J. Mol. Riol. (1979) 135, 565-580

Sequences of the l-672 g/cm3 Satellite DNA

of

Drosophila melanogaster DOUGLASBRUTLAG~AND W.J.

PEACOCK

and In&&al Research Organization Division of Plant Industry P.O. Box 1600, Canberra City, A.C.T., Australia 2601

Commonwealth

Acientific

(Received 28 Decewbber 1978, and in revised form

21 August 1979)

The 1.672 g/cm3 satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin D/CsCl gradient’, and a netropsin sulfate/C&l gradient. The resulting DNA was homoperleous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucl~otide sequence analysis indicates the presellco

of two

A-A-T-A-T-A-T

different

DNA FurtIler

species physiczd,

in tllis chemical

satellite.

and

poly

and trmplat,e

properties

of t,he

isolated complementary strands demonstrate that these t’wo repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. Those results further suggest that the sequence hetsrogeneit,y observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or cllromosomal region.

1. Introduction Analyses of highly-repeated satellite DNA fractions of several rodents and other mammalian species have indicated a high degree of heterogeneity among the repeat’ed sequence (Polli, et al., 1966; Flamm et al., 1969; Southern, 1970; Sutton & McCallum, 1971; Fry et al., 1973; Prosser et al., 1973; Biro et al., 1975). These sequence alterations have been detected by several methods including changes in thermal stability or buoyant density of renaturated DNA, rates of renaturation, direct sequence analysis and, more recently, by altered patterns in restriction nuclease digests (Horz et al., 1974 ; Southern. 1975 ; Cooke, 1975). Some of these techniques detect nucleotide mismatches formed upon renaturation, while others detect variations of sequence in native DNA. The nature of these sequence alterations suggests that t’hey may have arisen by random single base changes or mutation in some “basic” or primitive repeat,ing sequence (Southern, 1970). t Present address: Department of Biochemistry, Stanford University Medical Center, Stanford, Calif. 94305, U.S.,4. Author to whom reprint requests should be directed. 565 Q 1979 Academic Press Inc. (London) Ltd. 0022-2836/79/350565-16 $02.00/O

566

I). BRUTLAG

.I?; I) 12’. -1 I’E~\(iC)(‘li

In contrast to mammalian satellites. some of the satellite DIVAS of fkmyhila ;LIT very homogeneous when examined hy various criteria (Peacock et ab.. 1973: (Ml 8 Atherton. 1974: Endow ef al., 1975: Sederoff & Lowwst,ein. 1975). Three highI> repeated satellites of Droqhda virilis each contain a single repeating heptanuclcotitl~~ Fvhich accounts for over 95”/:, of t.hc sat,ellite DNA (Gall & Xt~herton. 1974). 1CIowc)vc~ the thermal stability and the buoyant densiCes of thr renaturrd satrllitc DS,Zh :I IX’ unchanged from the native state (Blumenfeld. 1973). X similar IlomogcwGty hah IKWI reportred for three of the satellites of I)rosoph%Zm nwlal~ogndrr Iutsed OII t,hp t ht~ m:~1 stability of renatured DNA (Endow rt rrl.. 1975: Krutlap pf rd.. 1977). If’ ttw 1). melanogaster satellites are allowed t’o renature close to their t,, thry do so \\.it h rapid satellitw (t,he 1.67%. 1%X6 kinetics and exhibit, a small At,. If these three fkosophila and 1.706 g/cm3 species) are renatured strictly at t, : --%“C. a limited anlollnt of’ p~~cdud, heterogeneity can 1~ detected as it At, of 5 to 11 deg. C in the rrtlaturrti These results imply a regular arrangement of wquenw heterogent~ity (Brrlt,lap & Peacock. 1975: Brutlag et al.. 1977). in this paper we report a, sequence analysis of one of t,hrw 1). nreZm?wgustrr satr~llitw (the I .672 g/cm” species) which demonstrates a regular a.rrangement of closely relatt~tl sequences. This apparently homogeneous DNA nctually cont’aitls two dist,inc+ I’(:peating

sequences : 60”,,,

2. Materials and Methods (a) Materials species m analytical C’sCl gradients a~lrl CsC’l gradients containitrg act,inolnycill I> or C’sCl gratl~c,l~is netropsill sulfatr (Peacock at al., 1!)73: Brutjlag et nl.. 1977). Alkaline saparatod t,he heav,y and light oomplelrwntary stratlds. C’alf t hylxlns DNA was p~wc~l~asetl from Sigma and yeast tRNA from Brit’ish Dr~lp Honscw. lildabeled nacleotidw I\C’I’I~ purchased from PL Biochemicals and were analyzed for purit,y prior to use by PEI t,llitllayer chromat,ography. (cr-32P)-labeled nucleosidn triphosphatj(~s were svrltllesized ucc~)rtling to the methods of Symons (I 974). All 4 drosy or riJ)(~~l11(‘1(:osidfl triphospbatos I\.C~IY’ ( IO mCi/pfrrol tirral synthesized in parallel from t.lre sa~rw sarnplt: of 1“‘l’lortl~op)lospl-rat.cl spec. acat. : Aust,ralia.n Atomic JGlrrpy (:ommissiorr ). c~!lntnitlg idclltical specific af't i\,itiw of each nucleotide for accurate, cptantitation of rclat i vc’ Inolar yields. DNA polyt~wt~ase I from Escherichia coli was electrophoret~ically Ilornogetwous fract,ion V11 purified according to Jovin et (21. (1!169). E. coli core RNA polymeraso \+‘a~ pllrificd tluwqxll the pllosplltrcellulose step according to Burgess (196!1). Spleezl phosphodiestrrasc, micrococcal nuc*leasr. venom phosphodiesteraso and pancrt:at,ic DNasc (RNas+fr~(*. DPFF) \vcr’e purchased from Worthington Biochemicals. ‘I’, RNasr, pancwatic RNasrt and lJ, RNase \vvrv purchased t,hrough Sigma. Whatman 3MM paper was used for paper electrophortxsis antI chromatography. Cellulose acrtat,e was purchased from Toxoid Ltd. PET-cellulosr plates on plastic backing were purchased from Machery-Nagel as were cellulose MN300 arrtl clrrornat~oprapl~y plat,es \I~H prcpawc~ DEAE-cellulose powders and DEAF:.ccll111osc according t,o Brownlee & Sanger ( IUA!)).

