Artefactual gene induction during preparation of Xenopus laevis animal cap explants.
The animal cap assay in Xenopus laevis was used to study the induction and regulation of the mesoderm-specific gene Xegr-1, a homolog of the mammalian egr-1 genes. Egr-1 is an immediate-early gene whose growth factor-stimulated transcriptional induction displays a transient activity profile and occurs independent of protein synthesis. The Xegr-1 promoter contains multiple serum response elements (SREs). In this paper we show that Xegr-1 is induced unspecifically during the process of animal cap preparation. Transcripts of Xegr-1 appear already 30 min after cutting of animal caps. Xfos, another SRE-regulated immediate-early gene, is induced with the same kinetics as Xegr-1. In contrast, the Xbra gene is not induced under the experimental conditions used. Xfos and Xegr-1 transcripts are not rapidly down-regulated after mechanical stimulation, but can be detected for up to 4 h later. Wounding-dependent Xegr-1 induction is reduced by injection of either mRNA coding for the dominant inhibitory forms of both the FGF receptor and the transcription factor Elk-1. Xegr-1 expression can be reinduced by mesoderm-inducing factors. These results led us to develop a new protocol for animal cap preparation, which circumvents the observed undesired artefactual gene activation events.
Krain
B
B
Institut für Molekularbiologie, Medizinische Hochschule Hannover, Germany.
Nordheim
A
A
D000818:Animals; D016477:Artifacts; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D004210:Dissection / Q000009:adverse effects*; D051766:Early Growth Response Protein 1; D004625:Embryo, Nonmammalian; D017874:Immediate-Early Proteins / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D020218:Response Elements; D020825:T-Box Domain Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis; D050807:ets-Domain Protein Elk-1
D004268:DNA-Binding Proteins; C486111:EGR1 protein, Xenopus; D051766:Early Growth Response Protein 1; D017874:Immediate-Early Proteins; D011518:Proto-Oncogene Proteins; D020825:T-Box Domain Proteins; C116765:TBXT protein, Xenopus; D014157:Transcription Factors; D029867:Xenopus Proteins; D050807:ets-Domain Protein Elk-1