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Anucleolate frog embryos contain ribosomal DNA sequences and a nucleolar antigen
DEVELOPMENTAL
BIOLOGY
102,409-416
(1984)
Anucleolate Frog Embryos Contain Ribosomal DNA Sequences and a Nucleolar Antigen ROBERTE.STEELE,PATRICIA ~.THOMAS,'AND RONALD H.REEDER Fred Hutchinson Cancer Research Center, 1124 Coluwzbia Street, Seattle, Washington Received July
19, 1983;
accepted in revised form October31,
98104
1983
Previous studies of Xenopus .!oeuis embryos homozygous for the nucleolar deletion mutation have concluded that these embryos contain few, if any, copies of the genes for the 18 S and 28 S ribosomal RNAs. Using hybridization to restriction endonuclease digests of DNA it is found, in fact, that a small amount of ribosomal DNA is still present in such embryos. The ribosomal DNA in these embryos appears to include a few normal repeats together with a variety of unusual fragments containing either spacer or gene sequences. An antibody found in the serum of a scleroderma patient reacts with an antigen localized in the nucleoli of wild-type embryos. In anucleolate embryos this antigen is found in the so-called pseudonucleoli and in many small bodies in the nuclei. INTRODUCTION
In 1958, Elsdale et al. described a recessive lethal mutation in the frog Xenop~ Levis in which the nucleolus is absent. Frogs homozygous for the mutation (0-nu) die at the swimming tadpole stage. Biochemical studies revealed that homozygous mutant embryos did not synthesize 18 S and 28 S ribosomal RNAs (rRNAs) (Brown and Gurdon, 1964). Wallace and Birnstiel(1966) provided the explanation for the absence of rRNA synthesis in 0-nu embryos by demonstrating that the mutation involved deletion of at least 95% of the genes for the 18 S and 28 S RNAs. Subsequently Brown and Weber (1968) measured rDNA content in 0-nu DNA which had been fractionated by buoyant density gradient centrifugation and showed that less than 1% of the normal amount of rDNA was present in the region of the density gradient where rDNA normally bands. Lacking the capacity for ribosome production, 0-nu embryos survive to the swimming tadpole stage by virtue of the large number of ribosomes provided by the egg (Brown and Gurdon, 1964). In addition to providing proof that the nucleolus organizer is the site of the genes encoding the 18 S and 28 S rRNAs, the anucleolate mutation of Xenopus has proved useful for studying how synthesis of the various components of the ribosome is coordinately regulated during development (Brown, 1967;Miller, 1973; Pierandrei-Amaldi et a& 1982). To obtain additional information on the consequences of the nucleolar deletion mutation in X laevis, we have examined the genome of 0-nu embryos for residual ribosomal DNA sequences which might have gone un‘Present address: Genetic Seattle, Wash. 98121.
Systems
Corporation,
3005 1st Ave.,
detected in the hybridization experiments of Brown and Weber (1968). In addition, we have examined what effect deletion of the nucleolus organizer region of the chromosome has on the occurrence and distribution of a nucleolar antigen which is detected by serum from a human patient with the autoimmune disease scleroderma. MATERIALS
AND
METHODS
Source of embryos. X laev& adults heterozygous for the nucleolar deletion were raised from embryos obtained by mating a heterozygous male (generously provided by Donald Brown) to a wild-type female. Embryos were screened for nucleolar number by phase-contrast microscopy of squashed tail tip samples. Embryos for the experiments described in this paper were obtained by mating the original heterozygous male to two of his heterozygous female offspring. Mating was induced by injection of frogs with human chorionic gonadotropin, and embryos were reared in 0.2~ modified Ringer’s solution (Busby and Reeder, 1982) containing 50 pg/ml gentamicin sulfate (Burns-Biotec). liolution of emho DNA. DNA was extracted from pools of 3-day-old 0-nu embryos and from individual heterozygous and wild-type tadpoles which were 18 days old. Embryos were rinsed in distilled water and homogenized in a glass-Teflon homogenizer in buffer containing 0.1 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM Na,EDTA, 0.5% SDS, 1 mg/ml proteinase K (EM Biochemicals). The homogenates were incubated at 50°C for at least 2 hr and in some cases overnight. After incubation, the homogenates were adjusted to 0.2 M sodium acetate and extracted twice with phenol and once with chloroform. DNA was recovered from the
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DEVELOPMENTAL BIOLOGY VOLUME102,1984
aqueous phase by ethanol precipitation and resuspended in buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM NazEDTA, and 10 pg/ml RNase A.
Restriction endonuckase digestions and gel electw phoresis. Embryo and plasmid DNAs were digested with
RESULTS Ribosomal DNA Sequences Are Present in
Anucleolate Embryos
The hybridization experiments of Brown and Weber restriction endonucleases (New England Biolabs or Be- (1968) showed that at least 99% of the 18 S and 28 S rRNA genes have been deleted in 0-nu embryos. Since thesda Research Laboratories) at 37°C in the buffers recommended by Maniatis et al. (1982). DNAs were elec- diploid cells of X Zuevis contain about 1000 copies of each of these genes (Brown and Weber, 1968), 0-nu emtrophoresed in 1% agarose gels (Seakem) in Tris-boratebryos could contain up to 10 copies of each. To test EDTA buffer (Maniatis et al, 1975b). Cloned DNA fragwhether 0-nu embryos completely lack ribosomal DNA ments to be used for hybridization probes were excised from plasmids by digestion with the appropriate re- sequences, DNA from each of three pools of ten 0-nu striction endonuclease and purified by fractionation in embryos each was digested with BamHI and hybridized a plasmid containing a agarose gels followed by electroelution (McDonell et aL, with the insert from pXlrlOlA, complete X Zaevis rDNA repeat cloned into the Hind111 1977). DNA blotting and hybridization, DNA was transferred site of pBR322 (Bakken et aL, 1982). As controls, DNAs from individual 1-nu and wild-type embryos from the from gels to nitrocellulose filters according to Southern (1975). After transfer, filters were dried in vacua at 80°C same mating were digested with BamHI and fractionated in the same gel with DNA from the 0-nu embryos. for 2 hr. DNA probes for hybridization were labeled with 32Pby nick-translation (Maniatis et ah, 1975a). HyThe results of this experiment are shown in Fig. 1. Surbridizations were carried out according to Wahl et aL prisingly, a number of fragments are labeled in 0-nu (1979). Following hybridization, filters were exposed to DNA. These include fragments identical in size to those found in the rDNA repeats of 1-nu and wild-type emXAR-5 film (Kodak) at -70°C with DuPont Cronex Hibryos as well as fragments which appear to be unique Plus or Lightning-Plus intensifying screens. Immunocytochemistry. For immunofluorescent stainto 0-nu embryos. Digestion of a normal X Levis rDNA ing, additional tail tip material (l-2 mm in length) was repeat with BamHI generates the four classes of fragremoved from embryos which had previously been scored ments labeled A, B, C, and D in Fig. 2. Fragment C, for nucleolar number. Samples were transferred in about which is derived from within the gene region, is constant 2 ~1 of phosphate-buffered saline (PBS) to a glass slide, in size (1.2 kb). Fragments A and B vary in size as a covered with a glass coverslip, and squashed by tapping result of heterogeneity in the spacer (Botchan et aL, with a pencil eraser until the individual cells were well 1977) but are on the order of 4-5 kb in length. The D separated. The squash preparations were frozen on a spacer fragments vary in both size and number from block of dry ice, coverslips were removed, and the prep- frog to frog (Botchan et aL, 1977). The map in Fig. 2 is arations were fixed in 95% ethanol at -20°C for 10 min. of the repeat cloned by Morgan et al. (1983) and designated pXlr164. This particular repeat contains four After fixation, the slides were air-dried. The y-globulin fraction (IgG) was isolated from serum BamHI D fragments. Hybridizing fragments correof a patient with scleroderma (EW) using protein Asponding to the A, B, C, and D BamHI fragments are Sepharose (Pharmacia). For assays, the IgG was used indicated in Fig. 1. 