Characterization of the 3' Region of the Human DNA Topoisomerase I Gene1

Previous studies suggest that topoisomerase I (Topo I) plays a critical role in cell growth. However, the structure of the Topo I gene has not yet been determined. Two complementary DNA (cDNA) clones for the human Topo I 4.1-kilobase mRNA were isolated independently from HeLa and KB cell cDNA libraries. These clones were identical and they contained 679 base pairs of coding and 1138 base pairs of noncoding sequences. The clones had a two-base difference in the 3' noncoding region compared to the Topo I cDNA from human placenta. The structure of the 3' end of the human Topo I gene from six human tumor cell lines was examined. The Topo I cONA recognized 16.5, 24.2, and 16.0 kilobases of genomic DNA restricted with £coRI, //im/III and /VI. respectively. The individual genomic fragments were ordered by double digestion and hybridization with cDNA subclones. The results indicate that the human Topo I gene contains several intervening sequences. The gene arrangement was similar in all six cell lines and no polymorphism was observed. However, each digestion con tained genomic fragments that hybridized with all the subclones, sug gesting that at least one Topo I pseudogene, or another Topo I gene with a different structure, was present in every cell line. As predicted, double digestions generated at 161 base pair fragment that indicates the presence of an intronless pseudogene. In contrast to the DNA topoisomerase I gene, the presumptive pseudogene(s) appears to be hypomethylated. In addition to the 4.1-kilobase Topo I mRNA, a larger 6-kilobase RNA was identified in human KB and HeLa cells which could be a processed Topo I mRNA intermediate. INTRODUCTION Topo I1 catalyzes topologica! alterations in DNA and has an important function in both replication and transcription (1-4). Recently, a role for Topo I in tubulin polymerization was postulated (5). Human Topo I was purified to homogeneity and was shown to be a M, 97,000 protein. Based on studies of human Topo I cDN A clones, this protein is structurally distinct from yeast Topo I (6, 7). Little is known about the structure of the human Topo I gene or its regulation, although others have recently reported that only a single copy is present in the human genome (7). Since Topo I is a potential target for the develop ment of cancer chemotherapeutic agents, a knowledge of the structure, and particularly the expression of the Topo I gene, is important. This paper describes the isolation and characteriza tion of Topo I cDNAs and their use in investigating the 3'terminal structure of the human Topo I gene in several cell lines derived from human tumors. MATERIALS AND METHODS Cell Lines. HeLa/S3, KB, Molt-4F (human T-lymphoblastic cell line), PLC/PRF/5 (human hepatoma cell lines), P3HR-1, and Raji (human B-lymphoblastic cell lines) were obtained from the American Type Culture Collection (Rockville, MD). HCT-116 (a human colon Received 10/31/88; accepted 4/7/89. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This study was support by Grant CA-44358 from the National Cancer Institute. 1To whom requests for reprints should be addressed. 1The abbreviations used are: Topo I. DNA topoisomerase 1; cDNA, comple mentary DNA; TBE. 89 mM Tris-borate. 89 m\i boric acid. 2m\t EDTA; SS PE, 149 m\t NaCI. 10 mM NaH:PO, H2O. l mM EDTA. pH 7.4. tumor cell line) was provided by Dr. Lan Bo Chen for the Dana-Farber Cancer Center, Boston, MA. All cell lines were maintained in RPMI 1640 containing 5% fetal bovine serum and 0.1 mg/ml kanamycin. Anti-mouse IgM-peroxidase conjugate and crude Torula yeast RNA were purchased from Sigma Chemical Co. (St. Louis, MO). The human HeLa cDNA library was obtained from Clontech (Palo Alto, CA). Restriction endonucleases and the cDNA synthesis kit were purchased from Bethesda Research Laboratories (Gaithersberg, MD). Xgtl 1 arms, package system, and Escherichia coli Y1090 were purchased from Promega (Madison, WI). The Topo I monoclonal antibody was de scribed previously (4, 8). All other chemicals were of standard reagent