In Vitro Expression of Human p53 cDNA Clones and Characterization of the Cloned Human p53 Gene

Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.

Section: Discussionmentioning

confidence: 99%

Characterization of the Human p53 Gene

Lamb

Crawford

1986

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

Chu

Copur

et al. 1999

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...

Molecular Basis for Heterogeneity of the Human p53 Protein

Harris

Brill

Shohat

et al. 1986

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-1 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.