Molecular cloning of human er1 cDNA and its differential expression in breast tumours and tumour-derived cell lines
Introduction
Mesoderm differentiation in Xenopus can be induced by a number of factors, including members of the fibroblast growth factor (FGF) family. Many of the early events in mesoderm induction have been investigated and shown to involve a number of well-characterized intracellular signal transduction pathways, including PLCγ and Ras [reviewed in Friesel and Maciag (1995)]. Little is known, however, about the genes that are the ultimate targets of these signal transduction pathways: the immediate–early genes. In a recent report, we described the cloning and characterization of one such gene, er1, from Xenopus embryos (Paterno et al., 1997). er1 is an immediate–early gene whose transcription is activated by FGF during mesoderm induction in Xenopus. ER1 protein was shown to be localized exclusively to the nucleus and to contain domains with potent transcriptional transactivation activity. The observation that er1 is an immediate–early transcription factor activated by FGF suggests that it may play a role in FGF-regulated cellular activities, including those associated with the pathology of human cancers.
In this report, we have isolated and characterized the cDNA for the human homologue of er1 and have investigated its expression pattern in normal and neoplastic tissue.
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cDNA cloning and sequencing of human er1
Using forward (F) and reverse (R) primers 5′-TCCGTTACACCAGGATGTAG-3′ (F) and 5′-GGCTGAAATTCCAGTTGGTA-3′ (R), designed according to the Xenopus er1 sequence, a 440-bp product was amplified from a human testis cDNA library (Clontech, Palo Alto, CA), as described previously (Paterno et al., 1997). The cDNA was cloned into pCR2.1 (Invitrogen) (Paterno et al., 1997), and the sequence for both strands of this er1 cDNA and all subsequent cDNA inserts was determined as in Gillespie et al. (1995). A
cDNA cloning of human er1
We recently described the cloning and characterization of er1, a novel immediate–early gene from Xenopus laevis whose expression levels were increased by FGF (Paterno et al., 1997). Using primers based on the Xenopus sequence, we amplified a similar sequence from a human testis cDNA library. The full-length human cDNA consisted of a single open reading frame (ORF) of 1296 bp bracketed by a 68-bp 5′ UTR containing an in-frame stop codon and a 210-bp 3′ UTR containing an 18-bp poly-A tail (Fig. 1
Conclusions
This report presents the cloning and expression analysis of the human homologue of er1. Comparison of the Xenopus and human ER1 proteins reveals a high degree of conservation between lower and higher vertebrates.
Human er1 mRNA expression was negligible in all normal tissues and breast cell lines examined, but was upregulated in breast carcinoma cell lines and in breast tumours, suggesting that expression of er1 is associated with the neoplastic state in human breast carcinomas.
Acknowledgements
The authors thank Paula Ryan and Wendy Winsor for technical assistance. This work was supported by a grant from the Cancer Research Society to L.L.G. and G.D.P.
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