Further studies on the characterization of repetitive Rhynchosciara DNA

Cell Differentiation 2, 131 - 141 (1973). © North-Holland Publishing Company

FURTHER STUDIES ON THE CHARACTERIZATION OF REPETITIVE RHYNCHOSCIARA DNA* J. BALSAMO, J.M. H I E R R O * * and F.J.S. L A R A Departamento de Bioqu{rnica, bzstituto de Qu{rnica, Universidade de Sao Paulo, Caixa Postal 20. 780. Sao Paulo, Brasil Accepted 23 March 1973 Comparison between the renaturation kinetics of DNA obtained from mitotic nuclei (embryos) and polytene nuclei (salivary glands) has shown that only about one third of the most repetitive sequences are normally replicated during the polytenization process, in Rhynchosciara angelae salivary glands. The same evidence was obtained by hybridization experiments, using RNA complementary to the most repetitive DNA sequences and DNA extracted from mitotic and polytene nuclei. Hybridization results obtained with DNA fractionated in CsC1 gradient and melting temperature profiles of the isolated repetitive fractions have shown that the most repetitive DNA sequences have a G - C content within the 17-27% range.

Flies of the genus Rhynchosciara have in c o m m o n with other Diptera, polytene chromosomes of large size and clearly defined m o r p h o l o g y in the cells of m a n y tissues. A specially interesting feature of polytenic systems is the differential synthesis of DNA in heterochromatin. It was suggested by Heitz (1934) that during p o l y t e n i z a t i o n h e t e r o c h r o m a t i n fails to replicate or replicates more slowly than euchromatin. This suggestion was later supported by several experimental results (Berendes et al., 1967; Rudkin. 1969; Gall et al., 1971 :Mutder et al.. 1971). It was also possible to localize certain repetitive DNA sequences in chromocenters of p o l y t e n e nuclei of Drosophila (Hennig et al, 1970; Jones et al., 1970: Rae, 1970; Gall et al., 1971) and one species ofRhynchosciara (Eckhardt et al., 1971). We have investigated the characteristics of DNA from polytenic tissues biochemically, studying its renaturation kinetics. In a previous paper (Balsamo et al.,

* Abbreviations: cRNA, RNA synthesized in vitro by means of RNA-polymerase and DNA template; SSC, 0.15 M NaCI, 0.015 M Na citrate, pH 7.2. ** Present address: Depto de Bioquimica, Facultad de Medicina, Universidad de la Republica Montevideo, Uruguay.

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1973a), we found that DNA from Rhynchosciara angelae* nuclei exhibit heterogeneous renaturation kinetics, similarly to what is found in many eukaryotic organisms (Britten et al., 1968). In salivary glands, we could detect at least three families of sequences; 86% of the genome probably represent 'unique' sequences, and about 14% represent repetitive DNA, distributed between two classes of sequences, namely the 'fast' and 'intermediate' fractions. Each of these repetitive fractions was shown to represent about 7% of the salivary gland genome, with reiteration values of 2 × 103 and 2 X 102 for the 'fast' and 'intermediate' fractions respectively. The renaturation kinetics of salivary gland DNA, as assayed by hydroxyapatite fractionation, indicated that only the 'intermediate' and 'slow' fractions are normally replicated in the latter stages of larval development (Balsamo et al., 1973a). This fact suggested the possible identity of the most repetitive DNA with the centromeric under-represented heterochromatin of polytene chromosomes. We now present further evidence for the under-replication of the 'fast' fraction. We also have further characterized the 'fast' and 'intermediate' DNA fractions, in terms of GC content and homogeneity.

