Factors influencing the yield of satellite DNA in extractions from Drosophila virilis and Drosophila melanogaster adults and embryos

154

Biochimica et Biophysica Acta, 432 (1976) 154--160 Q Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98590 F A C T O R S I N F L U E N C I N G THE YIELD OF S A T E L L I T E DNA IN E XTR AC TI ONS FROM DROSOPHILA VIRILIS AND DROSOPHILA M E L A N O G A S T E R ADULTS AND EMBRYOS

PETER M.M. RAE, THOMAS R. BARNETT and DONNA G. BABBITT Department of Biology, Yale University, New Haven, Conn. 06520 (U.S.A.) (Received October 20th, 1975)

Summary The application of different DNA extraction methods to identical batches of Drosophila virilis and Drosophila rnelanogaster flies or em bryos has revealed that the ionic strength of a homogenization medium is of critical importance if c h l o r o f o r m extractions are performed. The low yield o f satellite DNA after h o m o g en izatio n in low salt buffers is less severe if EDTA is included in the buffer. Phenol ex tr a ct i on procedures result in no such differential behavior of satellite and main band DNA, b u t under certain circumstances a particular satellite fraction o f Drosophila virilis DNA may be lost.

Introduction Satellite DNA is t hat fraction of an organism's genome t hat forms one or more bands in CsC1 density gradients at positions different from bulk DNA as a consequence o f differences in base composition between bulk DNA and such fractions. Satellite DNAs usually are composed of highly repeated sequences arranged in tandem, and belong to the class of "simple sequence" DNAs found in the genomes o f many eukaryotes. Ideally, DNA from adults of Drosophila rnelanogaster exhibits the following c o m p o n e n t s in CsC1 density gradients: a main band containing the bulk of the DNA; a heavy shoulder to the main band, comprising a b o u t 3% of the total DNA; two light satellites o f very similar density, each amounting to a b o u t 3% of the DNA; and a very light, quasi-poly[d(A-T)], band [1,2]. The nucleotide sequence of each of these satellite DNAs has been determined [3,4]. In Drosophila virilis there are three b u o y a n t density satellites on the light side o f the main band [1], which also have been sequenced [5]: satellite I contributes a b o u t 30% to the total DNA of cells from largely diploid tissues (larval brains and imaginal discs} or from embryos; satellites II and III each represent about 10% of the total DNA. In DNA from

155 adults, satellite values are reduced b y about one-third due to the presence in the organism of many cells with polytene nuclei, in which satellite DNA is under-replicated [ 1,6 ]. In mouse, 10% of nuclear DNA forms a satellite in CsCl. While a b o u t 80% or more of mouse DNA can be extracted from nuclei or chromosomes with 2 M NaC1, satellite DNA is quantitatively retained in insoluble material [7,8]. We have found a similar situation to obtain in Drosophila. Further, other commonly used methods for DNA extraction can result in relatively low yields, or the apparent absence, of satellite DNAs in Drosophila melanogaster and Droso-

