A Repetitive DNA Family (Sau3a Family) in Human Chromosomes

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). As examined DNA isolated from the lymphocytes from 10 seen in the figure, several hybridized bands with DNA individuals including 4 in the same family. As seen in Fig. 3, polymorphism were observed among the DNA samples, a considerable degree of RFLP was observed among these notably between the DNA samples of lymphocytes from the DNA samples and some of the polymorphic bands (indicated 1 2 3 4 5 kbp by arrows) transmit to the progeny. DNA in the r -A| " - origin Covalently Closed Circular Extrachromosomal A Fraction. We have found that DNA hybridized with pHeS3- - 9.4 - 6.6 A B C - 4.4 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 kbp A_ - origin - 2.3 - 2.0 - 9.4 - 6.6 - 4.4

2.3 - 0.56 - 2.0 B - ornin

- 0.56 2.0 = 1.9 - 1.6 - 1.4 - 0.95 - 0.83 FIG. 2. Hybridization of Sau3A family DNA to chromosomal - 0.56 DNA from human, chimpanzee, monkey, and mouse. Ten micro- grams of each DNA was digested with EcoRI and electrophoresed in a 0.7% agarose gel. A photograph (A) was taken after staining the gel with ethidium bromide (1 ug/ml). After transferring to a nylon FIG. 1. Southern blot analysis of human chromosomal DNA membrane, the DNA was hybridized at 650C overnight with 32P- using pHeS3-45 as a DNA probe. Ten micrograms of each purified labeled pHeS3-45. Before autoradiography, the membrane was chromosomal DNA was digested with EcoRI and Sau3AI, electro- washed three times in 3x SSC/0.1% NaDodSO4 at 650C for 30 min phoresed in agarose gels (A, 0.7%; B, 1.5%), transferred to nylon (B) and then washed once with 0.3x SSC/0.1% NaDodSO4 at 650C membranes, and hybridized with 32P-labeled pHeS3-45. (A) After for 3 hr (C). (A) Total DNA (fluorescence stained); (B and C) digestion with EcoRI; (B) after digestion with Sau3AI. Sources ofthe autoradiograph. Lanes: 1, HeLa cell DNA; 2, human (male, 49 yr DNA are as follows: lane 1, lymphocytes from a healthy individual old) lymphocyte DNA; 3, chimpanzee lymphocyte DNA; 4, monkey (Oriental, male, 49 yr old); lane 2, lymphocytes from a healthy (rhesus monkey) lymphocyte DNA; 5, DNA from cultured CV-1 individual (Oriental, male, 24 yr old); lane 3, human leukemia cells (African green monkey) cells; 6, DNA from cultured mouse (K-562); lane 4, human leukemia cells (HL-60); lane 5, HeLa cells. (erythroleukemia) cells. Lanes 4-6 are omitted in C. Downloaded by guest on September 29, 2021 Biochemistry: Kiyama et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4667

1 2 3 4 5 6 7 8 9 10 123456 kbp are a mixture of covalently closed circular oligomers. Essen- tially the same ladder-like pattern, although to a lesser degree, was observed in the extrachromosomal DNA fraction - 9.4 - 6.6 from human leukemia cells (HL-60) (Fig. 4, lane 2). The DNA that exhibited the ladder-like pattern was cova- - 4.4 lently closed circular DNA. The total extrachromosomal DNA from the Hirt supernatant from HeLa cells was first - 2.3 fractionated by CsCl/ethidium bromide density gradient U - 2.0 centrifugation, and each fraction was further fractionated by :;:*:: .- gel electrophoresis. The DNA was then hybridized with :se Lirs- s;

