Proc. Nati. Acad. Sci. USA Vol. 83, pp. 696-700, February 1986 Genetics Multiplicity of satellite DNA sequences in Drosophila melanogaster (repeated DNA/heterochromatin/clone instability) ALLAN R. LOHE* AND DOUGLAS L. BRUTLAGt Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305 Communicated by David S. Hogness, September 3, 1985 ABSTRACT Three Drosophila melanogaster satellite inserts of the same size ranges as the starting material, and DNAs (1.672, 1.686, and 1.705 g/ml in CsCl), each containing therefore showing no evidence for deletion, were sequenced a simple sequence repeated in tandem, were cloned in pBR322 (see Results). as small fragments about 500 base pairs long. This precaution We investigated the instability of clones derived from the minimized deletions, since inserts of the same size as the three simple satellite DNAs by varying the size of fragments fragments used for cloning were recovered in a stable form. A used for cloning. When small DNA segments (==500 bp) were homogeneous tandem array of one sequence type usually cloned in pBR322, fragments of the same size could be extended the length of the insert. Eleven distinct repeat recovered from the plasmids although the efficiency of sequences were discovered, but only one sequence was pre- cloning was low. Clones were sequenced to verify the fidelity dominant in each satellite preparation. The remaining classes of cloning and to identify the repeating sequence. Although were minor in amount. The repeat unit lengths were restricted each of the 1.672, 1.686, and 1.705 satellites is comprised to 5, 7, or 10 base pairs, with sequences closely related. Each primarily of one simple sequence arranged in extremely sequence conforms to the expression (RRN),(RN)., where R is homogeneous, tandem arrays, an unexpected finding was A or G. The multiplicity of simple repeated sequences revealed that numerous clones contained short repeating sequences despite the small sample size suggests that numerous repeat different from the predominant type of repeat. These repeats sequences reside in heterochromatin and that particular rules differ by nucleotide changes only at certain positions of the apply to the structure of the repeating sequence. repeat sequence and not at all in others. The results indicate that the complexity of repeated sequence types in hetero- Approximately 20% of the genome of Drosophila melano- chromatin is much greater than suggested by simply four gaster consists of highly repeated sequences called satellite major satellite DNAs and that certain rules govern the DNA (1). This DNA falls into four distinct buoyant-density sequence permissible in a tandem array of simple repeats. classes (1.672, 1.686, 1.688, 1.705 g/ml in CsCl) and each can be isolated with high purity as satellite peaks (2, 3). The MATERIALS AND METHODS sequences are arranged in long tandem arrays and are restricted to heterochromatin, chromosomal regions defi- Cloning of Satellite DNAs. Total DNA was isolated from cient in genes (4). The sequences of three satellite DNAs nuclei ofD. melanogaster Oregon R embryos of average age (1.672, 1.686, 1.705 satellites) have been inferred by analysis 8 hr (12). Satellite DNAs were isolated in antibiotic/CsCl of pyrimidine tracts or RNA synthesized from a satellite gradients and banded as single symmetrical peaks in the DNA template (1, 3, 5, 6). This showed that the major analytical ultracentrifuge (4). A partially purified preparation repeating unit of each satellite was extremely simple, only 5 of the 1.690-g/ml satellite (1) was obtained by centrifuging or 10 base pairs (bp) long. DNA banding on the light side of an actinomycin D/CsCl Confirmation of the sequences and close examination of gradient in netropsin sulfate/CsCl. Since most of the repeat- the organization ofrepeats by current cloning and sequencing ed DNA in the 1.690 preparation is homologous to 359-bp methods have been difficult for the simple-sequence satel- repeats of the 1.688 satellite (see Results), the 1.690 satellite lites, in contrast to the 1.688 satellite (7). Most ofthis satellite appears to result from gradient tailing of the 1.688 satellite. DNA is comprised of more complex 359-bp repeating units Purified preparations of either the 1.672 or the 1.686 also arranged in tandem for many kilobases (kb) (8). Whereas satellite (5 /ug) in 0.3 M NaOAc were sonicated at low plasmid clones of the 1.688 satellite can contain inserts of up temperature (MSE Sonicator, three times for 10 sec at to 8 kb, or 33 tandem repeats, without showing evidence of maximum output) and 300- to 600-bp fragments were isolated instability (7, 9), clones of the 1.672, 1.686, and 1.705 by electrophoresis in a 1.4% agarose gel. After treatment with satellites are highly unstable (10). Restriction enzyme digests nuclease S1 (Boehringer Mannheim, 5000 units) at 370C for 30 of such clones showed initially a smear of sub-molar-insert min and the large