Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1695-1699, March 1992 Biochemistry Highly conserved repetitive DNA sequences are present at human DEBORAH L. GRADY, ROBERT L. RATLIFF, DONNA L. ROBINSON, ERIN C. MCCANLIES, JULIANNE MEYNE, AND ROBERT K. MoYzIs* Center for Human Genome Studies and Life Sciences Division, Los Alamos National Laboratory, University of California, Los Alamos, NM 87545 Communicated by Alexander Rich, November 5, 1991

ABSTRACT Highly conserved repetitive DNA sequence DNA-Mobility-Shift Assays. Preparation ofprotein extracts clones, largely consisting of (GGAAT). repeats, have been and DNA-mobility-shift assays were conducted as described isolated from a human recombinant repetitive DNA library by by Strauss and Varshavsky (11). HeLa-cell 0.35 M NaCl high-stringency hybridization with rodent repetitive DNA. nuclear extracts (-3 pg) and 32P-end-labeled DNA (-0. 14 ng) This sequence, the predominant repetitive sequence in human were incubated in 50 mM NaCl at 370C for 1 h to allow binding satellites II and m, is similar to the essential core DNA of the prior to electrophoresis on a low-ionic-strength 6% polyacryl- Saceharomyces cerevisiae , centromere DNA ele- amide gel. Nonspecific protein binding was controlled by the ment (CDE) m. In situ hybridization to human telophase and addition of sheared Escherichia coli DNA or poly [d(I-C)]. Drosophila polytene chromosomes shows localization of the Quantitation of DNA-mobility-shift-gel autoradiographs was (GGAAT). sequence to centromeric regions. Hyperchromicity conducted using a Visage 110 image analysis system. studies indicate that the (GGAAT). sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has RESULTS the same thermal stability as the duplex. Hyperchromicity Clone Isolation. A search for additional highly conserved studies of synthetic DNA variants indicate that all sequences repetitive DNA sequences was conducted using the methods with the composition (AATGN). exhibit this unusual thermal used to identify the human sequence (TTAGGG)X stability. DNA-mobility-shift assays indicate that specific (5). The pHuR library (for plasmid human repeat) (5, 9) was HeLa-ceil nuclear proteins recognize this sequence with a rela- screened with either hamster or mouse repetitive DNA, tive affinity >105. The extreme evolutionary conservation ofthis under conditions allowing only 85-100%o identical sequences DNA sequence, its centromeric location, its unusual hydrogen (depending on length) to cross-hybridize (5). Positive clones bonding properties, its high affinity for specific nuclear proteins, were counter-screened with radiolabeled (GT)25 and and its similarity to functional centromeres isolated from yeast (TTAGGG)7 oligomers, to eliminate clones containing these suggest that this sequence may be a component of the functional previously identified conserved repeats (5). Three clones human centromere. were identified by screening with hamster repetitive DNA. One of these clones (pHuR98) has been reported (9). An Up to 10% of the DNA of human chromosomes consists of additional five clones were identified with high-stringency tandem arrays of repetitive sequences localized at the cen- hybridizations to mouse, rather than hamster, repetitive tromere (1). These DNA arrays are known to consist of DNA (GenBank accession nos. M77215-M77221). various copy numbers ofa (2), ,f satellite (3), and the The common sequence, shared by all eight clones, is the three classic satellites I, II, and III (4). Although some or all 5-nucleotide repeat (GGAAT), and diverged related se- of these repetitive sequences may be involved in centromeric quences. This sequence has been reported to be the core function, there is no evidence, as yet, that the functional component of human satellites II and III (4). In addition, human centromere has been isolated. perfect and diverged CATCATCGA(A/G)T and CAAC- Evolutionary conservation of a DNA sequence is a likely CCGA(A/G)T repeats, interspersed components of satellites indication of functional importance. The human telomere II and III, respectively (4), are present