Highly conserved repetitive DNA sequences are present at human

Proc. Natl. Acad. Sci. USA Vol. 89, pp. 1695-1699, March 1992 Biochemistry

Highly conserved repetitive DNA sequences are present at human centromeres 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

DNA-Mobility-Shift Assays. Preparation of protein extracts and DNA-mobility-shift assays were conducted as described by Strauss and Varshavsky (11). HeLa-cell 0.35 M NaCl nuclear extracts (-3 pg) and 32P-end-labeled DNA (-0. 14 ng) were incubated in 50 mM NaCl at 370C for 1 h to allow binding prior to electrophoresis on a low-ionic-strength 6% polyacrylamide gel. Nonspecific protein binding was controlled by the addition of sheared Escherichia coli DNA or poly [d(I-C)]. Quantitation of DNA-mobility-shift-gel autoradiographs was conducted using a Visage 110 image analysis system.

ABSTRACT Highly conserved repetitive DNA sequence clones, largely consisting of (GGAAT). repeats, have been isolated from a human recombinant repetitive DNA library by high-stringency hybridization with rodent repetitive DNA. This sequence, the predominant repetitive sequence in human satellites II and m, is similar to the essential core DNA of the Saceharomyces cerevisiae centromere, centromere DNA element (CDE) m. In situ hybridization to human telophase and Drosophila polytene chromosomes shows localization of the (GGAAT). sequence to centromeric regions. Hyperchromicity studies indicate that the (GGAAT). sequence exhibits unusual hydrogen bonding properties. The purine-rich strand alone has the same thermal stability as the duplex. Hyperchromicity studies of synthetic DNA variants indicate that all sequences with the composition (AATGN). exhibit this unusual thermal stability. DNA-mobility-shift assays indicate that specific HeLa-ceil nuclear proteins recognize this sequence with a relative affinity >105. The extreme evolutionary conservation ofthis DNA sequence, its centromeric location, its unusual hydrogen bonding properties, its high affinity for specific nuclear proteins, and its similarity to functional centromeres isolated from yeast suggest that this sequence may be a component of the functional human centromere.

RESULTS Clone Isolation. A search for additional highly conserved repetitive DNA sequences was conducted using the methods used to identify the human telomere sequence (TTAGGG)X (5). The pHuR library (for plasmid human repeat) (5, 9) was screened with either hamster or mouse repetitive DNA, under conditions allowing only 85-100%o identical sequences (depending on length) to cross-hybridize (5). Positive clones were counter-screened with radiolabeled (GT)25 and (TTAGGG)7 oligomers, to eliminate clones containing these previously identified conserved repeats (5). Three clones were identified by screening with hamster repetitive DNA. One of these clones (pHuR98) has been reported (9). An additional five clones were identified with high-stringency hybridizations to mouse, rather than hamster, repetitive DNA (GenBank accession nos. M77215-M77221). The common sequence, shared by all eight clones, is the 5-nucleotide repeat (GGAAT), and diverged related sequences. This sequence has been reported to be the core component of human satellites II and III (4). In addition, perfect and diverged CATCATCGA(A/G)T and CAACCCGA(A/G)T repeats, interspersed components of satellites II and III, respectively (4), are present in some of the clones (9). Zoo-blot analysis, using clone pHuR98 or synthetic consensus oligomers indicated that cross-hybridizing sequences are present in all higher eukaryotic DNAs examined (Fig. 1), including vertebrates, insects, and plants. Interestingly, this conserved satellite sequence is similar to the central region of the yeast centromere sequence (CDE) III (Fig. 2). CDE III is the most critical component of the yeast centromere, based on sequence homology and directed mutational analysis (12, 13). Point mutations of the cytidines indicated in Fig. 2 abolish mitotic function (13). Nine nucleotides of the critical region identified by mutational experiments are shown in Fig. 2, aligned with the similar regions of human satellites II and III. Along these nine core nucleotides, eight nucleotides are identical, with only a single thymidine missing in the human sequence. The probability of this short similarity occurring by chance is 6.1 x 10-5 or "5000 times in human DNA. What is intriguing is that these sequences are located at human centromeric regions (see below) and are present in "5000 times the expected abundance [1.2 x 108