0-nu embryos contain all of the at a dilution of 1 to 100 in 1% bovine serum albumin BamHI fragments found in normal rDNA repeats. It is unlikely that the residual rDNA seen in the O(BSA) in PBS. The diluted antibody was incubated with nu embryos is due to chance inclusion of a few l-nu or the squashed preparations for 20 min at room temperature. The antibody was then removed, and the slides wild-type embryos. In the first place, such contamination in each of three were washed in PBS four times for 4 min per wash step. would have had to occur uniformly separate pools of 0-nu embryos. An even stronger arA second step reagent, goat anti-human IgG (Antibodies, Inc.) conjugated with fluorescein isothiocyanate, was gument is the fact that 1-nu and wild-type embryos all used at a dilution of 1 to 100 in 1% BSA in PBS to detect contain a BamHI spacer fragment of 0.68 kb (indicated specifically bound human IgG. The second antibody was by the arrowhead in Fig. 1) which is completely absent incubated with the preparations for 20 min at room from the 0-nu embryos. Since only two nucleolus organizer bearing chromosomes were involved in this temperature and washed off as after incubation with mating, this 0.68-kb band must be a marker for the the first antibody. The slides were dried and mounted wild-type nucleolus organizer. Its total absence from with 90% glycerol in PBS and photographed using Kodak the 0-nu pools indicates that they are not contaminated Tri-X film (ASA 400) set for an ASA of 1600. The film was developed using Kodak HC-110 with 1.5 times usual by the wild-type nucleolus organizer. From results of dot-blot hybridization with the development time to achieve an effective ASA of 1600.
STEELE,
THOMAS,
AND
REEDER
Ribosd
1234567 kb - .23.7 - -9.46 - -6.66
AB[
- .4.26
- -2.30 - -1.96
- -0.59
FIG. 1. Ribosomal DNA patterns of 0-nu, 1-nu, and wild-type embryos. DNAs were digested with BumHI, electrophoresed in a 1% agarose gel, transferred to nitrocellulose, and probed with the q-labeled rDNA repeat from pXlrlOlA. Lanes 1, 2, and 3 contain DNA from three pools of ten O-W embryos each. Lanes 4, 5, and 6 contain DNA from three individual 1-nu embryos. Lane 7 contains DNA from a single wild-type embryo. All embryos were from the same mating. Arrowheads in lanes 1 and 3 indicate fragments which are not present in all three 0-nu pools. The other arrowhead identifies the 0.68-kb fragment discussed in the text. Lanes 4-7 have been underexposed to allow comparison to lanes l-3. A-D designate BumHI fragments or groups of BumHI fragments as indicated on the map in Fig. 2.
pXlrlOlA insert to 0-m DNA (data not shown) we estimate that the 0-nu genome contains rDNA in an amount about 5% of that of wild-type frogs. This value is the same as that obtained by Wallace and Birnstiel (1966) but is somewhat higher than the value of 1% determined by Brown and Weber (1968). This difference may indicate that the amount of rDNA in 0-w frogs can vary to some degree. Variation in rDNA amount in different individuals of a species has been shown for two amphibian species (Miller and Brown, 1969; Macgregor et al, 197’7). Another possibility is that the unusual rDNA fragments which we detect do not have the same buoyant density as normal ribosomal repeats. In fact, Brown and Weber (1968) did detect some hybridization of rRNA to main band DNA of 0-nu embryos. An unusual aspect of the hybridization results for 0-
DNA in Anucleolate
411
Frogs
nu embryo DNA is the apparent variability of the pattern. Arrowheads in lanes 1 and 3 of Fig. 1 indicate differences between the patterns for the three embryo pools. The source of this variability is unknown. All of the embryos in the three pools resulted from the same mating. Furthermore, both copies of the chromosome carrying the nucleolar deletion should be identical since they were derived from a single heterozygous male (see Materials and Methods). Although the chromosome carrying the deletion should be identical in all of the 0-nu embryos, some of the remaining chromosomes in these embryos will be maternally derived and others will be of paternal origin. Some of the rDNA sequences detected in 0-w embryos may be located on these other chromosomes. If that is the case, the variability in the patterns in the three pools could be the result of independent assortment of parental polymorphisms in these dispersed rDNA sequences. In order to define better the composition of the rDNA sequences in 0-nu embryos, we hybridized segments of a complete rDNA repeat to restriction endonuclease digests of DNA from 0-nu embryos. Figure 2 shows a complete rDNA repeat and the origins of the hybridization probes which were specific for spacer or coding sequences. DNA from a single pool of 101 0-nu embryos was digested with various restriction endonucleases and hybridized with each of these probes. Figure 3 shows the results of these hybridizations. Both probes hybridize to DNA from 0-nu embryos, but the patterns of hybridization for the two probes differ considerably. Hind111 cleaves normal rDNA repeats only once per repeat to give a fragment 11-16 kb in length (Wellauer et al, 1976). Both probes detect fragments in this size range as well as a variety of fragments ranging in length from about 6 kb to less than 2 kb in Hind111 cleaved Onu DNA. EcoRI cleaves rDNA to produce the 4.65-kb coding fragment and a larger fragment containing most of the 18 S gene, all of the spacer, and a small segment of the 28 S gene (Fig. 2). The larger fragment varies in I
I HindliT
18s A BornHI
1 kb
I I EcoRI HindlJI EcoRI 285 I 5.8s II 8 1 c 1 BamHI BamHI
FIG. 2. Structure of a single ribosomal DNA repeating unit. The thin line indicates the nontranscribed spacer sequence. The filled thick lines are transcribed spacer regions, and the open thick lines are coding sequences. The region of the nontranscribed spacer indicated by the bracket has been cloned in a plasmid designated pXlr264 (Morgan et al, 1983) and was used here as a hybridization probe for spacer sequences. The other bracket indicates the 4.65kb EcoRI fragment which was used as a probe for coding sequences. This fragment was isolated from the rDNA repeat cloned in pXlrlOlA (Bakken et d, 1982). Details of the BamHI pattern of an rDNA repeat are described in the text.