fragment of DNA polymerase I (2.5 units) bands which could be resolved to a single fragment of about at 370C for 5 min, fragments were ligated into the Cla I site 0.5 kb after repeated rounds of single-colony isolation (10, of pBR322, which had been made blunt with large-fragment 11). Since the starting size of the purified satellite DNA in DNA polymerase I and four deoxynucleoside triphosphates these experiments was about 10 kb, more than 95% of the (25 1LM each) and dephosphorylated with bacterial alkaline satellite DNA insert had been deleted during propagation. phosphatase (Sigma, 0.2 unit, 650C for 30 min). After trans- In addition, many ofthese clones contained nonrepresenta- formation of Escherichia coli DH1, ampicillin-resistant col- tive DNA sequences and other alterations apparently intro- onies were recovered. The blunt-ending of DNA subsequent duced subsequent to cloning (unpublished observation). to size-selection resulted in some fragments being less than There were frequent interruptions in the periodicity of the 300 bp, depending on the lengths of single-stranded DNA at repeat by additions or deletions and numerous base alter- ends following sonication, and ensured that a range of ations. These aberrations were not seen when clones carrying Abbreviations: bp, base pair(s); kb, kilobase(s). The publication costs of this article were defrayed in part by page charge *Present address: Division of Entomology, CSIRO, P.O. Box 1700, payment. This article must therefore be hereby marked "advertisement" Canberra City, A.C.T. 2601, Australia. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom correspondence should be addressed. 696 Downloaded by guest on October 3, 2021 Genetics: Lohe and Brutlag Proc. Natl. Acad. Sci. USA 83 (1986) 697 satellite lengths were cloned. Clones of the 1.705 satellite screened by colony hybridization using purified satellite were made similarly except fragments were blunt-ended DNA as a probe. before size-selection so that fragments were greater than 300 Regardless of which of the three satellites was used as bp, and the DNA was inserted between the HindIII and starting material, only about 5% ofthe colonies were positive BamHI sites ofpBR322 by use ofHindIII and BamHI linkers. by colony hybridization. These usually retained the insert, DNA was sequenced (13, 14) using the external EcoRI site without deletions, with the size range being the same as the on the left ofthe insert or the HindIII (1.672 and 1.686 clones) starting material (300-600 bp), and no deletion was evident or BamHI (1.705 clones) site on the right of the insert. Both during large-scale growth of these plasmid DNAs. However, strands were sequenced, using 80-cm gels which allowed up most of the remaining 95% of ampicillin-resistant colonies to 600 bp to be determined from a single labeled end. had deletions, often spanning the insert and vector DNA. Satellite DNA Buoyant Density and Quantitation. The buoy- Different plasmids had deletions of varying degrees. Since ant density of satellite DNAs was determined by filter Hae III cuts the vector many times but rarely the satellite hybridization of fractions spanning a CsCl gradient of total DNA insert, the end points ofthe deletions could be mapped. DNA (300 pg). The gradient was collected in 192 fractions Deletions started from the satellite insert and continued into from the top of a VTi5O tube (Beckman). DNA from each adjacent vector DNA in a unidirectional manner, through the fraction (5 1.l) was heat-denatured, spotted onto nitrocellu- tetracycline-resistance gene and sometimes extending to the lose filters (6-mm diameter), and baked for 1.5 hr at 80'C. The origin of replication. This may result from selection against filter-bound DNA samples were hybridized in 450 mM deletions extending leftward into the ampicillin-resistance NaCl/45 mM sodium citrate/50% (vol/vol) formamide in gene used to select colonies. The deletion event appeared to separate vials at room temperature for 16 hr. The probe was occur early in plasmid replication, possibly during the first satellite insert DNA, excised with EcoRI and HindIII or replication of the transformed DNA, because multiple bands BamHI and labeled at the termini to 5000 cpm/ng with characteristic of continued deletion were not observed. Rapid [a-32P]dTTP (3000 Ci/mmol; 1 Ci = 37 GBq) and unlabeled deletion was due to the repeated DNA originating from D. deoxynucleoside triphosphates, using large-fragment DNA melanogaster rather than E. coli and was independent of the polymerase I. After hybridization, filters were washed, particular sequence type or highly reiterated nature of the heated at 10'C below the Tm (Tm is mean melting temperature; simple satellite DNAs. Excision ofthe 357-bp fragment in clone Tms are sequence-specific, unpublished observations) and 1.705-42 (Table 1) and recloning in pBR322 gave 95% positive assayed for radioactivity in a scintillation counter.