1 2

B

c i 1.60

1.55

FIG. 4. Southern blot analysis of the extrachromosomnal DNA using pHeS3-45 1.50 as a DNA probe. The purified DNA from 6 x 108 cells was electrophoresed in an FIG. 5. Determination of the conformation of the extra- agarose gel (0.7%) and hybridized with chromosomal DNA. Low molecular weight DNA in the Hirt super- 32p-labeled pHeS3-45 probes. Lanes: 1, natant from HeLa cells (1 x 109 cells) was centrifuged (55, 000 rpm, ifextrachromosomnal DNA from HeLa 20 hr) in the presence of ethidium bromide (600 Mg/ml) in CsCl cells; 2, extrachromosomal DNA from solution (p = 1.3860). Fractions were collected from the bottom of HL-60 cells. Arrows indicate the oligom- the tubes and each fraction (in duplicate) was electrophoresed in ers, (monomer, dimer, trimer, etc.) of the 0.7% agarose gel. One sample was hybridized with 32P-labeled basic unit. Possible incomplete units pHeS3-45 probe (A). The other sample was hybridized with 32p- (1.2, 1.4, 1.6, and 1.8 units) between labeled human mitochondrial DNA (B). Arrow indicates the position monomers and dimers are indicated by of mitochondrial covalently closed circular DNA. Density of the longer arrows. fractions is shown in C. Downloaded by guest on September 29, 2021 4668 Biochemistry: Kiyama et al. Proc. Natl. Acad. Sci. USA 83 (1986)

ccc DNA 1 2 3 4 linear DNA per haploid genome) in human chromosomes. It is known that origin kbp the distribution of the DNA that hybridizes with Alu, Kpn I, penta 3.52 and alphoid satellite family among small polydisperse DNA tetra - t 2.02 is more or less proportional to the content in the total genomic tri-= t | |-t -~~~~~di~~ri 2.021.91 DNA (18). Thus, the repetitive DNA reported here seems to di - 1.58 represent an unusually unstable excision-prone DNA family mono -mono 0.83 in the human chromosome. Structure of the Repetitive DNA. Since the size of the DNA (=340 bp) originally cloned in pHeS3-45 was considerably smaller than that of the estimated basic unit (800-900 bp) of the extrachromosomal DNA, we recloned DNA with the FIG. 6. Southern blot analysis of Sau3AI-digested extrachromo- complete unit length from the extrachromosomal DNA frac- somal and chromosomal DNA from HeLa cells. Extrachromosomal tion of HeLa cells. Among the clones that hybridized with DNA from 1.2 x 108 HeLa cells was digested with Sau3AI and pHeS3-45, one clone (pSP3) contained the complete DNA electrophoresed in a 1.5% agarose gel (lane 2). Ten micrograms of sequence, which turned out to be --850 bp. The base chromosomal DNA was digested with Sau3AI. 32P-labeled pHeS345 sequencing of the insert in pSP3 was carried out and the total was used as the probe. Lanes 1 and 2, extrachromosomal DNA with base sequence (849 bp) has been elucidated (Fig. 7). As (lane 2) and without (lane 1) Sau3AI treatment. Lanes 3 and 4, expected, there is a single Sau3AI cutting site in the 849-bp chromosomal DNA with (lane 4) and without (lane 3) Sau3AI basic unit (dashed underlining in Fig. 7). treatment. The positions of oligomers in covalently closed circular sequence have become (ccc) form or linear form are indicated. EcoRI- and HindIII-digested Several unique features in the DNA X DNA were used as positional markers for linear DNA. clear from the sequencing data. As shown in Fig. 7, the sequence of the basic unit (849 bp) apparently consists of tandem repeats of five relatively homologous subunits with minimum unit (monomer) of the extrachromosomal DNA as 171, 171, 167, 169, and 171 bp, respectively. Consensus well as that of the repetitive sequence in the chromosome is sequence with .60% homology are also shown. Each of the estimated to be between 800 and 900 bp. subunits (--- 170 bp) may be divided into two regions, one with From the intensity of the hybridized bands, we have a relatively common DNA sequence among the subunits and estimated that =1% of the total DNA sequence that hybrid- the other with a more diverse DNA sequence. The region izes with pHeS3-45 is present extrachromosomally in HeLa with the common DNA sequence (region II in Fig. 7) is =150 cells. This is equivalent to =20 copies of the minimum unit bp long with a Hinfl cutting site at the same position (boxed (monomer) per cell, accounting for a substantial portion of by a solid line in Fig. 7), and the region with a diverse DNA the extrachromosomal polydisperse DNA in HeLa cells sequence is 22 bp long (region I in Fig. 7). We have tentatively whose total copy number ranges from 50 to 200 molecules per named these five subunits, Sau3A-sub 1, -sub 2, -sub 3, -sub cell (15, 16). The proportion (-1%) of the repetitive DNA 4, and -sub 5 as shown in Fig. 7. There are neither open present in the extrachromosomal fraction is almost 100-fold reading frames nor direct or inverted repeats in the DNA. higher than the average of the proportion of the total This DNA sequence does not coincide with any of the extrachromosomal small polydisperse DNA (<0.03% of the previously known repetitive sequences in human or primate total genomic DNA) (17). In substantiating this, we have chromosomes. The only sequences that have considerable found that a significant percentage (8%; 14 of 184) of the homology are those belonging to alphoid satellite DNA (19). clones obtained from Alu I digests of the extrachromosomal An average of 70.5% and 73.7% homology was detected DNA hybridized with pHeS3-45, while 18% (34 of 184) ofthe between the Sau3A family and %170 bp of subunit I and clones hybridized with the Alu I probe (Blur-8), despite the subunit II of the alphoid satellite DNA, respectively (6). fact that the Sau3A family (1000 copies per haploid genome) More recently, an alphoid repetitive DNA was reported (20). represents only 1/300th of the Alu I family (300,000 copies The DNA consists of five subunits =170 bp long and is