phila virilis. Methods and Results Adults of Drosophila melanogaster or Drosophila virilis were used fresh or after storage at either --40 or --70°C {flies stored at --15 to --20°C have not been good sources for high yields o f high molecular weight DNA). Fresh or frozen embryos of the t w o species have also been used for the preparation of DNA but, as noted below, while freezing appears to have no effect on the outcome of a DNA extraction from adults, the process is in certain circumstances detrimental to the yield of satellite DNA from embryos. The greatest absolute and relative yields of satellite DNA are obtained when small amounts of tissue are lysed in a buffered solution containing EDTA (a chelator of divalent cations) and the ionic detergent sodium dodecyl sarcosinate {Sarkosyl), the lysate is adjusted with CsC1 to a density of about 1.70 g/cm 3 , and the suspension is centrifuged at high speed until the DNA forms bands in the CsC1 density gradient. Sarkosyl is used instead of sodium dodecyl sulfate because it is soluble at high salt concentrations. While this technique has been used mostly for analytical ultracentrifugations (e.g. refs. 1 and 6), it can conveniently be scaled up to process several hundred micrograms of DNA. With this procedure, a b o u t 6% of the DNA from adult Drosophila melanogaster is contributed by the double light satellite, while the three light satellites in Drosophila virilis DNA comprise about 33 and a b o u t 50% of total DNA from adults and embryos, respectively. It is against these satellite DNA values obtained using the "direct lysate" procedure that yields resulting from the other DNA extraction procedures are judged in this report. Impetus for the study was provided b y an observation that Drosophila melanogaster DNA prepared by chloroform extraction according to Ritossa and Spiegelman [9,10] contained little detectable satellite DNA, while if phenol was used instead of chloroform to remove proteins, a substantial amount of purified DNA was satellite [11,2]. In these procedures, flies are homogenized in 0.35 M sucrose, 0.025 M KC1, 0.05 M Tris, pH 7.6, 0.005 M magnesium acetate, and a crude nuclear pellet is prepared from the homogenate by low speed centrifugation. This is suspended in 0.15 M NaC1, 0.1 M EDTA, pH 8.0, and sodium dodecyl sulfate is added to 2%. After heating to 60°C for 10 min, the lysate is made to 2 M NaC104 and proteins are removed with either chloroform or phenol. If chloroform is used, the yield of satellite is less than 2% of the total DNA, while with phenol, satellite represents about 6% of the extractable DNA.

156

MAIN BAND

SATELLITES

Tr

J~ J~
S Density

Fig. 1. C r u d e p r e p a r a t i o n s o f n u c l e i w e r e m a d e f r o m i d e n t i c a l b a t c h s o f Drosophila virilis a d u l t s w h i c h h a d b e e n h o m o g e n i z e d in 0 . 3 5 M sucrose, 0 . 0 5 M Tris, p H 7.8, 0 . 0 0 5 M m a g n e s i u m a c e t a t e plus e i t h e r 0 . 2 5 M KCI ( u p p e r ) o r 0 . 0 2 5 M KC1 ( l o w e r ) . D N A w a s p u r i f i e d f r o m b o t h in parallel w i t h c h l o r o f o r m e x t r a c t i o n s , and s a m p l e s w e r e c e n t r i f u g e d to e q u i l i b r i u m in a n a l y t i c a l CsC1 b u o y a n t d e n s i t y g r a d i e n t s . I n t h e u p p e r tracing, a b o u t 34% o f the D N A is r e p r e s e n t e d b y satellites, while in t h e l o w e r , t h e value is less t h a n 10%. T h e t r a c i n g s are p h o t o e l e c t r i c scans m a d e a f t e r 1 9 . 5 h o f c e n t r i f u g a t i o n a t 4 4 8 0 0 r e v . / m i n a n d 25°C in a n MSE C e n t r i s c a n 7 5 u l t r a c e n t r i f u g e . T h e r u n s w e r e m a d e s i m u l t a n e o u s l y w i t h e q u a l a m o u n t s of D N A . Essentially i d e n t i c a l p r o f i l e s w o u l d h a v e b e e n o b t a i n e d w e r e a b a t c h of flies, h o m o g e n i z e d in t h e 0 . 0 2 5 M KCI b u f f e r , split a n d e x t r a c t e d in o n e case w i t h p h e n o l ( u p p e r ) a n d in t h e o t h e r w i t h c h l o r o f o r m (lower).

Such a comparison, which can more strikingly be made using Drosophila virilis (Fig. 1), suggested that responsibility for differences in satellite DNA yield lay with the agent of protein denaturation and removal. That this is true only in part, however, was found when flies were homogenized directly in the 0.15 M NaC1, 0.1 M EDTA resuspension medium, and the sodium dodecyl sulfate lysate was subsequently extracted with chloroform (the method also used b y Schweber [12]). [n this case, maximum relative yields of satellite DNA were obtained, indicati~g that the ionic strength of the homogenization medium, and/or its content of divalent ion chelator, influences the yield of satellite.

