Isolation of twelve satellite DNAs from Drosophila hydei

Isolation of twelve satellite DNAs from

Drosophila hydei R. Renkawitz

Institut.Ji~r AIIgemeine Biologie, Universitiit Diis,~ehhnJ~ D-4000 DiissehloiJ~ West Germany

(Received 9 April 1979) The genome ~?]a Drosophila hydei genotype with a reduced amount ~?]heteroehromatin was.]i'actionated by three cycles ~ff preparative gradients: firstly in Ag +/Cs2S04, secondly in actinomyein D/CsCI, and finally in neutral CsCI. Using this method, tweh'e highly repetiti~e simple-sequence satellites were isolated. Ten o/them comprised only a mimn" amottnt ~?Jthe ffetiome ill contrast to the two major satellites ./ound earlier I (p = 1.696 and 1.714 g/crn3). These minor satellites were characterized by their banding in the gradient systems used, by their density in neutral CsCI, and by their rnehing point. Using these' characteristics, it was foumt that the.fi'aetions ~?/the A g +/CueSO,, gradient do m~t contain pur(fied sin qle components, heeause up to.five d([J~'rentsatellites hand in the same position ~ffthe Ag +/Cs2SO 4 gradient. It was possible to isolate a high number ¢?[ satellites et,en fl'om a genome with a reduced amount o[ heterochromatin. Thus, the D. hydei heterochromatin does mn contain one unique highly repetitit:e sequence DNA, hut is comprised ~/ many d(lfi,rent satellite sequences.

Introduction Ag+/CszSO4 gradients have often been used to isolate highly repetitive simple sequence DNA (satellites) -~5 and more complex DNA sequences 6 ~. By using a mutant of Drosophila hydei which has lost most of the X chromosomal heterochromatin and therefore most of its highly repetitive DNA 9, the problem arose as to whether it was possible to isolate any other fraction by Ag+/CszSO4 gradients, apart from the moderately repetitive DNA of the heterochromatin ~° and the major satellites described earlier ~. If it was possible, such a finding would have biological significance for further genetic studies. In addition, another problem was as to whether the DNA fractions isolated by Ag+/Cs2SO4 were pure single components or several different components. Isopycnic twin satellites have been described in CsCl 3 and we wished to know if different DNA components can also band in Ag+/Cs2SO4 at a singular position. To answer these questions DNA of D. hydei was fractionated by Ag+/CszSO4 gradients and further purification of the fractions was achieved by successive centrifugations in actinomycin D/CsCI and neutral CsC1 gradients. These fractions were further analysed by thermal denaturation.

Experimental DNA was isolated by a lysis method from frozen adult D. hydei flies of stock 703/33 (provided by O. Hess) as previously described ~. Most of the X heterochromatin of this stock (genotype Xn-X H /Y and X H • A/Y/Y) was deleted. Before fractionation, the isolated DNA was sheared with a syringe to molecular weights of about 2 × 106d to achieve reproducibility of the fractionatic;n. The Ag+/Cs2SO4 gradients were carried out with an Ag + to DNA P ratio (rl) of 0.27 1~ and the actinomycin D/CsC1 gradients with equal weight amounts of actinomycin to DNA ~2. Actinomycin was removed as described elsewhere 12 0141 8130/79/030133 0452.00 IPC Business Press

Analytical CsCI gradients were performed in a Beckman Model E ultracentrifuge and the cells were -scanned with an RS Dynograph Multiplexer at 262 nm. Thermal denaturation was achieved in 0.1 × SSC (standard saline citrate) by a Gilford 2527 thermoprogramer. The optical density was recorded with a Gilford 250 spectrophotometer. Results Fractionation

Nuclear DNA from an X H stock of D. hydei was isolated. This mutant was chosen as it is missing most of the X chromosomal satellite (p = 1.696 g/cm3), which is the major satellite of D. hydei 1. This stock was used to avoid masking of the minor DNA components by this prominent satellite (13'~i of the wild type genome). The DNA, sheared to a molecular weight of 2 × 106d and bound with Ag + (r;=0.27), was fractionated by centrifugation in Cs2SO 4 (Figure 1). Apart from a main band, three other components were also visible, a light component (fractions 1 3), a heavy shoulder of the main band (fraction 6), and a heavy peak (fractions 9 11). The light component bound little or no Ag +, as judged from its density position in a series of gradients with increasing Ag + amounts (rr=0.00-0.27). The following fractionations of the indicated fractions (Fiyure 1) were done to decide whether the isolated regions of the Ag+/Cs2SO4 gradient were single components, or whether they were mixtures of several different DNA sequences banding at similar positions. Fractions from several gradients were pooled and, after removing the Ag +, recentrifuged in actinomycin D/CsCI (Fiqure 2). Fracti onation of t he Ag +/Cs z SO4 components lighter than the main band are shown in Figure 2a, components heavier than the main band are shown in Figure 2b, however, the main band of the Ag+/CszSO,~ gradient was not further analysed. All of the actinomycin/CsCl gradients derived from Ag+/CszSO4 fractions exhibited a complex distribution

