In contrast to skeletal muscle, the mechanisms responsible for activation and maintenance of tissue-specific transcription in cardiac muscle remain poorly understood. A family of hormone-encoding genes is expressed in a highly specific manner in cardiac but not skeletal myocytes. This includes the A-and B-type natriuretic peptide (ANP and BNP) genes, which encode peptide hormones with crucial roles in the regulation of blood volume and pressure. Since these genes are markers of cardiac cells, we have used them to probe the mechanisms for cardiac muscle-specific transcription. Cloning and functional analysis of the rat BNP upstream sequences revealed unexpected structural resemblance to erythroid but not to muscle-specific promoters and enhancers, including a requirement for regulatory elements containing GATA motifs. A cDNA clone corresponding to a member of the GATA family of transcription factors was isolated from a cardiomyocyte cDNA library. Transcription of this GATA gene is restricted mostly to the heart and is undetectable in skeletal muscle. Within the heart, GATA transcripts are localized in ANP-and BNP-expressing myocytes, and forced expression of the GATA protein in heterologous cells markedly activates transcription from the natural cardiac muscle-specific ANP and BNP promoters. This GATA-dependent pathway defines the first mechanism for cardiac muscle-specific transcription. Moreover, the present findings reveal striking similarities between the mechanisms controlling gene expression in hematopoietic and cardiac cells and may have important implications for studies of cardiogenesis.The discovery of the MyoD family of myogenic factors has resulted in great advances in the understanding of the mechanisms of skeletal muscle commitment and differentiation (reviewed in references 48 and 68). In contrast, the mechanisms controlling cardiac determination and differentiation remain essentially unknown (49). So far, no MyoD-like factors have been detected in cardiac muscle (54), and mice homozygous for inactivated MyoD (53), Myf-5 (13), or myogenin (27) loci have a normal cardiac phenotype. These observations suggest that tissue-specific transcription and cell differentiation are controlled by distinct regulatory pathways in the two striated muscles. This would be consistent with the fact that skeletal and cardiac myocytes have distinct spatial and temporal origins in the developing embryo, although they both arise from mesoderm.Skeletal muscle cells originate from the somites of the dorsal (paraxial) mesoderm, whereas cardiac muscle cells are derived from the splanchnic mesenchyme of the anterior lateral plate mesoderm (10, 32). Commitment of mesodermal cells to the cardiac lineage occurs very early, when cells migrate to form the cardiogenic area at the beginning of the third week (days 16 to 18) of human embryonic development (or at 18 to 20 h in chicken embryogenesis [35]). By the end of the third week (days 21 to 22), the tubular-or primitive-heart is formed and joined by blood vessels. Thus, the heart ...

Previously, we identified a class of genes in Dictyostelium that are prespore cell-type specific in their expression in the multicellular aggregate and are inducible by cAMP acting through cell-surface cAMP receptors. In this paper, we report the cloning and analysis of the regulatory regions controlling the expression of one such gene that encodes a spore coat protein, SP60. By use of a fusion of the firefly luciferase gene and Escherichia coli lacZ [expresses [3-galactosidase (~-gal)], we have identified cis-acting regions required for proper spatial and temporal expression in multicellular aggregates and for cAMP induction in shaking cell culture. Deletion analysis suggests that a CA-rich element (CAE) and surrounding sequences present three times within the 5'-flanking sequence are required for proper regulation. SP60-IacZ fusions that include all three of these regions express lacZ only in the posterior -85% of migrating slugs (prespore zone). Studies show that SP60 is expressed during mid to late aggregation, and SP60-lacZ-positive cells are spatially localized as a doughnut-shaped ring within the forming aggregate. Cells within the skirt that surrounds the aggregate and that are still migrating into the aggregate do not stain. Sequential 5' deletions of CAEs and surrounding regions affect the expression level of SP60-luciferase in response to developmental signals and cAMP, as well as the spatial pattern of SP60-IacZ. Deletion of the first (most 5') of these regions restricts the spatial expression of SP60-IacZ fusions to the anterior of the prespore zone. When both the first and second regions are removed, the expression level drops, and the staining is restricted to the prespore/prestalk boundary. Furthermore, the staining pattern that is seen with these two deletions is present as a gradient from anterior to posterior within the prespore zone. Deletion of all three regions results in a loss of both cAMP and developmentally induced expression. These results suggest the presence of a gradient within the prespore zone that differentially affects the activity of promoters containing different numbers of response elements.