MATERIALS AND METHODS Animals and gland dissection The animals were raised under laboratory conditions as described previously (Lara et al., 1965). Embryo DNA was obtained from eggs collected soon after oviposition by fertilized females. Two eggs masses were enough to yield about 200 ~tg of DNA. The salivary glands and fat body were obtained from female larvae at the 4th instar of development by dissection in a solution of the same osmolarity and Na+/K+ ratio as the hemolymph of the larvae at this instar (Terra et al., 1972). DNA preparation DNA from salivary glands and fat body cells was obtained according to Meneghini et al., (1972), as described in detail elsewhere (Balsamo et al., 1973b). DNA from embryos and pupae was prepared from nuclei by a method developed by Birnstiel (1969, unpublished results). The tissues were homogenized in 0.50 M sucrose, 0.025 M citric acid, containing 1% mercaptoethanol, in a homogenizer, for 45 sec. The resulting suspension was filtered through gauze and centrifuged for 5 min at 8000 g. The pellet was resuspended in the same medium using a Dounce homogenizer, and recentrifuged at 7500 g, for 5 min. The pellet was washed once again, centrifuged and finally resuspended in a mixture of 2 M NaC1, 1 M Tris pH 8, 10 X SSC and water in the proportion 5 : 2 : 6: 4, containing 1% mercaptoethanol. * This species was recently reclassified as R. americana (Wiedeman, 1821) by Breuer (1969).

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Then, 1 mg/ml pronase and 1% SDS were added and the solution was incubated at 60°C for 10 rain, and 37°C for 1 hr. After two steps of deproteinization with chloroform-isoamyl alcohol (25 : 1), the nucleic acids were precipitated from the aqueous phase with two volumes of ethanol, collected by means of a stirring glass rod, and dissolved in 0.I X SSC. The solution was made 1 X SSC and RNase and a-amylase were added to a final concentration of 100 ~g/ml each. The incubation with the enzymes was for 30 rain at 37°C, followed by two cycles of shaking with chloroform-isoamyl alcohol. The DNA was obtained from the last aqueous phase, by adding 1.7 volumes of isopropyl alcohol, and dissolved in 0.01 M Tris- HC1, pH 7 8. Xanthomonas campestris DNA was prepared according to Marmur (1961). All the DNA preparations were purified by banding in CsC1 gradients.

CsCl density centriJhgations The procedure was essentially that described by Ftamm et al. (1966). When only DNA purification was desired, 4.271 g of CsC1 (Harshaw, optical grade) were added to 3.50 g of DNA solution in 0.01 M Tris, pH 7 8. This yields a solution with an initial density of 1.700 g/cm 3. The centrifugations were carried out at 42,000 rpm, for a minimum of 36 hr, at 20°C, in a rotor 50 Ti of Spinco L ultracentrifuge (Beckman). About 15 fractions were collected starting at tile bottom of tile tube and diluted with 0.5 ml of 0.01 M Tris, pH 7 8, for optical measurements. The fractions containing DNA were pooled and tile DNA precipitated by addition of two volumes of ethanol. When the purpose was to determine the density of the repetitive DNA (see Results), the initial density was 1.720 g/cm 3, and Xanthomonas eampestris DNA was added as a density marker. After tile centrifugation under the conditions described above, about 43 fractions of 0.1 ml were collected from the bottom of the tube. Before centrifugation, DNA was sheared to tile desired molecular weight by ultrasonic treatment.

DNA renaturation kinetics Renaturation measurements by optical methods were carried out in thermostatically controlled cells, in a Zeiss PMQII spectrophotometer. Samples of 1.8 ml of DNA solution, in 1.0 M NaC1, were denatured with 0.1 ml of 1 N NaOH, at 65°C. At the zero time the solution was neutralized by adding 0.2 ml of a solution made by mixing 1.0 M Tris CHI, pit 8.0 and 1 N NCI (1: 1), preheated to the same temperature as DNA solution. The drop in absorbance was then followed.

Hydroxyapatite fractionation Hydroxyapatite was prepared according to Miyazawa et al. (1965) and the fractionation procedure was basically that described by Flamm et al. (1969). DNA was