DNA was labeled %n &ro by a rlick-t,larlslatiotI react ioll \vittl I)NA polyrnerase 1 (Itigb~~ et al.. 1977). The react,ion \~as carried out, at 14°C to inlrihit de UO’UOsynthesis of poly[d(A-T)]. Tile extsnt of reaction was followed I-)y a&l precipitation until a plat,eau IINS reached (4 to 12 h) and the product was isolated by phenol extraction and Sephadw (:75 chromatography. To ensure accuratct fidelity of nick translat,iorr. t,he reactions wtwj

DROSOPHILA

SATELLITE

DNA

567

terminated at less than 20% replacement of the t,emplate nucleotides. Under these conditions neither de novo synthesis of poly[d(A-T)] nor net DNA synthesis occurred (see sequence analyses, Table 4). Base composition was determined by enzymatic hydrolysis to either 3’ or 5’ nucleotide monophosphates. The similarity of both analyses showed that the specific activities of the 4 nucleoside triphosphates were identical. Digestion to 3’ nucleotides was carried out in 0.1 ml of 10 mm-Tris.HCl (pH 8*4), 2 rnM-CaCl,, 30 pg calf thymus DNA/ml carrier with 2 pg of micrococcal nuclease. After 1 h at 37”C, 1 M-KH,PO, was added to a final concn of 20 mM, which reduced the pH to 6.4, and 2 pg of spleen phosphodiesterase was added for 1 h at 37°C. Digestion to 5’ nucleotides was carried out in 0.1 ml of 40 mM-Tris.HCl, 40 mM-glycylglycine (pH 7.4), 1.0 mM-MgCl,, 2 mmCaCl,, 30 pg calf thymus DNA carrier/ml with 1 pg pancreatic DNase. After 1 h at 37”C, 1.0 &INaOH was added to raise the pH to 9.0 and 10 pg of snake venom phosphodiesterase added for 1 h at 37°C. The nucleotides from either digest were separat,ed by electrophoresis at, pH 3.5 as described by Smith (1967). Unlabeled 3’ or 5’ nucleotides were added in order to observe the separated nucleotides by short wavelength ultraviolet irradiation. Pyrimidine tract analysis of labeled DNA was carried out according to Burton & Peterson (1960), and the tracts were separated by chromatography on PET-cellulose thin-layers according to Southern & Mitchell (1971). The individual tracts were located by autoradiography and quantitated by cutting the plastic-backed PEI-cellulose plates and counting each tract, directly in a gas-flow counter. Clomplementary RNA was synthesized with 5 pg DNA template in a 0. l-ml reactiorl containing 40 mx-Tris.HCl (pH 7.9), 10 mM-MgCl,, 160 mnl-KCl, 1 miv-dithiothreitol. ribonucleotide t,riphosphates and 5 pg of core RNA poly100 PM each of (a-32P)-labeled merase at, 37°C. The incorporation proceeded rapidly until the amount of RNA equalled the amount of DNA template. When incorporation stopped (30 to 60 min) the reaction was diluted to 0.5 ml with 0.05 M-Tris.HCl (pH 7.4), CaCl, was added to 0.2 mM- final concentration and pancreatic DNase added to 10 &ml. After 30 min at room temperature t,he reaction was terminated with EDTA, NaCl, sodium dodecyl sulfate and phenol, and the RNA was purified as outlined for nick-translated DNA. RNA prepared in this way was of broad size distribution with average chain length 2000 nucleotides as determined by eloctrophoresis in polyacrylamide gels in 5 M-urea at 60°C. Tire base composition of t’otal complementary RNA or of separated oligonucleot,ides uas carried out by llydrolysis of labeled RNA with 20 keg yeast transfer RNA carrier irl was analyzed either by elect,ro. 10(/o piperidine at 100°C for 90 min. The hydrolyzate phoresis at pH 3.5 as described above or by PEI-cellulose chromatography according to Randerath & Randeratb (1965). Nearest, neighbor analysis of DNA was performed by carrying out 4 nick-translation reactions in parallel, each having only one of the 4 nucleotides labeled. Each DNA sample was then isolated and degraded to 3’ nucleoside monophosphates as described above. Thn dinucleotide frequencies were calculated as described by Jesse et al. (1961). Nearest, neighbor analysis of cRNA to each complementary strand was performed by alkalinct ilydrolysis of 4 cRNA preparations each labeled with only one nucleotide. Calculation of dinucleotjide frequencies is the same as above except, eacll frequency in Table 1 is assigned to its complementary dinucleotide sequence present in tjhe template. Pancreatic RNase, T, RNase, Uz RNase and part.ial pancreatic RNase digests were all performed as described by Brownlee (1972). Each digest was incubated with 0.1 M-HCI to hydrolyze any remaining cyclic nucleotides to the 2’ or 3’ forms before chromatograplly. ‘1’110 complete digests were resolved on PEI-cellulose thin-layers using solvent I (1.4 finlittlium fnrmate, pH 3.4) and solvent, III (0.8 M-LiCl, pH 8.0) in the 2-dimensional systelns tlescribed by Mirzabekor bt Griffin (1972). Individual oligonllcleotides were located hl arttoradiography and counted directly. For further analysis, the spots were scraped and elnted wit11 30% triethylammonium bicarbonate (pH 10.0) as described by Brownlee (1972). The partial pancreatic RNAse digests were resolved by electrophoresis at pH 3.5 ou cellulose acetate, transferred to DEAE-cellulose thin-layer plates (7.5: l), and chromatographed with either homomix I or III of Brownlee & Sanger (1969). The thermal melting profile of the isolated strands was carried out in SSC (0.15 ~w-NaC’l. 0.0 15 M-sodium citrate) exactly as described by Mandel & Marmur (1968). The solvent was