Up to 10% of the DNA of human chromosomes consists of tandem arrays of repetitive sequences localized at the centromere (1). These DNA arrays are known to consist of various copy numbers of a satellite (2), ,f satellite (3), and the three classic satellites I, II, and III (4). Although some or all of these repetitive sequences may be involved in centromeric function, there is no evidence, as yet, that the functional human centromere has been isolated. Evolutionary conservation of a DNA sequence is a likely indication of functional importance. The human telomere sequence (TTAGGG),, was identified and cloned by screening for evolutionarily conserved repetitive DNA sequences (5). Further work on the human telomere indicated: (i) its extreme conservation, present at least through vertebrates (i.e., >400 million years old) (6); (ii) its occasional amplification, often at chromosome fusion points (7); and (iii) its ability to form unusual DNA structures (8). Like the telomere sequence, we reasoned that other important DNA regions, such as those involved in centromere function, would be conserved. We report here the identification of another class of highly conserved human repetitive DNA sequences that may be a component of the functional human centromere.

MATERIALS AND METHODS Construction of a Human Repetitive DNA Library, Library Screening, Sequencing, Oligomer Synthesis, Thermal Hyperchromicity, and in Situ Hybridization. All methods have been described (1, 5, 7, 9, 10). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Abbreviation: CDE, centromere DNA element. *To whom reprint requests should be addressed. 1695

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Proc. Natl. Acad. Sci. USA 89 (1992) o

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FIG. 1. Conservation of the (GGAAT), repetitive sequence. Representative DNAs from a variety of eukaryotic species were hybridized to a 32P-labeled synthetic CAACCCGAGT(GGAAT)6 deoxyoligonucleotide [Sat III consensus sequence (4)]. E. coli DNA was used as carrier DNA to prevent competition with conserved repetitive sequences (5). Primate DNA filters were placed in separate hybridization bags to avoid intrafilter competition. Hybridization conditions were 15-200C below the melting temperature for perfectly matched duplexes (5, 9). Positive hybridization was obtained with all 16 species tested, except Saccharomyces and E. coli. Pictured are hybridizations to human, orangutan, chicken, maize, Drosophila, sea urchin (Strongylocentrotus), and yeast (Saccharomyces) DNAs cut with either Sau3AI (left lanes of pairs) or Rsa I (right lanes of pairs), electrophoresed through a 1% agarose gel, and blotted to nitrocellulose (5, 9). Exposure time was 4 h for primate DNA filters and 24 h for all other filters. Similar results were obtained with radiolabeled pHuR98 DNA (9) or (GGAATCAT)5 or (GGAAT)6 oligomers. kb, Kilobase(s).

base pairs (bp) (4)]. It should be noted that all three classic human satellites have sequence similarities to yeast CDEs (Fig. 2) and that yeast CDE sequence similarities to other eukaryotic satellite DNAs were described after their initial identification (12). Chromosomal Localization of the Highly Conserved Repetitive Sequence. In situ hybridization with biotinylated satellite III consensus sequence oligomers gave prominent hybridization to the centromeric regions of human chromosomes, in addition to the adjacent heterochromatin regions of chromosomes 1, 9, 16, and Y (Fig. 3). By using synchronized cell populations, a greater fraction of telophase chromosomes was produced. As can be seen in Fig. 3A, the hybridization signals are directly at the centromeric constriction. Approximately 80%6 of the centromeres give distinct signals on metaphase or telophase chromosomes, similar to the efficiency obtained for human telomere sequences (5). Whether this represents random in situ hybridization efficiency, clearly the case for (TTAGGG)6 hybridization (5), or variable copy numbers of satellite II or III sequences on different human chromosomes is yet to be investigated. Previous in situ hybridization studies, using less-sensitive autoradiographic detection, indicated that at least half of the human chromosomes contain centromeric satellite II- or III-related sequences (15). A biotinylated (ATTCC)6 oligomer hybridized weakly to the chromocenter (centromeric) region of Drosophila polytene chromosomes (data not shown). PCR amplification of Drosophila DNA using (ATTCC)6 as an oligomeric primer yielded a number of discrete bands. Hybridization of these

FIG. 2. Diagrammatic representation of the Saccharomyces centromere and similar human repetitive DNA sequences. The 111- to 119-bp consensus yeast centromere region is diagrammed, as originally 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 mitotic stability 1000fold, small arrow) eliminate mitotic function.

