Localization of a Unique Gene by Direct Hybridization in Situ

Proc. NatL. Acad. Sci. USA Vol. 78, No. 6, pp. 3755-3759, June 1981 Genetics Localization of a unique gene by direct hybridization in situ (human a-globin/ 'I-labeled cDNA plasmid/metaphase chromosomes/probe network formation/dextran sulfate) DANIELA S. GERHARD, ERNEST S. KAWASAKI*, F. CARTER BANCROFT, AND PAUL SZABOt Memorial Sloan-Kettering Cancer Center, and Department of Genetics and Molecular Biology, Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University, New York, New York 10021 Communicated byJoseph G. Gall, March 2, 1981 ABSTRACT Previous determinations of the chromosomal lo- For most mRNA species, this is a goal that would be quite dif- cation ofunique genes have required that the chromosomes of in- ficult to achieve. terest be fractionated, either by species-specific chromosome loss We report here the development ofa method for employing from interspecies hybrids, or by physical fractionation proce- a hybrid plasmid containing a cDNA sequence to determine by dures. We have developed a general technique for the localization hybridization in situ the chromosomal location ofa unique gene. of a unique gene, which requires no prior chromosome fraction- To demonstrate that this method yields the correct result, we ation. The technique involves the use of a labeled hybrid cDNA have used it to localize the gene that codes for human a-globin, plasmid for direct hybridization in situ to metaphase cells from the whose chromosomal location has previously been determined organism under investigation. As a model system for development through the use of somatic cell hybrids plus molecular hybrid- of this technique, we have employed a human a-globin cDNA ization (8, 9). plasmid (JW1O1) to localize the corresponding gene cluster. To obtain a sufficiently large autoradiographic signal, we have both labeled this plasmid with "I to a high specific activity (109 dpm/ EXPERIMENTAL PROCEDURES ,ug) and taken advantage ofthe ability ofa double-stranded probe of to form networks. To obtain sufficient hybridization specificity, Precoating of Slides. Slides are incubated for a minimum various experimental procedures were used, the most important 5 hr in lOx Denhardt's solution (lx is 0.02% each of bovine of which was the choice from among a range of probe concentra- serum albumin, Ficoll, and polyvinylpyrrolidone) (10) at 60C tions of the highest that does not yield excessive background hy- to reduce binding ofthe probe to the glass slide (11). The slides bridization. We have shown that, with an autoradiographic ex- are then washed profusely in double-distilled water and air posure time of only 12 days, use of this technique correctly dried; they can be stored for several months after preparation. localized the human a-globin gene cluster to chromosome 16. This Preparation of Chromosomes. Penta X (69,XXXXX), a hu- technique should be generally applicable to the localization ofany man lymphoblastoid cell line, was kindly supplied by M. Sin- gene for which a corresponding cDNA hybrid plasmid is available. iscalco of this institute. Metaphase chromosomes are prepared as described (12). The slides are either used for hybridization An understanding of the mechanisms involved in the expression in situ within a day or stored with desiccant at -70'C. and regulation of any eukaryotic genes will ultimately require Preparation of 125I-Labeled dCTP. 125I-Labeled dCTP (spe- a determination of the location of its genetic elements within cific activity 300-1000 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) the genome of the corresponding organism. A first step in this is prepared by converting 5-mercuro-dCTP in a thallium-cat- determination is the localization ofthe gene to a particular chro- alyzed reaction using carrier-free Na'25I (D. Ward and W. Pren- mosome and, if possible, to a particular subchromosomal sky, personal communication). A standard reaction mixture con- region. sists of the following: 10 mCi of Na'25I (IMS300, Amersham) Early gene localization techniques involved the use ofeither neutralized by the addition ofa sufficient amount ofH2S04, 0. 