568

1). BRUTLAG

AND

W.

.I.

PEAC’OC’K

checked both for pH (7.3) and for conductivity. &XII DNA sample was ly against solvent, degassed and melted, the initial concentrat,ions heavy strand, 0.393 0.D.2eo; 1.672 g/cm3 light, strand, 0.455 O.D.Z~,,; 0.342 0.D.260. The absorbance at each t’emperaturr was correctsed for and normalized to the o.D.~~~ at 15°C.

dialyzed extellsi\.tswere: 1.672 p/crn~~ and poly[,d(A-T).], t,hermnl expansion

3. Results (a) Major

repeating oligmucleotidm

Several workers have carried out part’ial seyuencr analysis of the L+T-rich l-672 g/cm3 satellite DNA of 2). meZanogaster. Fan&r rt ccZ. (1970) and Gall & At’herton (1974) reported nearest neighbor analyses which indicat,ed that 830,; of the adenylate (A) and thymidylate (T) residues were in an alt~ernating arrangement. Analysis of the pyrimidine tracts as well as oligonucleotide isolated from complementary RNA (cRNA) confirmed this and indicated that the major repenting units consisted of ($:;t::)

and (E)

1974; Endow (;t:;:::;:)

m a ratio

of I : 143

(Peacocsk of al..

et al.. 1975). While thrsc data, suggestrd the

non-integral

ratio

indicated

that

1973: Gall bt Atherton.

a, short repeating a longer,

more

subunit

like

complicated

repeating unit was likely. Thr presencr of minor amounts of the p,vrimidinr tra,cts C,, C,-T, and C!,-T, suggested t,here were single base changes in an alternabing A-T rt:gion which, if regular, could result in even longer repeatring unibs. A complete nearest neighbor analysis of both the complementary strands of this sat,ellite indicates that most of t,hese minor oligonucleot,ides result, from contaminating sequences rat,her than variations within the repeated u&s of the I.672 g/cm” species itself. (I)) Complete t~ewest ttright)or ntra1,ysi.s Since previous nearest neighbor analysis had heen carried out, with only two labeled nucleotides and under conditions where preferential copying of A-T may have occurred (17-fold net’ synthesis ; Fansler et al.. 1970) we decided to perform a completcb nearest neighbor analysis under conditions of limit.ed nucleotide replacementI. Tht: four dinucleotides ApT. TpA, ApA. and TpT comprisca 78O;, of all t,hr dinucleotides in the native DNA (Table 1). Examination of thr dinucleot)ides containing G and (‘ indicates that these bases are most oRen located adjacent t,o an A or a T residue. Thea dinucleotides containing these rarer bases fall into two frequency classes with GpA. ApG. CpT and TpC all close t,o l+O{, and CpA, ApC. GpT irnd TpG all about’ 3.0”,,. This is the pattern of dinucleotide frequencies one would expect if the G and (’ residues were due to single ba,se changes in a region of alternating h and T. Il’ot example, were T-A + G-C transvrrsions t’o occur wit,h a frequency of l.SV& one would expect, equality of ApG, GpA, TpC: and CpT: ApTpA TpApT

trmsversion ApGpA -+ TpCpT

Similarly, A-T --f G-C transitions in an alternating A-7’ region at t’ht: level of 3”,, would explain the equality of the other four G and C-containing dinucleotides. These data are also consistent with the G. C base-pairs coming from contaminating DNA sequences which are related to the 1.672 g/ cm3 satellite sequence by transversions

DROSOPHILA

SATELLITE TABLE

,569

DNA

1

Nearest neighbor analysis

(mol

Native 1.672 g/cm3 O/o of each dinuoleotide)

TP A 29.0

APA 9-l

TPT 10.1

APT 29.5

TPG 3.3 TpC l-7

APG 1.4 APC 2.8

(mol

1.672 g/cm3 o/0 of template

DNA Totals

CPA 2.9 CpT 1.8

GPA l-8

CPG 0.9 cpc 0.9

GPG 0.9

GPT 2.8

GPC 1.0

heavy strand dinucleotide)

Totals

TPA 37.7

APA 1.9

CPA 0.6

GP A 0.4

TPT 17.5

APT 37.4

CPT 0.5

GPT 0.9

TPG o-7

APG 0.7

TPC 0.4

APC 0.6

CPG 0.1 CpC 0.2

GPG 0.2 GpC 0.2

(mol TPA 38.7 TPT 1.1 TPG 0.3 TPC 0.2

1.672 g/cm3 light strand “/b of template dinucleotide) APA 17.8 ApT 38.4

CPT 0.4

GpA 0.3 GpT 0.3

APG 0.4 ApC 1.2

CPG 0.0 CpC 0.0

GPG 0.0 GpC 0.0

(%)

