Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) for Cellular Retinoid-Binding Proteins
Cellular retinoid-binding proteins were first identified in the mid-1970s (1). Subsequent studies established the existence of two forms of cellular retinoic acid-binding proteins (CRABPI and II) (2, 3) and two forms of cellular retinol-binding proteins (CRBP I and II) (4), each encoded by a separate gene. These cellular retinoid-binding proteins are members of a family of related lipid-binding proteins in which the ligand is enclosed within a deep hydrophobic pocket formed by a 10-stranded β-barrel polypeptide structure (5, 6). CRBPs and CRABPs are expressed in a tissue-specific manner (7, 8, 9, 10), with CRBP II most limited in expression, being confined mainly to the small intestine (4, 11). Although the ligand-binding properties of these proteins are well-known, largely as a result of biochemical studies on protein purified from tissues or expressed in Escherichia coli (see Chapters 8– 10), their functions and interactions with other cellular proteins are still uncertain. Recombinant cellular retinoid binding proteins, expressed in E. coli or in eukaryotic cells, are important tools for understanding their role in the cellular uptake of retinol, the sequestration and metabolic degradation of retinoic acid or the transfer of retinoids to nuclear receptors. cDNA clones for the complete coding regions of the various binding proteins can be prepared by reverse transcriptase-polymerase chain reaction (RT-PCR), using available sequence information to design the forward and reverse primers. In addition, the regulation of expression of these genes may be studied by quantitative RT-PCR using primers optimized for specificity, as an alternative to conventional Northern blotting. This chapter is based on our experience in clonmg human CRABP II and CRBP II via RT-PCR, but similar conditions will be applicable to CRBP I and CRABP I. An alternative to clomng these genes via RT-PCR is to search the Expressed Sequence Tag (EST) database with the cellular retinoid binding protein sequence of interest via the Internet. EST clones from the I.M.A.G.E. Consortium (LLNL) with exact matches can be obtained from GenomeSystems (St Louis, MO), and sequenced to verify their identity and length. This may also prove to be a useful “armchair cloning” strategy to identify new members of this gene family.
KeywordsTermination Codon Complete Code Region Guanidine Thiocyanate cDNA Synthesis Reaction Expected Product Size
- 1.Chytil, F., and Ong, D. E (1984) Cellular retinoid binding proteins, in The Retinoids, vol 2 (Sporn, M B., Roberts, A B., and Goodman, D. S., eds), Academic, Orlando, FL, pp. 89–123.Google Scholar
- 5.Cowan, S. W., Newcomer, M. E., and Jones, T. A. (1993) Crystallographic studies on a family of cellular lipophilic transport proteins-refinement of P2 myelin protein and the structure determination and refinement of cellular retinol-binding protein in complex with all-trans-retinol J Mol Biol 230, 1225–1246PubMedCrossRefGoogle Scholar
- 13.Astrom, A, Tavakkol, A, Pettersson, U, Cromie, M, Elder, J. T., and Voorhees, J J (1991) Molecular-cloning of 2 human cellular retinoic acid-binding proteins (CRABP)—retinoic acid-induced expression of CRABP-II but not CRABP-I in adult human skin in vivo and in skin fibroblasts in vitro. J Biol Chem 266, 17,662–17,666PubMedGoogle Scholar
- 19.Wilkinson, M. (1988) RNA isolation-a mini-prep method. Nucl Acids Res 16, 10, 933.Google Scholar