A Rapid Gel-Shift Technique to Analyse DNA-Protein Interactions using Phastsystem™

Ramanujam, P.; Fogerty, Scott; Heiser, William C.; Jolly, James

doi:10.1007/978-3-0348-7561-5_16

Cited by 1 publication

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References 11 publications

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“…For gel retardation assays, a 32P-labeled oligonucleotide consisting of a 15-base random oligonucleotide flanked by defined terminal ends of 15 bases each was incubated with 0.3 ,ug of glutathione S-transferase fusion protein at room temperature for 20 min (14). The retarded DNA was excised, eluted, and extracted with phenol/chloroform.…”

Section: Methodsmentioning

confidence: 99%

Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity.

Kimura

1993

Proc. Natl. Acad. Sci. U.S.A.

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Schwannoma-derived growth factor (SDGF) is a mitogen and neurotrophic protein which belongs to the epidermal growth factor (EGF) family. There are two basic amino acid clusters in the SDGF molecule which are homologous to the nuclear targeting signal of the simian virus 40-encoded large tumor antigen. Mutational analysis of these clusters showed that they function as nuclear targeting signals, and a gel retardation assay showed that SDGF binds to A+T-rich DNA After binding and receptor activation, most receptor-growth factor complexes are thought to be internalized, transported to lysosomes, and degraded (1,2). Recently, however, examples have emerged where growth factors exert their effect directly in the nucleus. Exogenously applied platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) accumulate in the nucleus of target cells (3,4), and their nuclear targeting signals have been identified (5-7). bFGF regulates gene transcription in a cell-free system (8). These findings indicate that growth factors may function within the nucleus. Schwannoma-derived growth factor (SDGF) was initially isolated from a schwannoma cell line as a mitogen for astrocytes and fibroblasts (9) and is neurotrophic (10). The following data show that the transport of SDGF into the nucleus is required for it to induce its mitogenic activity. MATERIALS AND METHODSMutant genes were constructed by PCR. Each mutant has 27 bases encoding a 9-amino acid epitope (YPYDVPDYA) recognized by mouse monoclonal antibody 12CA5 (11). Transfections were done as described (9), and for staining, cells were fixed with 3% formaldehyde and treated with 0.5% Triton X-100. After 1 hr of incubation with antibody 12CA5, cells were incubated with fluorescently labeled goat antimouse antibody.Cell fractionation (12), Western blot analysis (13), and [3H]thymidine incorporation (9) were done as described. For gel retardation assays, a 32P-labeled oligonucleotide consisting of a 15-base random oligonucleotide flanked by defined terminal ends of 15 bases each was incubated with 0.3 ,ug of glutathione S-transferase fusion protein at room temperature for 20 min (14). The retarded DNA was excised, eluted, and extracted with phenol/chloroform. PCR amplification was carried out and the resultant fragments were cloned and sequenced (15). Competition binding assays were performed as described (16).To assay c-fos and nerve growth factor I-A (NGFI-A) induction, Swiss 3T3 cells were serum starved for 2 days, and RNA was extracted (17) 30 min after the addition of SDGF and subjected to Northern blot analysis. RESULTS AND DISCUSSIONSince SDGF has two basic amino acid clusters which are homologous to the nuclear targeting signal ofthe simian virus 40 (SV40)-encoded large tumor antigen (18)(19)(20), the roles of the secretory signal sequence and the nuclear localization sequences of SDGF were determined by mutational analysis. These experiments were carried out by transfecting COS cells (SV40-transformed monkey cells) with an expression vector conta...

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Section: Methodsmentioning

confidence: 99%

Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity.

Kimura

1993

Proc. Natl. Acad. Sci. U.S.A.

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show abstract

A Rapid Gel-Shift Technique to Analyse DNA-Protein Interactions using Phastsystem™

Cited by 1 publication

References 11 publications

Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity.

Schwannoma-derived growth factor must be transported into the nucleus to exert its mitogenic activity.

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