RETINOID RECEPTOR CONTROL IN CYTODIFFERENTIATION

拟议研究的一个初步目标是阐明 类维生素A结合蛋白（RBP）的完整基因组序列， 细胞视黄醇结合蛋白 （cRBP）、细胞视黄酸结合蛋白（cRABP）和 膜相关视黄酸结合蛋白（mRABP）。 RBP将 纯化至均一并对其氨基末端测序。 氨基 这些蛋白质的氨基酸序列和来自一个 已知的，高度同源的蛋白质将是合成 几种寡核苷酸。 这些寡核苷酸将用作主要的 探针筛选完整的小鼠cDNA文库;衍生的cDNA将 用于鉴定cRBP、cRABP和mRABP的完整基因组克隆。 逆转录病毒将被构建成含有正常或反向的 （反义）基因组序列（从或接近氨基末端 延伸3 prime以包括解析的RBP的任何主要内含子 基因. 这些逆转录病毒将被感染到细胞系中， （i）其RBP cRABP+、cRBP或cRABP+/cRBP+的表达，和 (ii)它们对相对低浓度的类维生素A的反应能力 视黄酸+或视黄醇+。 细胞特性的变化（例如， 增殖、转化、终末分化）， 和不存在（i）另外表达的RBP基因组序列或 反义信息;（ii）类维生素A，和（iii）肿瘤促进剂， 还将评价12-O-十四烷酰基佛波醇-13-乙酸酯。 这些 实验将（i）提供完整的氨基酸以及基因组， cRBP、cRABP和MRABP的序列;（ii）确定RBP的要求 在细胞分化的控制，（iii）允许RBP的表征 二级结构;及（iv）为预期发展提供数据 抗类维生素A和抗RBP化合物，这可能是显着的价值， 化学预防

An initial objective of the proposed research is the elucidation of the complete genomic sequences of the retinoid-binding proteins (RBPs), cellular retinol-binding protein (cRBP), cellular retinol-binding protein (cRBP), cellular retinoic acid-binding protein (cRABP) and membrane-associated retinoic acid-binding protein (mRABP). RBPs will be purified to homogeneity and their amino-terminal ends sequenced. The amino acid sequences of these proteins and a codon frequency derived from a known, highly homologous protein will be the basis for the synthesis of several oligonucleotides. These oligonucleotides will be used as primary probes in screening complete mouse cDNA libraries; the derived cDNAs will be used to identify the complete genomic clone of cRBP, cRABP, and mRABP. Retroviruses will be constructed that contain either a normal or inverted (anti-sense) genomic sequence (from or near the amino terminal end extending 3 prime to include any primary intron) of the resolved RBP genes. These retroviruses will be infected into cell lines that have been selected for (i) their expression of RBPs cRABP+, cRBP or cRABP+/cRBP+ and (ii) their ability to respond to relatively low concentrations of retinoids retinoic acid+, or retinol+. Changes in cell characteristics (e.g., proliferation, transformation, terminal differentiation) in the presence and absence of (i) an additionally expressed RBP genomic sequence or anti-sense message; (ii) retinoids, and (iii) the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, will also be evaluated. These experiments will (i) provide the complete amono acid as well as genomic sequence for cRBP, cRABP and MRABP; (ii) establish the requirement for RBPs in the control of cytodifferentiation, (iii) allow characterization of RBP secondary structures; and (iv) provide data for the prospective development of anti-retinoid and anti-RBP compounds that may be of significant value in chemoprevention.