1 pedigree analysis or somatic cell genetics. More recently, hy- mM NaOAc (pH 4.2), 0.8 mM thallic nitrate, and 0.2 mM Hg- bridization in situ has been employed to localize reiterated dCTP (P-L Biochemicals). The mixture is incubated at 60C for genes in various organisms (see ref. 1 and references therein) 5-10 min, the reaction is terminated by the addition of 50 vol and some unique genes in species containing polytene chro- of cold 5 mM triethylammonium carbonate (Et3NHCO3H) (pH mosomes (2). By contrast, the use of molecular hybridization 9.5), and the products are applied to a 0.25-ml DEAE-cel- procedures to localize unique genes in organisms without poly- lulose column equilibrated in 5 mM Et3NHCO3H. The column tene chromosomes has depended on prior fractionation of the is washed extensively with 0.1 M Et3NHCO3H to elute un- chromosomes under investigation. This has been achieved reacted Nal, and 125I-labeled dCTP is then eluted by the ad- either by constructing interspecies (human-mouse) cell hybrids dition ofa minimal volume of0.6 M Et3NHCO3H. The 125I-la- thatpreferentially lose human chromosomes (3, 4) or by physical beled dCTP is further purified by passage over a Bio-Gel P2 fractionation of chromosomes (5, 6). A more direct in situ hy- column (Bio-Rad) in 5 mM Et3NHCO3H. bridization technique, in which preformed networks tailed with Preparation of125I-Labeled Plasmid DNA. Human a-globin 5-bromodeoxyuridine are used to detect hybrids between chro- cDNA plasmid JW101, originally constructed by Wilson et al. mosomes and a polyadenylylated RNA probe, has been em- (13), was generously provided by A. Bank of Columbia Uni- ployed successfully to localize an integrated retrovirus on versity. Forlabeling, the nick-translation protocol ofNordstrom chicken chromosomes (7). However, the extension of this method to localize other genes would require the use ofprobe Abbreviation: NaCl/Cit, 0.15 M NaCI/0.015 M trisodium citrate (standard saline citrate). RNA preparations that are as highly purified as retroviral RNA. * Present address: Cetus Corporation, 600 Bancroft Way, Berkeley, CA 94710. The publication costs ofthis article were defrayed in part by page charge t Present address: c/o Dr. Giorgio Bernardi, Institut de Recherche en payment. This article must therefore be hereby marked "advertise- Biologie Moleculaire, Tour 43-2, Place Jussieu, 75221 Paris, Cedex ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 05, France. 3755 Downloaded by guest on September 26, 2021 3756 Genetics: Gerhard et al. Proc. Natl. Acad. Sci. USA 78 (1981) et al. (14) is followed exactly as described, employing '25I-la- we have employed the technique of Ward and Prensky to syn- beled dCTP at a final concentration of 5.0 AM and a reaction thesize '25I-labeled dCTP with a specific activity of 300-1000 volume of 10 jul. The reaction volume is then brought up to 0.5 Ci/mmol. In addition, it has been shown that recombinant ml with 10 mM Tris HCl (pH 7.6)/1 mM EDTA, and the mix- plasmids can be used to amplify the signal from a given hybrid ture is extracted with an equal volume of phenol/chloroform through the formation ofdouble-stranded networks. Wahl et al. (1:1 vol/vol). The phases are separated by centrifugation, and (18) have discussed the formation of such networks when ran- the aqueous phase is passed over a Sephadex G-50 column domly sheared double-stranded DNA probes are employed, (Pharmacia). The excluded peak is dialyzed against 1 liter of 1 and they have shown that the rate ofhybridization is increased M NaCl/ 10 mM Tris HCl (pH 7.6)/ 1.0 mM EDTA overnight. by addition of dextran sulfate to the hybridization buffer. In After addition of 1.0 mg each of poly(A) and poly(U) (P-L Bio- collaboration with L. Lederman we have recently shown that chemicals), the nucleic acids are precipitated by adding 2.5 vol the rate ofprobe hybridization to cytological preparations is also of 95% (vol/vol) ethanol and incubating for 1-2 hr at -20'C. increased 20- to 30-fold when dextran sulfate is present in the The precipitate is then collected by centrifugation (16,000 X hybridization buffer (unpublished data). g, 30 min), dried, and dissolved at a final concentration of 50 In the present studies, we have adapted the principle of ng/ml in hybridization buffer: 50% (vol/vol) formamide (Ko- probe network formation to the localization of a unique gene dak spectral grade), 2x NaCl/Cit (pH 7.0), lx Denhardt's by hybridization in situ. The technique employed is illustrated solution, alkali-sheared salmon sperm DNA at 200 Atg/ml, 0.1 schematically in Fig. 1. A recombinant plasmid that has been mM KI, 5 mM NaPO4, and 10% dextran sulfate (lx NaCl/Cit labeled with 125I to a high specific activity by nick-translation is 0.15 M NaCl/0.015 M trisodium citrate). Using "2I-labeled is randomly sheared and denatured, and then is annealed on dCTP of specific activity 1000 Ci/mmol, we produce an '25i- a cytological preparation. In the two extreme cases shown, labeled plasmid with specific activity of about 109 dpm/,g. either networks of probe molecules can form in solution and Hybridization in Situ. Endogenous RNA is eliminated by then hybridize to a homologous sequence in chromosomal DNA ribonuclease digestion with RNase A (type IIIA, Sigma) at 100 (pathway I), or networks can be built onto a free single-stranded ,g/ml in 2x NaCl/Cit at room temperature for 1 hr. The tail of a probe molecule that has hybridized to a homologous RNase reaction is stopped by carefully washing the slides with sequence in chromosomal DNA (pathway II).

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