Int. J. Biolog. Macromolecules, 1979, Vol 1, August

133

Drosophila satellites: R. Renkawitz

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of several DNA components (Fi~,lures 2a and h). Fractions of these gradients were isolated, the actinomycin was removed and the DNA centrifuged again in CsCI. To determine the buoyant densities of the DNA components isolated in this way, they were centrifuged in CsCI in an analytical ultracentrifuge and compared to the density of Micrococcus lysodeikticus DNA (p = 1.731 g/cm3). Most of the fractions from actinomycin/CsCl gradients were initially checked by centrifugation in an analytical ultracentrifuge to select those fractions which contained most of a specific DNA component.For example, a DNA component with the density of 1.706 g/cm "~ is contained in fractions 1/8, 2/8, and 3/8, but only fraction 1/8 was chosen for further preparative centrifugation. It is clear from the complexity of each actinomycin/CsCl gradient and from the distribution of the single DNA components over several actinomycin/CsCI gradients, that the Ag + fractionation alone cannot separate the DNA into unique components. This is shown in greater detail in a later section. Some actinomycin/CsCl fractions contained single DNA components, e.g., fraction 2/4 with component 1.696, whereas components with densities 1.680 and 1.693 or 1.698 and 1.704 were found together in fraction 1/6 or 3/5, respectively. Fractions 2 and 3 of actinomycin gradients 1 to 5 contained DNA components with densities between 1.700 and 1.702 g/cm 3, which have a higher sequence complexity than simple sequence DNA, as tested by melting and reannealing. Purification of fractions 2/3 and 4/3 is shown. 2/3 contains the "5 S" DNA ~3 and 4/3 contains a moderately repetitive DNA localized within the heterochromatin ~° Fractionation of the dense side of the Ag*/CszSO, , gradient is shown by gradients 6 to 11. Fraction 6/3 is a moderately repetitive component with the density 1.697 g/cm -~ situated in the nucleolus and chromocentrc of polytene chromosomes TM. Gradients 7 and 8 exhibit components which are also found either in gradient 6 or in gradients 9 to 11, except for fractions 8/3 and 8/4, which were the only ones containing components 1.692 and 1.694. Gradients 9 to 11 all included the following five DNA components: p=1.677, 1.682, 1.689, 1.696, and 1.705 g/cm 3, but in different amounts. Component 1.696 is

Figure 2 Preparative centrifugation of actinomycin D/CsCI and neutral CsCI gradients. The isolated fractions ( 1 11) of the Ag+/Cs2SO4 gradient (Fi,qure 1) were further fractionated by actinomycin/CsCl gradients. These in turn were purified by neutral CsCI gradients and the densities of isolated components measured by analytical CsCI gradients. The number of each gradient corresponds to the fraction number derived from

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Drosophila satellites: R. Renkawitz the highly repe.titive D N A of the X heterochromatin ~, which is found in a small amount of these X n- flies. The reproducibility of this method was tested b y repeated fracti0nations using the same methods which gave the same results. As a control for the correct density determination the G C (guanine+cytasine) content was calculated from the density value ~'~ and from the melting point ~5. This is only possible for non-satellite fractions, as satellites are often aberrant in this respect. The components with the densities 1.697 and 1.702 g/cm 3 are not highly repetitive ~°. The G C values calculated from their densities are 38 and 43'~,, and calculated from their melting points 34 and 45%, respectively. This is within thc error of this method. Great care had been taken to avoid contamination of the nuclei by the mitochondria. One isolated DNA c o m p o n e n t (1.689}, however, had a density similar to mitochondrial D N A ~ (1.688 g/cm3). But, as shown in the next section, its melting profile exhibits the characteristics