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Fig. 1. Hybridization of repetitive cRNA with CsCl-fractionated DNA - DNA extracted from pupae was sheared and then centrifuged in CsCI together with Xanthomonas campestris DNA, which was used as a marker. The fractions containing DNA were used for hybridization with 2 ug/ml of RNA complementary to repetitive DNA fractions (1 X 10 6 cpm/#g) in 6 X SSC, for 2 or 4 hr, at 60°C. (o o) Absorbances at 260 nm; (o . . . . o) radioactivity in the hybrid after 2 hr; (A A) radioactivity in the hybrid after 4 hr. sheared by ultrasonic treatment to a molecular weight or 105 (fig. 4) or 106 (fig. 1) daltons. DNA denaturation was obtained by adding 0.1 N NaOH to a final concentration of 0.05 N, to the DNA solution in 0.01 M Tris HC1, pH 7 8. The DNA concentration was adjusted to the desired value (about 120 ~g/ml) by adding water preheated to 65°C. At zero time, sodium phosphate buffer, pH 6.8, was added to obtain a final phosphate concentration of 0.12 M. The renaturation was followed at 65°C, until a Cot* value of 0.020 was attained. At this Cot value, almost all of the 'fast' fraction is reassociated (Balsamo et al., 1973a) and was bound to hydroxyapatite. This material was eluted and dialysed against 0.001 M EDTA to eliminate the residues of hydroxyapatite. After another dialysis step, against 0.001 M Tris HC1 pH 7 8, the DNA solution was concentrated and the DNA used as template for E. coil DNA dependent-RNA polymerase. Thermal denaturation Melting temperature determinations were carried out in standard SSC. The increase in absorbance was monitored in a Zeiss PMQII spectrophotometer, in thermostatically controlled cells. The temperatures were measured using a thermo-

*Cot = product of DNA concentration in moles nucleotide/! and time of annealing Britten et al., 1966.

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electric couple, directly immersed in the blank solution. All data were corrected for thermal expansion.

Preparation of complementary RNA RNA polymerase was prepared from E. coli strain A-19. as described by Burgess (1969) and used after DEAE cellulose column chromatography, without further purification. The transcription was carried out in 0.25 ml of buffer containing 4 units of enzyme, 10/lg DNA, 60 m/l moles GTP and 20/aCi (fig. 4) or 100/~Ci (fig. 1) each of 3H-CTP (20.1 Ci/mmole), 3H-UTP (20.1 Ci/mmole) and 3H-ATP (24.5 Ci/ mmole) from New England Nuclear. The incubation was performed for 30 rain at 37°C and the buffer composition was that described by Pardue et al. (1970) (0.04 M Tris, pH 7.9, 0.15 M KC1, 0.0046 M MgC12, 0.002 M MnC12, 7 X 10 -5 M EDTA, and 0.0058 M/3-mercaptoethanol). The reaction was stopped by adding 40 ~g/ml of DNase l (Worthington) and digestion was carried out for 10 rain at 37°C. The cRNA was extracted according to Bishop et al. (1969).

Molecular hybridization experiments The method of Gillespie et al. (1965) as slightly modified by Gambarini et al. (1972) was followed. DNA was denature by adding an equal volume of 1 M NaOH and maintained for at least 10 min at room temperature, before neutralizing with 4 vol. of a solution containing 0.25 M Tris HC1, pH 8.0, 0.25 M HC1 and 1 M NaC1. The DNA concentration was made about 1 ~tg/ml and the solution was loaded on nitrocellulose membrane filters (Millipore GS, 13 mm diameter presoaked for a minimum of 2 hr, in 2 X SSC), at low temperature (about 4°C) and under slight pressure. The filters were dried overnight in a desiccator and for 2 hr in an oven. at 80°C. The hybridization reaction was carried out in 2 ml of 6 X SSC, for the time and temperature indicated in the legen of the figures.

RESULTS

Hybridization of CsCl-fractionated DNA with cRNA transcribed from repetitive DNA sequences In a previous paper we determined the buoyant density of the 'intermediate' fraction, taking advantage of the fact that the 'fast' fraction in underreplicated in polytenic tissues of Rhynchosciara (Balsamo et al., 1973a). This was done by denaturing fractions from a CsCI banded 3H-labeled salivary gland DNA and allowing them to renature under conditions that allow for the exclusive renaturation of the repetitive sequences. Two bands of renatured DNA were obtained: the major one had a density of 1.697 g/cm 3 , corresponding to a GC content of 38.7% (Schildkraut et al., 1962); the second, smaller, component appeared in the light zone of

