The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-117 and the cloned p53 gene indicated the presence of seven major exons.

A linear simian virus 40 origin-containing DNA fragment replicated in monkey COS cells, generating tandemly repeated (head-to-tail) structures. Electron microscopy revealed circle-and-tail configurations characteristic of rolling-circle replication intermediates. Circularization of the same DNA before transfection led to a theta type of replication which generated supercoiled DNA molecules.We have documented elsewhere that origin-containing (ori+) linear forms of simian virus 40 (SV40) DNA, when added to transfection mixtures containing circular SV40 and 4X174 replicative form I DNAs, enhance the frequency of SV4O/4X174 recombination as measured by infectiouscenter in situ plaque hybridization in monkey BSC-1 cells (4). A key structural feature of such recombinants is the interspersion of 4X174 DNA within tandem head-to-tail repeats of sequences derived from the ori+ linear SV40 DNA. It was also shown that the tandem repeats are generated by a replication-related process rather than by homologous recombination. To account for these findings, we postulated that the recombination-enhancing properties of linear SV40 ori+ DNA are associated with the entry of that DNA into a rolling-circle type of replication which generates recombinogenic intermediates (4). The proposed model envisages that 4X174 replicative form I DNA recombines with the circular template of the intermediate (5) such that continuation of rolling-circle replication generates tandemly repeated segments containing both SV40 ori+ and 4~X174 DNAs. The model is based on the assumptions that linear SV40 DNA can enter a rolling-circle type of replication and, compared with circular SV40 DNA (whose replication is predominantly of the theta type; reviewed in reference 15), the linear forms exhibit a propensity for rolling-circle replication. In this report we present evidence for both assumptions.The 910-base-pair (bp) pSVK1H RsaI B fragment (Fig. 1), which contains the SV40 origin and regulatory region (nucleotides 5171-5243/0-294) linked to 545 bp of pML2K plasmid DNA, displays the recombination-enhancing properties noted above (4). To study the replication of this SV40 ori+ DNA fragment, we transfected monkey cells of the COS line (which produce T-antigen constitutively; 6) in the presence of DEAE-dextran (10) with 1 ,ug of pSVK1H RsaI B fragment DNA per ml or, as a control, with the analogous p6-1K1H RsaI B fragment DNA (Fig. 1) in which the SV40 replication origin was rendered inactive by a six-bp deletion. Low-molecular-weight DNA was then extracted (9) from the nuclei (11) of the transfected cells, digested with BglI or ClaI, electrophoresed on a 1% agarose gel, and blot-hybridized (13, 14) with plasmid pML2K [32P]DNA. At 24 h post-transfection, the major band hybridizing to pML2K DNA was taken as the input fragment DNA on the basis of * Corresponding author.

The human p53 gene was cloned and characterized by using a battery of p53 DNA clones. A series of human cDNA clones of various sizes and relative localizations to the mRNA molecule were isolated by using the human p53-H14 (2.35-kilobase) cDNA probe which we previously cloned. One such isolate, clone p53-H7 (2.65 kilobases), spans the entire human mature p53 mRNA molecule. Construction of the human cDNA clones in the pSP65 RNA transcription vector facilitated the generation of p53 transcripts by the SP6 bacteriophage RNA polymerase. The p53-specific RNA transcripts obtained without further processing were translated into p53 proteins in a cell-free system. By using this rapid in vitro transcription-translation assay, we found that whereas clone p53-H7 (2.65 kilobases) coded for a mature-sized p53 protein, a shorter cDNA clone, p53-H13 (1.8 kilobases), dictated the synthesis of a smaller-sized p53 protein (45 kilodaltons). The p53 proteins synthesized in vitro immunoprecipitated efficiently with human-specific anti-p53 antibodies. Genomic analysis of human DNA revealed the presence of a single p53 gene residing within two EcoRI fragments. Heteroduplex analysis between the full-length cDNA clone p53-H7 and the cloned p53 gene indicated the presence of seven major exons.

A linear simian virus 40 origin-containing DNA fragment replicated in monkey COS cells, generating tandemly repeated (head-to-tail) structures. Electron microscopy revealed circle-and-tail configurations characteristic of rolling-circle replication intermediates. Circularization of the same DNA before transfection led to a theta type of replication which generated supercoiled DNA molecules.