Nucleic Acids Research

The separated Hand L-strands of the a-satellite of the guinea pig, Cavea porcellus, recovered from centrifugation in alkaline CsCl gradients, form complexes with 7 different double-stranded (ds) DNAs including those of 1 bacteriophage, 2 prokaryotes, 2 invertebrates and 2 mammals. The complexes are not artifacts due to in vitro labeling of the satellite, methods of collection, the presence of divalent cations, or the fact that trace amounts of single-stranded (ss) DNAs are used. More complex ssDNAs, such as that recovered from nicked RF M13, do not associate with dsDNAs. INTRODUCTION Satellite DNAs are ubiquitous in eukaryotes 12; some of those isolated and characterized have been localized by cytological hybridization to specific regions of one or 'more chromosomes2. The primary nucleotide sequences of several have been determined3'4'5. Physical characterization of satellites is quite advanced; by contrast to such studies, ideas concerning their functions are still largely speculative. These include the provocative but still unproved suggestions as to their roles in "chromosome housekeeping"6,7 or as "filler" to provide the requisite amount of DNA needed to accompany male-specific DNA and form a discrete Y chromosome8 (see also9 ). With the exception of a few redundant DNAs that form distinct bands in CsCl gradients and code for specific proteins there is very little known with certainty concerning the biological functions of satellite DNAs. We report here data that show the potential for molecular interactions of satellite DNAs. Specifically, we show that the separated strands of the a-satellite of the guinea pig, Cavea porcellus1, form complexes with dsDNAs, some of which are considerably less highly repeated or even unique. We describe the conditions under which such associaticns occur. In addition, we have characterized the complexes and present evidence that they are not experimental artifacts. The individual strands of the a-satellite of the guinea pig show a C Information Retrieval Limited 1 Falconberg Court LondonWl V 5FG England 43 Nucleic Acids Research Volume 4 no. 1 January 1977 Nucleic Acids Research marked bias in base composition, the L-strand containing 36% C residues and the H-strand 36% G residues11'12. Almost 60% of the sequences in the L-strand are -C-C-C-T-A-A-, the remainder being considered as variations caused by one or two base changes per hexamer; the H-strand is the comple13 mentary sequence The separated strands of another satellite, present as a minor component in the DNA of the hermit crab, Pagurus pollicaris, form similar com14 plexesl. We have done the more extensive studies described here on the guinea pig satellite because of its greater availability; it comprises 9% of the total DNA15 and is isolable in milligram quantities. Several brief summaries of this work have been reported14'16. MATERIALS AND METHODS Purification of DNA. Satellite DNA of the guinea pig (Cavea porceZls) was prepared as described previously11,2. Human thymus DNA, prepared batchwise by adsorption to hydroxyapatite17 (HAP; Bio-Gel HTP, DNA grade, Bio-Rad Lab, Richmond, California) was freed of satellites by centrifugation in Cs2S04 in the presence of Hg2 at a ratio of O.1/DNA phosphate18. E. coli [14C]-DNA was the gift of W. L. Carrier; M. Zuteus DNA was purchased from Miles Laboratories, Inc.; both DNAs were further purified by passage through HAP before use. M13 RF[32P]-DNA, the gift of S. Mitra, was "nicked" by treatment with S1 nuclease (Sigma; see below) purified by passage through Sephadex G 100 to remove dsDNase activity. T7 DNA was prepared according 19 to Richardson In vitro Labeling of DNA. a. Iodination: Two to ten micrograms of DNA was labeled with 125I using the method of Commerford20. Treatment with Na2S03 was omitted and dialysis was carried out at 40 and 600 21 When native E. coli DNA was labeled, the specific activity of the product was approximately half that obtained when denatured E. coli DNA was labeled under identical conditions. b. DNA polymerase I: DNA was labeled with E. coli polymerase I (Boehringer) (3.3 units/vg DNA) in a reaction volume of 0.1 ml containing 0.04 m Ci [methyl-3H]dTTP, specific activity 26 Ci/ mmol. Concentrations of unlabeled nucleoside triphosphates were 0.1 mM dGTP and dATP and 0.25 mM dCTP. The reaction mixture was 0.05 M in sodium phosphate (PB), pH 7.55 and 0.01 M in MgCl2. After incubation for 3 hr at 150 , the reaction was stopped by adding EDTA to 0.05 M. The volume was brought to 1 ml and protein was extracted by shaking with an equal volume of freshly distilled phenol (equilibrated with Tris buffer, pH 7.8, followed by distilled water) for 15 min at 30 The aqueous layer was