10 20 30 40 50 60 70 80 90 consensus CCTTCNTTNGAAACGGGAANANCTTCACANAAAAACTANACGGAAGCATTCTCAGAAACTTCTTTGTGATGATTGCATTCAACTCACAGA 1 . 90 sub 1 *.--GTCC-CC - A--A- -ATC.- A-.-- G-T. A.- G...... 172 261 C A C-.------G----A-.... sub 2 343*--A-TG-A-T--A--A--T-A-.---TCT-.--- ...... A---C-A-.---A.-....429 sub 3 AT-- C -GC-.------TA-A--- C.---*..C.0---C------G*...... -T--C-...... 510 599 sub 4 * .. C- -C.- T-T-T.- TC- - ... G--A.. TG- -TCC. G- . 679 768 sub 5 AT --G--GO...... TTC-T-.-----G-.------A--A------...... A...... -T--TG-T C --TCA -*< ~ I ------N- <0 11

100 110 120 130 140 150 160 170 consensus GTTGAACATTCCTNTNGATAGAGCAGNTTTNAAACNCTCTTTTTGTAGAATCTGCAAGTGGANATTTGGACCNCTTTGAGG 91'- 171 sub 1 ..*-- A-A ...... G--GT. -.--AA ...... O.TO....TO...... TO . 262 342 sub 2 -.--.-*C- - G--T-A -G-C --T--CC --C-AC...... TC...... C.. .. 430 509--- sub 3 ...... -C--G--T--C---.-TT---C...C...-A A....T.A...... A . T-- 600 678 sub 4 0G ------A-...... AAT.---GCT ...... A. T--G---A- 769 849 sub 5 A...... T.----C-T- -C.---- C-C-G-....C...... -C-- C 000....GGG-----..--