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Fig. 3. The probe in Fig. 1 was the entire rDNA repeat, and thus it will detect all rDNA fragments. EcoRV does not cleave the normal rDNA repeat in X laevis. With both probes, however, a number of fragments are detected migrating below the size of limiting mobility in the EcoRV treated samples. A particularly strong signal is seen at about 8.5 kb with the spacer probe. Only a faint signal is seen at this position with the EcoRI probe. The strength of the signal obtained with the spacer probe suggests that the 8.5-kb fragment may be a repeated element containing spacer and little or no coding sequence. Both probes label significant amounts of DNA at limiting mobility in the EcoRV digested samples. DNA of this size which hybridizes with both probes has the properties expected of complete rDNA repeats arranged in a tandem array. Nuclei of Anucleolate Embryos Contain a Nucleolar Antigen
FIG. 3. Distribution pattern of rDNA spacer and coding sequences in 0-nu embryos. All samples were electrophoresed in the same gel. After transfer of the DNA, the nitrocellulose filter was cut into two pieces which were probed with the two fragments shown in Fig. 2. Panel A shows the hybridization pattern obtained with the coding sequence probe, and Panel B shows the result obtained with the spacer probe. The enzymes used were BumHI (lane 1); Hind111 (lane 2); EcoRI (lane 3); EcoRV (lane 4).
length from 6 to 11 kb (Wellauer et uL, 1976). When probed with the 4.65-kb EcoRI fragment, the EcoRI digest of 0-nu DNA shows a prominent fragment of the expected size. The spacer probe labels several EcoRI fragments in the size range expected for the larger EcoRI fragment of an rDNA repeat, with a prominent fragment 6.4 kb in length. Both probes detect a number of EcoRI fragments of sizes distinct from those contained in normal rDNA repeats. Although the majority of the BumHI fragments seen in Fig. 1 are present in Fig. 3, there are some minor differences between the two patterns. This was to be expected since the embryos in the two experiments were derived from matings of the same male to two different females. Furthermore, the two probes used in Fig. 3 do not completely cover the rDNA repeat (Fig. 2). Therefore, fragments of the rDNA corresponding to the regions not covered by these probes will not be seen in
Sera from patients with the autoimmune disease scleroderma often contain an antibody which binds to an antigen normally localized in the nucleolus (Berstein et aL, 1982). Previous experiments (Thomas, unpublished) have shown that scleroderma sera cross-react with nucleoli from a variety of vertebrate species, including X lamis. Since the antigen detected by scleroderma serum is normally restricted to the nucleolus, it was of interest to determine the fate of this antigen when a normal nucleolus is absent. Tail tip samples from 0-nu, I-nu, and wild-type embryos were fixed, squashed on slides, and treated with serum from a scleroderma patient which has previously been determined to give nucleolar staining. The human antibody binding patterns were visualized with a fluorescent goat anti-human antibody. Figure 4 shows the results of antibody staining of samples from embryos with different nucleolar numbers. As expected, staining in wild-type and I-nu embryos was localized in the nucleolus (panels A and B). In addition to nucleolar staining, nuclei of these embryos showed low level staining of dispersed particulate material. The nuclei of 0-nu embryos (panels C and D) show a staining pattern which is markedly different from those for I-nu and wild-type embryos. Substantial quantities of the antigen are present in 0-nu nuclei but these nuclei show a staining pattern composed of various sized aggregates of stained material. The largest aggregates can be seen to correspond to bodies visible in the phase-contrast photographs. These bodies are the so-called pseudonucleoli which are visible at late developmental stages in 0-nu embryos (Esper and Barr, 1964; Hay and Gurdon, 1967).
STEELE, THOMAS, AND REEDER
Ribosd
DISCUSSION
The anucleolate mutation of X Levis was first detected in a single female frog by virtue of the fact that half of her offspring possessed only one nucleolus in each cell (Elsdale et uZ.,1958). Miller and Gurdon (1970) have shown that partial or complete deletions of the nucleolus organizer arise relatively frequently in X Zaewia They have found both types of deletions among the progeny of adults with two normal sized nucleoli. The simplest mechanism for these alterations in the size of the nucleolus organizer is unequal crossing-over within the rDNA cluster during meiosis. If unequal crossing-over is responsible for all classes of nucleolar deletions in frogs, the so-called “anucleolate” condition would actually be a case of extremely unequal crossing-over which deletes so much of the rDNA that a visible secondary constriction and nucleolus are absent. In such a case, a small cluster of rDNA repeats should remain. We have shown that the genome of an anucleolate embryo is not completely devoid of rDNA sequences. Sequences homologous to both the gene and spacer regions of rDNA are present. Several of the results obtained here suggest that some of the rDNA in the anucleolate embryo is organized into a tandem array of normal repeats. The anucleolate embryos contain the expected array of BamHI rDNA fragments (Fig. 1). In addition, an EcoRI fragment of the expected size is labeled when the 4.65-kb EcoRI fragment from the rDNA repeat is used as a probe (Fig. 2). Finally, EcoRV is the only enzyme which leaves a significant amount of rDNA at the limiting mobility which also reacts with both gene and spacer probes (Fig. 3). EcoRV is the only one of the enzymes used which does not cleave rDNA and therefore would be expected to give this result if some of the rDNA in the anucleolate embryo consists of normal rDNA repeats arranged in a tandem cluster. We have evidence, therefore, that a remnant of the normal rDNA cluster remains in anucleolate embryos as would be expected if elimination of most of the repeats occurred by unequal crossing-over. In addition to the apparently normal repeats, we have detected a variety of unusual rDNA sequences in 0-m embryos. These sequences often contain only spacer or only coding sequences. We do not know whether normal
DNA in An&lute
Frogs
413
embryos also contain such unusual rDNA fragments. If they are present, we would expect them to be obscured by the overwhelming strength of the hybridization signal from the normal rDNA repeats. The origin of the unusual rDNA fragments in 0-nu embryos is unknown. They could arise from rearrangements involving normal rDNA repeats, transpositions of other sequences into rDNA repeats, or point mutations which generate new restriction sites or delete preexisting ones. At late stages in development large bodies, which have been termed pseudonucleoli, are visible in the nuclei of many of the cells in 0-nu embryos (Esper and Barr, 1964; Hay and Gurdon, 1967). These pseudonucleoli stain positively for RNA and contain a granular component with the same fine structure as the granular component of normal nucleoli, although the amount of the granular component in pseudonucleoli is much less than in normal nucleoli (Esper and Barr, 1964; Hay and Gurdon, 1967). The granular component in normal nucleoli is thought to be composed of preribosomal particles (Warner, 1974). We have shown here that pseudonucleoli also contain an antigen localized in normal nucleoli. Pseudonucleoli thus share a number of properties with normal nucleoli. The presence of RNA and a granular component in pseudonucleoli suggests that they are associated with active ribosomal RNA genes. It seems reasonable to conclude that pseudonucleoli mark the locations of the few normal rDNA repeats which we have detected by hybridization and that these repeats are transcriptionally active. That these repeats are located at the normal nucleolus organizer site is indicated by the observation that only one or two pseudonucleoli are formed in a cell (Hay and Gurdon, 1967; Fig. 4). Previous attempts to detect newly synthesized 18 S and 28 S RNAs in 0-nu embryos gave negative results (Brown and Gurdon, 1964). This negative result was probably due to a lack of sufficient sensitivity to detect the low levels of RNA which would be made by the small number of genes in 0-nu embryos. In addition to binding to the pseudonucleoli, the scleroderma antibody reacts strongly with a number of small bodies in nuclei of anucleolate embryos. Hay and Gurdon (1967) have described multiple small nucleolar bodies which are present in both normal and anucleolate embryos until the end of cleavage. These bodies are then
FIG. 4. Immunofluorescent staining patterns of cells from O-n%,1-nu, and wild-type embryos treated with EW scleroderma serum. Tail tip samples from the three types of embryos were treated as described under Materials and Methods and photographed with phase-contrast and fluorescence optics. Panels A-D show fluorescence patterns, and panels E-H show the corresponding phase-contrast views. Panels A and E show wild-type embryos; panels B and F show 1-nu embryos; panels C, D, G, and H show 0-nu embryos. The nuclei of 0-nu embryos contain bodies called pseudonucleoli at stages late in development (Esper and Barr, 1964, Hay and Gurdon, 1967). Embryos used in this work were scored for nucleolar number prior to the appearance of pseudonucleoli. For the immunofluorescence experiments, scored embryos were allowed to develop for an additional day before samples were taken for staining with antibody. Pseudonucleoli appeared during the period between scoring and staining.
