Am. J. Hum. Genet. 46:784-788, 1990

A Human Alpha Satellite DNA Subset Specific for Chromosome 12 Antonio Baldini, * Mariano Rocchi, *tT Nicoletta Archidiacono,t Orlando J. Miller,* and Dorothy A. Miller* I

*Department of Molecular Biology and Genetics, and Center for Molecular Biology, Wayne State University, Detroit; and

tLaboratorio di Genetica Molecolare, Istituto G. Gaslini, Genoa

Summary We have isolated a DNA clone (pBR12, locus D12Z3) which identifies an alphoid subset specific for chromosome 12. This alphoid subset has an EcoRI periodicity of 680 bp and is characterized by a higherorder repeat of about 1.4 kb (eight basic units of about 170 bp each) as revealed by several restriction enzymes. The sequence analysis confirmed the alphoid nature of pBR12 and the dimeric organization.

Introduction

Alphoid DNA is a complex family of satellite DNAs found in primate genomes and localized to the pericentromeric region of chromosomes (Maio 1971; Kurnit and Maio 1973; Manuelidis and Wu 1978; Rosenberg et al. 1978). Although the basic organization is always characterized by monomers of about 170 bp tandemly repeated, the nucleotide sequence and the monomeric organization may be considerably divergent from one chromosome to another (Willard 1985). Therefore, under appropriate stringency conditions, an alphoid clone may recognize a single chromosome. The chromosomespecific alphoid subsets are in general characterized by a higher-order repeat composed of n diverged monomers which, in turn, is tandemly repeated many times to form long arrays (Willard and Waye 1987). Chromosome-specific subsets have been described for most of the human chromosomes (reviewed in Willard and Waye 1987). In a few cases multiple subsets are present on a single chromosome (e.g., see Waye et al. 1987; Baldini et al. 1989). Chromosome-specific probes detecting repetitive DNA have been used in a variety of studies, such as Received August 16, 1989; revision received October 20, 1989. Address for correspondence and reprints: Dorothy A. Miller, Department of Molecular Biology and Genetics, Wayne State University, Scott Hall Room 3126, 540 East Canfield, Detroit, MI 48201. 1. Present address: Department of Genetics, Yale University School of Medicine, New Haven, CT. i) 1990 by The American Society of Human Genetics. All rights reserved. 0002-9297/90/4604-0016$02.00

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detection of polymorphisms (Willard et al. 1986), identification of human chromosomes in somatic hybrids, and detection of aneuploidies in interphase cells (e.g., see Pinkel et al. 1986; van Dekken and Bauman 1988). In order to extend the usefulness of such probes, we have been studying chromosomes whose alphoid subsets have not been described in detail. We report here the isolation and characterization of a clone which identifies an alphoid subset specific for chromosome 12. Material and Methods Southern Blot Analysis

High-molecular-weight genomic DNAs from somatic cell hybrids and from human lymphoblastoid cell lines were prepared by following standard procedures (Maniatis et al. 1982). Complete endonuclease digestions were performed using a fourfold excess of enzyme under the conditions suggested by the suppliers. Gel electrophoresis was performed in 1 x TAE (1 x TAE = 40 mM Tris-acetate, 1 mM EDTA). Genomic DNAs were run in 0.8% agarose gel for 16-18 h, denatured, and DNA transferred to Hybond membrane (Amersham), using as transfer buffer 20 x SSC (1 x SSC = 150 mM sodium chloride, 15 mM sodium citrate, pH 7). Clone inserts (50 ng) were labeled with 32P-dCTP (3,000 Ci/mmol; Amersham) by using random oligomer priming. Specific activities were 3-6 x 108 cpm/pg of DNA. We used two kinds of stringency conditions: (1) Less

