GADD34 is one of a subset of proteins induced after DNA damage or cell growth arrest. To examine the function of GADD34, we used the yeast two-hybrid system to clone the protein that interacts with murine GADD34. As bait we used the product of the partial GADD34 cDNA, including the regions rich in proline, glutamic acid, serine and threonine (PEST) and γ 1 34.5 regions. A cDNA clone, named GAHSP40, which is a mouse DnaJ family protein with a high similarity to human HLJ1 was cloned. The interaction between GADD34 and GAHSP40 in cultured cells was confirmed by a co-immunoprecipitation experiment and in NIH 3T3 cells by two-hybrid analysis in vivo . For binding of the two proteins, the γ 1 34.5-similar region of GADD34 was necessary; however, the PEST region was also involved and the C-terminus of GAHSP40, but not the J-domain, was important. GAHSP40 was detected in all mouse tissues examined, but a different transcript was found in the testis. Both GADD34 mRNA and GAHSP40 mRNA were significantly elevated by treatment with methyl methanesulphonate, although the time courses were different. In addition, both GAHSP40 and GADD34 mRNA were induced by heat shock.

We have used the yeast two-hybrid system to clone the protein that interacts with the BFCOL1 (binding factor of a type-I collagen promoter) zinc-finger transcription factor that was cloned previously as the factor that binds to the two mouse proximal promoters of the type-I collagen genes. We utilized as bait the N-terminal domain of BFCOL1 that includes the zinc-finger DNA-binding domain. One cDNA contained a potential open reading frame for a polypeptide of 392 amino acids and was identical to PTRF (polymerase I and transcript-release factor), which is involved in transcription termination of the RNA polymerase I reaction. Northern-blot analysis revealed that the pattern of mRNA expression was similar to that of the type-I collagen gene. In addition, we detected the mRNA expression only in a fibroblast cell line and two bone cell lines, but not in other blood and neuronal cell lines. Recombinant protein was shown to enhance the binding of BFCOL1 to its binding site in the mouse proα2(I) collagen proximal promoter in vitro . The transient-transfection experiment showed that PTRF had a suppressive effect on the mouse proα2(I) collagen proximal promoter activity. We speculate that PTRF might play a role in the RNA polymerase II reaction as well as that of RNA polymerase I.

Oestrogen (E2) regulates the expression of its target genes at transcriptional and post-transcriptional levels. To clarify the mechanism of E2-induced post-transcriptional regulation, with attention to the involvement of the oestrogen receptor (ER), we studied the effect of tamoxifen (TAM), a synthetic E2 antagonist that inhibits ER-mediated transcription, on E2-induced transcriptional and post-transcriptional regulation of the chicken ovalbumin (OVA) gene in chick oviducts. Run-on analysis with oviduct nuclei isolated from E2-treated chicks showed that TAM treatment completely blocked E2-induced transcription of the OVA gene within 24 h without affecting ER gene expression. Likewise, the rate of transcription fell to below the limit of detection after E2 withdrawal from the chicks. Reflecting the transcription rate, OVA mRNA accumulated linearly in E2-treated chicks, and E2 withdrawal caused a rapid loss of OVA mRNA. However, in the chicks treated with TAM and E2, OVA mRNA was degraded slowly over 48 h with a half-life of 24 h, suggesting that TAM does not inhibit E2-induced mRNA stabilization. Moreover, E2-induced mRNA stabilization was observed even when transcription of the OVA gene was blocked by a transcription inhibitor. Western-blot analysis showed that the remaining OVA mRNA was translatable. Thus the present study indicates that E2 regulates expression of the OVA gene via distinct pathways at transcriptional and post-transcriptional levels.