. The re- sults of probing with this EcoRI-PuuII fragment are shown in Figure 5D. Fragment I 1 and fragment IIL
764 R. A. Cole and K. L. Williams
1
2
3
4
kb
-
150-
loo-
50FIGURE 6.-Probing of a Psfl digest of genomic DNA from HU2551 (lane I), X22 (lane 2) and band 2 of strain X22 (lane 3) on OFAGE with the IO-kb Sal1 fragment of terminal rDNA. A, Ethidium bromide-stained OFAGE gel conditions: 8 sec pulses, 6.75 V/cm for 16 h. B.
Probing of gel A with the '*P-labeled IO-kb Sal1 rDNA fragment. The numbers 1-4 refer to the rDNA fragments after a Psfl restriction digest (for their position in the rDNA palindrome see Figure 8C). Lambda oligomers (lane 4) are in 50-kb increments.
ering there is less fragment I I L than fragment
I1
(Figure 5A; lane 3), this result suggests that the probe has more sequences in common with fragment EcoRI I I L than EcoRI fragment 11. If this is true then the 2.9 kb end of EcoRI fragment I1 (which adjoins EcoRIfragment V) should not hybridize to fragment IIL. T o test this, we digested HU2551 with PvuII, then gel purified and radioactively labelled a fragment of 4.5 kb. This fragment encompasses 2.9 kb of EcoRI
fragment I 1 and about 1.6 kb of EcoRI fragment V. Figure 5F shows that this probe hybridized to frag- ment I1 and V strongly. However, it failed to hybridize to fragment I I L in X22 (Figure 5F, lane 3). There- fore, EcoRI fragment I I L must consist of DNA se- quence complementary to the 7.1-kb EcoRI-PvuII fragment that adjoins EcoRI fragment IV. Consider- ing that it has already been established that EcoRI fragments IV and I are involved in the DNA dupli- cated in the concatamers, the addition of part of fragment I1 adjoining fragment I V seems logical if the concatamer is to arise by a simple mechanism. However fragment I I L is 8 kb and it is clear that 2.9 kb of EcoRI fragment I1 (10 kb) does not probe to this fragment. Therefore no more that 7 kb of frag- ment I1 DNA could be found in fragment IIL, leaving 1 kb of sequence unexplained. One possibility is that fragment I I L could be a dimer of the 4 kb of EcoRI
fragment I 1 adjoining fragment IV.
Insertion site of DNA concatamer in the rDNA molecule: After establishing the identity of the in- serted DNA in each concatamer, the next question was: where in the rDNA molecule does the insertion occur? One possibility was that the inserted DNA was
progressively added to the ends of the rDNA mole- cule. T o explore this hypothesis a PstI digest was performed on HU2551 and X22 DNA. PstI generates four fragments (50, 10, 6 and 4 kb; Figure 8C). The IO-kb PstI fragment (labeled 2 in Figure 8C) is the terminal section of the rDNA palindrome (COCKRURN, TAYLOR and FIRTEL 1978). I f the DNA insert was progressively added in this region, a molecular weight shift from 10 to 44 kb (34-kb DNA insert plus 10-kb fragment) would be expected in the genomic PstI digest of X22 and rDNA band 2 of X22 (Figure 6A, lanes 2 and 3) as there are no PstI sites in the 34 kb insert region. There is no 44-kb fragment evident in X22 (Figure 6A, lane 2). T h e extraction method used on rDNA band 2 from an OFAGE gel was too harsh to obtain DNA above 40 kb (Figure 6A, lane 3, arrow). Therefore to confirm this result we probed with a gel purified 10 kb SalI fragment of HU2551. This fragment is also the terminal region of the rDNA palindrome. Figure 6B shows that the SalI fragment hybridized to the 10-kb PstI fragment and there was no hybridization at 44 kb in the PstI digest of genomic X22 DNA (lane 2) or the PstI digest of rDNA band 2 (lane 3). This confirms that the 34 kb of DNA must insert within the rDNA molecule.