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the gradient. We discussed that this light peak might represent sequences of 'fast' fraction undergoing replication, which would be concentrated in that zone of the gradient, allowing their detection. We now have tried to further characterize the 'fast' and 'intermediate' DNA fractions by hybridizing DNA obtained after banding in CsC1 gradient with RNA transcribed in vitro on a template of repetitive DNA. For this purpose, DNA enriched in 'fast' fraction was separated by the hydroxyapatite procedure and transcribed with E. coli RNA polymerase as described under Materials and Methods. Because of the procedure used in the hydroxyapatite fractionation, it is likely that the cRNA obtained contains sequences complementary to both repetitive fractions. The hybridization results after 2 and 4 hr at 60°C in 6 X SSC are shown in fig. 1. After 2 hr the hybridization with the less repetitive DNA may be expected to be severely underrepresented; so, the complementary DNA identified in this way must represent mainly 'fast' fraction. In fig. 1 we can see that after 2 hr of hybridization, the radioactivity appears as a symmetrical peak at the light zone of the main band DNA, with a density of about 1.687 g/cm 3. A second smaller component is apparent at the heavy side of the absorbance DNA peak, which is more evident after 4 hr of hybridization and which shows a buoyant density very similar to that previously obtained for the principal component of the 'intermediate' fraction (1.697 g/cm 3 - Balsamo et al., 1973a). The peak at the lower density side of the gradient remains unchanged after 4 hr of hybridization; however, after this time, hybridization of a still less denser DNA becomes evident and appears as a small shoulder with a buoyant density rather similar to the minor component detected in the renaturation experiment of 'intermediate' fraction, quoted above, (Balsamo et al., 1973a). Taking these new results into account, we think that this less dense component may in fact represent DNA fro the 'intermediate' fraction.

Melting profiles of DNA from 'fast'and 'intermediate'fractions A further evaluation of the homogeneity and GC content of repetitive DNA fractions can be obtained from their thermal denaturation profiles. The differential melting profiles for the 'fast' and 'intermediate' fractions, isolated by the hydroxyapatite procedure from a sonicated DNA sample, are plotted in fig. 2. The 'fast' fraction shows a sharp profile with a Tm at 76.5°C. From the density value in fig. 1, a Tm of 80.3°C can be estimated. Although this difference could be due to mispairing in the renatured duplex (Bolton et al., 1965), it could also be due to the imprecise determination of the buoyant density value. In fact, the shape of the melting curve of the 'fast' DNA (Balsamo et al., 1973a) indicated a high precision of base pairing in renaturation (Britten et al., 1966). Taking into account these two Tm values, we may attribute to the 'fast' fraction a GC content within the 17-27% range (Marmur et al., 1962). The 'intermediate' fraction melts over a broader temperature range than the 'fast' one (Balsamo et al., 1973a). When these results are plotted in a differential

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T(°C) Fig. 2. Differential melting curves of isolated 'fast' and 'intermediate' fractions - ZXArepresents (At2-Atl), where, At 2 and Atl are the 260 nm absorbances (corrected for water expansion) at temperatures t 2 and tl, respectively. The abscissa represents the mean temperature of the intervals and is equal to (t 2 + tl)/2. The denaturation conditions were 1 X SSC and the solution was maintained 10 min at each temperature, before reading the absorbance value. The DNA fractions were obtained as described previously (Balsomo et al., 1973a). (o o) 'fast' fraction: (e e) 'intermediate' fraction. form, one observes a curve with two inflections, as seen in fig. 2. The second inflection, at about 82.5°C, should represent the major component of the 'intermediate' fraction, which has a density of 1.697 g/cm 3, corresponding to a native Tm value of 85.2°C (Balsamo et al., 1973a). This difference between these two values could be due to imperfect base pairing in the renatured duplex and also to the fact that the isolation procedure leads to a DNA with very low molecular weight. The other component has a melting temperature of 72°C (fig. 2); it could represent a contamination of 'fast' fraction sequences. However, the hybridization experiment shown in fig. 1 suggested an apparent heterogeneity in base composition of the 'intermediate' fraction and the Tm of 72°C may likely represent the less dense component. This latter supposition implies that the 'intermediate' fraction retains the two components in the renatured form, suggesting that these two components are unique in GC content and that renatured DNA is in good register.

