Abstract
The recently cloned fli-1 gene is a member of the ets oncogene family that is preferentially expressed in hematopoietic cells. It is a target of dysregulation by Friend leukemia virus insertion and translocation in Ewing's sarcoma and neuroepithelioma. In this report, we have studied the function and regulation of both murine and human fli-1. Analysis of the human and mouse fli-1 proteins showed that fli-1 binds to specific DNA sequences highly related to m-ets-2 binding sites. Methylation protection experiments showed that fli-1 and m-ets-2 contacted the same nucleotides in two different binding sites. The fli-1 protein was shown to be a transcriptional activator in co-transfection studies. Stimulation of murine bone marrow macrophages by mediators of inflammation, such as lipopolysaccharide, phorbol 12-myristate 13-acetate, interleukin-1, and interferon-gamma resulted in the reduced expression of fli-1 mRNA. fli-1 was only expressed in a defined subset of human erythroleukemia cell lines.
Highlights
The recently cloned fli-1 gene is a memberof the ets oncogene family that is preferentiallyexpressed in hematopoietic cells
Initial work on PU.1 and ets-1 demonstrated that ets family members were sequence-specific DNA binding proteins, that they bound to similar sequences, and that DNA binding by these two proteins required the region of amino acid identity [6, 13]
The fact that these two proteins bound to similar sequences with a unique protein structure defined a new DNA binding motif termed the ETS-domain [14].The PU.l protein was shown by co-transfection studies to be a transcriptional activator [6]
Summary
Gel Electrophoresis DNA Binding Assay-Bandshift analysis was performed, as described [6]. fli-1, PU.l, and m-ets-2 proteins were produced by in vitro transcription/translation, as described [6].Tmncations of these genes were created by polymerase chain reaction. Fli-1, PU.l, and m-ets-2 proteins were produced by in vitro transcription/translation, as described [6].Tmncations of these genes were created by polymerase chain reaction. Gel Electrophoresis DNA Binding Assay-Bandshift analysis was performed, as described [6]. Fragments of these genes containing the ETS-domain were subcloned into Bluescript KS’ and sequenced prior to production of protein. In oitro-translated human fli-1 protein bound to the ets-2 consensus pi-1 Transfection Assays-The coding region of the mouse fli-1 DNA bindingsequence (E.18),but did not bindto the PU.l consensus cDNA [17]was subcloned into the EcoRIIKpnI sitoef the expression DNA bindingsequence (P.23).The ets-2 consensussequence is shown vector PJ6 [21].
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