Model: Based on the information accumulated so
far, a model and mechanism for concatamer forma- tion is presented in Figure 7. Figure 7A depicts a
-+
5s 26s 17s
rDNA Duplication in D. discoideum
L 4
17s 26s 5s
765
A
B
80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80
kb
L I I I I 1 I I I I I I I I I I
FIGURE 7.--Scheme for the formation of rDNA concatamers (band 2 and 3 from an OFAGE gel). 7A. Band 1: wild-type rDNA molecule reproduced from COCKBURN. TAYLOR and FIRTEL (1978) with EcoRl restriction sites shown. Hatched arrows at the top of the figure are the transcribed regions. The ori is the origin of replication in fragment 1. Replication proceeds from the center of the molecule (black arrow). T h e black diamonds indicate the /‘vu11 site in EcoRl fragment 11. It is proposed that band 2 is formed by a recombination between two wild- type rDNA molecules, which generates a new recombinant molecule (-1 22 kb) with two origins of replication. Fragment I and two fragments of IV have been duplicated and a new fragment I 1 (light) formed. Fragment I1 (light) is a dimer of the 4 kb of fragment I 1 adjacent to fragment IV. The second molecule formed by a recombination event does not have an origin and is therefore lost. Note that band 2 has a different hinge region to the wild-type 88-kb band 1 . B, Band 3 is formed by an identical recombination event between band 2 and a 88-kb wild-type rDNA molecule. This generates a recombinant molecule (about 160 kb) with three origins of replication, three fragments of I , six fragments of 1V and two fragments of I1 (light). The second molecule, with no origin, is lost.
duces a 122-kb rDNA molecule containing two copies of fragment I, four of fragment IV and a new frag- ment IIL. Fragment IIL is 8 kb in length, consisting of an inverted dimer of the 4 kb of EcoRI fragment
I1 that adjoins EcoRI fragment IV. T h e second recom- binant molecule is about 50 kb in length and has the remaining 6 kb of the EcoRI fragment 11. This mole- cule has no origin of replication and therefore does not survive, unlike the 122-kb molecule which has two origins.
Figure
7B
shows the formation of rDNA band 3 by the same mechanism described for the formation of r D N A band 2, except that the recombining molecules are band 2 and a wild-type rDNA molecule (band 1). T h e resulting rDNA molecule is 156 kb with three copies of fragment I , six copies of fragment IV and two copies of fragment IIL. Fragment I is reestab- lished as the hinge of the rDNA. Again the second molecule, which lacks an origin of replication, is lost. Since band 4 also shows the same fragmentation pat- tern as band 2 and 3 (Figure 4), and is a multimer(Figure 3) it is probably formed by recombination between band 3 and a wild-type rDNA molecule, although other recombination events can be envis- aged. Presumably the other multimers (Figure 3, lanes 2 and 4) would also follow this pattern.
One way of confirming this model is to use it to predict different fragmentation and hybridization pat- terns when genomic DNA is digested with different restriction enzymes. PstI (Figure 8A, lanes 1 and 2) and Hind111 (Figure 8A, lanes 5 and 6) were used to digest X22 (lanes 2 and 6) and HU2551 (lanes 1 and 5), then the digested DNA was OFAGE electropho- resed, southern blotted and probed with gel purified fragment I1 from an EcoRI digest of NP2. Probing with an EcoRI fragment 11, which is part of the DNA insert, should tell us its position within the rDNA molecule. T h e model (Figure
7 )
predicts that an 84-kb DNA fragment (50 and 34 kb) would be generated if band 2 from X22, which is the first rDNA conca- tamer on an OFAGE gel, was digested with PstI.
766 R. A. Cole and K . L. Williams
A
E
U C.-
I
1 2
3 4
5
6
B
1 2
3 4
5
6
Band
1
2
I
3 141 1 141 3I
2 PslIc
WIHl
A C FF C A H B Hindmm x w v
-
U IV I Iv-
U V V U I X Ul EcoRlBand
2
2 3 141 1 141 3
I
2D
BP k l
Pst 1
n A FC CF A P C C F A
1441
B H m d mm x w v
-
D Iv I Iv DOight) N I N-
U v w ID EcoRIFIGURE &-Probing of PsfI and Hind111 genomic digests of HU2551 and X22 on OFAGE with "P-labeled I:'coRI fragment 11 from NP2. A , Ethidium bromide-stained OFAGE gel: PstI digest (HU2551, lane 1; X22, lane 2) and Hind111 digest (HU2551, lane .i; X22, lane 6). Lambda oligomers (kb) and lambda/HindIII (kb) markers are in lanes 3 and 4, respectively. B, Probing of gel A with "P-labeled GcoRI rDNA fragment 11 from NP2. T h e arrows refer to bands that hybridize to the probe. C and D, Restriction maps of wild-type rDNA band 1, and hand 2, respectively, showing PsfI fragments (1-4), Hind111 fragments (A-H) and EcoRl fragments (1-IX). T h e thick black and gray lines are the parts of bands 1 and 2 that are predicted to hybridize when probed with EcoRI, fragment 11.
size DNA intact (SCHWARTZ and CANTOR 1984). This can be clearly seen in Figure 8A. T h e revertant
HU2551 (lane l ) , which has only wild-type band 1 rDNA, shows a strong band at about 50 kb. In con- trast, X22 shows a smear, indicating t.hat there was a fragment much larger than 50 kb (white arrow, lane 2). When this gel was probed with rDNA EcoRI frag- ment 11, HU2551 (Figure 8B, lane 1) showed a strong band at 50 kb. In addition to this band, X22 showed a weaker, higher molecular weight band (black arrow), indicating a second larger DNA fragment was present,
as predicted.