in some of the clones sequence (TTAGGG),, was identified and cloned by screening (9). Zoo-blot analysis, using clone pHuR98 or synthetic for evolutionarily conserved repetitive DNA sequences (5). consensus oligomers indicated that cross-hybridizing se- Further work on the human telomere indicated: (i) its ex- quences are present in all higher eukaryotic examined treme conservation, present at least through vertebrates (i.e., (Fig. 1), including vertebrates, insects, and plants. >400 million years old) (6); (ii) its occasional amplification, Interestingly, this conserved satellite sequence is similar to often at chromosome fusion points (7); and (iii) its ability to the central region of the yeast centromere sequence (CDE) form unusual DNA structures (8). Like the telomere se- III (Fig. 2). CDE III is the most critical component of the quence, we reasoned that other important DNA regions, such yeast centromere, based on sequence homology and directed as those involved in centromere function, would be con- mutational analysis (12, 13). Point mutations of the cytidines served. We report here the identification of another class of indicated in Fig. 2 abolish mitotic function (13). Nine nucle- highly conserved human repetitive DNA sequences that may otides of the critical region identified by mutational experi- be a component of the functional human centromere. ments are shown in Fig. 2, aligned with the similar regions of human satellites II and III. Along these nine core nucleotides, MATERIALS AND METHODS eight nucleotides are identical, with only a single thymidine Construction of a Human Repetitive DNA Library, Library missing in the human sequence. The probability of this short Screening, Sequencing, Oligomer Synthesis, Thermal Hyper- similarity occurring by chance is 6.1 x 10-5 or "5000 times chromicity, and in Situ Hybridization. All methods have been in human DNA. What is intriguing is that these sequences are described (1, 5, 7, 9, 10). located at human centromeric regions (see below) and are present in "5000 times the expected abundance [1.2 x 108 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviation: CDE, centromere DNA element. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

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o .a a SAT 11 SAT SAT II RELATED HUMAN .C VI SAT III SEQUENCE 0.

c . CDE I CDE 11 CDE III YEAST E -0a _ N o in CENTROMERE o. F * ---/ kb / \ 78 -86 bp 87 -95%AT X \ \ 8.0- I 1\ /G C - - O III 4.0- 2 &. C ATCAC TG -' CDE I TGATTTCC CDE xe eu E - ATTCCATG - SAT 11 - ATGA - TTCC - SAT 11 2.0- IA logo logo - TTGA-TTCC - SAT III 1.0- .4z FIG. 2. Diagrammatic representation ofthe Saccharomyces cen- 0.5- tromere and similar human repetitive DNA sequences. The 111- to 119-bp consensus yeast centromere region is diagrammed, as origi- _' nally determined by sequence similarity (12, 13). Three centromere DNA elements, designated CDE I (8 bp), CDE 11 (78-86 bp), and CDE III (25 bp) are shown, aligned along human repetitive DNAs with similar DNA sequences. Functional mutational analysis of the yeast centromere has indicated that CDE I plays a minor role in mitotic stability (13). Mutations in CDE I (open arrows) reduce FIG. 1. Conservation of the (GGAAT), repetitive sequence. mitotic stability <10-fold (i.e., 10-5 to 10-a). Only a portion of the Representative DNAs from a variety of eukaryotic species were 25-bp CDE III region, defined by sequence similarity, is essential for hybridized to a 32P-labeled synthetic CAACCCGAGT(GGAAT)6 mitotic function (13). This central "core" region is (T/A)TG(A/ deoxyoligonucleotide [Sat III consensus sequence (4)]. E. coli DNA T)TTTCCGAA, similar to the originally defined CDE III element was used as carrier DNA to prevent competition with conserved (12). The remaining "conserved" nucleotides outside this "core" repetitive sequences (5). Primate DNA filters were placed in separate region appear to be functionally less important (13). Mutations in the hybridization bags to avoid intrafilter competition. Hybridization two cytidine residues totally (large arrow) or significantly (>1000- conditions