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replaced by definitive nucleoli in normal cells. In some cells of anucleolate embryos these- nucleolar bodies may persist until the embryo dies, while in other cells they are partly or entirely replaced by pseudonucleoli (Hay and Gurdon, 1967).These bodies are probably responsible for the punctate antibody binding pattern seen in the nuclei of 0-nu embryos (Fig. 4). The derivation of nucleolar bodies is unknown. Determination of the biochemical nature of the antigen recognized by the scleroderma antibody should offer additional insight into the origins of these bodies. This work was supported by Grant GM26624 from the National Institutes of Health. R.E.S. was supported by National Research Service Award GM67569 from the NIH. We thank Grace Maloney and Judy Roan for technical assistance, Sandra Bizak for typing the manuscript, and members of the laboratory for their helpful comments. We are grateful to Dr. Bruce Guilliland of the Seattle Public Health Hospital for supplying sera from scleroderma patients. REFERENCES BAK~EN, A. H., MORGAN,G., SOLLNER-WEBB,B., ROAN,J., BUSBY,S., and REEDER,R. H. (1982). Mapping of transcription initiation and termination signals on Xw Levis ribosomal DNA. Proc. Nat1 Acud Sci USA 79, 56-60. BERSTEIN,R., STEIGERWALD,J., and TAN, E. M. (1982). Association of anti-nuclear and anti-nucleolar antibodies in progressive systemic sclerosis. Clix Exp. Imm. 48, 43-51. BOTCHAN,P., REEDER,R. H., and DAWID, I. B. (1977). Restriction analysis of the nontranscribed spacers of Xenopus laevis ribosomal DNA. Cdl 11,599~667. BROWN,D. D. (1967). The genes for ribosomal RNA and their transcription during amphibian development. Curr. Top. Dev. Bid 2, 47-73. BROWN,D. D., and GURDON,J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus Levis. Proc Nat1 Ad Sti USA 51,139-146. BROWN,D. D., and WEBER,C. S. (1968). Gene linkage by RNA-DNA hybridization. I. Unique DNA sequences homologous to 4s RNA, 5s RNA, and ribosomal RNA. J. Mol. Bid 34, 661-686. BUSBY,S. J., and REEDER,R. H. (1982). Fate of amplified nucleoli in Xenspus lam-is embryos. Deu Bid 91, 458-467. ELSDALE,T. R., FISCHBERG,M., and SMITH, S. (1958). A mutation that reduces nucleolar number in Xenopus Levis. Exp. CeURes. 14,642643. ESPER,H., and BARR,H. J. (1964).A study of the developmental cytology
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of a mutation affecting nucleoli in Xaqpus embryos. Den BioL 10, 105-121. HAY, E. D., and GURDON,J. B. (1967). Fine structure of the nucleolus in normal and mutant Xenopus embryos. J. CeU Sci. 2,151-162. MACGREGOR, H. C., VLAD, M., and BARNETT,L. (1977). An investigation of some problems concerning nucleolus organizers in salamanders. Chrumosoma (Berlin) 59,283~299. MANIATIS, T., FRITSCH,E. F., and SAMBROOK,J. (1982). “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. MANIATIS, T., JEFFREY,A., and KLEID, D. G. (1975a). Nucleotide sequence of the rightward operator of phage X. Proc NatL Awd Sci USA 72,1184-1188. MANIATIS,T., JEFFREY,A., and VANDESANDE,H. (1975b). Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14,3787-3794. MCDONELL,M. W., SIMON,M. N., and STUDIER,F. W. (1977). Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Afd BioL 110.110-146. MILLER, L. (1973). Control of 5s RNA synthesis during early development of anucleolate and partial nucleolate mutants of Xcnopas laevis. J. CelL BioL 59, 624-632. MILLER, L., and BROWN,D. D. (1969). Variation in the activity of nucleolar organizers and their ribosomal gene content. Chrwnwsoma (Berlin) 28,430-444. MILLER, L., and GURDON,J. B. (1970). Mutations affecting the size of the nucleolus in Xewqw kwvis. Nature (London) 227,1108-1110. MORGAN,G. T., REEDER,R. H., and BAKKEN,A. H. (1983).Transcription in cloned spacers of Xm Levis rDNA. Proc NatL Acad Sk USA, 6490-6494. PIERANDREI-AMALDI,P., CAMPIONI,N., BECCARI,E., BOZZONI,I., and AMALDI, F. (1982).Expression of ribosomal-protein genes in Xenupus Zucvis development. Cell 30,163-171. SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. MoL Bid 93, 503517. WAHL, G. M., STERN,M., and STARK, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethylpaper and rapid hybridization by using dextran sulfate. Proc NatL Acad Sci. USA 76, 3683-3687. WALLACE,H. R., and BIRNSTIEL,M. L. (1966). Ribosomal cistrons and the nucleolar organizer. Biochim Biophys Acta 114,296-310. WARNER,J. R. (1974). The assembly of ribosomes in eukaryotes. In “Ribosomes” (M. Nomura, A. Tissieres, and P. Lengyel, eds.), pp. 461-488. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. WELLAUER, P. K., DAWID, I. B., BROWN,D. D., and REEDER, R. H. (1976). The molecular basis for length heterogeneity in ribosomal DNA from Xaopu.s Zueuia J. MoL BioL 105,461-486.
