This proposal intends to examine the mechanism by which cell-transforming proteins come to reside in the nucleus. The adenovirus E1A gene product is required for the transcription of other adenovirus genes and is involved in cell transformation and immortalization. This protein acts at the level of transcription and has been shown to be concentrated in the nucleus of cells. We have used the microinjected Xenopus laevis oocyte to examine transcriptional activity and nuclear targeting of this protein. When overproduced in bacteria to yield a bona fide (nonfusion) protein, E1A stimulates the transcription of an adenovirus gene in injected oocytes. When examined for its ability to enter oocyte nuclei following cytoplasmic injection, however, we show that the protein undergoes a modification such that its apparent molecular weight increases by about 2,000 daltons. Only this modified form of the protein is found in nuclei. A second transforming nuclear protein, that encoded by the human c-myc gene, also undergoes a similar modification in the cytoplasm before it can enter the nucleus.
The specific aims of this proposal are to determine the nature of the modification, the amino acid residue(s) it modifies, and its relationship to nuclear targeting and possible transcriptional regulation. The modification will be determined by oocyte microinjection of small E1A peptides, followed by HPLC and amino acid sequencing. Further studies include the construction of fusion proteins between E1A and myc and other nonnuclear proteins via recombinant DNA technology. The ability of the fusion proteins to be modified and enter nuclei will be assessed. The long-term goals of this project are to determine the signal on proteins which target them to the nucleus and to investigate determinants on proteins which might be responsible for transcriptional regulation and transformation. Thus, these studies are of basic importance in cellular and molecular as well as developmental biology. The gene products which are under investigation have been implicated in the two-step model for cancer. Thus, this proposed project has a direct bearing on our ultimate understanding of cancer. (K)

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Molecular Cytology Study Section (CTY)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Worcester Foundation for Biomedical Research
United States
Zip Code
Wu, L; Good, P J; Richter, J D (1997) The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins. Mol Cell Biol 17:6402-9
Villarreal, X C; Richter, J D (1995) Analysis of ATF2 gene expression during early Xenopus laevis development. Gene 153:225-9
Gebauer, F; Xu, W; Cooper, G M et al. (1994) Translational control by cytoplasmic polyadenylation of c-mos mRNA is necessary for oocyte maturation in the mouse. EMBO J 13:5712-20
Standiford, D M; Richter, J D (1992) Analysis of a developmentally regulated nuclear localization signal in Xenopus. J Cell Biol 118:991-1002
Paris, J; Swenson, K; Piwnica-Worms, H et al. (1991) Maturation-specific polyadenylation: in vitro activation by p34cdc2 and phosphorylation of a 58-kD CPE-binding protein. Genes Dev 5:1697-708
Simon, R; Richter, J D (1990) The degradation sequence of adenovirus E1A consists of the amino-terminal tetrapeptide Met-Arg-His-Ile. Mol Cell Biol 10:5609-15
Slavicek, J M; Jones, N C; Richter, J D (1989) A karyophilic signal sequence in adenovirus type 5 E1A is functional in Xenopus oocytes but not in somatic cells. J Virol 63:4047-50
McGrew, L L; Dworkin-Rastl, E; Dworkin, M B et al. (1989) Poly(A) elongation during Xenopus oocyte maturation is required for translational recruitment and is mediated by a short sequence element. Genes Dev 3:803-15
Richter, J D (1989) In vivo photocrosslinking reveals that transcription factor binding to the mammalian ATF recognition sequence is required for E1A-induced transactivation in injected Xenopus laevis oocytes. Nucleic Acids Res 17:4503-16
McGrew, L L; Richter, J D (1989) Xenopus oocyte poly(A) RNAs that hybridize to a cloned interspersed repeat sequence are not translatable. Dev Biol 134:267-70

Showing the most recent 10 out of 17 publications