were 15-200C below the melting temperature for perfectly fold, small arrow) eliminate mitotic function. matched duplexes (5, 9). Positive hybridization was obtained with all 16 species tested, except Saccharomyces and E. coli. Pictured are PCR-amplified bands to Drosophila polytene chromosomes hybridizations to human, orangutan, chicken, , Drosophila, gave distinct hybridization to the chromocenter (Fig. 3B). sea urchin (Strongylocentrotus), and yeast (Saccharomyces) DNAs Thermal Stability. The G/C-strand asymmetry in cut with either Sau3AI (left lanes of pairs) or Rsa I (right lanes of (GGAAT), is reminiscent oftelomeric repeats that are capable pairs), electrophoresed through a 1% agarose gel, and blotted to of forming stable G G base pairs (5, 8). Melting curves of the nitrocellulose (5, 9). Exposure time was 4 h for primate DNA filters (GGAAT)6 repeat exhibit unusual properties (Fig. 4 and Table and 24 h for all other filters. Similar results were obtained with 1). The purine-rich strand alone has the same thermal stability radiolabeled pHuR98 DNA (9) or (GGAATCAT)5 or (GGAAT)6 as the duplex (Fig. 4). Gel electrophoresis studies indicate that oligomers. kb, Kilobase(s). the (GGAAT)6 oligomer migrates between the single-strand base pairs (bp) (4)]. It should be noted that all three classic (ATTCC)6 oligomer and (GGAAT)6-(ATTCC)6 duplex, sug- human satellites have sequence similarities to yeast CDEs gesting that a fold-back or multistrand structure is present (Fig. 2) and that yeast CDE sequence similarities to other (data not shown). Oligodeoxynucleotides with substitutions of eukaryotic satellite DNAs were described after their initial alternative nucleotides in the (GGAAT) repeating unit were identification (12). synthesized and melting curves were determined (Table 1). For most oligomers, either no melting curve or a gradual Chromosomal Localization of the Highly Conserved Repet- itive In situ with biotinylated satellite increase in absorbance as the temperature was increased, due Sequence. hybridization to purine intrastrand unstacking, was observed (Table 1 and III consensus sequence oligomers gave prominent hybridiza- Fig. 4). The only substituted oligomers that exhibit similar tion to the centromeric regions of human chromosomes, in thermal stabilities were (GCAAT)6, (GAAAT)6, (GTAAT)6, addition to the adjacent regions of chromo- (CGAAT)4, and (GGCAT)6 (Table 1), the later two expected somes 1, 9, 16, and Y (Fig. 3). By using synchronized cell to be stably self-complimentary utilizing normal G C and APT populations, a greater fraction of telophase chromosomes base pairs. Interestingly, these variants represent the most was produced. As can be seen in Fig. 3A, the hybridization frequent variants actually found in cloned human satellite signals are directly at the centromeric constriction. Approx- DNAs (GenBank release 66), accounting for >70%o of the imately 80%6 of the centromeres give distinct signals on single-base variants. The probability that this observed fre- metaphase or telophase chromosomes, similar to the effi- quency of base changes in satellite II and III sequences is ciency obtained for human telomere sequences (5). Whether random is extremely unlikely (X2 test; P = 0.001). this represents random in situ hybridization efficiency, Since (GCAAT)6, (GAAAT)6, and (GTAAT)6 exhibit high clearly the case for (TTAGGG)6 hybridization (5), or variable thermal stability, a mixed oligomer (GNAAT)6 was synthe- copy numbers of satellite II or III sequences on different sized and its thermal stability was determined. This mixed human chromosomes is yet to be investigated. Previous in oligomer exhibited the unusual stability originally obtained situ hybridization studies, using less-sensitive autoradio- for (GGAAT)6 (Table 1). All combinations of base mismatch graphic detection, indicated that at least half of the human and pairing at the N position in the (AATGN)" repeat appear chromosomes contain centromeric satellite II- or III-related to be compatible with the observed thermal stability. A sequences (15). tandem array ofthe conserved