BIOLOGY
102,409-416
(1984)
Anucleolate Frog Embryos Contain Ribosomal DNA Sequences and a Nucleolar Antigen ROBERTE.STEELE,PATRICIA ~.THOMAS,'AND RONALD H.REEDER Fred Hutchinson Cancer Research Center, 1124 Coluwzbia Street, Seattle, Washington Received July
19, 1983;
accepted in revised form October31,
98104
1983
Previous studies of Xenopus .!oeuis embryos homozygous for the nucleolar deletion mutation have concluded that these embryos contain few, if any, copies of the genes for the 18 S and 28 S ribosomal RNAs. Using hybridization to restriction endonuclease digests of DNA it is found, in fact, that a small amount of ribosomal DNA is still present in such embryos. The ribosomal DNA in these embryos appears to include a few normal repeats together with a variety of unusual fragments containing either spacer or gene sequences. An antibody found in the serum of a scleroderma patient reacts with an antigen localized in the nucleoli of wild-type embryos. In anucleolate embryos this antigen is found in the so-called pseudonucleoli and in many small bodies in the nuclei. INTRODUCTION
In 1958, Elsdale et al. described a recessive lethal mutation in the frog Xenop~ Levis in which the nucleolus is absent. Frogs homozygous for the mutation (0-nu) die at the swimming tadpole stage. Biochemical studies revealed that homozygous mutant embryos did not synthesize 18 S and 28 S ribosomal RNAs (rRNAs) (Brown and Gurdon, 1964). Wallace and Birnstiel(1966) provided the explanation for the absence of rRNA synthesis in 0-nu embryos by demonstrating that the mutation involved deletion of at least 95% of the genes for the 18 S and 28 S RNAs. Subsequently Brown and Weber (1968) measured rDNA content in 0-nu DNA which had been fractionated by buoyant density gradient centrifugation and showed that less than 1% of the normal amount of rDNA was present in the region of the density gradient where rDNA normally bands. Lacking the capacity for ribosome production, 0-nu embryos survive to the swimming tadpole stage by virtue of the large number of ribosomes provided by the egg (Brown and Gurdon, 1964). In addition to providing proof that the nucleolus organizer is the site of the genes encoding the 18 S and 28 S rRNAs, the anucleolate mutation of Xenopus has proved useful for studying how synthesis of the various components of the ribosome is coordinately regulated during development (Brown, 1967;Miller, 1973; Pierandrei-Amaldi et a& 1982). To obtain additional information on the consequences of the nucleolar deletion mutation in X laevis, we have examined the genome of 0-nu embryos for residual ribosomal DNA sequences which might have gone un‘Present address: Genetic Seattle, Wash. 98121.
Systems
Corporation,
3005 1st Ave.,
detected in the hybridization experiments of Brown and Weber (1968). In addition, we have examined what effect deletion of the nucleolus organizer region of the chromosome has on the occurrence and distribution of a nucleolar antigen which is detected by serum from a human patient with the autoimmune disease scleroderma. MATERIALS
AND
METHODS
Source of embryos. X laev& adults heterozygous for the nucleolar deletion were raised from embryos obtained by mating a heterozygous male (generously provided by Donald Brown) to a wild-type female. Embryos were screened for nucleolar number by phase-contrast microscopy of squashed tail tip samples. Embryos for the experiments described in this paper were obtained by mating the original heterozygous male to two of his heterozygous female offspring. Mating was induced by injection of frogs with human chorionic gonadotropin, and embryos were reared in 0.2~ modified Ringer’s solution (Busby and Reeder, 1982) containing 50 pg/ml gentamicin sulfate (Burns-Biotec). liolution of emho DNA. DNA was extracted from pools of 3-day-old 0-nu embryos and from individual heterozygous and wild-type tadpoles which were 18 days old. Embryos were rinsed in distilled water and homogenized in a glass-Teflon homogenizer in buffer containing 0.1 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM Na,EDTA, 0.5% SDS, 1 mg/ml proteinase K (EM Biochemicals). The homogenates were incubated at 50°C for at least 2 hr and in some cases overnight. After incubation, the homogenates were adjusted to 0.2 M sodium acetate and extracted twice with phenol and once with chloroform. DNA was recovered from the
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aqueous phase by ethanol precipitation and resuspended in buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM NazEDTA, and 10 pg/ml RNase A.
Restriction endonuckase digestions and gel electw phoresis. Embryo and plasmid DNAs were digested with
RESULTS Ribosomal DNA Sequences Are Present in
Anucleolate Embryos
The hybridization experiments of Brown and Weber restriction endonucleases (New England Biolabs or Be- (1968) showed that at least 99% of the 18 S and 28 S rRNA genes have been deleted in 0-nu embryos. Since thesda Research Laboratories) at 37°C in the buffers recommended by Maniatis et al. (1982). DNAs were elec- diploid cells of X Zuevis contain about 1000 copies of each of these genes (Brown and Weber, 1968), 0-nu emtrophoresed in 1% agarose gels (Seakem) in Tris-boratebryos could contain up to 10 copies of each. To test EDTA buffer (Maniatis et al, 1975b). Cloned DNA fragwhether 0-nu embryos completely lack ribosomal DNA ments to be used for hybridization probes were excised from plasmids by digestion with the appropriate re- sequences, DNA from each of three pools of ten 0-nu striction endonuclease and purified by fractionation in embryos each was digested with BamHI and hybridized a plasmid containing a agarose gels followed by electroelution (McDonell et aL, with the insert from pXlrlOlA, complete X Zaevis rDNA repeat cloned into the Hind111 1977). DNA blotting and hybridization, DNA was transferred site of pBR322 (Bakken et aL, 1982). As controls, DNAs from individual 1-nu and wild-type embryos from the from gels to nitrocellulose filters according to Southern (1975). After transfer, filters were dried in vacua at 80°C same mating were digested with BamHI and fractionated in the same gel with DNA from the 0-nu embryos. for 2 hr. DNA probes for hybridization were labeled with 32Pby nick-translation (Maniatis et ah, 1975a). HyThe results of this experiment are shown in Fig. 1. Surbridizations were carried out according to Wahl et aL prisingly, a number of fragments are labeled in 0-nu (1979). Following hybridization, filters were exposed to DNA. These include fragments identical in size to those found in the rDNA repeats of 1-nu and wild-type emXAR-5 film (Kodak) at -70°C with DuPont Cronex Hibryos as well as fragments which appear to be unique Plus or Lightning-Plus intensifying screens. Immunocytochemistry. For immunofluorescent stainto 0-nu embryos. Digestion of a normal X Levis rDNA ing, additional tail tip material (l-2 mm in length) was repeat with BamHI generates the four classes of fragremoved from embryos which had previously been scored ments labeled A, B, C, and D in Fig. 2. Fragment C, for nucleolar number. Samples were transferred in about which is derived from within the gene region, is constant 2 ~1 of phosphate-buffered saline (PBS) to a glass slide, in size (1.2 kb). Fragments A and B vary in size as a covered with a glass coverslip, and squashed by tapping result of heterogeneity in the spacer (Botchan et aL, with a pencil eraser until the individual cells were well 1977) but are on the order of 4-5 kb in length. The D separated. The squash preparations were frozen on a spacer fragments vary in both size and number from block of dry ice, coverslips were removed, and the prep- frog to frog (Botchan et aL, 1977). The map in Fig. 2 is arations were fixed in 95% ethanol at -20°C for 10 min. of the repeat cloned by Morgan et al. (1983) and designated pXlr164. This particular repeat contains four After fixation, the slides were air-dried. The y-globulin fraction (IgG) was isolated from serum BamHI D fragments. Hybridizing fragments correof a patient with scleroderma (EW) using protein Asponding to the A, B, C, and D BamHI fragments are Sepharose (Pharmacia). For assays, the IgG was used indicated in Fig. 1. 