The mercuric ion-resistance operons of plasmid R100 (originay from Shigella) and transposon TnSOI (originally from a plamid isolated in Pseudomonas) have been compared by DNA sequence analysis. The sequences for the first 1340 base pairs of TnSO1 are given with the best alignment with the comparable 1319 base pairs of R100. The homology between the two sequences starts at base 58 after the end of the insertion sequence IS-1 of R100. The sequences include the transcriptional regulatory region, and the homology is particularly strong in regions just upstream from potential transcriptional initiation sites. The trans-acting regulatory gene merR consists of 180 base pairs in both cases and codes for a highly basic polypeptide of 60 amino acids, which is also rich in serine. The TnSOI and R100 merR genes differ in 25 of the 180 base positions, and the resulting polypeptides differ in seven amino acids. The regulatory region before the major transcription initiation site contains potential -35 and -10 sequences and dyad symmetrical sequences, which may be the merR binding sites for transcriptional regulation. The first structural gene, merT, encodes a highly hydrophobic polypeptide of 116 amino acids. The R10 and Tn5OI merT genes differ in 17% of their positions, leading to 14 (12%) amino acid changes. This region had previously been shown to encode a protein governing membrane transport of mercuric ions. The second structural gene, merC, would give a 91 amino acid polypeptide with a hydrophobic amino-terminal segment. The Tn501 and R100 nerC genes differ at 37 base positions, leading to 10 amino acid changes.Mercuric ion resistance, like many other resistances to toxic heavy metals in prokaryotes, is governed by genes on plasmids and transposons (1-3). Plasmid R100 is the initial antibiotic-resistance plasmid that appeared in 1956 in a Shigella flexneri strain in Japan (4, 5), and its total size is 90 kilobase pairs (kb). It encodes resistance to chloramphenicol, fusidic acid, tetracycline, sulfonamide, and streptomycin, as well as to mercuric ions (6). The last three resistances are encoded on the 19.9-kb transposon Tn2l (7,8) that occurs between flanking 768-base-pair insertion elements IS-la and IS-lb (7, 9), which are not part of the transposon (7). Transposon TnSOJ is an 8.5-kb transposable element that encodes resistance to mercuric ions in addition to transposition functions (10). It was first found in a Pseudomonas aeruginosa strain (11), and it was moved into Escherichia coli for subsequent study (10,12). The organization of the mercuric ion resistance determinant of plasmid R100 is well known. Transposon mutagenesis studies (13)(14)(15) and cloning studies (16,17) determined the orientation of the operon and that the mercuric ion-resistance determinant starts close to the right boundary of IS-1. The operon was initially (13, 16) shown to consist of a regulatory gene (merR) that encodes a trans-acting positive/negative regulatory product, followed by an operator-promoter region, and then by a large t...

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The rat bone Gla protein (BGP, osteocalcin) gene was isolated from a rat genomic library and sequenced. BGP is a 5.8-kD noncollagenous protein secreted by calcified tissues whose expression is regulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The BGP gene consists of 4 exons separated by 3 introns and spans almost 1 kb of DNA. The 5'-flanking region of the gene contains CCAAT and TATAAA elements common to eukaryotic genes. An analysis of approximately 600 bp of 5'-flanking sequence of this gene revealed sequences homologous to regulatory elements for glucocorticoids, metal ions, and cAMP. The latter is especially significant since recent evidence suggests that the rat BGP gene can be regulated by cAMP. This region of the gene also contains numerous pairs of inverted repeat sequences (imperfect palindromes). The sequence of the rat BGP gene was compared to that of the recently published human BGP gene (Celeste et al., EMBO J. 5, 1885, 1986). The coding regions of these two genes share 77% sequence identity, and several regions of the 5'-flanking sequences are also well conserved. Knowledge of the sequence of the rat BGP gene will allow studies of its regulation by 1,25(OH)2D3, cAMP, and other trans-acting transcriptional factors, and identification of the regulatory sequence elements involved.