Buffer Buffer Buffer Buffer Buffer Buffer Buffer

0 . 0 1 5 M NaCI, B u f f e r A 0 , 0 1 5 M NaCI, B u f f e r A 0 . 1 5 M NaCl, B u f f e r A 0 . 1 5 M NaCI, 0.1 M d i s o d i u m E D T A , p H 8 0 . 0 1 5 M NaC1, 0 . 0 1 M d i s o d i u m E D T A , p H 8 0.1 M d i s o d i n m E D T A , B u f f e r B 0.1 M d i s o d i u m E D T A , B u f f e r B D D C C C D D

C C C C C C

Buffer Buffer Buffer Buffer

0 . I 5 M NaCI, Buffer A 0 . 1 5 M NaCI, 0 , I M d i s o d i u m E D T A , p H 8 0.1 M d i s o d i u m E D T A , B u f f e r B 0.1 M d i s o d i u m E D T A , B u f f e r B C C D D

Buffer C Buffer C

0 . 0 2 5 M KCI, B u f f e r A 0 . 0 2 5 M KC1 B u f f e r A

Drosophila melanogas ter a d u l t s

Buffer Buffer Buffer Buffer Buffer Buffer

Drosophila uirilis a d u l t s

Lysis

0 . 0 2 5 M KC1, B u f f e r A 0 . 0 2 5 M KCL B u f f e r B 0 . 0 2 5 M KC1, 5 m M MnCI2, B u f f e r B 0 . 0 2 5 M KCL 5 m M d i s o d i u m E D T A , B u f f e r B 0 . 2 5 M KC1, B u f f e r A 0 . 0 2 5 M KCL B u f f e r A

Homogenization

chloroform chloroform chloroform phenol

chloroform phenol

chloroform CsC1 g r a d i e n t chloroform chloroform chloroform chloroform phenol

chloroform chloroform chloroform chloroform chloroform Phenol

Extraction

4--5 6 6 6

2 4--5

12--15 30 13 32 21 25 34

12 13 15 21 33 32

A p p r o x i m a t e yield o f satellites (percent total DNA)

1

12

9, 10 II

12

11

9

Reference to procedure

CsCl g r a d i e n t s , c o n t a i n i n g 1 - - 2 p g D N A , w e r e c e n t r i f u g e d f o r a b o u t 2 0 h in a n MSE C e n t r i s c a n 7 5 ull~acentri:~uge as d e s c r i b e d in ttie l e g e n d t o Fig. 1, o r in a B e c k m a n Model E u l t r a c e n t r i f u g e at 4 4 770 r e v . / m i n a n d 20°C. C u t - o u t s w e r e m a d e o f p h o t o e l e c t r i c s c a n s f r o m t h e f o r m e r , a n d of t r a c i n g s o f u l t r a v i o l e t p h o t o g r a p h s from the latter after verification that e x t r e m e s of the image on the negative feb within the linear range of the film's p h o t o g r a p h i c resPonse. For d e t e r m i n a t i o n of t h e a m o u n t of satellite D N A in a p r e p a r a t i o n , t h e a r e a u n d e r a p r o f i l e w a s w e i g h e d , t h e n a p e r p e n d i c u l a r w a s d r a w n f r o m t h e p e a k o f t h e m a i n b a n d t o t h e b a s e l i n e . T h e half o f m a i n b a n d w h o s e c u r v e w a s n o t i n f l u e n c e d b y t h e p r e s e n c e o f satellite w a s c u t o u t a n d w e i g h e d , t h e n t h i s v a l u e w a s m u l t i p l i e d b y t w o a n d s u b t r a c t e d f r o m t h e t o t a l T h e b a l a n c e in e a c h case r e p r e s e n t e d t h e c o n t r i b u t i o n o f satellite D N A , and is e x p r e s s e d in t h e T a b l e as a p e r c e n t o f t h e t o t a l w e i g h t . T h e a b b r e v i a t i o n s u s e d are: B u f f e r A, 0 . 3 5 M s u c r o s e , 0 . 0 5 M T r i s • HC1, p H 7.8, 0 . 0 0 5 M m a g n e s i u m a c e t a t e ; B u f f e r B, 0 . 3 5 M s u c r o s e , 0 . 0 5 M T r i s - HC1, p H 7 , 8 ; B u f f e r C. 0 . 1 5 M NaC1, 0,1 M d i s o d i u m E D T A , p H 8, 2% s o d i u m d o d e c y l s u l f a t e ; B u f f e r D, 0.1 M d i s o d i u m E D T A . 0 . 0 5 M T r i s - HCI, p H 7.8, 1% s o d i u m d o d e c y l s a r c o s i n a t e (Sarkosyl).