0-nu embryos contain all of the at a dilution of 1 to 100 in 1% bovine serum albumin BamHI fragments found in normal rDNA repeats. It is unlikely that the residual rDNA seen in the O(BSA) in PBS. The diluted antibody was incubated with nu embryos is due to chance inclusion of a few l-nu or the squashed preparations for 20 min at room temperature. The antibody was then removed, and the slides wild-type embryos. In the first place, such contamination in each of three were washed in PBS four times for 4 min per wash step. would have had to occur uniformly separate pools of 0-nu embryos. An even stronger arA second step reagent, goat anti-human IgG (Antibodies, Inc.) conjugated with fluorescein isothiocyanate, was gument is the fact that 1-nu and wild-type embryos all used at a dilution of 1 to 100 in 1% BSA in PBS to detect contain a BamHI spacer fragment of 0.68 kb (indicated specifically bound human IgG. The second antibody was by the arrowhead in Fig. 1) which is completely absent incubated with the preparations for 20 min at room from the 0-nu embryos. Since only two nucleolus organizer bearing chromosomes were involved in this temperature and washed off as after incubation with mating, this 0.68-kb band must be a marker for the the first antibody. The slides were dried and mounted wild-type nucleolus organizer. Its total absence from with 90% glycerol in PBS and photographed using Kodak the 0-nu pools indicates that they are not contaminated Tri-X film (ASA 400) set for an ASA of 1600. The film was developed using Kodak HC-110 with 1.5 times usual by the wild-type nucleolus organizer. From results of dot-blot hybridization with the development time to achieve an effective ASA of 1600.
STEELE,
THOMAS,
AND
REEDER
Ribosd
1234567 kb - .23.7 - -9.46 - -6.66
AB[
- .4.26
- -2.30 - -1.96
- -0.59
FIG. 1. Ribosomal DNA patterns of 0-nu, 1-nu, and wild-type embryos. DNAs were digested with BumHI, electrophoresed in a 1% agarose gel, transferred to nitrocellulose, and probed with the q-labeled rDNA repeat from pXlrlOlA. Lanes 1, 2, and 3 contain DNA from three pools of ten O-W embryos each. Lanes 4, 5, and 6 contain DNA from three individual 1-nu embryos. Lane 7 contains DNA from a single wild-type embryo. All embryos were from the same mating. Arrowheads in lanes 1 and 3 indicate fragments which are not present in all three 0-nu pools. The other arrowhead identifies the 0.68-kb fragment discussed in the text. Lanes 4-7 have been underexposed to allow comparison to lanes l-3. A-D designate BumHI fragments or groups of BumHI fragments as indicated on the map in Fig. 2.
pXlrlOlA insert to 0-m DNA (data not shown) we estimate that the 0-nu genome contains rDNA in an amount about 5% of that of wild-type frogs. This value is the same as that obtained by Wallace and Birnstiel (1966) but is somewhat higher than the value of 1% determined by Brown and Weber (1968). This difference may indicate that the amount of rDNA in 0-w frogs can vary to some degree. Variation in rDNA amount in different individuals of a species has been shown for two amphibian species (Miller and Brown, 1969; Macgregor et al, 197’7). Another possibility is that the unusual rDNA fragments which we detect do not have the same buoyant density as normal ribosomal repeats. In fact, Brown and Weber (1968) did detect some hybridization of rRNA to main band DNA of 0-nu embryos. An unusual aspect of the hybridization results for 0-
DNA in Anucleolate
411
Frogs
nu embryo DNA is the apparent variability of the pattern. Arrowheads in lanes 1 and 3 of Fig. 1 indicate differences between the patterns for the three embryo pools. The source of this variability is unknown. All of the embryos in the three pools resulted from the same mating. Furthermore, both copies of the chromosome carrying the nucleolar deletion should be identical since they were derived from a single heterozygous male (see Materials and Methods). Although the chromosome carrying the deletion should be identical in all of the 0-nu embryos, some of the remaining chromosomes in these embryos will be maternally derived and others will be of paternal origin. Some of the rDNA sequences detected in 0-w embryos may be located on these other chromosomes. If that is the case, the variability in the patterns in the three pools could be the result of independent assortment of parental polymorphisms in these dispersed rDNA sequences. In order to define better the composition of the rDNA sequences in 0-nu embryos, we hybridized segments of a complete rDNA repeat to restriction endonuclease digests of DNA from 0-nu embryos. Figure 2 shows a complete rDNA repeat and the origins of the hybridization probes which were specific for spacer or coding sequences. DNA from a single pool of 101 0-nu embryos was digested with various restriction endonucleases and hybridized with each of these probes. Figure 3 shows the results of these hybridizations. Both probes hybridize to DNA from 0-nu embryos, but the patterns of hybridization for the two probes differ considerably. Hind111 cleaves normal rDNA repeats only once per repeat to give a fragment 11-16 kb in length (Wellauer et al, 1976). Both probes detect fragments in this size range as well as a variety of fragments ranging in length from about 6 kb to less than 2 kb in Hind111 cleaved Onu DNA. EcoRI cleaves rDNA to produce the 4.65-kb coding fragment and a larger fragment containing most of the 18 S gene, all of the spacer, and a small segment of the 28 S gene (Fig. 2). The larger fragment varies in I
I HindliT
18s A BornHI
1 kb
I I EcoRI HindlJI EcoRI 285 I 5.8s II 8 1 c 1 BamHI BamHI
FIG. 2. Structure of a single ribosomal DNA repeating unit. The thin line indicates the nontranscribed spacer sequence. The filled thick lines are transcribed spacer regions, and the open thick lines are coding sequences. The region of the nontranscribed spacer indicated by the bracket has been cloned in a plasmid designated pXlr264 (Morgan et al, 1983) and was used here as a hybridization probe for spacer sequences. The other bracket indicates the 4.65kb EcoRI fragment which was used as a probe for coding sequences. This fragment was isolated from the rDNA repeat cloned in pXlrlOlA (Bakken et d, 1982). Details of the BamHI pattern of an rDNA repeat are described in the text.