Underreplication of the 'fast'fraction in polytene tissues Previous results (Balsamo et al., 1973a) indicated that DNA from the 'fast' fraction is underreplicated during the polytenization process in salivary gland cells of R. angelae. If these results were correct, we might expect that DNA obtained

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from R. angelae, embryos, with very few, if any polytenization degree, would show a different renaturation pattern. In fig. 3 we present the renaturation data for DNA obtained from embryos of R. angelae, as determined by the UV method (Wetmur et al., 1968). Three fractions can be observed, similarly to salivary gland DNA; however, the 'unique' sequences comprise 73% of the genome, in contrast with the 86% obtained in salivary glands. The remaining 27% is distributed between the two reiterated fractions, 8% corresponding to the 'intermediate' and 19% to the 'fast' one. For the 'fast' fraction of embryo DNA, a reiteration value of 8 X 103 can be calculated, quite different from that of 2 X 103 obtained for salivary glands. These results indicate that about two thirds of the fast renaturing component present in embryo DNA is underrepresented in salivary gland DNA. A further approach to investigate the underreplication of the fast renaturing DNA sequences in polytene cells was to compare the amount of DNA homologous to RNA copied from these sequences in polytene and non-polytene tissues. We have chosen salivary glands as typical polytene cells and fertilized eggs, collected before hatching of the larvae, representative for non-polytene diploid cells. The cytology of fat body cells has not been worked out. Their nuclei are, however, much smaller than salivary glands and if there is some degree of polyteny, this must be much lower than that in salivary glands

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Fig. 4. Hybridization of 'fast' RNA to embryo, fat body and salivary glands DNA-3H-RNA complementary to 'fast' fraction was prepared as described in Materials and Methods (30,000 cpm//ag). The hybridization reaction was for 10 hr, at 6 X SSC, at 60°C. (A_ ~') Salivary gland DNA: lc; ,u) fat body DNA; (o -e) embryo DNA.

The fast renaturing DNA fraction was isolated by hydroxyapatite fractionation. purified, and used as template for E. coli RNA-polymerase. The complementary RNA obtained ('fast' RNA) was purified and hybridized to the three different DNA preparations. The saturation curves obtained are presented in fig. 4. The differences in hybridization level attained show that the embryo genome has more sequences of 'fast' fraction than the polytene genome from salivary glands. The fat body cell genome must have a proportion for 'fast' fraction intermediate between that of embryos and that of salivary glands.

DISCUSSION Our results on the DNA renaturation kinetics have shown that only about one third of tile most repetitive DNA of R. angelae is normally replicated in polytene chromosomes. The results also show that the underreplicated DNA fraction differs in base composition from the rest of tile nuclear DNA, having a GC content in the range 17 27% in contrast to tile 33% o f total DNA (Meneghini et al., 1971). It is very likely that this fraction corresponds to the satellite DNA reported by Eckhardt et al. (1971 ) in male adult R. hollaenderi. Tiffs satellite was shown to be located in heterochromatic regions of tile polytene chromosomes. The situation is very similar to that observed in Drosophila, by Dickson et al.

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(1971). They argue that the sequences o f the most repetitive D N A fraction that undergo replication must be located p r e d o m i n a n t l y in the e u c h r o m a t i c chromosome arms, whereas the non-replicating one w o u l d be located in the c e n t r o m e r i c h e t e r o c h r o m a t i n . This special localization was discussed in terms o f the functional properties o f h e t e r o c h r o m a t i c D N A by several authors (Heitz, 1934; Rudkin et al., 1961; Dickson et al., 1971; Gall et al., 1971). We also have found that some sequences from the 'fast' fraction, in Rhynchosciara salivary glands, are active in transcription of R N A classes largely confined to the cell nucleus (Balsamo et al., 1973a, b). We m a y suggest that the same sequences active in transcription are also active in replication. The ' i n t e r m e d i a t e ' fraction was d e t e c t e d in the same a m o u n t , b o t h in salivary glands and e m b r y o genome. The data indicating the presence o f two c o m p o n e n t s differing in GC c o n t e n t confirms our earlier suggestion (Balsamo et al., 1973a) that this fraction must be heterogeneous, being c o m p o s e d o f different D N A families with reiteration degrees too similar to allow their individualization in renaturation experiments.

ACKNOWLEDGEMENTS This work was supported by funds from the 'Funda~o de Amparo h Pesquisa do Estado de S~.o Paulo (Projeto BIOQ-FAPESP)' and from The Multinational Program of tile Organization of American States, J.M. Hierro was a visiting Investigator under this Program. We are grateful to tile Drafting Section of tile lnstituto Adolpho Lutz, S'ao Paulo, for making tile graphs and to Mr. Jose Reis Coelho for ttle maintenance of the Rhynchosciara cultures.

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