PCR-amplified bands to Drosophila polytene chromosomes gave distinct hybridization to the chromocenter (Fig. 3B). Thermal Stability. The G/C-strand asymmetry in (GGAAT), is reminiscent of telomeric repeats that are capable of forming stable G G base pairs (5, 8). Melting curves of the (GGAAT)6 repeat exhibit unusual properties (Fig. 4 and Table 1). The purine-rich strand alone has the same thermal stability as the duplex (Fig. 4). Gel electrophoresis studies indicate that the (GGAAT)6 oligomer migrates between the single-strand (ATTCC)6 oligomer and (GGAAT)6-(ATTCC)6 duplex, suggesting that a fold-back or multistrand structure is present (data not shown). Oligodeoxynucleotides with substitutions of alternative nucleotides in the (GGAAT) repeating unit were synthesized and melting curves were determined (Table 1). For most oligomers, either no melting curve or a gradual increase in absorbance as the temperature was increased, due to purine intrastrand unstacking, was observed (Table 1 and Fig. 4). The only substituted oligomers that exhibit similar thermal stabilities were (GCAAT)6, (GAAAT)6, (GTAAT)6, (CGAAT)4, and (GGCAT)6 (Table 1), the later two expected to be stably self-complimentary utilizing normal G C and APT base pairs. Interestingly, these variants represent the most frequent variants actually found in cloned human satellite DNAs (GenBank release 66), accounting for >70%o of the single-base variants. The probability that this observed frequency of base changes in satellite II and III sequences is random is extremely unlikely (X2 test; P = 0.001). Since (GCAAT)6, (GAAAT)6, and (GTAAT)6 exhibit high thermal stability, a mixed oligomer (GNAAT)6 was synthesized and its thermal stability was determined. This mixed oligomer exhibited the unusual stability originally obtained for (GGAAT)6 (Table 1). All combinations of base mismatch and pairing at the N position in the (AATGN)" repeat appear to be compatible with the observed thermal stability. A tandem array of the conserved unit ofthis repeat (AATG) was synthesized [i.e., (GAAT)8 (Table 1)]. It also exhibits high thermal stability. A possible structure for (AATGN),, consistent with this data, involves G-A base pairing between nucleotides on one or multiple strands. Base pairing in a helical turn would

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Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 3. In situ hybridization to human telophase and Drosophila polytene chromosomes. In situ hybridization was conducted in 2 x 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].

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FIG. 4. Thermal hyperchromicity profiles of synthetic oligodeoxynucleotides. Hyperchromicity profiles of (GGAAT)6 (solid line), (ATTCC)6 (dashed and dotted line), (GGAAT)6-(ATTCC)6 duplexes (dashed line), and (GGATT)6 (dotted line) in 50 mM NaCl are shown (6). A/Ao is the ratio ofobserved absorption to initial absorption at 260 nm.

DNA-Mobility-Shift Assays. The clone pHuR98 consists of three divergent CAACCCGA(G/A)T sequences interspersed with GGAAT repeats (9). DNA-mobility-shift assays were conducted to determine if this sequence binds nuclear proteins in a sequence-specific manner. Two discrete DNAprotein complexes were observed (Fig. 5). Evidence that these shifted DNA bands result from DNA-protein interactions included (i) incubating with decreasing amounts of extract that led to decreased signals and (ii) digesting with proteinase K prior to DNA-protein binding that eliminated the shifted bands (data not shown). Kinetic analysis of the formation of these two DNAmobility-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 band occurs. These results, as well as the salt and temperature 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 complexes in the presence of increasing amounts of E. coli, or poly [d(I-C)] DNA can be seen in Fig. SB. The HeLa 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 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 protein(s). Cloned Alu (1), a satellite (2), or telomere (5) repetitive DNA sequences do not compete for this protein(s)

(data not shown).