Y I E L D S O F S A T E L L I T E D N A AS A F R A C T I O N O F M A I N B A N D P L U S S A T E L L I T E A F T E R V A R I O U S E X T R A C T I O N P R O C E D U R E S

TABLE I

158 A systematic study, summarized in Table I, revealed t h a t when chloroform extractions are used, highest possible relative yields of satellite DNA are obtained only if flies are homogenized in a medium containing greater than about 0.2 M salt. The presence or absence of divalent cations (Mg 2., Mn 2÷ or Ca 2÷) in the homogenization buffer does not appreciably affect the percentage of satellite in a preparation, although the inclusion of between 5 and 100 mM disodium EDTA results in a yield of satellite intermediate between the greatest and the least. The various extractions outlined in Table I were performed on a number of different collections of flies, and in most instances a given collection was split into two or more batches. These were then subjected to different DNA extraction procedures, with replicates of a particular condition being performed with different batches. Two or more procedures, each with a different single variable, were usually carried out simultaneously with uniform homogenization conditions and buffer volumes. Typically, between i and 5 g of flies were homogenized in 10--50 volumes of a homogenization buffer, and detergenthigh salt lysates of crude nuclear preparations had a volume of 10--20 ml. We noticed no effect of the ratio of buffer volume to tissue weight, within these limits, on the relative yields of satellite DNA in the various preparations. The conditions of subsequent extractions, ethanol precipitations, and RNAase and a-amylase digestion were as described in the cited references. Emphasizing the importance of the ionic strength of the buffer in which organisms are initially homogenized for a chloroform extraction is the fact that if flies are disrupted in 0.25 M KC1 and the salt concentration is immediately reduced 10-fold with buffer lacking KC1, the expected high yield of satellite is still obtained. Essential irreversibility of the event that inhibits the release of satellite after homogenization in low salt buffers is indicated by the fact that the effect cannot be reversed during subsequent salt treatments in the extraction procedure (addition of NaC1 to 0.15 M and disodium EDTA to 0.1 M, further addition of NaC104 to 2 M) or by the addition of sodium dodecyl sulfate or Sarkosyl to 2% before chloroform extraction. The direct addition of CsC1 to a Sarkosyl lysate (to 5.5 M) followed by ultracentrifugation does result in a good recovery of satellite, but if the CsC1 suspension is extracted with chloroform before ultracentrifugation, the lower yields of satellite are obtained. This result suggests a trapping of satellite DNA in protein that is denatured by chloroform. Kurnit [13] reported that the use of chloroform for the preparation of mammalian DNA may result in the appearance of DNA aggregates that rapidly form bands in CsC1 gradients, and that a consequence of this can be the preferential loss of DNA banding in the position of mouse satellite DNA in equilbrium density gradients. Since it was possible that tl~e loss of satellite DNA we have observed after the chloroform extraction of low salt homogenates could have been due to a similar phenomenon, we extracted DNA from a batch of flies in such a way that half the preparation would be rich in satellite DNA and half would be lacking in these sequences. This was accomplished through the extraction of half of a low salt homogenate with phenol and half with chloroform. Aliquots of DNA were then centrifuged in CsC1 solutions having an initial density of about 1.700 g/cm 3, and photographs were taken at about 4, 6, and 17 h into the run at 44 770 rev./min. In both instances, only normal band