A 40.6 T 56.3 G 1.7 c 1.4

Totals

CpA I.0

(%)

A 42.8 T 44.2 G 6.5 C 6.4

(%)

9 57.8 T 40.2 G 0.7 c 1.4

The nearest neighbor dinucleotide frequencies of native 1.672 g/cm3 DNA were determined as described in Materials and Methods, section (b). The frequencies in the separated strands were determined from cRNA. The frequency of each dinucleotide in the template was determined from the frequency of the complementary dinucleotide in the cRNA. As control experiments on our nick-translation we also measure dinucleotide frequencies of calf thymus DNA and total DNA In calf thymus DNA our data agreed well with published literature (Jossr from D. melanognster. et nl., 1961), but we were surprised to note that the frequency of CpG in Drosophila DNA was not depressed as in most other eukaryotes (Swartz et ctl., 1962). The frequency of CpG (3.1%) was nearly the same as GpC (4.3%).

and t,ransitions. The nearest neighbor analysis of the isolated complementary strands is most consistent with this latter hypothesis. The more highly purified complementary strands contain much lower levels of the G and C-containing dinucleotides (Table l), and there is no obvious correlation of the complementary dinucleotides between the two strands which would be expected if the G-C base-pairs were due to sequence variations within the satellite DNA. The possible exception to this is the relatively high level of ApC and CpA in the light strand (1.2% and 1.0%) and the complementary sequences TpG and GpT in the heavy strand (0.7% and 0.9%).

570

D.

RRUTLAG

AND

(C) ~Sryuf?ncP UttKLl~ys%sfrortt

\V.

.J.

l’Er1COCK

(!ottl~ubuttlrtlta,g~~!4 12iV.4

The absence of’ rest’riction sites within t.his satellite IJN:\ has made styutww analysis difficult. We have chosen to follow the m&hods rrported by Gall li- .It,twtorl (1974) of sequencing complementary RNA synthesized from t~it~lrt~rtht> natiw 1%iz! g/cm3 satellite DNA or one of its complementa,ry strwlds. hc~wvor \\ CLhave la hrleti the cRKA uniformly by having identical specific wtivities of all four ril)olluc~lt~ositlt~ triphosphates. This method allows accura,te tlrt,c-rminatioii of’ the It~vols of minor oligonucleotides since a11 phosphates are labelt~d ctqunlly ;md it also aHo\\.> OIIP to isolate longer partial RNase digestion products and to dct~wminr tht,ir styuc~lct’ 1)~. pa,rtial exonuclease digestion (Galibert et nl.. 1974). Complt+ digestion of t hews cRNAs with pancreatic R&‘axe garc t,he smw ratios of thv major oligolluc:lt,otirlt,s :I:, ha.d hecn determined carlitxr from pyrimidinr t’rac*t analysis or from cRNA Intwlt~l at’ only a single hasr. RSA from the heavy strand ttmplai(~ yicldcd ,1-X-l’ and A- \in a 1 : I .44 ratio (+O.OS. tt ~= 8) while RNA from thca light strand cwntaint~d primaril!~ U and A-lT in the ratio I :2.57 mI0.15 (tt X. Tahlv 2 and IJig, I ). The arrangement of t’hc, rnajo~’ oligolluoleotitlrs A- I’ ~1~1 &\-.\-I- in the hva~vy strand cRNA \s’as determirwd by partial digestion with panwcatic~ K,Naw. ‘IX* t \I.() IllilsjOl partial products were (A-A-U),(A-I!), and (A-A-l’),(A-I’), (wt~ Fig. 2 and Table 3). Analysis of each of these major parbial produc+ Iby partial cxonrrckwse digwtion with spleen phosphodiesterasc shobred t#hat, all possi hlv isomers c,f’ thtastb oliponucleotides were prewnt, (data not shown). That is. the oli~oi~uclcotitlc of composition (A-L4-1T)l(A-U), contained both 5’ A-AU-L4-l’ 3 and -5’ .\-I’-;1-X-IT ~3’ n-hilt, thv oligonucleot,ide (A-A-lr),(A-CT), conbained thrre diffcrwt scqutwws (5’ &,1-l’-:\I‘3’ and 6’ .L\-l~-A-li-;\-:\-1; 3’). It is also apparrllt frollj 1 I)(, A-U 3’> .5’ 8-T-A-A-1:--\-U partial pancreatic RKase digest, that the olig(,“uclt~otic~~~ L\-l’ is rlloat oft,cn i3.Clj:l(Y’tlt to it’self resulting it) the partia,l product, (A-L’),. ‘1’1wrta iire r;trr~l~~tlrrw :\-IT units in ;I row (set: Pig. 2(a)). Similarly. the very lob\. amount of (*\--1-I’), in t Iw partia1 di,gesl suggests t’hat the oligonnclt,otidt: X-A-I: is asual ly Hankt:d 1)). an X-I- 011 twitcher side. Taken together these data suggest, tha,t 5’ A-A-I’-X-1’ 3’ and 5’ :l-L1-l’-.~-l--A-l’ 3’

I.4

Y-

Lithium

formote

g/cm’

(pli

Heavy

3.4)

B

strand

FIG. I. Pancreatic RNase digest of cRNA from the complementary pancreatic RNase and chromatographed as described in Materials The relative molar amounts composition after alkaline hydrolysis. chromatogram accurately reflects all sequences present in the cRNA. oligonuclcotides. Hyphens omitted for clarity.