DISCUSSION Highly conserved human repetitive DNA sequences were isolated from a library constructed from randomly sheared and reassociated DNA (5, 9). This library contains a greater

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representation of sequences that are cut infrequently with restriction enzymes, such as centromeric repeats (9). Other than the (GT), and (TTAGGG),, sequences reported previously (5), eight clones consisting predominantly of (GGAAT),, repeats were also obtained. These clones were isolated at high stringency with either mouse or hamster DNA, indicating that related sequences are present in these two rodent genomes. Southern blot, PCR, and in situ hybridization analyses confirmed the conservation of this sequence among diverse species (Figs. 1 and 3). These results indicate that this or closely related sequences are >1 billion years old, the last branch point between lineages of the organisms examined. This is the most conserved DNA component found at the human centromere, since a satellite sequences are present only in primates (18). In situ hybridization analysis localized the major clusters of this sequence to the centromeric region ofhuman and Drosophila chromosomes (Fig. 3). In addition to the small signals at centro-

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FIG. 5. DNA-mobility-shift analysis. 32P-end-labeled pHuR98 insert DNA (158 nucleotides long) was incubated with 0.35 M NaCl extracts of HeLa-cell nuclear proteins (11). InA, increasing incubation times (lanes; A, 0 time; B, 30 s; C, 1.5 min; D, 3 min; E, 7.5 min; F, 15 min; G, 30 min; H, 1h) yielded two discrete bands shifted to higher molecularweight. Salt concentrations >50 mM reduced the amount of the lower band with a concomitant increase of the upper band. Temperatures 105) is comparable to other highly selective protein-DNA interactions, such as the lac repressor-operator DNA interaction (17) or the binding of the yeast CBF3 protein complex to CDE III (27). Whether the DNA sequence itself or a potentially unusual DNA structure is being recognized in these interactions is yet to be determined. Fig. 6 is a schematic diagram of the centromeric region of human chromosomes (23). It should be noted that a number of functional domains are present in this region, and each may contain hundreds of thousands of base pairs of DNA. For example, the kinetochore domain stretches along the entire outer surface of the centromere region (Fig. 6 and refs. 23 and 28). It is not known how the chromatin domains in the centromeric region are coiled or folded. It is thought, however, that only a component of the chromatin fiber extends out of the centromere region to the kinetochore plate (22). While DNA sequences responsible for interacting with the kinetochore may be interspersed with other "spacer" DNA present in the central region (Fig. 6 Upper right and ref. 28), it is also possible that sequences adjacent to the spacer DNA can be folded in such a manner as to be on the outside of the centromere region (Fig. 6 Lower right). Positioned as such, they would be ideally located to interact with kinetochore proteins. The latter model is consistent with the known linear molecular DNA organization of a satellite and classical satellite sequences yet accounts for the observed concordant metaphase in situ hybridization patterns of these arrays (Fig. 3 and refs. 9 and 23). Alternatively, small clusters of satellite sequences, difficult to detect by hybridization in the presence of large amplified satellite blocks, may be interspersed in a-satellite arrays. Such simple-sequence satellites would not be cut by enzymes that free the a-satellite sequences as discrete blocks. Both models predict that the bulk of DNA sequences present in the centromeric region may only be important to "space" the kinetochore and pairing DNA domains in the correct orientation. Either model is consistent with the rapid

FIG. 6. Diagrammatic representation of human centromere domains. DNA regions responsible for interacting with the kinetochore may be 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 kinetochore proteins.