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Human Chromosome 12-specific Alphoid DNA stringent conditions that permit cross-hybridization of different families of alphoid DNA were used for the analysis of the genomic DNA from the hybrid and library screening. The reaction was carried out in 50% formamide, 6 x SSC, 5 x Denhardt's solution, 0.5% SDS, 10 mM EDTA pH 8, and 100 ig sonicated herringsperm DNA/ml at 420C for 16-18 h. Washing was done twice at 650C for 15 min in 2 x SSC, 0.1% SDS. (2) High-stringency conditions that permit hybridization only of high homologous sequences were also used. In contrast to conditions in (1), we made these changes: hybridization in 3 x SSC, 50% formamide at 52°C; washing at 69°C; and final washing three times for 15 min each in 0.1 x SSC, 0.1% SDS. Filters were exposed to XAR-5 film (Kodak) under intensifying screen at -700C for 16-24 h. Isolation and Characterization of the Clone pBRI2

The human-hamster somatic cell line YE2iTC contains chromosome 12 as the only cytologically detectable human component. A Southern blot containing HindIII-cut genomic DNA from this line was hybridized, under low-stringency conditions, to the alphoid clone pC1.8, which we have shown contains EcoRI dimers (Baldini et al. 1989). An intense band of about 1.4 kb was identified. HindIII restriction fragments of 1.2-1.6 kb were isolated from low-melting-temperature agarose and ligated into pBS plasmid vector (Stratagene). The recombinants were propagated in Escherichia coli strain JM101, and the bacterial colonies were screened with pC1.8. One positive clone was identified and isolated and named pBR12. The insert of this clone was 680 bp long, although all the other recombinants tested contained inserts of the expected length (1.2-1.6 kb). In situ hybridization on metaphase chromosome spreads from a human female donor was performed using 3H-labeled pBR12 (specific activity as 2.7 x 107 cpm/pg of DNA). The hybridization was performed at 390C in 2 x SSCP (1 x SSCP = 120 mM sodium chloride, 15 mM sodium citrate, 20 mM sodium phosphate, pH 6), 50% (v/v) formamide; 10% (w/v) dextran sulfate, and 1 mg sonicated herring-sperm DNA/ml. Post-hybridization washing was at 500C in 2 x SSC, 50% formamide three times and in 2 x SSC five times. The slides were then air-dried and dipped in Ilford K2 nuclear emulsion. The exposure was carried out at 4°C for 5-7 d, and the slides were then developed in D-19 (Kodak). Chromosomes were identified by Q-banding after the slides were developed.

pBR12 was also hybridized, under high-stringency conditions, to a standard somatic cell hybrid panel composed of 18 cell lines which have been characterized elsewhere (Rocchi et al. 1986). The nucleotide sequence of pBR12 (not shown) was determined with the dideoxy method using T7 DNA polymerase (Pharmacia) by following the procedure suggested by the supplier. Results

Clone pBR12, a 680-bp HindIII fragment, was isolated from a subgenomic library constructed from a somatic cell hybrid containing only human chromosome 12. In situ hybridization showed that this clone, under moderate-stringency conditions, hybridizes mostly to the pericentromeric region of chromosome 12 (52% of the total number of autoradiographic grains). Minor percentages of grains were observed on the pericentromeric regions of chromosome 7 (5%), chromosome 1 (5%), chromosome 3 (3%), chromosome 16 (3%), and chromosome 19 (3%) (fig. 1). The chromosome specificity of this done was confirmed by high-stringency hybridization of pBR12 to a Southern blot containing DNAs from 18 somatic cell hybrids (human-hamster) of a panel that has been characterized elsewhere (Rocchi et al. 1986). In this case only those hybrids containing chromosome 12 gave a hybridization signal (fig. 2), indicating that, under these conditions, pBR12 does not cross-hybridize to any other chromosome. A Southern blot analysis was performed to study the genomic organization of the alphoid subset identified by pBR12. Human genomic DNA was cut with several restriction enzymes and hybridized, under high-stringency conditions, to pBR12 (fig. 3A). Three enzymes (HindIII, TaqI, and PvuII) gave an intense band of about 1.4 kb, indicating that this subset is organized in highorder units composed of eight monomers of 170 bp each. The great majority of the EcoRI restriction sites are spaced every 680 bp, while Hinfl sites are present every 340 bp. DraI, PstI, and RsaI cut this DNA very infrequently. In order to reconstruct the restriction map ofthe chromosome 12 alphoid higher-order repeat, we performed a Southern blot analysis ofdouble digestions of human genomic DNA (not shown). The restriction map derived in this way is shown in figure 3B, along with the restriction map of pBR12 (derived from the sequence analysis). The comparison of these two restriction maps suggests that the cloned segment lacks an EcoRI frag-

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Histogram showing distribution of autoradiographic grains on the pericentromeric region of chromosomes after in situ hybridization under moderate-stringency conditions. A total of 251 grains were scored on 34 metaphases. Most (85%) of the total grains were located on the pericentromeric region of chromosomes.

