Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line

volume 9 Number 16 1981 Nucleic Acids Research Characterization of a gene encoding a 115 K super T antigen expressed by a SV40-transformed rat cell line Evelyne May , Jean-Marc Jeltsch** and Frank Gannon ** *Institut de Recherches Scientifiques sur le Cancer, B.P. n° 8, 94800 Villejuif, and Labora: toire de Ge"netique Moleculaire des Eucaryotes du CNRS, Unite 184 de Biologie Moleculaire de Genie Genetique de 1'INSERM, Faculte de Medecine, Strasbourg, France Received 17 June 1981 ABSTRACT It has been reported that SV40 -transformed V 11 F 1 clone 1 subclone 7 rat cells (subclone 7) produce a super T antigen of 115,000 M . This super T antigen is entirely SV40 coded and is synthesized by translation of an elongated form of SV40 early mRNA (May, E., Kress, M., DayaGrosjean, L., Monier, R. and May, P. (1981) J. Virol., 37, 24-35). The results reported here show that there is only one independent insertion of viral DNA in the cellular genome of subclone 7 cells. When DNA from subclone 7 cells was cleaved with Bam HI endonuclease two distinct SV40 sequence containing fragments were generated with sizes of 5 Kb and 10 Kb, respectively. Two recombinant cosmids were constructed by insertion of the 5 Kb and 10 Kb fragments, respectively, into cosmid pHC 79. Using restriction map analysis and nucleotide sequencing, we showed that the 5 Kb fragment actually contained the complete sequence of a gene encoding super T antigen. As compared to the normal SV40 early gene, the sequence of super T gene showed the following rearrangements : (i) The segment between nucleotides 411B - 3544 was duplicated in a direct order and (ii) these two copies of 573 nucleotide sequence were separated by a 93 nucleotide tract which was a nearly perfect inverted Tepeat of the segment located between nucleotides 4868 and 4770 (nucleotide numbering used here = Weissmann number + 1 7 ) . INTRODUCTION Multiple species of T antigens with molecular weight distinct from normal sized large T antigen can be identified in a variety of SV40 transformed rat and mouse cell lines. Species of T antigen with a molecular weight considerably larger than that of normal-sized large T antigen are widespread in SV40-transformed rat or mouse cells and are referred to as super T antigen (1, 2, 3, 4 ) . As already reported (5) SV40 transformed rat cell line V 11 F 1 clone 1 subclone 7 produces a single species of super T antigen of 115,000 M f in the absence of detectable trace of large T antigen (86,000 M ) . We have previously shown that this super T antigen (super T) is an elongated form of large T antigen, which contains a duplication of that part of large © IRL Press Umited, 1 Falconberg Court, London W1V 5FG, U.K. 41 1 ' Nucleic Acids Research MATERIAL AND METHODS Cells The rat cell line V 11 F 1 cl.1 subclone 7 has been previously described (5, 6) . DNA extraction High molecular weight cellular DNA was isolated as described by Birg (8). Cells were grown to confluence in 10-cm petri dishes. After removal of the medium, the cells were rinsed twice with phosphate buffered saline (PBS) and then lysed at room temperature for 10 minutes with 1 ml of 10 mM Tris-HCl pH 7.9, 5 mM EDTA, 10 mM NaCl, 0.5 % SDS and 1 mg/ml pronase preheated at 37°C (Calbiochem). The lysate was transferred to an erlenmeyer flask and left overnight at room temperature. The lysate was then gently shaken with an equal volume of phenol saturated with Tris-HCl 1 M, pH 7.9 and then the mixture was allowed to stand until the two phases became separated. The aqueous phase, containing the nucleic acids was shaken as before with an equal volume of phenol-chloroform-isoamylalcohol 50 : 48 : 2. The (aqueous) upper layer obtained was dialyzed overnight against 100 volumes of 10 mM Tris-HCl pH 7.9, 5 mM EDTA, 1 M NaCl and then against numerous changes of 10 mM Tris-HCl pH 7.9, 5 mM EDTA. Pancreatic RNAse (Choay, France), previously heated at 80°C for 10 minutes to inactivate DNAse ac- 4112 T antigen corresponding to the genome region extending approximately from 0.46 to 0.35 map units, as determined by fingerprint analysis of super T and by S1 mapping analysis of 115 K super T mRNA (6). The duplicated sequence virtually corresponds to that region of SV40 genome in which 12 of 13 tsA mutation sites are clustered (7). To gain a better understanding of the biogenesis of super T antigens, we undertook the analysis of the arrangement of the integrated viral DNA sequences in subclone 7 cells. The integrated viral sequences with the flanking cellular sequences at both extremities have been cloned in a cosmid. Sequencing data reported in this paper show that the sequence duplication in 115 K super T mRNA is a consequence of a homologous duplication of a 573 nucleotide sequence in the viral DNA integrated in subclone 7 cells. In addition, this analysis reveals that the two copies of the 573 nucleotide repeated sequence bracket a 93 nucleotide sequence which is a nearly perfect inverted repeat of a viral segment located farther than 1200 nucleotides in the early viral region. The biological implications of this complex integration pattern of SV40 DNA are discussed. Nucleic Acids Research 4113 tivity, was added to a final concentration of 10 jig/ml and MgCl- was added to a final concentration of 5 mM. The resulting solution was incubated at 37°C for 2 h. Following removal of the RNAse by phenol extraction, the DNA was dialyzed exhaustively as described above. Transfer of high molecular weight DNA from agarose gel to nitrocellulose, hybridization and detection of specific DNA sequences. After digestion with restriction enzymes electrophoresis of DNA (20 ug/slot) was carried out on a 1 % agarose vertical slab gel in a buffer containing 40 mM Tris-HCl pH 7.8, 5 mM sodium acetate and 4 mM EDTA at 2 V/cm for 14 h. After electrophoresis the DNA was partially acid-depurinated, alkali-denatured and transferred to a nitrocellulose filter (Schleicher and Schull, BA 85) by the method of Southern (9) slightly modified by Wahl and al. (10). Highly purified SV40 DNA form I was labeled in vitro by the nicktranslation reaction (11). Hybridization to 32P-labeled SV40 DNA (107 cpm/ blot) and the washing of the filter were performed as described by Breathnach et al. (12). The filters were then dried and exposed for autoradiography with Royal X Omat film at -70°C by using a Dupont cronex hi plus intensifying screen. Cloning procedures DNA extracted from V 11 F 1 cl.1 subclone 7 was cloned in the cosmid vector pHC 79 (13) essentially as described by Gannon et al. (14). The cellular DNA was digested to completion by the restriction enzyme Bam HI and the fragments of approximately 5 and 10 kilobases (Kb) were selected after a sucrose gradient. Twelve ug of 5 Kb and 14 ug of 10 Kb DNA were ligated in a volume of 20 ul with respectively 10 jjg and 5 \ig of the cosmid pHC 79 DNA digested with Bam HI and used to transduce the E. Coli strain 1106, using the packaging method and stra (...truncated)