FIG. 7. Nucleotide sequence of the basic unit of the Sau3AI family DNA. One of the clones (pSP3) containing the whole length of the unit was used for DNA sequencing. The total 849-bp sequence is arranged to show the presence offive subunits, each -170 bp long. Spaces produced by arranging the subunits are hyphenated. The unique Sau3AI site in the basic unit (dashed underlining) exists in the sub 2 sequence. Common Hinfl sites present in each subunit are boxed and a x-like sequence (GCTGATGG) is underlined with a solid line. The consensus sequence was deduced from the base sequences of the five subunits. Homology of <601% (three of five) is shown by the letter N. The total 171 bp of the consensus sequence is divided into two regions according to their homology of base sequences among subunits. The low homologous region (I) and the high homologous region (II) are shown. Downloaded by guest on September 29, 2021 Biochemistry: Kiyama et al. Proc. Natl. Acad. Sci. USA 83 (1986) 4669 apparently enriched in the extrachromosomal fraction, as is any homologous DNA sequence between the family mem- the Sau3A family. Comparison of the base sequences with bers. Recently, several short stretches of DNA sequence those of the Sau3A family has revealed -78% homology have been implicated as the recombination-prone DNA between them. sequence in human chromosomes (8). We failed to detect any From these DNA sequence data, one may speculate on the such DNA sequences in our repetitive DNA. However, it mechanism ofgeneration ofthe repetitive DNA and the DNA may be worth mentioning that there is one x-like sequence polymorphism associated with the DNA sequence. It appears (GCTGATGG) (22) present in the basic unit (underlined in that the 170-bp subunit (a prototype) was first amplified 5-fold Fig. 7). The X sequence (GCTGGTGG) is the first DNA to generate the basic unit and then further amplified =1000- sequence demonstrated as a signal sequence for generalized fold to form the tandemly repetitive DNA sequences that are recombination catalyzed by recBC-DNase in prokaryotes now present in the human chromosomes. During the first (23, 24). stage of amplification, diversification of the DNA sequence The biological significance of the repetitive DNA family in the subunits, especially at the diverse region ofthe subunit, (Sau3A family) is unclear at present. Nevertheless, the DNA must have occurred as deduced from the DNA sequencing family reported here may provide a unique system in which data ofthe subunits. It is also likely that some ofthe amplified one can study the mechanism of chromosomal instability and basic units have been translocated to other locations in the homologous recombination in human cells. The extrachro- chromosomes. The extrachromosomal circular DNAs re- mosomal DNA will be a convenient biochemical marker to ported here may have served as the intermediates to form analyze the dynamic state of chromosomes. The repetitive another group of repetitive DNA belonging to the same DNA sequence may be used as a probe to detect DNA family. The diagrammatic representation of the process is polymorphism among individuals. shown in Fig. 8. Mechanism of Formation of the Extrachromosomal DNA. We thank Ms. M. Harada for editing the manuscript. We also thank Recently, Jones and Potter demonstrated that small Drs. N. Takahashi, K. Okumura, and H. Ishikawa for their assist- polydisperse circular DNA is generated via intrastranded ance in conducting experiments. Help by Drs. M. Shimizu and Y. Sakaki for the preparation of human lymphocytes and DNA from homologous recombination (21). Thus, the presence of a chimpanzee and monkey is highly appreciated. This work was substantial amount of the repetitive DNA in