unit ofthis repeat (AATG) was A biotinylated (ATTCC)6 oligomer hybridized weakly to synthesized [i.e., (GAAT)8 (Table 1)]. It also exhibits high the chromocenter (centromeric) region of Drosophila poly- thermal stability. tene chromosomes (data not shown). PCR amplification of A possible structure for (AATGN),, consistent with this Drosophila DNA using (ATTCC)6 as an oligomeric primer data, involves G-A base pairing between nucleotides on one yielded a number of discrete bands. Hybridization of these or multiple strands. Base pairing in a helical turn would Downloaded by guest on September 28, 2021 Biochemistry: Grady et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1697

FIG. 3. In situ hybridization to human telophase and Drosophila polytene chromosomes. In situ hybridization was conducted in 2x standard saline citrate/30%o (vol/vol) formamide at 370C as described (5, 6, 9). Chromosomes were counterstained with propidium iodide (orange) after incubation with fluorescein-labeled avidin and one amplification with avidin antibody, to detect the biotinylated DNA (yellow). (A) Hybridization of biotinylated (GGAAT)6 to human telophase chromosomes. (B) Hybridization of biotinylated Drosophila DNA PCR products primed with (ATTCC)6 to Drosophila polytene chromosomes. PCR conditions were 30 cycles at 940C for 1 minm, 55C for 2 min, and 720C for 2 min with 5 /AM primer and using standard protocols (GeneAmp Kit, Perkin-Elmer). The annealing temperature (550C) was 4100C below the melting temperature for perfectly matched duplexes (Table 1) and 415°C above the heteroduplex of this primer with known Drosophila centromeric satellite sequences [i.e., (GAGAA)"; Table 1 and ref. 14]. involve G'A base pairs bracketing two A-T base pairs, as DNA-Mobility-Shift Assays. The clone pHuR98 consists of follows: three divergent CAACCCGA(G/A)T sequences interspersed with GGAAT repeats (9). DNA-mobility-shift assays were 5'-AATGGAATGG-3' conducted to determine if this sequence binds nuclear pro- * 1111 l-1111 teins in a sequence-specific manner. Two discrete DNA- 3'-GTAAGGTAAG-5' protein complexes were observed (Fig. 5). Evidence that these shifted DNA bands result from DNA-protein interac- The stability of the (GAAT)8 repeat (Tablel) can be ade- tions included (i) incubating with decreasing amounts of quately explained by the presence of such stable G-A base extract that led to decreased signals and (ii) digesting with pairs, since oligomers with adjacent alternating G-A base proteinase K prior to DNA-protein binding that eliminated pairs have been shown to be as stable as normal Watson- the shifted bands (data not shown). Crick duplexes (16). Whether this structure or more exotic Kinetic analysis of the formation of these two DNA- alternatives are correct remains to be determined. mobility-shift complexes can be seen in Fig. 5A. After a 30-sec incubation only the lower DNA-protein band has formed. With time, formation of the higher molecular weight 1.3 band occurs. These results, as well as the salt and temper- ature dependence of complex formation (Fig. 5), suggest that the slower-migrating complex may be a multimer of the faster-migrating complex. The relative formation of these specific DNA-protein 1.2 complexes in the presence of increasing amounts of E. coli, or poly [d(I-C)] DNA can be seen in Fig. SB. The HeLa 0 nuclear protein(s) responsible for the observed DNA mobility shift has a 10,400-fold greater affinity for the pHuR98 DNA sequence. If there is 1 to a maximum of 25 binding sites per 1.1 pHuR98 DNA (assuming each GGAAT repeat is capable of binding a single protein) (9) and if there are no pseudosites in E. coli DNA, then the actual relative affinity is greater than 105 to 2 x 106 (17). Competition experiments using another clone consisting primarily of GGAAT repeats (pHuR94) show that this sequence can compete for the pHuR98 binding 1.0 40 60 80 protein(s). Cloned Alu (1), a satellite (2), or telomere (5) repetitive DNA sequences do not compete for this protein(s) TEMPERATURE (°C) (data not shown). FIG. 4. Thermal hyperchromicity profiles of synthetic