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Since different D N A components may band at the same position in the density gradients, it was necessary to characterize the DNA fractions by a second physical property. For this purpose the melting temperature and melting range (20-, measured between 17 and 83'!. of the hyperchromicity) of the isolated D N A fractions in 0.1 x SSC were chosen to distinguish between the highly repetitive simple sequences and the more complex sequences. As seen in Fi,qure 3, most of the fractions exhibited a very narrow melting transition with a melting range of ,2.3 C or less, indicating simple sequence DNA {Table 1}. Two samples with higher complexities are also shown {p = 1.697 and 1.702 g/cm3j; their melting ranges are 7.2 and 8.1 C, respectively. Components with a unimodal sharp transition are 1.680, 1.696, 1.702, and 1.714. Fraction 2/4/2 with the density of 1.696 has a different T,, (74.9 CI than sample 11"8/2 with the same density (T,,=62.7 C). Sample 10/6/2 contains components 1.677, 1.682, and some of 1.689, as shown by analytical centrifugation {Figure 2). This is confirmed by the melting curve: 1.677 melts at 56.7 C and 1.689 at 66.1 C in addition to the transition caused by component 1.682. The fraction 8/3 contains mainly component 1.694 (T,,=66.7 C) and a minor amount of 1.682. Fraction 3/5/3 has its density peak at 1.704, but the melting curve shows that in addition to the transition at 74.2 C, there is also a transition at 68.4 C corresponding to the component 1.698. Similarly, components 1.693 and 1.696 (fractions 1/6/2 and 2/4/2) show transitions of contaminating DNA. These data are summarized in TaMe 1. Twelve of the investigated D N A components showed a narrow melting range (from which nine had a range of less than 1.1 C and three less than 2.3 C). Only one of these sharp melting components (p = 1.696 g/cm3; T,, = 62.7'C) could be detected in unfractionated D N A of wild type

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Table 1 Densities, melting temperatures and distribution in the

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Density (g/cm 3) 1.677 1.680 1.682 1.689 1.693 1.694 1.696 1.696 1.697 1.698 1.702 1.702 1.704 1.714

Temperature Range (C) (C) 56.7 57. I 62.6 66.1 69.6 66.7 74.9 62.7 67.7 68.4 69.1 72.2 74.2 76.9

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+ + =Highly repetitive simple sequence DNA o o = Moderately repetitive DNA

Int. J. Biolog. Macromolecules, 1979, Vol 1, August

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Drosophila satellites: R. R e n k a w i t z

flies. After lowering the temperature they reanneal rapidly, even in 0.1 × SSC, again indicating highly repetitive simple sequence DNA. Usually 0.1 × SSC is too low a salt concentration to support bimolecular annealing ~7, but highly repetitive D N A does reanneal in 0.0195 M Na + (0.1 × SSC) as demonstrated by Cordeiro-Stone and Lee ~8 D N A distribution in the A g + / C s z S 0 4

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T a b l e I summarizes all isolated DNA components. On the left of this Table those Ag+/Cs2SO4 fractions containing a particular D N A component are shown. These twelve satellite components and two moderately repetitive fractions differ from each other by at least one of the following criteria ( T a b l e l): banding in Ag+/Cs2SO,~, density in CsCI, melting point, and/or melting range, As satellite DNAs are often aberrant in the relationship between melting point, GC-content and density; different satellites can be found which have the same T,.-value or the same density. For example, component 1.696 ( 7",, = 74.9 C) and component 1.704 ( T , , = 74.2 C) have a very similar melting point, but they differ in their densities in CsCI and their banding in Ag+/Cs2SO,~. Also, two components with the density of 1.696 g/cm 3 have very different melting points (74.9 and 62.7C). Therefore, all of these components are separable from each other. Some are separable owing to their individual isolation. Others, however, appear together in one fraction, but their amounts relative to each other vary depending upon how small and accurate a fraction is isolated. This indicates that they are not physically linked in the molecular weight range tested. None of the isolated components, amounts to more than about 2'~0 of the genome (except for the earlier described satellites 1.714 and 1.696, which comprise about 4 and 13% of wild type D. hydei I ). Estimated percentages of each satellite are not given, because after three cycles of preparative centrifugations and fractionations the recovered DNA does no longer reflect the relative amounts within the genome. The most striking observation is that none of the Ag +/Cs2SO4 fractions contains just one DNA component, but up to five different components band at one position, These components which are isopycnic in Ag+/CszSO4 do not have similar densities in neutral CsCI; their densities can differ from each other by as much as 22 mg/cm 3 (fraction 2). This indicates that there is no relationship between the density of a component in CsCI and its density in Ag+/CszSO4 . For example, two different D N A components, which have the same density in neutral CsCI (1,696), bind very different amounts of Ag +ions. One component does not bind any Ag + (fraction 1 3), whereas the other is among those binding most of the Ag + (fraction 6 11). A further observation is that even homogeneous satellite DNA with a sharp unimodal melting curve and a sharp banding in C s C I can have a very broad banding in Ag+/Cs2SO4 (e.g., component 1.696 in fractions 6 11J. This latter phenomenon might be caused by minor sequence heterogeneities or by other sequences being attached to the satellite DNA.

