Retinoic acid has profound effects on vertebrate limb morphogenesis (refs 1-6, reviewed in refs 7-9), including in the mouse, where it can act as a teratogen generating phocomelia and bone defects. A retinoic acid gradient, possibly amplified by a graded distribution of cellular retinoic acid-binding protein (CRABP), could provide positional information across the antero-posterior axis of the chick limb bud. The discovery of nuclear retinoic acid receptors (RARs) acting as retinoic acid-inducible enhancer factors provided a basis for understanding how retinoic acid signals could be transduced at the level of gene expression. We have now used in situ hybridization to study the distribution of messenger RNA transcripts of the three murine receptors (mRARs) and CRABP during mouse limb development. Both mRAR alpha and mRAR gamma transcripts, but not those for mRAR beta, are present and uniformly distributed in the limb bud at day 10 post-coitum, whereas CRABP transcripts have a graded proximo-distal distribution, indicating that differential expression of CRABP, but not of mRAR alpha or mRAR gamma, could participate in the establishment of the morphogenetic field. At later stages, mRAR gamma transcripts become specific to the cartilage cell lineage and to the differentiating skin and mRAR beta transcripts are mostly restricted to the interdigital mesenchyme. CRABP transcripts, however, are excluded from regions expressing mRAR gamma and mRAR beta. These results indicate that all three RARs and CRABP have specific functions during morphogenesis and differentiation of the mouse limb.

The locations of DNA binding by the proteins involved with positive and negative regulation of transcription initiation of the L-arabinose operon in Escherichia coli have been determined by the DNase I protection method. Two cyclic AMP receptor protein sites were found, at positions -78 to -107 and -121 to -146, an araCprotein-arabinose binding site was found at position -40 to -78, and an araC protein-fucose binding site was found at position -106 to -144. These locations, combined with in vivo data on induction of the two divergently oriented arabinose promoters, suggest the following regulatory mechanism: induction of the araBAD operon occurs when cyclic AMP receptor protein, araC protein, and RNA polymerase are all present and able to bind to DNA. Negative regulation is accomplished by the repressing form of araC protein binding to a site in the regulatory region such that it simultaneously blocks access of cyclic AMP receptor protein to two sites on the DNA, one site of which serves each of the two promoters. Thus, from a single operator site, the negative regulator represses the two outwardly oriented ara promoters. This regulatory mechanism explains the known positive and negative regulatory properties of the ara promoters.Studies on the L-arabinose operon of Escherichia coli have established the following important facts on regulation of the divergently oriented ara promoters PC and PBAD (see Fig. 1).The activity of both promoters is stimulated by the cyclic AMP (cAMP) receptor protein (CRP) in the presence of cyclic AMP (1-4). The promoter PBAD is positively regulated by araC protein in the presence of arabinose-i.e., the protein is required for activity of the promoter (1, 2, 5). Under noninducing conditions, the araC protein instead acts negatively to repress both the PC and PBAD promoters (1-3, 6). At least part of the DNA site necessary for repression of PBAD lies upstream from all of the sites necessary for induction of PBAD, as shown by the existence of deletions entering the ara regulatory region from the Pc side that abolish repression of PBAD without affecting induction of PBAD (6, 7).The requisite components are now available for in vitro studies of the mechanism of regulation of the ara operon. The regulatory region DNA has been isolated, and its nucleotide sequence has been determined (8,9) and found to contain elements similar to the RNA polymerase-binding sites seen in other E. coli promoters at about 10 and 35 bases before the start sites of transcription (8). The sequence also contains several stretches that resemble the CRP-binding site in the gal operon (10). Also available are the proteins involved in the regulation of the ara operon: araC protein (11), CRP (12), and RNA polymerase (13).In the work reported here, we have utilized the DNA se- MATERIALS AND METHODS DNA fragments for sequence determination and protection were obtained from plasmid pMB9ara440 (8) by EcoRI endonuclease digestion and polyethylene glycol precipitation (16). CRP was purified as described (12), and ara...

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