Abstract
Orphan nuclear receptors have been identified using a variety of methods over the years. The first were identified by low-stringency hybridization using known receptors as probes. This strategy has been successful because members of the steroid receptor superfamily contain a conserved DNA-binding domain and share regions of similarity in the ligand-binding domain. These conserved regions may also be used to design polymerase chain reaction primers that have been used to identify new receptors, primarily members of known families. The recent explosive increase in DNA sequences from EST and genomic sequencing projects has also allowed the identification of new family members. The Caenorhabditis elegans genome has recently been sequenced and shown to contain a large variety of putative nuclear receptor genes, some of which may be represented in mammalian genomes. The question remains of how to identify potentially highly divergent mammalian homologs. One possibility is to wait until such sequences appear in the rapidly growing sequence databases from rodent and human genome projects. This method has been used to identify a novel member of the steroid receptor superfamily (1–3) and may ultimately result in the identification of others. For those who do not wish to wait, or who work on model organisms whose genome projects are not well advanced (e.g., Xenopus), there is no substitute for directly isolating the relevant cDNAs.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kliewer, S. A., Moore, J. T., Wade, L., Staudinger, J. L., Jones, M. A., McKee, D. D., et al. (1998) Orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92, 73–82.
Lehmann, J. M., McKee, D. D., Watson, M. A., Willson, T. M., Moore, J. T., and Kliewer, S. A. (1998) Human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J. Clin. Invest. 102, 1016–1023.
Bertilsson, G., Heidrich, J., Svensson, K., Asman, M., Jendeberg, L., Sydow-Backman, M., et al. (1998) Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc. Natl. Acad. Sci. USA 95, 12,208–12,213.
Ausubel, F. M. (1987) Current Protocols in Molecular Biology. Greene and Wiley-Interscience. J. Wiley, New York.
Melchior, W. B. J. and Von Hippel, P. H. (1973) Alteration of the relative stability of dA:dT and dG:dC base pairs in DNA. Proc. Natl. Acad. Sci. USA 70, 298–302.
Wood, W. I., Gitschier, J., Lasky, L. A., and Lawn, R. M. (1985) Base composition-independent hybridization in tetramethylammonium chloride: a method for oligonucleotide screening of highly complex gene libraries. Proc. Natl. Acad. Sci. USA 82, 1585–1588.
Blumberg, B., Mangelsdorf, D. J., Dyck, J., Bittner, D. A., Evans, R. M., and De Robertis, E. M. (1992) Multiple retinoid-responsive receptors in a single cell: families of RXRs and RARs in the Xenopus egg. Proc. Natl. Acad. Sci. USA 89, 2321–2325.
Greene, M. E., Blumberg, B., McBride, O. W., Yi, H. F., Kronquist, K., Kwan, K., et al. (1995) Isolation of the human peroxisome proliferator activated receptor g cDNA: expression in hematopoietic cells and chromosomal mapping. Gene Exp. 4, 281–299.
Kliewer, S. A., Forman, B. M., Blumberg, B., Ong, E. S., Borgmeyer, U., Mangelsdorf, D. J., Umesono, K., and Evans, R. M. (1994) Differential expression and activation of a family of murine peroxisome proliferator-activated receptors. Proc. Natl. Acad. Sci. USA 91, 7355–7359.
Blumberg, B., Wright, C. V. E., De Robertis, E. M., and Cho, K. W. Y. (1991) Organizer-specific homeobox genes in Xenopus laevis embryos. Science 253, 194–196.
Bürglin, T. R., Finney, M., Coulson, A., and Ruvkun, G. (1989) Caenorhabditis elegans has scores of homeobox-containing genes. Nature 341, 239–243.
Gont, L. K., Steinbeisser, H., Blumberg, B., and de Robertis, E. M. (1993) Tail formation as a continuation of gastrulation: the multiple cell populations of the Xenopus tailbud derive from the late blastopore lip. Development 119, 991–1004.
Gont, L. K., Fainsod, A., Kim, S. H., and De Robertis, E. M. (1996) Overexpression of the homeobox gene Xnot-2 leads to notochord formation in Xenopus. Dev. Biol. 174, 174–178.
Hawley, S. H., Wunnenberg-Stapleton, K., Hashimoto, C., Laurent, M. N., Watabe, T., Blumberg, B., and Cho, K. W. Y. (1995) Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct nerual induction. Genes Dev. 9, 2923–2935.
Gabriel, W., Blumberg, B., Sutton, S., Place, A., and Lance, V. (2001) Aromatase expression in alligator embryos at male and female incubation temperatures, in press.
Umesono, K. and Evans, R. M. (1989) Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell 57, 1139–1146.
Bürglin, T. R. (1988) Yeast regulatory gene PHO2 encodes a homeo box. Cell 53, 339–340.
Meinkoth, J. and Wahl, G. (1984) Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267–284.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2001 Humana Press Inc.
About this protocol
Cite this protocol
Blumberg, B. (2001). Identification of Nuclear Hormone Receptor Homologs by Screening Libraries with Highly Degenerate Oligonucleotide Probes. In: Lieberman, B.A. (eds) Steroid Receptor Methods. Methods in Molecular Biology™, vol 176. Humana Press. https://doi.org/10.1385/1-59259-115-9:119
Download citation
DOI: https://doi.org/10.1385/1-59259-115-9:119
Publisher Name: Humana Press
Print ISBN: 978-0-89603-754-0
Online ISBN: 978-1-59259-115-2
eBook Packages: Springer Protocols