Cloning of a novel orphan receptor (GCNF) expressed during germ cell development.

Abstract

We have cloned a novel member of the nuclear receptor superfamily that has been identified from complementary DNA libraries derived from mouse tissues using a low stringency cross hybridization strategy. The deduced protein sequence contains 495 amino acids and consists of the characteristic DNA-binding and ligand-binding domains of the nuclear receptor superfamily. The primary sequence of this new orphan is distinct from those of previously cloned members and subgroups. Analysis of the DNA-binding properties of the in vitro synthesized protein revealed that this new orphan receptor binds to the sequence TCAAGGTCA that includes the steroidogenic factor-1 half-site and direct repeat with 0 bp spacing elements. Northern blot and ribonuclease protection assays showed that the receptor was predominantly expressed in the testis. Results from in situ hybridization experiments confirmed this observation and showed it to be located in the spermatogenic cells. High level expression was also detected in developing oocytes in the ovary. Thus, high level expression of this gene is restricted to developing germ cells, the oocytes and spermatogenic cells. We speculate that this orphan receptor may be a molecule involved in regulating some aspect of meiosis, and that the major function of this factor is likely to be involved in the regulation of gene expression in germ cell development during gametogenesis. It has been designated germ cell nuclear factor.

The Embryonic Function of Germ Cell Nuclear Factor Is Dependent on the DNA Binding Domain

“…It has been shown that GCNF is not only expressed in mouse and Xenopus embryos after the onset of gastrulation (14,(21)(22)(23)(24) but also in the gametogenic cells of adult vertebrates (3,4,11,18,(25)(26)(27). This expression pattern indicates that GCNF may play a role in gametogenesis and normal embryonic development during gastrulation.…”

“…GCNF was initially cloned by our laboratory using low stringency screening with a DNA binding domain (DBD) probe (3) and subsequently cloned by other laboratories and given other names, e.g. RTR (retinoid receptor-related testis-specific receptor (4)) and NCNF (neuronal cell nuclear factor (5)).…”

“…This high amino acid identity within the DBD and LBD across species indicates the functional conservation of GCNF protein in binding to its DNAresponse element and its putative ligand during evolution. Indeed, the DNA binding specificity of GCNF is conserved among mouse, human and Xenopus (3,(12)(13)(14)(15).…”

“…GCNF can specifically bind to either a DR0 element, a direct repeat of the estrogen receptor half-site (AGGTCA) with zero base pair spacing between the half-sites, as a homodimer (3,12,13,(15)(16)(17), or an extended DR0 half-site (TCAAGGTCA) either as a monomer (12), a homodimer (13,17), or both a monomer and homodimer (17). Recently, two dimerization motifs in mouse GCNF, one located in the DBD including the adjacent TA box and the other in ␣-helix 3 of the LBD, have been characterized (17).…”

Get effective polyclonal antisera in one month

“…As shown in Fig 2, the molecular weight of the three different antigens spanned about ten folds i.e. the 55KD GCNF/RTR (germ cell nuclear factor/retinoid receptor-related testis-associated receptor) [8], [9] and a 38 KD newly discovered gene product Bin2a fragment expressed in E. Coli and the 5.9 KD ILP(insulin like peptide) [10] fragment expressed in yeast. The modified immunization method was paralleled with the tradi Fig 2. The SDS-PAGE pattern of the three purified proteins expressed from E.coli or yeast 1 # .…”