0

i-672

Y - Lithium

formate

g/cm’

(pIi

3.4)

Light

-

strand

The cRNA was synthesized, isolated, treated with strands of the 1.672 g/ cm3 satellite. and Methods. Each labeled oligonucleotide was &ted and further analyzed for base of the oligonucleotides are given in Table 2. Since the RNA is uniformly labeled this This autoradiograph is intentionally overexposed to show the minor G and C-containing

I.4

I.672

DROSOPHILA

SATELLfTE

5931

DXA

TABLE 3 Partial pancreatic RNase products of cKNA from the lG2 Base composition of oligonucleotide

Molar A-U/A-A-U

ratio of measured

g/cm”

heavy strand

Oligonucleotide Composition

> 94% AU 1.1 2.0 0.7 2.9 1.1 3.1 1.7 0.96 2.3 1.2

The base composition of the first 11 oligonucleotides of Fig. 2(b) was determined bot’h from t,he length and position of the oligonucleotide on the 2.dimensional chromatogram and from a complete pancreatic digestion analysis. Both the base composition and oligonucleotide composition of the subsequent 6 oligonucleotides were inferred from the length and position on the ohromatogram based on the repeating pattern generated by the fir& 11. Hyphens omitted from sequences for clarity.

the major partial products, may constitute the basic repeating sequences in 1,672 g/cm3 cRNA. We examined even longer oligonucleotides to arrange t,hese major repeating sequences (Fig. 2(b)). Since two A-A-U sequences are rarely adjacent,, t’he presence of (A-A-U),(A-U), and (A-A-U),(A-U), in the partial digest suggested that two and three copies of 5’ A-A-U-A-U 3’ are contiguous. Similarly, since (A-U), is a rare sequence, the presence of (A-A-U),(A-U), and (A-A-U),(A-U), suggests that two and three copies of 5’ A-A-U-A-U-A-U 3’ are present in tandem. While the presence of these longer partial products does not eliminate the possibility of an alternating

2. Partial pancreatic RNaso digests of RNA complementary to the 1.672 g/cm3 heavy 1.672 g/cm3 heavy strand cRNA was partially digested with pancreatic RNase and the products resolved by electrophorosis on cellulose acetate followed by homochromatography as described in Materials and Methods. Electrophoresis resolves the oligonucleotides based on composition with U-rich ones migrating faster. Resolution by homochromatography is based almost exclusively on the length of the oligonucleotide. A plot of migration PXWUB chain length is nearly linear for these chromatograms. Two different homomixtures resolve either shorter (a) 01 longer (b) oligonucleotides. (a) Shows the final digestion products A-U and A-A-U as well as the major partial products (A-A-U), (A-U), and (A-A-U)1 (A-U),. The relative absence of oligonucleotide 6 bases long, i.e. (A-U), and (A-4-U), is most apparent in (a). Longer oligonucleotides are identified in (b). A complete pancreatic RNase digest of the first 11 partial products is described in Table 3. In addition to the major spots labeled here many minor oligonucleotides t’o the right of the major spots were shown to derive from cRNA from contaminating light-strand type sequences in that they were U-rich and released free U upon complete digestion. The minor oligonucleotides to the left of the major series were highly enriched in minor sequences such as ;\-.4-/-U and A-A-B-A-U which are very rare components of 1.672 g/cm3 DN,4. FIG.

strand.

674

I). HRUTLAG

ANI)

W. J. I’Ii:.I(,‘O(‘li

arrangement of 5’ A-*%-U-A-U 3’ and 5’ A-A-U-A-(‘-AI‘ 3’. it does rule out this as t.ho exclusive pattern. If the sequences 5’ A-A-U-A-L’ 3’ alld 5’ .A-A-U-A-lr-A-l’ 3’ \v(:ro (~.~cm’~satellil~~ cvrlsisttxl never adjacent t,o ea.& ot,her, it. would imply t.hat. the I.ti’i2 hj of two distinct DNA species with different repeating subunits.

strand of the five-nucleotide repeat would form a duplcs wit,h onf tnismatched nucleotide per five base-pairs. Similarly. hoth strands of t,hr sc.vc,n-nuclrotidr repeat would anneal with one mismatch every seven base-pairs. The evidence that both complementary &an& can form a hairpin duplex is several-fold. Electron microscopy of the isolated strands sho\vs a highly- branched duplex DNA indistinguishable from poly[d(A-T) ] (Rrntlag K- Peacock. 1975). If native 1.672 g/cm3 DNA is heat denatured and quickly cooled rtt IOH. ionic st,rength (0.01 l/r-Tris. HCI, pH 7.4) the two strands can he separated directly in a CsCI gradient into a light at neutral pH. The denatured 1.672 g/ cm3 DiYA resolves predominantly &and (1.667 g/cm3) and a heavy strand (l-705 g/ cm3) with a small amount of DXA distributed between these two major peaks (Peacook rj/ al.. 1973). Each strand of 1.672 g/cm3 DNA citn also serve as a template for E’. co& DNA polymerase 1 without the addition of any oligonucleotides as primers (SW Table 4. Mow). Finally. tiach strand can be thermally denatured (Fig. 4). Upon heating. k)otJ~ the heavy zmd Iight strands display a large hypochromicity at 260 nm typical of a duplex DNA. Mweover. each strand shows two distinct transitions, indicating the prcsenoe of’ two different DNA species. The two transitions in the heavy sOrand occur at 24 and 15 deg. (y belo\\ bhe melting temperature for poly[d(A-T)]. These temperaturcti are very close to those expected for a d(A-T) polymer with one mismatch in five base-pairs (2Oq/,) or one mis-

E’lG. 4. Thermal denaturation of the complementary strands of the 1.672 g/cm3 satellite. Each &and melts with a hyperchromicity typical of duplex DNA and shows 1,minor and 2 major transitions. The t, and relative amount of hyperchromicity of each component of the heavy strand (0, 1.672 H) is 72% with t, = 41.6”C, 21% with t, = 50°C and 7% with a t, of 66.5”C. For the light strand (0, 1.672 L) the 1st component yields 67% of the hyperchromicity at t,, -x 39.5’C, the 2nd component 24% at t, = 49’C and the 3rd 9yh at t, = 66°C. In the same experiment synthetic poly[d(A-T)] (a) melted as a single transition at 65°C.

