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Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53
Abstract:
Three clones for the human tumor antigen p53 were isolated from a cDNA library prepared from A431 cells. One of these clones, pR4-2, contains the entire coding region for human p53. This clone directs the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of an authentic p53 molecule in an in vitro transcription-translation reaction. Although the pR4-2 clone contains the coding region for p53, it is not a full-length copy of the human p53 mRNA. Northern analysis showed that the p53 mRNA is approximately 2,500 nucleotides long, whereas the pR4-2 insert is only 1,760 base pairs in length. Analysis of the DNA sequence of this clone suggests that the human p53 polypeptide has 393 amino acids. We compared the predicted amino acid sequence of the pR4-2 clone with similar clones for the mouse p53 and found long regions of amino acid homology between these two molecules.
Insights
Researchers isolated a clone, pR4-2, containing the full coding region for human p53 (tumor antigen). This clone synthesizes a functional p53 polypeptide, showing homology with mouse p53, aiding cancer research.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- The p53 tumor antigen is a critical regulator of cell growth and a key target in cancer research.
- Understanding the genetic and molecular characteristics of human p53 is essential for developing targeted cancer therapies.
Purpose of the Study:
- To isolate and characterize clones encoding the human tumor antigen p53.
- To confirm the functional integrity and sequence homology of the isolated p53 clone.
Main Methods:
- Isolation of cDNA clones from an A431 cell line library.
- In vitro transcription-translation assays to assess polypeptide synthesis.
- Northern blot analysis to determine mRNA length.
- DNA sequencing and comparative amino acid sequence analysis with mouse p53.
Main Results:
- Three human p53 clones were isolated, with clone pR4-2 containing the entire coding region.
- pR4-2 directed the synthesis of a polypeptide with the correct molecular weight and immunological epitopes of human p53.
- Northern analysis indicated the p53 mRNA is ~2,500 nucleotides, while the pR4-2 insert is 1,760 base pairs.
- Sequence analysis predicted a 393-amino acid human p53 polypeptide with significant homology to mouse p53.
Conclusions:
- The pR4-2 clone represents a valuable tool for studying human p53 function and its role in tumorigenesis.
- The identified homology between human and mouse p53 suggests conserved functional importance across species.
- Further research using this clone can advance the understanding of p53-mediated cellular processes and cancer development.