Wild-type rDNA has a number of HindIII restric- tion sites (Figure 8C). T h e model for inserted DNA in the bander strains predicts new HindIII fragments when EcoRI fragment I1 is used for a probe (Figure 8D). In the wild-type rDNA (e.g., HU2551) 21-kb fragment A (Figure 8C) hybridized as expected (Fig-
ure 8B, lane 5, arrowhead). X22 (Figure 8B), in addition to the 21-kb fragment (lane 6), should have a second weaker 12-kb band, which contains one copy of fragment IIL (Figure 8D). As predicted by the model (Figure 8B, lane 6) a second band of 12 kb was observed in X22. Therefore, two predictions of the model proposed in Figure
7 ,
have been satisfied by the experiments shown in Figure 8.DISCUSSION
rDNA Duplication in D. discoideum 767
1985). Under pulsed field electrophoresis conditions we have demonstrated another, more dramatic
change. Varying amounts of concatamerization of rDNA in some strains has resulted in regularly spaced banding patterns from about 0.1 to 0 . 3 mb on OF- AGE gels. In reviewing the earlier results it seemed probable that the rDNA352 allele identified by WELKER, HIRTH and WILLIAMS (1985) could result from the concatamerization observed in this report.
In most eukaryotes rDNA genes are present in the chromosomes as tandem repeats (WELLAUER and DAWID 1977; BOSELEY et al. 1979; SAFRANY and HID-
VEGI 1989). In the protozoan Tetrahymena, however,
a single chromosomal rDNA copy exists in the mi- cronucleus which is amplified during the life cycle into multiple copies of a 22-kb extrachromosomal palin- drome in the macronucleus (YAO and GALL 1977; YAO, BLACKBURN and GALL 1978). Slime molds, and Physarum polycephalum apparently lack any chromo- somal copies of rDNA, all copies existing as linear extrachromosomal palindromes of 88 kb and 66 kb, respectively (COCKBURN, TAYLOR and FIRTEL 1978; VOGT and BRAUN 1976). On OFAGE gels the 88-kb rDNA of
D.
discoideum can be visualized as an in- tensely staining band in wild-type strains (Figure IA), while some laboratory strains show a ladder of higher molecular weight rDNA bands. We show here that the genetic basis of this banding phenotype can in- volve alterations in genes on at least two different chromosomes (linkage group I1 and IV). By excising the rDNA-related bands from OFAGE gels, purifying, digesting with restriction enzymes and electrophores- ing them, concatamerization of a 34-kb, nontran- scribed spacer region of rDNA has been identified as causing the ladder. Southern hybridization analysis has revealed that the 34-kb fragment results from recombination at a specific site in EcoRI fragment I1 of the rDNA molecule.Studies with Saccharomyces cerevisiae have identified two noncontiguous DNA fragments ( H O T ] ) in the rDNA cluster that stimulate recombination (KEIL and ROEDER 1984; VOELKEL-MEIMAN, KEIL and ROEDER 1987). One of these fragments corresponds to the transcription initiation site of RNA polymerase I (VOELKEL-MEIMAN, KEIL and ROEDER 1987), and has been shown to stimulate recombination when pro- moting transcription (STEWART and ROEDER 1989). VOELKEL-MEIMAN and ROEDER (1990) found that
HOT2 stimulates recombination of a distant marker
more than a nearby marker in the same gene. LIU and WANG (1 987) have postulated that negative and posi- tive supercoiling behind and in front of the transcrip- tion complex during transcription causes the build up
of torsional stress which is relieved by topoisomerases. Based on this model VOELKEL-MEIMAN and ROEDER ( 1990) have suggested that transcriptionally stimu-
lated recombination is a consequence of supercoiling induced by transcription. Strong supporting evidence has linked mutations in chromosomal topoisomerase genes to an increase in rDNA recombination in yeast (CHRISTMAN, DIETRICH and FINK 1988; KIM and WANG 1989). WALLIS et al. (1989) have proposed that failure by topoisomerases to relieve this torsional stress could cause an increase in recombination.