159 formation occurred, and we have concluded that aggregative phenomena as observed by Kurnit [13] are not the cause of low satellite DNA yields in our preparations. We have performed several extraction experiments on post-blastoderm embryos of Drosophila virilis. In constrast to the results obtained using adults, we have found that frozen embryos subjected to procedures involving chloroform give essentially uniformly low yields of satellite, while this, again, is not the case if phenol is used for removal of protein. The reason for this behavior is not known. As noted, phenol is superior to chloroform for DNA extractions where high relative yields of satellite are desired; it generally also results in greater recoveries of total DNA. It has been reported, however, that poly[d(A-T)] is soluble in phenol [14,15], and we have found t h a t if phenol which has not been freshly distilled or preserved with 0.5% 8-hydroxyquinoline is used for any step in the purification of Drosophila virilis DNA, large amounts of satellite II, poly[d(A-T-A-A-A-C-T)/(T-A-T-T-T-G-A)] (ref. 5), may be preferentially lost from the aqueous phase. This behavior is particularly curious because satellite II is a positional isomer of satellite III, poly[d(A-C-A-A-A-T-T)/T-G-T-T-T-A-A)], which is not so affected. Smith et al. [16] have shown t h a t the solubility of poly[d(A-T) • d(A-T)] in phenol is a function of salt concentration and pH. Our observations on the behavior of satellite II of Drosophila virilis, however, have been obtained with various salt concentrations between 0.015 and 2 M, and with pH values between 6.8 and 9.0. Finally, mention must be made of our observation that inclusion of the nonionic detergent Triton X-100 in homogenization buffers can lead to the loss of satellite DNA when chloroform is used for extraction. Again, the reason for this is not known, although the phenomenon may reflect a differential interaction between the detergent and certain chromosomal proteins [ 17]. Discussion

Our experiments were designed to localize the step in a chloroform extraction procedure for DNA that is critical to the yield of satellite in the final purified DNA preparation. We concentrated on Drosophila virilis because up to about 34% of the total DNA from adults (which are a mixture of diploid and polytene cells) can be satellite in this species, and the differences in yield of satellite between extraction methods is very dramatic (Fig. 1). Many different extractions were performed, and the yields of satellite DNA relative to main band DNA were determined from analytical CsC1 density gradients. We have concluded that the event which prevents the release of satellite DNA along with the rest occurs when the organisms are first homogenized for the preparation of a crude nuclear fraction. At this step, fresh or frozen flies are ground in buffer in a mortar or disrupted in a blendor, filtered through nylon screen, then homogenized further in a Dounce homogenizer. A crude nuclear pellet is prepared from the homogenate by low speed centrifugation, lysed with either sodium dodecyl sulfate or Sarkosyl, and the lysate is made 2 M in NaC104 before nucleic acids are extracted with chloroform. If homogenization is done in salt concentrations below about 0.2 M, sate]lite