turnover of most DNA sequences at the centromere [i.e., a satellite at human centromeres (2), mouse satellite at mouse centromeres (29), telomeric (TTAGGG),, tracts at many other vertebrate centromeres (7), etc.] yet proposes that a "core" of conserved sequences is maintained that represent the actual kinetochore (and possibly pairing) domains (Fig. 6). We propose that the conserved (GGAAT), repeat and/or its interspersed CATCATCGA(A/G)T and CAACCCGA(A/ G)T sequences may represent such a component. This work was supported by grants from the U.S. Department of Energy to R.K.M. 1. Moyzis, R. K., Torney, D. C., Meyne, J., Buckingham, J. M., Wu, J.-R., Burks, C., Sirotkin, K. M. & Goad, W. B. (1989) Genomics 4, 273-289. 2. Willard, H. F. & Waye, J. S. (1987) Trends Genet. 3, 192-198. 3. Waye, J. S. & Willard, H. F. (1989) Proc. Natl. Acad. Sci. USA 86, 6250-6254. 4. Prosser, J., Frommer, J., Paul, C. & Vincent, P. C. (1986) J. Mol. Biol. 187, 145-155. 5. Moyzis, R. K., Buckingham, J. M., Cram, L. S., Dani, M., Deaven, L. L., Jones, M. D., Meyne, J., Ratliff, R. L. & Wu, J.-R. (1988) Proc. Natl. Acad. Sci. USA 85, 6622-6626. 6. Meyne, J., Ratliff, R. L. & Moyzis, R. K. (1989) Proc. Natl. Acad. Sci. USA 86, 7049-7053. 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. (1990) Chromosoma 99, 3-10. 8. Williamson, J. R., Raghuraman, M. K. & Cech, T. R. (1989) Cell 59, 871-880. 9. Moyzis, R. K., Albright, K. L., Bartholdi, M. F., Cram, L. S., Deaven, L. L., Hildebrand, C. E., Joste, N. E., Longmire, J. L., Meyne, J. & Schwarzacher-Robinson, T. (1987) Chromosoma 95, 375-386. 10. Riethman, H. C., Moyzis, R. K., Meyne, J., Burke, D. T. & Olson, M. V. (1989) Proc. Nat!. Acad. Sci. USA 86, 6240-6244. 11. Strauss, F. & Varshavsky, A. (1984) Cell 37, 889-901. 12. Fitzgerald-Hayes, M., Clarke, L. & Carbon, J. (1982) Cell 29, 235-244. 13. Carbon, J. & Clarke, L. (1990) New Biol. 2, 10-19. 14. Lohe, A. R. & Brutlag, D. L. (1986) Proc. Nat!. Acad. Sci. USA 83, 696-700. 15. Gosden, J. R., Mitchell, A. R., Buckland, R. A., Clayton, R. P. & Evans, H. J. (1975) Exp. Cell Res. 92, 148-158. 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, 1608-1612. 18. Maio, J. J., Brown, F. L. & Musich, P. R. (1981) Chromosoma 83, 103-125. 19. Miller, D. A. (1977) Science 198, 1116-1124. 20. John, B. & Miklos, G. L. G. (1979) Int. Rev. Cytol. 58, 1-114. 21. Bove, A., Bove, J. & Gropp, A. (1984) Adv. Hum. Genet. 14, 1-57. 22. Rattner, J. B. (1987) Chromosoma 95, 175-181. 23. Pluta, A. F., Cooke, C. A. & Earnshaw, W. C. (1990) Trends Biochem.

Sci. 15, 181-185. 24. Prive, G. G., Heinemann, U., Chandrasegaran, S., Kan, L. S., Kopka, M. L. & Dickerson, R. E. (1987) Science 238, 498-504. 25. Li, Y., Zon, G. & Wilson, W. D. (1991) Proc. Natl. Acad. Sci. USA 88, 26-30. 26. Rich, A. & RajBhandary, U. L. (1976)Annu. Rev. Biochem. 45, 805-860. 27. Lechner, J. & Carbon, J. (1991) Cell 64, 717-725. 28. Zinkowski, R. P., Meyne, J. & Brinkley, B. R. (1991) J. Cell Biol. 13, 1091-1110. 29. Pardue, M. L. & Gall, J. G. (1970) Science 168, 1356-1358.