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Fig. 3. The probe in Fig. 1 was the entire rDNA repeat, and thus it will detect all rDNA fragments. EcoRV does not cleave the normal rDNA repeat in X laevis. With both probes, however, a number of fragments are detected migrating below the size of limiting mobility in the EcoRV treated samples. A particularly strong signal is seen at about 8.5 kb with the spacer probe. Only a faint signal is seen at this position with the EcoRI probe. The strength of the signal obtained with the spacer probe suggests that the 8.5-kb fragment may be a repeated element containing spacer and little or no coding sequence. Both probes label significant amounts of DNA at limiting mobility in the EcoRV digested samples. DNA of this size which hybridizes with both probes has the properties expected of complete rDNA repeats arranged in a tandem array. Nuclei of Anucleolate Embryos Contain a Nucleolar Antigen
FIG. 3. Distribution pattern of rDNA spacer and coding sequences in 0-nu embryos. All samples were electrophoresed in the same gel. After transfer of the DNA, the nitrocellulose filter was cut into two pieces which were probed with the two fragments shown in Fig. 2. Panel A shows the hybridization pattern obtained with the coding sequence probe, and Panel B shows the result obtained with the spacer probe. The enzymes used were BumHI (lane 1); Hind111 (lane 2); EcoRI (lane 3); EcoRV (lane 4).
length from 6 to 11 kb (Wellauer et uL, 1976). When probed with the 4.65-kb EcoRI fragment, the EcoRI digest of 0-nu DNA shows a prominent fragment of the expected size. The spacer probe labels several EcoRI fragments in the size range expected for the larger EcoRI fragment of an rDNA repeat, with a prominent fragment 6.4 kb in length. Both probes detect a number of EcoRI fragments of sizes distinct from those contained in normal rDNA repeats. Although the majority of the BumHI fragments seen in Fig. 1 are present in Fig. 3, there are some minor differences between the two patterns. This was to be expected since the embryos in the two experiments were derived from matings of the same male to two different females. Furthermore, the two probes used in Fig. 3 do not completely cover the rDNA repeat (Fig. 2). Therefore, fragments of the rDNA corresponding to the regions not covered by these probes will not be seen in
Sera from patients with the autoimmune disease scleroderma often contain an antibody which binds to an antigen normally localized in the nucleolus (Berstein et aL, 1982). Previous experiments (Thomas, unpublished) have shown that scleroderma sera cross-react with nucleoli from a variety of vertebrate species, including X lamis. Since the antigen detected by scleroderma serum is normally restricted to the nucleolus, it was of interest to determine the fate of this antigen when a normal nucleolus is absent. Tail tip samples from 0-nu, I-nu, and wild-type embryos were fixed, squashed on slides, and treated with serum from a scleroderma patient which has previously been determined to give nucleolar staining. The human antibody binding patterns were visualized with a fluorescent goat anti-human antibody. Figure 4 shows the results of antibody staining of samples from embryos with different nucleolar numbers. As expected, staining in wild-type and I-nu embryos was localized in the nucleolus (panels A and B). In addition to nucleolar staining, nuclei of these embryos showed low level staining of dispersed particulate material. The nuclei of 0-nu embryos (panels C and D) show a staining pattern which is markedly different from those for I-nu and wild-type embryos. Substantial quantities of the antigen are present in 0-nu nuclei but these nuclei show a staining pattern composed of various sized aggregates of stained material. The largest aggregates can be seen to correspond to bodies visible in the phase-contrast photographs. These bodies are the so-called pseudonucleoli which are visible at late developmental stages in 0-nu embryos (Esper and Barr, 1964; Hay and Gurdon, 1967).
STEELE, THOMAS, AND REEDER
Ribosd
DISCUSSION
The anucleolate mutation of X Levis was first detected in a single female frog by virtue of the fact that half of her offspring possessed only one nucleolus in each cell (Elsdale et uZ.,1958). Miller and Gurdon (1970) have shown that partial or complete deletions of the nucleolus organizer arise relatively frequently in X Zaewia They have found both types of deletions among the progeny of adults with two normal sized nucleoli. The simplest mechanism for these alterations in the size of the nucleolus organizer is unequal crossing-over within the rDNA cluster during meiosis. If unequal crossing-over is responsible for all classes of nucleolar deletions in frogs, the so-called “anucleolate” condition would actually be a case of extremely unequal crossing-over which deletes so much of the rDNA that a visible secondary constriction and nucleolus are absent. In such a case, a small cluster of rDNA repeats should remain. We have shown that the genome of an anucleolate embryo is not completely devoid of rDNA sequences. Sequences homologous to both the gene and spacer regions of rDNA are present. Several of the results obtained here suggest that some of the rDNA in the anucleolate embryo is organized into a tandem array of normal repeats. The anucleolate embryos contain the expected array of BamHI rDNA fragments (Fig. 1). In addition, an EcoRI fragment of the expected size is labeled when the 4.65-kb EcoRI fragment from the rDNA repeat is used as a probe (Fig. 2). Finally, EcoRV is the only enzyme which leaves a significant amount of rDNA at the limiting mobility which also reacts with both gene and spacer probes (Fig. 3). EcoRV is the only one of the enzymes used which does not cleave rDNA and therefore would be expected to give this result if some of the rDNA in the anucleolate embryo consists of normal rDNA repeats arranged in a tandem cluster. We have evidence, therefore, that a remnant of the normal rDNA cluster remains in anucleolate embryos as would be expected if elimination of most of the repeats occurred by unequal crossing-over. In addition to the apparently normal repeats, we have detected a variety of unusual rDNA sequences in 0-m embryos. These sequences often contain only spacer or only coding sequences. We do not know whether normal
DNA in An&lute
Frogs
413
embryos also contain such unusual rDNA fragments. If they are present, we would expect them to be obscured by the overwhelming strength of the hybridization signal from the normal rDNA repeats. The origin of the unusual rDNA fragments in 0-nu embryos is unknown. They could arise from rearrangements involving normal rDNA repeats, transpositions of other sequences into rDNA repeats, or point mutations which generate new restriction sites or delete preexisting ones. At late stages in development large bodies, which have been termed pseudonucleoli, are visible in the nuclei of many of the cells in 0-nu embryos (Esper and Barr, 1964; Hay and Gurdon, 1967). These pseudonucleoli stain positively for RNA and contain a granular component with the same fine structure as the granular component of normal nucleoli, although the amount of the granular component in pseudonucleoli is much less than in normal nucleoli (Esper and Barr, 1964; Hay and Gurdon, 1967). The granular component in normal nucleoli is thought to be composed of preribosomal particles (Warner, 1974). We have shown here that pseudonucleoli also contain an antigen localized in normal nucleoli. Pseudonucleoli thus share a number of properties with normal nucleoli. The presence of RNA and a granular component in pseudonucleoli suggests that they are associated with active ribosomal RNA genes. It seems reasonable to conclude that pseudonucleoli mark the locations of the few normal rDNA repeats which we have detected by hybridization and that these repeats are transcriptionally active. That these repeats are located at the normal nucleolus organizer site is indicated by the observation that only one or two pseudonucleoli are formed in a cell (Hay and Gurdon, 1967; Fig. 4). Previous attempts to detect newly synthesized 18 S and 28 S RNAs in 0-nu embryos gave negative results (Brown and Gurdon, 1964). This negative result was probably due to a lack of sufficient sensitivity to detect the low levels of RNA which would be made by the small number of genes in 0-nu embryos. In addition to binding to the pseudonucleoli, the scleroderma antibody reacts strongly with a number of small bodies in nuclei of anucleolate embryos. Hay and Gurdon (1967) have described multiple small nucleolar bodies which are present in both normal and anucleolate embryos until the end of cleavage. These bodies are then
FIG. 4. Immunofluorescent staining patterns of cells from O-n%,1-nu, and wild-type embryos treated with EW scleroderma serum. Tail tip samples from the three types of embryos were treated as described under Materials and Methods and photographed with phase-contrast and fluorescence optics. Panels A-D show fluorescence patterns, and panels E-H show the corresponding phase-contrast views. Panels A and E show wild-type embryos; panels B and F show 1-nu embryos; panels C, D, G, and H show 0-nu embryos. The nuclei of 0-nu embryos contain bodies called pseudonucleoli at stages late in development (Esper and Barr, 1964, Hay and Gurdon, 1967). Embryos used in this work were scored for nucleolar number prior to the appearance of pseudonucleoli. For the immunofluorescence experiments, scored embryos were allowed to develop for an additional day before samples were taken for staining with antibody. Pseudonucleoli appeared during the period between scoring and staining.