1.672 g/cm3 complementary strands are efective for Dh’A polymerase I 1.672 g/cm3 Nucleotides

present

Template (nmol) Product (nmol) Pyrimidine tracts Tl T,

T, T4 C1T3

All 2.72 0.18

94.4 5.0 0.3 0.1 0.2

heavy dATP,

1.672

strand

All

dTTP only 2.72 0.06 (Relative 96.5 3.4 0.1 0.0 0.0

primer-templates

2.42 0.34 molar

‘23 ) 57.8 40.5 0.6 0.6 0.6

g/cm3

light dATP,

straw1 dTTP only 2.42 0.15 48.2 48.3 2.9 0.6 0.0

The individual complementary strands served as template for DNA polymerase under thr conditions described for nick-translation except that pancreatic DNase was not included. This limited synthesis to translation of internal nicks in a branched molecule to the end of that branch at which point the duplex would contain a flush 5’,3’ end. This incorporation was over in 60 min. ln a control experiment with the heavy strand of 1.686 g/cm3 satellites as a template-primer thr amount of product was less than 1 96 of the template. Only dTTP was labeled in thcso rxpnrirncrnt~ so t,hat molar amounts of each pyrimidine oligonucleotide were determined by dividing the counts in each h,~ the number of dTMP residues in that tract. Less than 1.8% of all the counts appearrxl at the orlgiu or in other longer oligonucleotides in each of these analyses. The analysis of the> pyrimidine tracts was performed twice and the results shown aw the average of hoth dott,rminations.

match in seven base-pairs (14%). The two transitions in the light strand occur at slightly lower temperatures (26 and 17 deg. C below poly[d(A-T)], suggesting that A-A mismatches destabilize DNA slightly more than T-T mismatches. Both the heavy and light strands show a minor third transition at the temperature expected for the melting of native 1.672 g/cm3 DNA. This transition could result either from

576

D. RRUTLAG

ANI)

W.

d. I’EA.CC)(‘K

some perfect invertred repeat sequence in the satellite DNA (such as a region of poly[d(A-T)]) or, more likely. from cross-strand cont’amination. The amount of hea.vJ strand contaminating the light strand or vice terra can be estimated from the amount of A-A-U present, in cRNA from the light strand. or from thtb amount of frtxtt I’ released from cRNA to the light strand on digestion with pancreatic RNase (Table 2j. These estimates from sequence analysis are consistttnt I\-ith the amount of the third melting transition.

Since the 1.672 g/cm3 heavy strand showed two thermal transitions, \\:e att’emptcd to resolve the two DNA species by thermal elution of I.672 g/cm3 DNA from hydroxyapatite (McCallum & Walker. 1967). We were surprised to find that the hea.v.v at, any tcmperaturr in 0.15 11. strand DNA lvould not adsorb t’o hydroxyapatite potassium phosphate buffer (pH 6.8). The heavy or light strands of l-672 g/cm” DSA would adsorb only at, concentrations of phosphate below O-05 hi. ‘l’h(lp eluted from hydroxyapatite as single-stranded DNA does using either heat or phosphat,r etution (0.05 to 0.10 “). No resolut,ion of either complementar,v strand Mas achieved. (“ontrol experiments with native or denatured E. colt’ J)NA \\.t:rt’ pthrformed in all casts. Apparently one mismatjch in five or s(‘ven nucleotides disrupts the DNA structural sufficiently to prewnt t,hra normal association of duptcs DNA wit II tl~drox?-;lI)atit,(, crystals. Partial rt~solution of t)Iir five and seven-tiuoleotitl? sequetrc~c~s 0 iIs achit~vett I)>chromatography of cRNA to the heavy strand on BND-cellulost~. This c~olumn t,akeh advantage of the differing degree of mismat,ch in the t \I,o s~~yuenccs and has I)een used to resolve tRNA species of differing secondary structurca. Tht~ initial cRNA has a molar ratio of A-U/A-A-C of 1.43 (Table 2). The fivrl-nucleotide sclyuence should have a ratio of A-c/A-A-U : 1.0 while the st,vt~n-tluc:l(~otitlr repeat w~,uld giv,~ A-U/A-A-I: ~~~2.0. All of the cRNA bound to BKD-cellutosr in 0.3 m-SaCI. and 3-S”,, was released bvith 1 M-K&t the conccnt~ration at which comylet~~ly duplex DNA 01’ RNA usually etutcs. This RNA had a molar ratio 14-c’/-I-~I-l~ 1.7X indicating that it is enriched for the seven-nucleotide sequenc(~. ‘l’hc~ rclmaining RN;\ (52”,,) L\X. t .I I clut,ed with W5”,, caffeine in 1 M-N&I and had a tnolar ratio of .hI:iA-At‘ indicating that it is enriched for the five-nuctrotide sey11mw. ‘I’tNw~ lYwltts SllO\\, tt1;tt RNA col*;plernent,al,~ to the seven-nucleotide repeat (5’ AA I’-,2-V-A-V 3’) chromategraphs more like intact duplex RNA than the RNA oonlI’lernental.! to the tint-nucleotide sequence (5’ A-A-U-A-U 3’). Attempts to resolve t’htl heavy or light strand DNA itself into the two sequences on BND-cellulose ha\T(l not hecn successful (Nelson & Brutlag, unpublished data). (f) Seyuenw