the extrachro- supported in part by a special coordination fund for studies on aging mosomal fraction strongly suggests that the DNA sequence from the Japanese Science and Technology Agency and by a in the chromosomes is constantly subjected to active homol- Grant-in-Aid from the Ministry of Education of Japan. ogous recombinational events between the tandemly repeat- ed DNA sequences. The ladder-like pattern indicates that 1. Houck, C. M., Rinehart, F. P. & Schmid, C. W. (1979) J. Mol. such recombinational events occur between the two homol- Biol. 132, 289-306. ogous repetitive DNA sequences. It should be noted that 2. Jelinek, W. R. & Schmid, C. W. (1982) Annu. Rev. Biochem. several minor DNA bands that hybridize with pHeS3-45 have 51, 813-844. always been observed between the major extrachromosomal 3. Adams, J. W., Kaufman, R. E., Kretschmer, P. J., Harrison, M. & Nienhuis, A. W. (1980) Nucleic Acids Res. 8, 6113-6128. circular DNA. For example, we observed four such bands 4. Shafit-Zagardo, B., Maio, J. J. & Brown, F. L. (1982) Nucleic between the monomer and the dimer (Fig. 4, lane 1; Fig. 6, Acids Res. 10, 3175-3193. lane 1). These minor bands may well be the excision products 5. Kurnit, D. M. & Maio, J. J. (1974) Chromosoma 45, 387-400. of the recombination between the DNA sequences with 6. Wu, J. C. & Manuelidis, L. (1980) J. Mol. Biol. 142, 363-386. incomplete homology. Since this DNA family consists offive 7. Shimizu, Y., Yoshida, K., Ren, C.-S., Fujinaga, K., Rajago- palan, S. & Chinnadurai, G. (1983) Nature (London) 302, subunits that contain a relatively common DNA sequence 587-590. among them, recombination between any of these subunits, 8. Jeffreys, A. J., Wilson, V. & Thein, S. L. (1985) Nature ifit should occur, would result in the formation ofthe circular (London) 314, 67-73. DNA with incomplete unit numbers such as 1.2, 1.4, 1.6, and 9. Hirt, B. (1967) J. Mol. Biol. 26, 365-369. 10. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 1.8, which are located between the major bands. 1513-1523. At present, we still do not know whether such recombi- 11. Southern, E. M. (1975) J. Mol. Biol. 98, 503-517. national events occur either at a particular recombination- 12. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) prone DNA sequence within a unit of the DNA family or at J. Mol. Biol. 113, 237-251. 13. Vieira, J. & Messing, J. (1982) Gene 19, 259-268. 14. Wallace, R. B., Johnson, M. J., Suggs, S. V., Miyoshi, K., Bhatt, R. & Itakura, K. (1981) Gene 16, 21-26. 15. Bertelsen, A. H., Humayun, M. Z., Karfopoulos, S. G. & 1 2 3 4 5 Rush, M. G. (1982) Biochemistry 21, 2076-2085. 16. Smith, C. A. & Vinograd, J. (1972) J. Mol. Biol. 69, 163-178. 1 2 . J.. mEhmmbmmbm m X F 1 2 3 4 5 1 2 3 4 5 1 'r 5 17. Yamagishi, H., Kunisada, T., Iwakura, Y., Nishimune, Y., Ogiso, Y. & Matsushiro, I. (1983) Dev. Growth Differ. 25, 3 563-569. 18. Kunisada, T. & Yamagishi, H. (1984) Gene 31, 213-223. CI P 19. Rosenberg, H., Singer, M. & Rosenberg, M. (1978) Science I 200, 394-402. 20. Jones, R. S. & Potter, S. S. (1985) Nucleic Acids Res. 13, 1027-1042. 1' 2' 3' 4' 5' 21. Jones, R. S. & Potter, S. S. (1985) Proc. Natl. Acad. Sci. USA i Amplification 82, 1989-1993. .l/ M 6 A _. /,. 22. Smith, G. R., Kunes, S. H., Schultz, D. W., Taylor, A. & 951 5' 1' 2' 3' 4' 5' 1' 2' 3' 4' 5'1' 2' 3' 4' 5' 1 ' 5' Triman, K. L. (1981) Cell 24, 429-436. 23. McMilin, K. D., Stahl, M. M. & Stahl, F. W. (1974) Genetics FIG. 8. A proposed mechanism of generation of Sau3A family. 77, 409-423. Numbers 1-5 indicate the amplified and diverse subunits. Rear- 24. Stahl, F. W., Crasemann, J. M. & Stahl, M. M. (1975) J. Mol. ranged subunits are shown as 1'-5'. Biol. 94, 203-212. Downloaded by guest on September 29, 2021