Topoisomerase activity has also been associated with the upstream non-transcribed spacer regions of actively transcribing rDNA of
D.
discoideum (NESS, PARISH and KOLLER 1986; NESS, KOLLER and THOMA 1988). It is in this upstream spacer that recombination occurs between two wild-type rDNA molecules (see Figure7),
causing the banding phenotype. It is con- ceivable that under conditions of rapid transcription, such as those inD.
discoideum amoebae, if the topoiso- merases associated with this region are defective ( i e . , fail to reduce torsional stress), this could create the conditions for illegitimate recombination. Homolo- gous recombination between two wild-type rDNA molecules was excluded from the model because it would not generate the 8-kb fragment IIL (Figure 7A). Reciprocal crossovers would generate 2 mol of fragment I1 in the EcoRI restriction digest of band 2. This was never observed. On the other hand, it is conceivable there is a specific homologous exchange between inverted repeats in the EcoRI fragment I1 region of two rDNA molecules.T h e above-mentioned studies indicate that recom- bination events involving topoisomerases in the non- transcribed spacer regions of rDNA are well docu- mented and remain a possible explanation for the banding phenotype we have observed. If a strain containing a mutated topoisomerase gene was crossed with a wild strain, the banding defect would be cor- rected. This could explain the recessive mutation (rrcA350) observed on linkage group 11. Conversely, the dominant mutation on linkage group IV requires that the product of this gene negatively interacts with the wild-type gene product, perhaps as part of a pro- tein dimer.
T h e bander strain X22 has very little wild-type 88- kb rDNA. Most of its rDNA exists in concatamer form with multimers of a 34-kb DNA insert. Despite the extra DNA burden this must impose on the cell during replication, this strain was able to be maintained in continuous culture in the laboratory for many months without significant change to its banding phenotype. Furthermore, there was no significant difference in growth rate in liquid culture between the revertant strain (HU2551) compared to X22. This implies not only that the concatamer rDNA has a negligible effect
768 R. A. Cole and K. L. Williams
A possible mechanism for this maintenance can be seen in the ciliate Tetrahymena thermophila. Like
D.
discoideum, this organism has extrachromosomal pal- indromic rDNA. Yu and BLACKBURN (1989) found that a microinjected plasmid containing one rRNA gene, a tandem repeat of the replication origin and a mutation in one of its two RNA promoters had a replication advantage over the endogeneous2
1-kb rDNA molecule which had two RNA genes, replica- tion origins and promoters. T h e microinjected plas- mid, after several cell generations, largely replaced the parental rDNA. Yu and BLACKBURN (1989) con- cluded that removal or inactivation of the repeated promoter with retention of the repeated replication origin was necessary to retain the circular rDNA plasmid. YU and BLACKBURN (1 990), on microinject- ing this recombinant plasmid into other strains of T. thermophila, found that in one strain it was rapidly replaced by successively longer linear rDNAs that eventually contained 30 tandem 1.9-kb repeats, re- sulting from homologous but unequal crossovers be- tween the l .9-kb repeats. They explain this phenom- enon as resulting from a recombination hot spot in the nontranscribed spacer region of the rDNA mole- cule. Recombination produces rDNA molecules with increasing numbers of origins which rapidly out com- pete and replace the rDNA forms containing fewer origin regions. T h e 34-kb spacer region that has been duplicated in the concatamers of theD.
discoideumbander strains also contains the replication origin of rDNA. Thus each progressive concatamer has an ex- tra origin of replication.
In the acellular slime mold P . polycephalum rDNA replication is unscheduled. That is, rDNA molecules can be randomly selected for replication any number of times during a mitotic cycle (VOGT and BRAUN
1977). If
D.
discoideum rDNA replication is also un- scheduled, then rDNA molecules containing two or more origins may have a selective advantage over the 88-kb wild-type rDNA molecule, thus maintaining their numbers in the banding strains. However, ribo- somal DNA molecules which have high numbers of DNA inserts and therefore consume more of the cell’s resources than the wild-type rDNA molecule, may be at a competitive disadvantage. Thus, one might expect a decline in the amount of rDNA concatamers with increasing size of the concatamer. This prediction is borne out by our results (Figure 1A).Although heterogeneity of spacer regions of rDNA is not uncommon, in the case reported here in
D.
discoideum the rDNA molecules are easily isolated and characterized by pulsed field electrophoresis tech- niques; this has made it possible to observe the specific and progressive nature of the rDNA concatameriza- tion. The uniformity in size of the inserted DNA and the site of insertion, points to a specific recombinationmechanism, which involves chromosomal genes. Fur- ther studies will focus on clarifying the sequences involved in recombination and determining the mech- anisms which have caused this banding phenotype.
We would like to thank ROLF MARSCHALEK of the Universitat Erlangen-Nurnberg, Germany, and one of the reviewers for critical comments about the model and RICK PAULIN for DNA from D.
discoideum strain NP2. This work was supported by an Australian Research Council program grant awarded to KEITH WILLIAMS.
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