Cloning and structural organization of the gene encoding the murine nuclear receptor transcription factor, NURR1
Introduction
We previously reported the isolation of a murine orphan receptor NURR1 (also termed RNR-1 and NOT) that is expressed predominantly in the main regions of the central nervous system (Law et al., 1992; Scearce et al., 1993; Mages et al., 1994; Saucedo-Cardenas and Conneely, 1996). NURR1 is a member of the nuclear receptor superfamily. This family comprises a group of structurally related transcription factors that program developmental, physiological and behavioral responses to a variety of chemical signals. The family includes receptors for steroids, certain vitamins and thyroid hormone (Evans, 1988; Beato, 1989; Tsai and O'Malley, 1994) in addition to a growing number of orphan members whose physiological function and cognate ligand, if any, remain to be established (O'Malley and Conneely, 1992).
NURR1 cDNA encodes a 66-kilodalton protein and exhibits close structural relationship to previously characterized orphan nuclear receptors, NGFI-B/NUR77/N10/NAK-1 (Milbrandt, 1988; Hazel et al., 1988; Ryseck et al., 1989; Nakai et al., 1990) and NOR-1/MINOR/TEC (Ohkura et al., 1994; Hedvat and Irving, 1995; Labelle et al., 1995). These receptors are part of a subclass of nuclear receptors that are immediate early gene products and function as constitutively active transcription factors that do not appear to require binding of a ligand for activity (Chalepakis et al., 1988; Davis et al., 1991; Wilson et al., 1991). In addition, these three transcription factors are able to bind to the same cis-acting enhancer DNA sequence to regulate target gene expression (Chalepakis et al., 1988; Ohkura et al., 1994; Hedvat and Irving, 1995; Wilson et al., 1993a, Wilson et al., 1993b; Murphy et al., 1995). These observations suggest that these transcription factors may regulate overlapping gene networks.
To understand the regulation of the NURR1 gene and its relationship to other members of the nuclear receptor superfamily, we have determined its structural organization. The exon-intron arrangement is similar to that of the NGF1-B/NUR77 nuclear receptor supporting the conclusion that this subclass of gene products have a close evolutionary relationship within the nuclear receptor superfamily. Analysis of the proximal promoter region of NURR1 reveals at least one potential cis-acting sequence that may participate in signal induced transcription of this immediate early gene.
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Isolation of the NURR1 gene
Two mouse 129/SvEv genomic libraries prepared in λDash II (Stratagene) and P1 (Genome Systems Inc) were used to isolate the NURR1 gene. Screening of the λDash II library was performed (Grunstein and Hogness, 1975) using as probe a DraII-PstI (680-bp) fragment from the N-terminal region of NURR1 cDNA (Law et al., 1992). The probe was radiolabeled using [32P] dCTP to a specific activity of 2×109 cpm/μg. Using this approach, one positive clone was identified from the λDASH II genomic library. Two
Acknowledgements
The authors would like to thank Irene Harrison for her excellent secretarial assistance in typing the manuscript. O. S.-C. is a recipient of a fellowship from the National Council of Science and Technology (CONACYT) of Mexico.
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