Differential expression of genes encoding alpha, beta and gamma retinoic acid receptors and CRABP in the developing limbs of the mouse.

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.

Mouse cellular retinoic acid binding protein: cloning, complementary DNA sequence, and messenger RNA expression during the retinoic acid-induced differentiation of F9 wild type and RA-3-10 mutant teratocarcinoma cells.

Retinoic acid, a natural derivative of vitamin A (retinol), induces mouse F9 teratocarcinoma stem cells to differentiate into nontumorigenic parietal endoderm cells. The mouse cellular retinoic acid binding protein (CRABP) has been implicated in the mechanism of action of retinoic acid (RA), since a mutant F9 cell line, RA-3-10, which possesses less than 5% of the wild type level of [3H]RA:CRABP binding activity, fails to differentiate in response to RA. In order to study the CRABP in this RA-induced differentiation process, we have cloned and sequenced the full-length mouse CRABP complementary DNA and have characterized its expression in wild type F9 and mutant cells. The mouse CRABP mRNA is a single, low abundant mRNA approximately 800 bases in length. The steady state level of the CRABP mRNA was measured in untreated stem cells and after the addition of RA alone, dibutyryl cyclic AMP plus theophylline (CT), or retinoic acid, dibutyryl cyclic AMP and theophylline (RACT) to F9 wild type and the mutant RA-3-10 cells. The CRABP mRNA was present in wild type F9 stem cells, and the level of its expression was changed by RA. When RA was added to F9 wild type cells, the steady state level of CRABP mRNA decreased 2- to 3-fold. When RACT was added to wild type cells, the level of CRABP mRNA increased and then decreased, resulting in a peak of CRABP mRNA expression between 24 and 48 h. In contrast, untreated mutant RA-3-10 cells had a lower level of CRABP mRNA than wild type stem cells, and the mutant cells responded quite differently to the addition of RA and RACT. The addition of RA caused an impressive 60-fold increase in the steady state level of CRABP mRNA in RA-3-10 cells by 120 h. One interpretation of this result is that there is negative regulation of CRABP mRNA expression, mediated directly or indirectly by the wild type functional CRABP protein, and that this regulation is aberrant in the RA-3-10 cells.

Transcription start site and induction kinetics of the araC regulatory gene in Escherichia coli K-12.

The in vivo transcription start site of the araC message was determined by S1 nuclease mapping of hybrids formed between labeled DNA, and RNA extracted from cells grown under a variety of physiological conditions, including the interval of transient derepression following arabinose addition. Under all conditions tested, transcription initiated from the same nucleotide position at -148.

The Escherichia coli L-arabinose operon: binding sites of the regulatory proteins and a mechanism of positive and negative regulation.

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 araC protein--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 stimultaneously 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.