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replaced by definitive nucleoli in normal cells. In some cells of anucleolate embryos these- nucleolar bodies may persist until the embryo dies, while in other cells they are partly or entirely replaced by pseudonucleoli (Hay and Gurdon, 1967).These bodies are probably responsible for the punctate antibody binding pattern seen in the nuclei of 0-nu embryos (Fig. 4). The derivation of nucleolar bodies is unknown. Determination of the biochemical nature of the antigen recognized by the scleroderma antibody should offer additional insight into the origins of these bodies. This work was supported by Grant GM26624 from the National Institutes of Health. R.E.S. was supported by National Research Service Award GM67569 from the NIH. We thank Grace Maloney and Judy Roan for technical assistance, Sandra Bizak for typing the manuscript, and members of the laboratory for their helpful comments. We are grateful to Dr. Bruce Guilliland of the Seattle Public Health Hospital for supplying sera from scleroderma patients. REFERENCES BAK~EN, A. H., MORGAN,G., SOLLNER-WEBB,B., ROAN,J., BUSBY,S., and REEDER,R. H. (1982). Mapping of transcription initiation and termination signals on Xw Levis ribosomal DNA. Proc. Nat1 Acud Sci USA 79, 56-60. BERSTEIN,R., STEIGERWALD,J., and TAN, E. M. (1982). Association of anti-nuclear and anti-nucleolar antibodies in progressive systemic sclerosis. Clix Exp. Imm. 48, 43-51. BOTCHAN,P., REEDER,R. H., and DAWID, I. B. (1977). Restriction analysis of the nontranscribed spacers of Xenopus laevis ribosomal DNA. Cdl 11,599~667. BROWN,D. D. (1967). The genes for ribosomal RNA and their transcription during amphibian development. Curr. Top. Dev. Bid 2, 47-73. BROWN,D. D., and GURDON,J. B. (1964). Absence of ribosomal RNA synthesis in the anucleolate mutant of Xenopus Levis. Proc Nat1 Ad Sti USA 51,139-146. BROWN,D. D., and WEBER,C. S. (1968). Gene linkage by RNA-DNA hybridization. I. Unique DNA sequences homologous to 4s RNA, 5s RNA, and ribosomal RNA. J. Mol. Bid 34, 661-686. BUSBY,S. J., and REEDER,R. H. (1982). Fate of amplified nucleoli in Xenspus lam-is embryos. Deu Bid 91, 458-467. ELSDALE,T. R., FISCHBERG,M., and SMITH, S. (1958). A mutation that reduces nucleolar number in Xenopus Levis. Exp. CeURes. 14,642643. ESPER,H., and BARR,H. J. (1964).A study of the developmental cytology
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of a mutation affecting nucleoli in Xaqpus embryos. Den BioL 10, 105-121. HAY, E. D., and GURDON,J. B. (1967). Fine structure of the nucleolus in normal and mutant Xenopus embryos. J. CeU Sci. 2,151-162. MACGREGOR, H. C., VLAD, M., and BARNETT,L. (1977). An investigation of some problems concerning nucleolus organizers in salamanders. Chrumosoma (Berlin) 59,283~299. MANIATIS, T., FRITSCH,E. F., and SAMBROOK,J. (1982). “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. MANIATIS, T., JEFFREY,A., and KLEID, D. G. (1975a). Nucleotide sequence of the rightward operator of phage X. Proc NatL Awd Sci USA 72,1184-1188. MANIATIS,T., JEFFREY,A., and VANDESANDE,H. (1975b). Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry 14,3787-3794. MCDONELL,M. W., SIMON,M. N., and STUDIER,F. W. (1977). Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J. Afd BioL 110.110-146. MILLER, L. (1973). Control of 5s RNA synthesis during early development of anucleolate and partial nucleolate mutants of Xcnopas laevis. J. CelL BioL 59, 624-632. MILLER, L., and BROWN,D. D. (1969). Variation in the activity of nucleolar organizers and their ribosomal gene content. Chrwnwsoma (Berlin) 28,430-444. MILLER, L., and GURDON,J. B. (1970). Mutations affecting the size of the nucleolus in Xewqw kwvis. Nature (London) 227,1108-1110. MORGAN,G. T., REEDER,R. H., and BAKKEN,A. H. (1983).Transcription in cloned spacers of Xm Levis rDNA. Proc NatL Acad Sk USA, 6490-6494. PIERANDREI-AMALDI,P., CAMPIONI,N., BECCARI,E., BOZZONI,I., and AMALDI, F. (1982).Expression of ribosomal-protein genes in Xenupus Zucvis development. Cell 30,163-171. SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. MoL Bid 93, 503517. WAHL, G. M., STERN,M., and STARK, G. R. (1979). Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethylpaper and rapid hybridization by using dextran sulfate. Proc NatL Acad Sci. USA 76, 3683-3687. WALLACE,H. R., and BIRNSTIEL,M. L. (1966). Ribosomal cistrons and the nucleolar organizer. Biochim Biophys Acta 114,296-310. WARNER,J. R. (1974). The assembly of ribosomes in eukaryotes. In “Ribosomes” (M. Nomura, A. Tissieres, and P. Lengyel, eds.), pp. 461-488. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. WELLAUER, P. K., DAWID, I. B., BROWN,D. D., and REEDER, R. H. (1976). The molecular basis for length heterogeneity in ribosomal DNA from Xaopu.s Zueuia J. MoL BioL 105,461-486.