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Figure 2 Autoradiograph of Southern blot containing HindIII-cut DNAs from 18 hybrid cell lines of a standard panel (lanes 1-18) and total human genomic DNA (lane 19). The filter was hybridized to pBR12 under high-stringency conditions. Hybridization signals appeared only on the hybrids which contained chromosome 12 (lanes 1-3, 8, 10, and 16-18). In addition to the band of 1.4 kb, there are bands spaced 340 bp apart. After a long exposure, all these bands are concordant with the 1.4-kb band.

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A, Southern blot analysis of human genomic DNA

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high-stringency conditions. D =DraI; E = EcoRI; Ha =HaeIII; = HindIll; Hi = Hinfl; Ps =PMt; Pv = PvuII; R =Rsal; T = Taql. The 1.4-kb higher-order repeat is revealed by HindIII, PvuII, and Taql. B, Partial restriction map of the chromosome 12-specific alpha satellite repeat of genomic DNA (derived from double-digestion analysis), compared with the partial restriction map of pBR12 (derived from the sequence analysis). The discrepancies between these

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ment of 680 bp. It might have been caused by a rearrangement. Alternatively, the genomic fragments used to construct the library may have been contaminated with smaller fragments. Present in the genome in low copy number are 680-bp HindIII fragments of this alphoid subset (fig. 2). The sequence analysis (not shown) revealed that pBR12 is composed of two imperfect repeats of 340 bp that show as much as 87% sequence similarity to the consensus sequence of the RI repeats (Wu and Manuelidis 1980). These data, along with the data on the genomic organization, indicate that pBR12-like DNA is a diverged subset of the alphoid dimeric family. The nucleotide sequence of pBR12 has been submitted to the EMBL/GenBank Databases (accession no. M28221).

787 Discussion

Chromosome-specific alphoid subsets have been described for most of the human chromosomes. With the present report we add a newly identified specific subset from chromosome 12. This subset was studied using the clone pBR12 (locus D12Z3) isolated from a somatic cell hybrid containing only human chromosome 12. The chromosome derivation and the specificity of this clone have been demonstrated using hybridization in situ to chromosomes and to DNAs from a somatic cell hybrid panel. In the latter case we showed that, under the conditions tested, pBR12 does not cross-hybridize to the alphoid subset of any other chromosome. The chromosome-specific alphoid subset identified by pBR12 is characterized by a basic dimeric organization which is revealed by Hinfl restriction periodicity. Dimers are organized in tetramers, each one containing a unique EcoRI restriction site. A third level of hierarchical organization is revealed by HindIII, PvuII, and TaqI, whose restriction sites are repeated every octamer. The octameric unit is the higher-order repeat of this subset. Chromosome-specific alphoid subsets with a basic dimeric organization have been described for chromosome 7 (Jorgensen et al. 1986; Waye et al. 1987), chromosome 10 (Devilee et al. 1988), and chromosome 3 (Yurov et al. 1986; Delattre et al. 1988; Waye and Willard 1989). Uncloned EcoRI dimers were mapped on chromosome 1, chromosome 3, chromosome 5, chromosome 6, chromosome 7, chromosome 10, and chromosome 19 (Manuelidis 1978). A possible common ancestral origin of the alphoid subsets of this group of chromosomes has been discussed elsewhere (Alexandrov et al. 1988). Additional chromosomes 12 have been found in chronic B-lymphocytic leukemia and in small cell B-lymphocytic lymphomas (reviewed in Yunis 1986). The availability of a chromosome 12-specific centromeric probe may allow the rapid screening of cells for this chromosome in any phase of the cell cycle.

Acknowledgment This work was supported by March of Dimes-Birth Defects Foundation grant I-950 to D.A.M. and by National Institutes of Health grants GM 39788 and GM 34959 to OJ.M.

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