A cell committed to the adipocytic pathway begins a multi-step process that proceeds through an unknown number of steps to recognizable adipogenesis and ends with the cell becoming a terminally differentiated, fat-filled adipocyte. We have cloned a 1200bp genomic DNA sequence (clone A) that has the capacity to commit 3T3-C2 cells, precrisis mouse skin fibroblasts and 10T1/1 mouse cells to enter adipogenesis upon later stimulation by serum, insulin and confluence, following kinetics that mimic the response of the 3T3-derived preadipocyte cell line 3T3-F442A to confluence and serum. Clone A is functional in the absence of an exogenous promoter and any other exogenous regulatory sequence. We plant to subject Clone A to rigorous molecular analysis. Our specific aims are as follows: ?1! We will determine the minimal size of the DNA with AC activity by cloning subfragments of Clone A and testing these for AC activity. ?2! We will complete the sequencing of Clone A by obtaining complete sequences from the second strand. From sequencing of the first strand we have found that at the level of 95% certainty the 1200 bp Clone A is not present in Genbank. ?3! Preliminary sequence data show an open reading frame flanked by a TATA box and a termination signal, and containing one pair of consensus splice junctions. If the complete sequence of Clone A contains a true open reading frame with proper consensus start signals, we will create a stop-codon mutation in Clone A and determine whether or not it blocks the Clone A's activity in mouse skin fibroblasts. ?4! At this point we cannot exclude the possibility that Clone A does not encode a protein. While the 1200 bp fragment is a complete gene in the sense that it has the capacity to confer a novel phenotype upon transfection, Northern blots with 1200 bp sequence as probe do not reveal an AC-specific messenger RNA in cultured preadipocytes. We will use an assay of greater sensitivity (RNA-PCR) to search for transcription products of AC DNA in transfected cells, embryonic preadipocytes and in cultured preadipocytes at different times after induction of adipogenesis. ?5! In the unlikely event that the final sequence lacks a coding sequence, or if mutants tested under Aim 3 prove equally active to unmutated Clone A DNA, we will undertake to determine whether Clone A's activity is the result of a specifically encoded and novel RNA, or whether Clone A serves directly as a DNA within the transfected cell, perhaps by acting as a binding site for a negative-regulatory protein. We will determine whether specific proteins from committed fibroblasts complex with clone A DNA and protein, by gel mobility shift assay. We will use mutated Clone-A DNA in the gel shift assay to gain a more precise map of DNA A-binding response elements within Clone A, and we will use standard chromatography techniques to purify protein?s! specifically binding to Clone A response elements. The adipocytic pathway has not been fully characterized at either the physiological or the molecular level. This work will initiate such a study at the molecular level.***//