variatims

are located

primarily

itt the nevett-tt ucleofitle

.~ec~u,ettw

Most of the G and C nucleotides present in the nativ,, 1.672 gjcrnj DNA ~v(‘rt’ removed upon purification of the complementary strands of this satellite (Table 1). We wished to know whether the residual 0.5 to 0.9’)!,, G xnd C residues in each strand were a,tso due to contaminating DNA or whether they were sequent:~ variations located within the satellite DNA itself. To determine this \IY’ used each compkmentary strand as a template for DNA polymerase I in the presence of all four, deoxynucleoside triphosphates or with only dATP and dTTP. Since each st’rand is double-helical it should serve as an effective template-primer for DNA polymerasth.

DROSOPHILA

SATELLITE

DNA

577

If the residual G and C nucleotides are due to contaminating DNA then the strands should be effective templates even in the absence of dGTP and dCTP. If there are G and C residues dispersed within the satellite DNA then the template ability of the complementary strands should be reduced. Table 4 shows that both heavy and light strands are excellent templates with up to 7% of the heavy strand and 15”/” of the light strand being replicated by DNA polymerase I. The amount of template copied in each case decreased 50 to 60% in the absence of dGTP and dCTP, indicating that either occasional G and C residues were present in about half of the template or that DNA polymerase encountered on average one G or C residue on each template molecule. To distinguish between these possibilities, pyrimidine tract analysis of the products of each reaction was carried out. The T-rich heavy strand gave rise to DNA containing pTp as the primary pyrimidine tract. The A-rich strand template yielded a product containing both pTp and pTpTp in a molar ratio of 1.43 to 1 (Table 4). This ratio suggests that both the sequences (5’A-A-T-A-T 3’ and 5’ A-A-T-A-T-A-T 3’) are being copied. When dGTP and dCTP were omitted from the reaction the molar ratio of pTy to pTpTp decreased to 1.00 (Table 4). This indicates that the sevennucleotide repeated sequence is not copied in the absence of dCTP and dGTP. The DNA made in the absence of these nucleotides appears to be exclusively the fivenucleotide repeat. The decreased amount of DNA made in the absence of dGTP and dCTP is quantitatively consistent with none of the seven-nucleotide sequence being copied and most of the five-nucleotide repeat still serving as effective template. From t,he single-strand length of the template (3 x lo3 base-pairs) and the fraction of 5’ (A-A-T-A-T), 3’ template copied (14% with and 120,/, without dGTP or dCTP) we calculate that each DNA polymerase synthesized on average 360 to 420 nucleotides per molecule of template. This suggests that sequence variations in the five-nucleotide repeat are very rare. Copying the complementary strands in the absence of dGTP and dCTP also provides a radiochemical way of separating the t,wo DNA sequences.

4. Discussion Nucleotide sequence analysis of the 1.672 g/ cm3 satellite DNA shows that it contains two distinct DNA species with different repeating subunits. The two DNAs,

properties that they cannot be resolved by classical procedures (Peacock et al.. 1973 ; Endow et al., 1975 : Brutlag et al.. 1977). The presence of two sequences within a single satellite suggests that sequence variation detected in satellite DNAs may result in part from mixtures of closely related sequences. Similar mixtures of DNA species have been resolved in crab sat.ellite DNA (Skinner et al.. 1970 ; Skinner & Beattic. 1973,1974). Several DNAs in a single satellite might be so closely related that they might cross-hybridize giving rise to mismatched nucleotides and the appearance of sequence variation in a single DNA (see Blumenfeld, 1973). Resolution of these closely related components of a satellite into individual repeated sequences is now possible by the use of molecular cloning (Fry & Brutlag, 1979). Tn addition to this mixture of sequences in 1.672 g/cm3 DNA we have investigated two other forms of sequence variation. Since both basic repeating sequences contain only A and T residues, variant sequences containing G. C pairs are readily detected. Na,tive 1.672 g/cm3 DNA contains a large fraction of G + C (5 to 7%). While

D. BRUTLAC:

578

AND

W. J. PEACOCJi

these G and C residues appear by nearest neighbor analysis t.o be single base changes in the 1.672 g/cm3 sequence, the removal of the bulk of the G and C bases after purification of the complementary strands indicates that they arise from contamina,nts which have a closely related sequence. The most, obvious contaminating sequenceh which could give rise to these apparent sequence variants art’: I:705 g/cm” satellitcpoly-($:t:i:$$

and poly( ,“~$~~~$:“,%~$;

and thv 1 Mti

g/cm” satellitt-.

