A Repetitive DNA Family (Sau3a Family) in Human Chromosomes
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Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4665-4669, July 1986 Biochemistry A repetitive DNA family (Sau3A family) in human chromosomes: Extrachromosomal DNA and DNA polymorphism (covalently closed circular DNA/restriction fragment length polymorphism/recombination) RYoITi KIYAMA, HIDEKI MATSUI, AND MICHIO OISHI Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Communicated by Franklin W. Stahl, March 7, 1986 ABSTRACT In this paper, we report a tandemly repeated 849-bp unit in cultured human cells, suggesting that the DNA sequence found in human chromosomes. The DNA Sau3A family DNA represents an unstable DNA sequence. sequence, which is present at -1000 copies per haploid genome, consists of a basic unit 849 base pairs (bp) long with MATERIALS AND METHODS a single specific restriction enzyme (Sau3AI) cutting site. The unit is further composed of five subunits, each -170 bp long. Materials. Ficoll-Hypaque solution (Lymphoprep) was When DNAs from various sources were examined by Southern purchased from Nyegaard (Oslo). Eagle's minimal essential hybridization using the repetitive DNA as a probe, a consid- medium and RPMI 1640 medium were obtained from Nissui erable degree ofrestriction fragment length polymorphism was Seiyaku (Tokyo). Fetal calf serum and newborn calf serum observed. Furthermore, a substantial percentage (=1.0%) of were purchased from Flow Laboratories and Irvine Scien- the same DNA sequence was also found extrachromosomally in tific, respectively. the cultured human (HeLa) cells as monomers and oligomers of Cell Culture. HeLa cells (S3) were cultured in minimal the basic unit in the form of covalently closed circular DNA. essential medium (calcium free) with 5% newborn calf serum These results suggest that the repetitive DNA is unstable and at 370C in a CO2 incubator. K-562 and HL-60 cells were prone to be excised from the chromosomes through homologous cultured in the same way but in RPMI 1640 medium supple- recombination. mented with 10% and 15% fetal calf serum, respectively. All of these cells were grown in suspension in 4-liter culture Human cell chromosomes contain a variety of repetitive flasks with constant stirring. CV-1 (African green monkey) cells were grown in Petri dishes (diameter, 10 cm) in minimal DNA families. Among them, Alu family is specific to primate essential medium with 10% newborn calf serum. chromosomes and consists of a basic unit of =300 base pairs DNA Isolation. The cultured cells were harvested at late (bp), which is interspersed along the chromosomes at as high exponential phase at a cell density of 3 x 105 to 5 x 105 cells as 300,000 copies per haploid genome (1, 2). The Kpn I family per ml. The chromosomal DNA was prepared from the Hirt is also interspersed throughout the human chromosomes precipitates (9) by the standard phenol procedure. Extra- - 10,000 times (3, 4). Alphoid satellite DNA generally consist chromosomal DNA was prepared from the Hirt supernatant of a tandemly repeated 340- or 680-bp basic unit (with -170 in the same way, but it was further purified by three bp subunits) and occupy ==2% ofthe human chromosomes (5, successive CsCl/ethidium bromide centrifugations. DNA 6). Repetitive DNAs with structural characteristics similar to from individuals was isolated from lymphocytes in fresh transposable elements have recently been reported (7). peripheral blood. The lymphocytes were prepared by Ficoll- It has been suggested that some of the repetitive DNA Hypaque gradient centrifugation, and the total DNA was sequences are the cause of chromosome instability and DNA extracted by the phenol procedure. DNA from chimpanzee polymorphism in human chromosomes. Jeffreys et al. (8) and monkey lymphocytes was prepared by Y. Sakaki. showed that several tandem-repetitive DNA sequences in Cloning. Extrachromosomal DNA from HeLa cells (4 x human chromosomes are unstable, which results in the DNA 109 cells, 7-liter culture) was subjected to sucrose gradient polymorphism associated with the DNA sequences. Howev- (10-40%) centrifugation (30,000 rpm, 20 hr, 40C, TST41.14 er, even in the hypervariable region, the frequencies of the rotor, Kontron) to remove mitochondrial DNA. Smaller formation of the varied DNA sequences, detected as restric- molecular weight DNA fractions were pooled and treated tion fragment length polymorphism (RFLP), are too low to with alkali (10). After having neutralized the samples, the allow more dynamic molecular biological studies on the DNA was digested with Alu I. The Alu I-digested DNA was mechanism DNA and rearrangement in human ligated into the Sca I site of pBR322. On this ligation, new of instability Hinfl sites were generated at both of the junctions. After the chromosomes. transformation of Escherichia coli (LE392), a total of 184 In this paper, we report a class of repetitive DNA family, clones