Two of the satellites represent the bulk of the highly repetitive D N A and have been described earlier ~. The other ten satellites represent only a minor proportion of the genome. Other still minor D N A sequences were found, but only those which gave enough D N A to allow melting studies were considered. Hence, the genome of D. hydei, even with reduced heterochromatin, contains many different simple sequence DNA stretches which are at least 2 x 10~'d long, as this is the size of the investigated DNA. No attempts were made to isolate the same fractions from DNA of higher molecular weight to determine the full length of contiguous satellite DNAs. As previously shown, DNA of higher complexity can also be isolated, e.g., the 5S RNA genes and moderately repetitive DNA sequences localized within the heterochromatin ~J Jo The Ag +/CszSO 4 gradients alone are not sufficient for the isolation of single DNA components, since many different DNA sequences band at the same position. But the method of using three different procedures in subsequent steps seems to be very useful to yield purified single components. A similar approach was done with calf thymus DNA~9 by using a combination of BAMD/Cs2SO. ~ and Ag +/Cs2SO.~ gradients, which led to the isolation of eight satellites. Using such a fractionation procedure as a first step of sequence selection, followed by cloning techniques, the effort of making a complete gene library could be avoided. On the other hand, isolated fractions can be used as a probe to screen a gene library for complementary clones. Acknowledgements The author would like to thank Drs W. Kunz and S. A. Gerbi for helpful discussions and the D F G for support by a grant given to Dr W. Kunz (Ku 282/5). References 1 2 3 4 5 6 7 8 9 10 I1 12 13 14 15

Discussion The investigation of the Drosophila hydei genome by three cycles of preparative fractionations (Ag +/Cs2SO,~, actinomycin D/CsCI, neutral CsCI) made it possible to isolate 12 highly repetitive simple sequence D N A fractions (satellites) in addition to D N A fractions of higher complexity.

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Rcnkawitz,R. ('hronu,.soma 1978, 66, 237 Corneo,G., Ginelli, E. and Polli, E. J. Mol. Biol. 1970,48, 319 Skinner,D. M. and Bcany,W. G. Proc. Nat. Acad. Sci. USA 1973, 70, 3108 Eilipski,J., Thiery, J. P. and Bcrnardi. G. J. Mol. Biol. 1973,80, 177 Endow,S. A., Polan, M. L. and Gall, J. G. J. Mol. Biol. 1975.96. 665 Brown,D. D., Wensink, P. C. and Jordan, E. Proc. Nat. Ac,d. Sci. USA 1971,68, 3175 Clarckson,S. G., Birnstiel, M. L. and Purdom, I. E. J. Mcd. Biol. 1973, 79, 411 Kedes,L. H. and Birnstiel, M. L. Nature (London) New Biol. 1971. 230, 165 Hennig,W. 9'. Mol. Biol. 1972, 71,407 Renkawitz,R. Chromo.soma 1978, 66, 225 Corneo,G., Ginelli, E., Soave, C. and Bernardi, G. Biochemistry 1968, 7, 4373 Shun,R. H. and Kedes, L. H. Cell 1974, 3, 283 Renkawitz-Pohl, R. and Renkawitz, R. Hoppe-Seyler's Z. Physiol. Chem. 1976, 357, 330 Schildkraut,C, L., Marmur, J. and Doty, P. J. Mol. Biol. 1962,4. 430 Mandel,M. and Marmur, J. in ~Methodsin enzymology',(Eds.L. Grossman and K. Moldave), Academic Press, New York, 1968, Vol 12B, 195 Polan,M. L., Friedman, S., Gall, J. G. and Gehring, W. J. Cell Biol. 1973, 56, 580 Wetmur,J. G. and Davidson, N. J. Mol. Biol. 1968, 31, 349 Cordeiro-Stone,M. and Lee, C. S. J. Mol. Biol. 1976, 104, 1 Macaya,G., Cortadas, J. and Bernardi, G, Eur. J. Biochem. 1978. 84, 179