The ubiquitous nature of the progesterone receptor binding factor‐1 (RBF‐1) in avian tissues

Abstract

The avian oviduct receptor binding factor-1 (RBF-1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF-1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF-1. The heart and pancreas had the highest and lowest RBF-1 levels, respectively; the concentration ranging by approximately 50-fold in these tissues. The 10 kDa size of the RBF-1 detected in all tissues suggests no significant tissue-specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF-1 have identical amino-terminal sequence. Using a recently isolated cDNA to RBF-1, the levels of RBF-1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue-specific differences in concentration. The presence of RBF-1 in non-progesterone responsive tissues suggests the possibility that RBF-1 may not be specifically involved in PR-DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor.

A DNA-binding Element for a Steroid Receptor-binding Factor Is Flanked by Dual Nuclear Matrix DNA Attachment Sites in the c-myc Gene Promoter

“…Using immunohistochemical techniques with antibodies against the purified RBF, the RBF was localized to the nuclei of selected cell types in certain avian and rat tissues (27,28). In similar studies, colocalizations of RBF and PR in the same cell types in the avian oviduct and rat reproductive organs were also shown.…”

“…The full-length cDNA to RBF has been cloned and used to identify a 0.7-kilobase mRNA in specific avian tissues. The mRNA levels in these same tissues correlate with the RBF protein levels by Western blot analyses (28). A homologous RBF-like mRNA species was identified in human tissues.…”

“…Bands encompassing RBF were excised, and the protein was eluted from the membrane in 70% (v/v) 2-propanol, 5% trifluoroacetic acid (v/v) overnight; dialyzed against 5% (v/v) acetonitrile/H 2 O, 0.25 mM PMSF, and lyophilized. This protocol was tested and reported in detail (22,28).…”

“…Chromosomal proteins were size-fractionated by molecular sieve chromatography as described previously (46). Fractions containing proteins in the 4 -20-kDa size range were pooled and separated by preparative SDS-polyacrylamide gel electrophoresis in a Tris-Tricine buffer as described previously (28). The gel was transferred to six sheets of polyvinylidene difluoride membrane using a CAPS buffer system (10 mM CAPS ϩ 10% methanol, pH 11.0).…”

“…The cDNA for RBF was obtained using polymerase chain reaction procedures, as described previously (28). Briefly, poly (A) ϩ mRNA from chicken oviduct was reverse transcribed using an oligo(dT) primer to obtain the first strand cDNA.…”