In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

Wolf, D.; Laver-Rudich, Z; Rotter, Varda

doi:10.1128/mcb.5.8.1887

Cited by 38 publications

(18 citation statements)

References 35 publications

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“…Earlier work had shown that wild-type human p53 mRNA is an approximately 2.8-kb species with a heterogeneous 5Ј untranslated region (UTR) of 161 to 246 nts and a 3Ј UTR of approximately 1,200 nts (42,51,52,69,70). While the p53 mRNA sequence investigated in the present study is only 1,587 nts long, our in vitro binding studies identified a 489-nt sequence within the protein-coding region as being one binding site for TS.…”

Section: Discussionmentioning

confidence: 43%

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

Chu

Copur

et al. 1999

Molecular and Cellular Biology

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Thymidylate synthase (TS) catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to generate thymidylate and dihydrofolate. This enzymatic reaction provides for the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis. As a result, TS remains a critical target enzyme in cancer chemotherapy (25, 60a).In addition to its role in enzyme catalysis, there is evidence that TS also functions as an RNA binding protein (5-7). Studies from this laboratory have demonstrated that the translation of human TS mRNA is regulated by its own protein product via a negative autoregulatory mechanism (5). The repression of TS mRNA translation by TS is mediated by specific binding of the protein to at least two distinct cis-acting sequences on its own mRNA (6). The first site corresponds to a 188-nucleotide (nt) sequence that includes the translational start site, while the second site is contained within a 100-nt sequence in the protein-coding region. However, in the presence of the nucleotide substrate dUMP or 5-fluoro-2Ј-deoxyuridine-5Ј-monophosphate (FdUMP), TS is unable to directly interact with its own mRNA, thus allowing for the synthesis of new protein to proceed (5, 7). Several in vitro studies have shown that shortterm exposure of human colon and breast cancer cells to TSinhibitory compounds, such as 5-fluorouracil (5-FU) or the antifolate analog ZD1694, is associated with an increased level of TS protein expression but no corresponding change in the levels of TS mRNA (8,9,35). These findings are consistent with earlier observations that both the RNA binding and the translational inhibition activities of TS are impaired in the presence of either nucleotide and/or folate substrates. Thus, the ability to regulate the expression of TS at the translational level in the setting of acute cytotoxic stress suggests that this regulatory event has biological relevance. Moreover, this process may represent an important mechanism by which normal cellular synthetic function can be controlled and a mechanism for the rapid development of cellular resistance in response to exposure to nucleotide inhibitors of TS, such as 5-FU, and antifolate inhibitors of TS, such as ZD1694, LY231514, and AG331.An immunoprecipitation-reverse transcription (RT)-PCR technique was recently developed to isolate from an intact cultured human colon cancer cell line a TS-ribonucleoprotein (RNP) complex made up of TS protein and TS mRNA (10). In addition to complexing with its own mRNA, TS formed an RNP complex with the mRNA of the c-myc transcription factor. Subsequent studies with RNA electrophoretic gel mobility shift assays (EMSAs) confirmed that the interaction between TS and c-myc mRNA was specific and identified the C-terminal coding region as being an important cis-acting regulatory element (11). Furthermore, in vitro translation experiments demonstrated that TS protein specifically repressed the translation of c-myc mRNA (11). Recent work has shown that TS is * Corresponding author. Mailing ad...

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Section: Discussionmentioning

confidence: 43%

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

Chu

Copur

et al. 1999

Molecular and Cellular Biology

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“…The cDNA for p53 was first cloned in 1984; [1][2][3] approximately 2 years later, a mobility shift in p53 protein was identified as a sequence polymorphism at amino acid 72, changing proline to arginine. [4][5][6] Unfortunately, at the time of its discovery, researchers were inadvertently studying a tumor-derived mutant form of p53, with the belief that TRP53 was an oncogene.…”

Section: Polymorphisms In Tp53: the Codon 72 Polymorphismmentioning

confidence: 99%

Polymorphic variants in the p53 pathway

Murphy

2006

Cell Death Differ

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“…When human primary tumors were screened for p53 with anti-p53 monoclonal antibodies, they too were found to contain several p53 species (27). The study of human p53 has been facilitated by the cloning of human cDNAs (7,17,34,36,37). Analysis of human genomic DNA with these p53 cDNAs revealed a single p53 gene (7,17,36,37 A cDNA library derived from the SV-80 cell line, which expresses in vivo two discrete forms, was therefore screened to isolate the individual cDNAs, each of which encodes one of the two p53 proteins.…”

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confidence: 99%

Molecular basis for heterogeneity of the human p53 protein.

Harris¹,

Brill²,

Shohat³

et al. 1986

Mol. Cell. Biol.

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168

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The human p53 tumor antigen comprises several physically distinct proteins. Two p53 proteins, separable by polyacrylamide gel electrophoresis, are expressed by the human transformed cell line SV-80. The individual cDNAs which code for these proteins were isolated and constructed into the SP6 transcription vector. The proteins encoded by these clones were identified by in vitro transcription with the SP6 vector and translation in a cell-free system. p53-H-1 and p53-H-19 cDNA clones code for the faster-and slower-migrating p53 protein species, respectively, of SV-80. The in vitro-expressed proteins of p53-H-1 and p53-H-19 had the same antigenic determinants and were structurally indistinguishable from their in vivo counterparts. By expressing defined restricted cDNA fragments in vitro, the region of heterogeneity between the respective cDNAs was located at the 5' end of the cDNAs. Exchanging the 5' fragments of interest and expressing the chimeric clones in vitro confirmed that the DNA heterogeneity was responsible for the difference in the electrophoretic mobility of these proteins. The sequences of the two cDNAs revealed a single base pair difference (G versus C) in the coding region of the clones. This sequence difference resulted in an arginine being coded for in clone p53-H-i and a proline being coded for at the equivalent position in clone p53-H-19. This variation accounted for the change in the electrophoretic mobility of the individual p53 protein species.The cellular protein p53 is expressed at low levels in nontransformed cells. When quiescent cultures of such cells are stimulated to proliferate, the amount of p53 is elevated transiently (19,(21)(22)(23)28). In contrast, various types of transformed cells express the protein at elevated levels constitutively (3,4,9,12,16,29,30), which suggested that the protein is involved in transformation.p53 was shown to be a transforming protein by using the L12 nonproducer murine cell line (32,33,36). These cells induce tumors in syngeneic hosts, which subsequently regress. When p53 expression was reconstituted in the cells by the transfection of a functional p53 gene, it induced lethal tumors in the hosts (32, 33). p53 was therefore necessary for expression of the fully transformed phenotype of the cell Iiiie. The protein was also shown to complement an activated Ha-ras gene in the tumorigenic conversion of rat embryo fibroblasts (5, 25) and rat adult chondrocytes (10), demonstrating another facet of the oncogenic character of p53.p53 expression is also enhanced in a number of human transformed cell lines. A salient feature of these cell lines is that they express more than one discrete p53 protein (3). When human primary tumors were screened for p53 with anti-p53 monoclonal antibodies, they too were found to contain several p53 species (27). The study of human p53 has been facilitated by the cloning of human cDNAs (7,17,34,36,37). Analysis of human genomic DNA with these p53 cDNAs revealed a single p53 gene (7,17,36,37 A cDNA library derived from the SV-80 cell li...

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In vitro expression of human p53 cDNA clones and characterization of the cloned human p53 gene.

Cited by 38 publications

References 35 publications

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

Polymorphic variants in the p53 pathway

Molecular basis for heterogeneity of the human p53 protein.

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