puly-

A-A-T-A-A-C-A-T-A-G (Brutlag &. Peacock, 1975: Endow rt uE.. 1975: Sederoff &. T-T-A-T-T-G-T-A-T-C Lowenstein. 1975 : Endow. 1977 ; Brutlag et al.. 1977 : Fry & Brublag. 1979). Thtx (i. (’ of transversions irl thtb base-pairs in the I.705 g/cm3 sequence,,1~:consist exclusivelv five-base-pair and seven-base-pair repeat,s of 1.672 ,Iw cm3 Dh’ A, while one of the G * (‘. pairs in the 1.686 g/cm3 sequence would appear as a t,ransvcrsion and the other ah w transition. The contamination of one satellite with anot~her is not. a trivial artifact of puritication. The I-672 and l-705 g/cm3 DNAs are t,he most. widely resolved satellit,e species in all of the gra.dients used to isolate them. and yet 1.672 g/cm” DNA is still the major of al.. 1977). ‘lb contaminant of 1.705 g/cm3 DNA (Goldring et al.. 1974: Hrntlag adjacent arrangements of closely related DKAs makes the d&trmination of sequrncc~ variation by classical sequence analysis unreliable. We have used several methods to analyze the distribution of tbc remaining (i.(’ base-pairs within the I.672 g/cm3 DNA. Since DNA polymerase replicates the tivtlnucleotide repea.ts for several hundred nucleotides in the absence of dCTP and dGTP. G*C subst,itutions must be rare in t,his DNA. A similar analysis of the 1.705 g/cm: has been nr:tdr

sequence

I)\: Kirnboim

& Spdrroff

(1975). Sinc*c

one strand of this sequence is entirely pyrimidines. t be length of pyrimidine tra& from 1.705 g/cm3 DNA gives a measure of variations whic~h clist’urb this sc~~u~n~:~~ organization. The\- recovered tracts of 750 nucleot~idrs ;ivcaraptb size. indicating long regions homogeneous in their pyrimidine bias. Thus both the sequences pal>,-

conserved

or very

recent

in the evolutionary

sense*.

The seven-nucleot’ide repeat, on the other hand. must contain some interspersed G. C pairs, as it will not serve as a template without these nucleotides. The amount of G + C remaining in the isolated complementary st,rands indicates that less than onta G.C pair per 50 base-pairs is present in the seven-base-pair sequence. To det~ermint* the distribution of t,he interspersed G residue, q we treated cRNA from the heavy or light strands with T, RNase (data not shown). Tho cRNA, initially a hroad distribut$ion with 1700 nucleotides average length, was degraded into a small discrete fra,gmrnt 60 nucleotides in length (100/o of the RNA), while the bulk of th(t RNA was dcgradrd to a heterogeneous collection of RNA of 500 nucleotides avt:ra,ge length. Incubation of the RNAs in the absence of T1 RNase resulted in t.hn degradation of the RXA but from chemical only the 500 base-pair heterogeneous size was present (resulting cleavage, Brownlee, 1972). Th’ IS suggests that some of the G-C base-pairs may t)(x distributed in a regular way within the seven-nucleotide fraction of thn 1.672 g/cm” DNA.

DROSOPHILA

SATELLITE

DNA

579

The presence of similar but discrete sequences within a satellite DNA has several important biological and evolutionary implications. Most satellite DNAs have been localized on several chromosomes (Jones, 1970; Pardue & Gall, 1970; Gall et al., 1971; Kurnit et al., 1973; Prescott et al., 1973). While the various satellites of D. melanoyaster are also located on several chromosomes, each has a unique distribution (Blumenfeld & Forrest, 1971; Peacock & Steffenson, 1975; Peacock et al., 1977). Distinct sequence components of satellite DNA might. therefore, have even more limited chromosomal distributions. The localization of the same satellite DNA on more than one chromosome does not necessarily imply that identical sequences are present on each chromosome. A unique distribution of heterochromatic DNA sequences would be consistent with many of the proposed roles of heterochromatin in homologous interactions such as meiotic pairing. We thank Dr G. Grigg and Dr H. Thrum for their generous gifts of netropsin

slllfatca.

REFERENCES

Hirnboim, H. C. & Sederoff, R. (1975). Cell, 5, 173-181. Riro, P. A., Carr-Brown, A., Southern, E. M. & Walker,

P. M. R. (1975). ,J. Mol.

Riol.

94, 71-86.

Blumenfeld, M. (1973). Cold Spring Harbor Symp. &ant. Biol. 38, 423-427. Blumenfeld, M. & Forrest, H. S. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 3145-3150. Brownlee, G. G. (1972). In Determination of Sequences in RNA in Laboratory Techniques (Work, T. S. & Work, E., eds), North-Holland/American Elsevier Press, New York. Brownlee, G. G. & Sanger, F. (1969). Eur. J. Biochem. 11, 395.-405. Brutlag, D. L. & Peacock, W. J. (1975). In The Eukaryote Chromosome (Peacock, W. ,f. & Brock, R. D., eds), pp. 35-45, Australian National University Press, Canberra. Brutlag, D., Appels, R., Dennis, E. S. & Peacock, W. J. (1977). .J. Mol. Biol. 112. 31 47. Burgess, R. R. (1969). J. RioZ. Chem. 244, 6160-6167. Burton, K. & Peterson, G. B. (1960). Biochem. J. 75, 17 27. Cooke, H. J. (1975). J. Mol. BioZ. 94, 87-100. Endow, S. A. (1977). J. Mol. BioZ. 114, 441-449. Endow, S. A., Polan, M. L. & Gall, J. G. (1975). J. Mol. BioZ. 96, 665-692. Yansler, B. S., Travaglini, E. C., Loeb, L. A. & Schultz, ,1. (1970). Biochem. Biophys. Res. Commun. 40, 1266-1272. Flamm, W. G., Walker, P. M. B. & McCallum, M. (1969). J. Mol. BioZ. 42, 441-455. Fry, K. & Brutlag, D. (1979). ,J. Mol. Biol. 135, 581-593. Pry. K.. Poon, R., Whitcome, P., Idriss, J., Salser, W., Mazrimas, J. & Hatch, F. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 2642-2646. Galibert’, F., Sedat, J. & Ziff, E. (1974). J. Mol. BioZ. 87, 377-407. Gall,