oligodeox- ynucleotides. Hyperchromicity profiles of (GGAAT)6 (solid line), (AT- DISCUSSION TCC)6 (dashed and dotted line), (GGAAT)6-(ATTCC)6 duplexes Highly conserved human repetitive DNA sequences were (dashed line), and (GGATT)6 (dotted line) in 50 mM NaCl are shown (6). isolated from a library constructed from randomly sheared A/Ao is the ratio ofobserved absorption to initial absorption at 260 nm. and reassociated DNA (5, 9). This library contains a greater Downloaded by guest on September 28, 2021 1698 Biochemistry: Grady et A Proc. Nati. Acad. Sci. USA 89 (1992) Table 1. Hyperchromicity of synthetic oligodeoxynucleotides A Oligomer Sequence Tm, 0C A B C D E F G H (GGAAT)6 Human satellite DNA 65 (Fig. 4) (ATTCC)6 Human satellite DNA - (Fig. 4) (GGAAT)6iATTCC)6 Human satellite DNA duplex 65 (Fig. 4) (AATGG)6 Human satellite DNA 65 (GcAAT)6 Human satellite DNA variant 65 (GAAAT)6 Human satellite DNA variant 60 (GTAAT)6 Human satellite DNA variant 56 (GNAAT)6 Mixed oligomer 62 (GNAAT)C(AT7NC)6 Mismatched duplex 58 (GGAAA)6 (GGAAc)6 (GGAAG)6 B (GGAGT)6 44 1.5 0 (GGATT)6 - (Fig. 4) (GGAcT)6 52 (GGGAT)6 CD *0 0 z 0 *~~a0 (GGTAT)6 o 1.0 F (GGcAT)6 69 z (GGTAA)6 wUl (GGAAT)4 Human satellite DNA 65 4c -J (AGAAT)4 w 0.5 i (AcAAT)4 @0 (ATAAT)4 (AAAAT)4 (cGAAT)4 58 ...... (CAAAT)4 100 1,000 10,000 100,000 (ccAAT)4 MASS EXCESS COMPETITOR DNA i-r_tCTlAA A'r1)4 FIG. 5. DNA-mobility-shift analysis. 32P-end-labeled pHuR98 insert (TGAAT)4 DNA (158 nucleotides long) was incubated with 0.35 M NaCl extracts of (TcAAT)4 HeLa-cell nuclearproteins (11). InA, increasing incubation times (lanes; (rrAAT)4 A, 0 time; B, 30 s; C, 1.5 min; D, 3 min; E, 7.5 min; F, 15 min; G, 30 min; (TAAAT)4 H, 1h) yielded two discrete bands shifted to highermolecularweight. Salt (GGAA)8 38 concentrations >50 mM reduced the amount of the lower band with a (GGAA)8-(TTCC)8 Duplex 66 concomitant increase of the upper band. Temperatures <37°( had the (GAAT)8 56 opposite effect. In B, quantitation of the amount of DNA-protein (GATA)8 32 complex, at various competitor DNA concentrations, was obtained and normalized to the optimum conditions shown in A. e, E. coli DNA (GTAG)8 competitor; o, poly [d(I-)] competitor. (GGTA)8 (GAGAA)6 Drosophila satellite DNA meric regions, major hybridization signals at the large hetero- (GAGAA)6.(ATTCC)6 Drosophila-human mismatched 40 chromatic blocks on human chromosomes 1, 9, 16, and Y were duplex observed (Fig. 3). These regions are known to consist of large (GGAAAT)5 Yeast CDE III polymer 46 amplified blocks of the (GGAAT), repeat (9). Some of these (GGAAAT)5.(ATTTCC)s Yeast CDE III duplex 62 heterochromatic regions are recent amplifications duringprimate Oligodeoxynucleotides 18-51 nucleotides long were synthesized, speciation, not present in homologous chimpanzee and gorilla hybridized, and denatured in 50 mM NaCl. The thermal denaturation chromosomes (19). Telomeric (TTAGGG)" repeats can undergo temperature (Tm) is taken at the last linear height increase in such periodic amplifications without apparent disruption of cel- hyperchromicity (see Fig. 4). Nucleotide variations from the con- lular function (7). A "core" of functional sequences is still served repeat are in small uppercase letters. (GGAAT) represented present on each chromosome, even in the presence of presum- ably nonfunctional amplified blocks of the same sequence representation of sequences that are cut infrequently with (7). Therefore, large variations in DNA copy number between spe- restriction enzymes, such as centromeric repeats (9). Other cies (or chromosomes) cannot be used as an argument for a lack than the and (TTAGGG),, sequences reported previ- (GT), ofbiological function, as has been suggested (20). Itis interesting ously (5), eight clones consisting predominantly of to note that the heterochromatic regions that contain amplified (GGAAT),, repeats were also obtained. These clones were degenerate (GGAAT)0 repeats (9, 14, 19) are both adjacent to the isolated at high stringency with either mouse or hamster centromeric constriction, which also contains this sequence (Fig. DNA, indicating that