grade. Library Screening and cDNA Characterization. Total cell RNA from KB-6b cells, a methotrexate-resistant subclone (9), was isolated as described below, and polyadenylated RNA was fractionated on a 10 to 30% sucrose gradient as described elsewhere (10). The fractions con taining the 3to 5-kilobase mRNAs were used with a kit to generate a cDNA library. This library and the commercial Xgtl 1 HeLa cell cDNA library were screened with Topo I monoclonal antibody using standard procedures (11). For immunostaining, nitrocellulose fillers were blocked with 20% (v/v) IgG-free calf serum in 0.015% (v/v) Tween 20150 miviNaCI-10 mM Tris buffer, pH 8.0. Anti-mouse IgM-peroxidase conjugate was used as a second antibody. Positive clones were amplified, the DNA was restricted with £coRI,and the insert was purified from agarose gel using electroelution (12). For DNA sequencing, the 1.8kilobase insert was subcloned into M13-mpl8 (13) and the fragments were obtained by using the "cyclone" sequencing strategy (14). The DNA sequences were generated using the method described by Sanger étal. (15) and analyzed with the Beckman Microgenie Sequence Analy sis Program. Isolation of Genomic DNA, Total Cell RNA, and Filter Hybridization Procedures. Genomic DNA was prepared as described previously (16). Briefly, cells were washed in phosphate-buffered saline, decanted, and then lysed in a buffer containing 1% (w/v) sodium dodecyl sulfate, 10 mM Tris (pH 7.6), 10 mM EDTA, and 400 mM NaCI. After an overnight proteinase K treatment (100 /jg/ml), phenyl chloroform extraction, and ethanol precipitation, RNA was digested for 2 h at 37°Cwith 100 ¿ig/ ml RNase A and then further digested with 0.5 mg/ml proteinase K overnight. After phenyl chloroform extraction and ethanol precipita tion, DNA was redissolved in 10 mM Tris (pH 7.6)-1 mM EDTA. The samples were restricted with a 5-fold excess of enzyme for several hours using conditions recommended by the supplier. Following further digestion overnight with a fresh 5-fold excess of enzyme, the restricted genomic fragments were separated on 0.7 to 1% agarose gels in TBE buffer. The gel was denatured, neutralized, and transferred by vacuum to nitrocellulose using a Vacublot apparatus (American Bionetics, Hayward, CA). The hybridization conditions were similar to those described previously (16). To reduce the background, the hybrid ization buffer also contained Torula yeast RNA purified by organic extractions at 200 Mg/ml and 2 x Denhardt's solution (17). The filters were washed for 20 minutes at 50°Cin a 0.1 x SSPE-0.5% (w/v) sodium dodecyl sulfate, before air-drying and autoradiography at -70°C with intensifier screens. Total cellular RNA was prepared as described previously (18). Po lyadenylated RNA was enriched using oligodeoxythymidylate cellulose (19). The hybridization analysis was similar to that used by Bastow et al. (18); RNA was denatured and fractionated on 1% agarose gel containing 1.5% formaldehyde. This facilitated visualization of RNA without destaining. After exposure to short-wave UV for several min (UV Transilluminator, San Gabriel, CA), RNA was transferred to nitrocellulose by diffusion blotting (20), which was complete after 24 h (18). Conditions for hybridization were the same as described above. All the probes were labeled by the random primer labeling procedure (21) using a random primed labeling kit from Boehringer Mannheim. 3922 American Association for Cancer Research Copyright © 1989 on March 1, 2013 cancerres.aacrjournals.org Downloaded from 3' REGION OF HUMAN DNA TOPO1SOMERASE l GENE DNA fragments obtained by restriction endonuclease digestion (ap proximately 1 Mg/fragment) were fractionated on 1% agarose gels and excised using a sterile scalpel blade. The gel slice was dissolved in 3fold (w/v) boiling sterile distilled water. The samples were then stored at -20°C.In order to radiolabel DNA, the agarose/DNA mixtures were maintained at 47°C.Aliquots were added to the random labeling reaction mix, vortexed rapidly, and incubated overnight at room tem perature. Labeled DNA was purified by the spun column procedure (22).