were analyzed in detail. Three clones hybridized with designated as Sau3A family, in human chromosomes. The mitochondrial DNA and 34 hybridized with human Alu family DNA family, with a basic unit 849 bp long, which further DNA. The remaining 147 clones were further analyzed by consists of five homologous subunits '170 bp long, exists in comparing their hybridization patterns with extrachromo- human chromosomes at ==1000 copies per haploid genome. somal and chromosomal DNA from HeLa cells. A clone Upon restriction enzyme digestion, a considerable degree of (pHeS3-45) that contains one ofAlu I fragments of the Sau3A DNA polymorphism was found to be associated with this family sequence (nucleotides 455-793 in Fig. 7) was selected sequence. Most strikingly, substantial portions of the DNA on the basis of its unique hybridization pattern (see Fig. 4, sequence are present extrachromosomally as monomers and lane 1). To obtain a clone that had the full length of the basic oligomers of covalently closed circular DNA of the basic unit (pSP3), the clones obtained from partially digested DNA with Alu 1 (5 min at 4°C with 1 unit of Alu I per ,ug of DNA) The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: bp, base pair(s); RFLP, restriction fragment length in accordance with 18 U.S.C. §1734 solely to indicate this fact. polymorphism. 4665 Downloaded by guest on September 29, 2021 4666 Biochemistry: Kiyama et al. Proc. Natl. Acad. Sci. USA 83 (1986) were screened by colony hybridization with pHeS3-45 as a two individuals. On the other hand, when the DNA was probe. digested with a restriction enzyme (Sau3AI) before electro- Southern Hybridization. DNA was transferred to nylon phoresis, the hybridizable DNA bands were converged to one membranes (Pall Biodyne) from agarose gels. The hybridiza- major band =850 bp long, although one of the samples (lane tion was performed according to Southern (11). After hybrid- 2) derived from the lymphocytes exhibited one extra band ization, membranes were washed three times in 2 x SSC (1 x with a smaller molecular size (Fig. 1B). These results suggest SSC = 0.15 M NaCl/0.015 M sodium citrate) with NaDodSO4 that unique DNA sequences hybridized with pHeS3-45 are (0.1%) at 650C for 30 min, and once in 0.5x SSC, 0.1% located in human chromosomes at a limited number ofplaces NaDodSO4. In one case (see Fig. 2), a more relaxed as well as tandemly repetitive units, each of them with a specific as a more stringent condition was used (see legend of Fig. 2). restriction enzyme (Sau3AI) cutting site. From the intensity For the hybridization with pHeS3-45 as a probe, pHeS3-45 of the bands hybridized with the probe, we have estimated was digested with Hinfi and the fragment of nucleotides the number of the repetitive unit to be =1000 copies per 477-647 (see Fig. 7) was recovered and nick-translated with haploid genome. We have tentatively designated the repeti- [32P]dCTP (ICN Radiochemicals) (12). Autoradiographs tive DNA as Sau3A family. In addition to human DNA, the were taken using Kodak X-Omat S films. pHeS3-45 DNA hybridized with DNA from chimpanzee, DNA Sequencing. The sequence ofpSP3 was determined by giving almost a similar pattern and hybridization efficiency the dideoxynucleotide chain-termination method using (Fig. 2B, lane 3) as with human DNA. However, the hybrid- pUC19 as a vector (13, 14). ized DNA with chimpanzee DNA was dissociated when the filter was washed under a more stringent washing condition AND DISCUSSION (Fig. 2C), suggesting the DNA sequences in chimpanzee are RESULTS not identical with those in human chromosomes. On the other A Repetitive DNA Sequence in Human Chromosomes and hand, the Sau3A DNA family did not hybridize at all with Associated DNA Polymorphism. In search of another class of DNA from monkeys such as rhesus monkey (Macaca human DNA family, we have screened a considerable num- mulatta) (Fig. 2B, lane 4) and Formosan monkey (Macaca ber of DNA clones by examining their patterns of Southern cyclopis) (data not shown) as well as with DNA from cultured hybridization with human DNA digested with restriction African green monkey (Cercopithecus sabaeus) cells (CV-1) enzymes. When one of the clones (pHeS3-45) isolated from (Fig. 2B, lane 5) and mouse erythroleukemia (Friend) cells Alu I digests of DNA from HeLa cells was used as a probe, (Fig. 2B, lane 6). Thus, there seems to be a line that distinct patterns of repetitive DNA sequence with some distinguishes humans and apes from Old World monkeys and degree ofRFLP emerged among DNA from various sources. other mammals in respect to the conservation of the Sau3A Fig. LA shows examples of the RFLP observed among family DNA. EcoRI-digested DNA isolated from lymphocytes from two To confirm the RFLP (restriction fragment length poly- healthy individuals (lanes 1 and 2), two human leukemia cells morphism) observed among the DNA samples, we have (K-562, lane 3; HL-60, lane 4), and HeLa cells (lane 5).