related sequences are present in these 3), and are found on chromosomes with a high incidence of two rodent genomes. Southern blot, PCR, and in situ hybrid- nondisjunction and other mitotic/meiotic abnormalities (21). If ization analyses confirmed the conservation of this sequence the (GGAAT)1 sequence is a component ofthe functional human among diverse species (Figs. 1 and 3). These results indicate centromere, then sporadic anomalous amplification might be that this or closely related sequences are >1 billion years old, expected to lead to these two results. Without an efficient the last branch point between lineages of the organisms mechanism to remove such amplifications (clearly lacking in the examined. This is the most conserved DNA component case of telomeric repeats; ref. 7), the cell might tolerate such found at the human centromere, since a satellite sequences "rusting hulks," especially since DNA "bulk" surrounding the are present only in primates (18). centromere seems to be necessary for higher eukaryotic chro- In situ hybridization analysis localized the major clusters of mosome function (22, 23). this sequence to the centromeric region ofhuman andDrosophila The unusual thermal stability ofthe (GGAAT)" repeat may chromosomes (Fig. 3). In addition to the small signals at centro- be the result of unusually stable G-A base pairs (Fig. 4). The Downloaded by guest on September 28, 2021 Biochemistry: Grady et al. Proc. Natl. Acad. Sci. USA 89 (1992) 1699 Centromere DNA Domains Domains Pairing FIG. 6. Diagrammatic representation of hu- man centromere domains. DNA regions respon- malm sible for interacting with the kinetochore may be Kinetochore either interspersed with central-domain DNA (Upper right) or adjacent to central-domain DNA (Lower right). In either case, chromatin folding in the centromeric region could place these DNA regions on the outer surface of the chromosome, in place to interact with kineto- Central chore proteins. hyperchromicity experiments with variants ofthe (GGAAT),, turnover of most DNA sequences at the centromere [i.e., a repeat (Table 1) favor this interpretation. Stable G-A base satellite at human centromeres (2), mouse satellite at mouse pairing in other oligodeoxynucleotides has been reported (16, centromeres (29), telomeric (TTAGGG),, tracts at many other 24, 25) and is a common structural motif participating in the vertebrate centromeres (7), etc.] yet proposes that a "core" three-dimensional structure of tRNA (26). How stretches of of conserved sequences is maintained that represent the this sequence can faithfully replicate, with the natural duplex actual kinetochore (and possibly pairing) domains (Fig. 6). having comparable stability to the purine-rich strand (Fig. 4), We propose that the conserved (GGAAT), repeat and/or its or whether a true duplex even exists under all physiological interspersed CATCATCGA(A/G)T and CAACCCGA(A/ conditions are questions yet to be answered. Like telomeric G)T sequences may represent such a component. sequences (5), this unusual DNA structure may represent This work was supported by grants from the U.S. Department of another "code" utilized for an important biological function. Energy to R.K.M. The ability of nuclear proteins to recognize this sequence with high specificity is consistent with this speculation (Fig. 1. Moyzis, R. K., Torney, D. C., Meyne, J., Buckingham, J. M., Wu, 5). The relative specificity observed in crude HeLa nuclear J.-R., Burks, C., Sirotkin, K. M. & Goad, W. B. (1989) Genomics 4, 273-289. extracts (>105) is comparable to other highly selective pro- 2. Willard, H. F. & Waye, J. S. (1987) Trends Genet. 3, 192-198. tein-DNA interactions, such as the lac repressor-operator 3. Waye, J. S. & Willard, H. F. (1989) Proc. Natl. Acad. Sci. USA 86, DNA interaction (17) or the binding ofthe yeast CBF3 protein 6250-6254. 4. Prosser, J., Frommer, J., Paul, C. & Vincent, P. C. (1986) J. Mol. Biol. complex to CDE III (27). Whether the DNA sequence itself 187, 145-155. or a potentially unusual DNA structure is being recognized in 5. Moyzis, R. K., Buckingham, J. M., Cram, L. S., Dani, M., Deaven, these interactions is yet to be determined. L. L., Jones, M. D., Meyne, J., Ratliff, R. L. & Wu, J.