160 DNA yield is very low, while above this molarity the expected large a m o u n t of satellite is obtained. The effect is not reversed by the subsequent addition of salt to high levels, by detergent, or by deproteinization with chloroform. It is, however, overcome if phenol is used as a protein denaturant instead of chloroform, or if a low salt lysate is made directly to about 5.5 M CsC1 for a density gradient. Further, an a m o u n t of satellite intermediate between the poorest and best yields can be had if 5 mM or more EDTA is included in the low salt buffer. These data suggest that, at least in the two Drosophila species we have examined, chromatin that contains satellite DNA is rather selectively sensitive to ionic environment, and that it is possibly precipitating or undergoing some other rearrangement, with the likely participation of endogenous divalent cations, when it is confronted with low ionic strength. The phenomenon is evidently a stable one, since even drastic elevation of the salt concentration, and the addition of EDTA to 0.1 M and sodium dodecyl sulfate to 2% does not dissociate the material once it has formed. The effectiveness of phenol at disrupting the complexes, and the absence of reasonable amounts of satellite DNA in the aqueous phase after a chloroform extraction, is likely due to the fact that chloroform is a surface denaturant, so that undissociated nucleoprotein will remain so, and the satellite DNA will be trapped. The results suggest that satellite DNA, which has been found to be located exclusively in constitutive heterochromatin [2], either is bound to chromosomal proteins in a manner different from the binding of other DNA, or is bound to special protein not c o m m o n in other chromatin. An investigation of these altervatives is underway. Acknowledgements A portion of this work was performed while P.M.M.R. and T.R.B. were at the Max-Planck-Institut fi]r Biologie, Abteilung Beermann, Tfibingen, Germany, and we are grateful to Professor W. Beermann for his support and interest. The rest was supported by an institutional grant from the American Cancer Society (IN 31 N9) and a grant from the National Science Foundation (GB 43821). References 1 Gall, J . G . , C o h e n , E.H. and Polan, M.L. ( 1 9 7 1 ) C h r o m o s o m a 33, 3 1 9 - - 3 4 4 2 Rae, P.M.M. ( 1 9 7 2 ) Adv. Cell Mol. Biol. 2, 1 0 9 - - 1 4 9 3 P e a c o c k , W.J., Brutlag, D., G o l d r i n g , E., Appels, R., H i n t o n , C.W. and Lindsley, D. ( 1 9 7 3 ) Cold Spring H a r b o r Syrup. Q u a n t . Biol. 38, 405---416 4 E n d o w , S.A., Polan, M.L. and Gall, J . G . ( 1 9 7 5 ) J. Mol. Biol. 96, 6 6 5 - - 6 9 2 5 Gall, J . G . a n d A t h e r t o n , D. ( 1 9 7 4 ) J. Mol. Biol. 85, 6 3 3 - - 6 6 4 6 E n d o w , S.A. and Gall, J . G . ( 1 9 7 5 ) C h r o m o s o m a 50, 1 7 5 - - 1 9 2 7 S c h i l d k r a u t , C.L. and Maio, J.J. ( 1 9 6 8 ) Biochim. B i o p h y ~ A c t a 161, 7 6 - - 9 3 8 Maio, J.J. a n d S c h i l d k r a u t , C.L. ( 1 9 6 9 ) J. Mol. Biol. 40, 2 0 3 - - 2 1 6 9 Ritossa, F.M. a n d S p i e g e l m a n , S. ( 1 9 6 5 ) Proc. Natl. A c a d . Sci. U.S. 53, 7 3 7 - - 7 4 5 10 K r a m , R., B o t c h a n , M. a n d H e a r s t , J.E. ( 1 9 7 2 ) J. Mol. Biol. 64, 1 0 3 - - 1 1 7 11 Laird, C.D. and M c C a r t h y , B.J. ( 1 9 6 8 ) G e n e t i c s 60, 3 0 3 - - 3 2 2 12 S c h w e b e r , M.S. ( 1 9 7 4 ) C h r o m o s o m a 44, 3 7 1 - - 3 8 2 13 K u r n i t , D.M. ( 1 9 7 4 ) Nucleic A c i d s Res. 1, 4 5 5 - 4 6 0 14 S k i n n e r , D.M. and T r i p l e t t , L.L. ( 1 9 6 7 ) B i o c h e m . Biophys. Res. C o m m u n . 28, 8 9 2 - - 8 9 7 15 Bhorjee, J., J a n i o n , C. and L a s k o w s k i , M. ( 1 9 7 2 ) Biochim. Biophys. A e t a 2 6 2 , 1 1 - - 1 7 16 S m i t h , D . A . , Martinez, A.M. and Ratliff, R . L . ( 1 9 7 0 ) Anal. B i o c h e m . 38, 8 5 - - 8 9 17 A l f a g e m e , C.R., Z w e i d l e r , A., M a h o w a l d , A. and C o h e n , L.H. ( 1 9 7 4 ) J. Biol. C h e m . 249, 3 7 2 9 - - 3 7 3 6