-R. (1988) Proc. Natl. Acad. Sci. USA 85, 6622-6626. Fig. 6 is a schematic diagram of the centromeric region of 6. Meyne, J., Ratliff, R. L. & Moyzis, R. K. (1989) Proc. Natl. Acad. Sci. human chromosomes (23). It should be noted that a number USA 86, 7049-7053. offunctional domains are present in this region, and each may 7. Meyne, J., Baker, R. J., Hobart, H. H., Hsu, T. C., Ryder, 0. A., Ward, 0. G., Wiley, J. E., Wurster-Hill, D. H., Yates, T. L. & Moyzis, R. K. contain hundreds of thousands of base pairs of DNA. For (1990) Chromosoma 99, 3-10. example, the kinetochore domain stretches along the entire 8. Williamson, J. R., Raghuraman, M. K. & Cech, T. R. (1989) Cell 59, outer surface ofthe centromere region (Fig. 6 and refs. 23 and 871-880. 9. Moyzis, R. K., Albright, K. L., Bartholdi, M. F., Cram, L. S., Deaven, 28). It is not known how the chromatin domains in the L. L., Hildebrand, C. E., Joste, N. E., Longmire, J. L., Meyne, J. & centromeric region are coiled or folded. It is thought, how- Schwarzacher-Robinson, T. (1987) Chromosoma 95, 375-386. ever, that only a component of the chromatin fiber extends 10. Riethman, H. C., Moyzis, R. K., Meyne, J., Burke, D. T. & Olson, out of the centromere to the kinetochore M. V. (1989) Proc. Nat!. Acad. Sci. USA 86, 6240-6244. region plate (22). 11. Strauss, F. & Varshavsky, A. (1984) Cell 37, 889-901. While DNA sequences responsible for interacting with the 12. Fitzgerald-Hayes, M., Clarke, L. & Carbon, J. (1982) Cell 29, 235-244. kinetochore may be interspersed with other "spacer" DNA 13. Carbon, J. & Clarke, L. (1990) New Biol. 2, 10-19. present in the central region (Fig. 6 Upper right and ref. 28), 14. Lohe, A. R. & Brutlag, D. L. (1986) Proc. Nat!. Acad. Sci. USA 83, 696-700. it is also possible that sequences adjacent to the spacer DNA 15. Gosden, J. R., Mitchell, A. R., Buckland, R. A., Clayton, R. P. & can be folded in such a manner as to be on the outside of the Evans, H. J. (1975) Exp. Cell Res. 92, 148-158. centromere region (Fig. 6 Lower right). Positioned as such, 16. Li, Y., Zon, G. & Wilson, W. D. (1991) Biochemistry 30, 7566-7572. 17. von Hippel, P. H. & Berg, 0. G. (1986) Proc. Natl. Acad. Sci. USA 83, they would be ideally located to interact with kinetochore 1608-1612. proteins. The latter model is consistent with the known linear 18. Maio, J. J., Brown, F. L. & Musich, P. R. (1981) Chromosoma 83, molecular DNA organization of a satellite and classical 103-125. satellite sequences yet accounts for the observed concordant 19. Miller, D. A. (1977) Science 198, 1116-1124. 20. John, B. & Miklos, G. L. G. (1979) Int. Rev. Cytol. 58, 1-114. metaphase in situ hybridization patterns of these arrays (Fig. 21. Bove, A., Bove, J. & Gropp, A. (1984) Adv. Hum. Genet. 14, 1-57. 3 and refs. 9 and 23). 22. Rattner, J. B. (1987) Chromosoma 95, 175-181. Alternatively, small clusters of satellite sequences, diffi- 23. Pluta, A. F., Cooke, C. A. & Earnshaw, W. C. (1990) Trends Biochem. cult to detect by hybridization in the presence of large Sci. 15, 181-185. 24. Prive, G. G., Heinemann, U., Chandrasegaran, S., Kan, L. S., Kopka, amplified satellite blocks, may be interspersed in a-satellite M. L. & Dickerson, R. E. (1987) Science 238, 498-504. arrays. Such simple-sequence satellites would not be cut by 25. Li, Y., Zon, G. & Wilson, W. D. (1991) Proc. Natl. Acad. Sci. USA 88, enzymes that free the a-satellite sequences as discrete 26-30. blocks. Both models predict that the bulk of DNA sequences 26. Rich, A. & RajBhandary, U. L. (1976)Annu. Rev. Biochem. 45, 805-860. 27. Lechner, J. & Carbon, J. (1991) Cell 64, 717-725. present in the centromeric region may only be important to 28. Zinkowski, R. P., Meyne, J. & Brinkley, B. R. (1991) J. Cell Biol. 13, "space" the kinetochore and pairing DNA domains in the 1091-1110. correct orientation. Either model is consistent with the rapid 29. Pardue, M. L. & Gall, J. G. (1970) Science 168, 1356-1358. Downloaded by guest on September 28, 2021