A systematic mutagenesis of the SV40 enhancer indicates that it spans -100 bp and is composed of at least two distinct DNA domains which exhibit very little enhancing activity on their own. Their association results in a 400-fold enhancement of transcription, virtually irrespective of their relative orientation and, to some extent, of the distance between them. Enhancer activity can also be generated by duplication of either domain. We show also that the activity of each domain is due to the presence of several specific sequence motifs. These motifs are found assorted in different combinations in other viral and cellular enhancers. Key words: transcription/site-directed mutagenesis/promoter/ RNA polymerase B(ll)/simian virus 40 1983;Lusky et al., 1983;Hearing and Shenk, 1983;Veldman et al., 1985). However, such enhancer consensus sequences occur in DNA segments without enhancer properties, and enhancer activity can be generated by duplicating DNA sequences without enhancer activity on their own (Weber et al., 1984;Swimmer and Shenk, 1984), thus questioning the real functional significance of these consensus sequences.Therefore, we decided to determine, at the nucleotide level, the DNA sequences essential for the activity of the prototype SV40 enhancer. Systematic deletions and point mutations have been constructed throughout the enhancer region and their effect on SV40 early transcription investigated in vivo, using a transient expression assay in HeLa cells. We show here that the SV40 enhancer encompasses a large DNA segment of -100 nucleotides containing the 72-bp sequence, but also extending further upstream. Furthermore, the present study reveals that the enhancer is composed of at least two distinct DNA domains which exhibit very little enhancing activity on their own. However, their association results in a dramatic 400-fold enhancement of transcription, virtually irrespective of their relative orientation and, to some extent, of the distance between them. Enhancer activity can also be generated by duplication of either domain. In addition, we show that the activity of each domain is due to the presence of several specific sequence motifs. Various assortments of these motifs are found in other viral and cellular enhancers, suggesting that enhancers are mosaics of a limited number of basic evolutionary related sequence motifs.

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A 1536-nucleotide-long sequence that carries the ampC fi-lactamase gene of the Escherichia coli K-12 chromosome has been determined. This gene codes for a protein of 377 amino acids, of which the first 19 amino acids form a signal peptide. The molecular weight of the mature enzyme was determined to be 39,600. The ampC P-lactamase with a substrate specificity for cephalosporins showed no significant sequence homologies with (-lactamases of the penicillinase type or with D-alanine carboxypeptidases. However, because the region around serine-80 of the ampC f3-lactamase has extensive homology with an active-site fragment ofthe Pseudomonas aeruginosa cephalosporinase, we suggest that the ampC cephalosporinase as well as related cephalosporinases form a distinct group of serine P-lactamases that have an evolutionary origin different from that of the serine penicillinases and thus constitute a new class of 13-lactamases. f3Lactamases of chromosomal or plasmid origin have been found in a large number of Gram-positive and Gram-negative bacteria (1). These enzymes have been classified according to such properties as substrate profile, isoelectric point, and molecular weight. The protein sequence has been determined for ,-lactamases from Staphylococcus aureus PC1 (2), Bacillus ltcheniformis 749/C (3, 4), and Escherichia coli/R6K, R-TEM (5).Nearly the entire sequence ofthe B. cereus 569/H P-lactamase has also been elucidated (6, 7). In addition, the amino acid sequence of the ,B-lactamase encoded by the plasmid pBR322 has been deduced from its nucleotide sequence (8). The two plasmid-mediated TEM P-lactamases from Gram-negative species differ only by one amino acid residue (5, 8).These 3-lactamases of known sequence all show substrate specificity for penicillins and have therefore been termed "penicillinases" (1). The molecular weight ofthese enzymes is around 29,000 (7). They show significant sequence homologies with each other (7) as well as with regions of D-alanine carboxypeptidases from B. stearothermophilus and B. subtilis (9). By the use of substrate analogues, the active site has been determined for three penicillinases and two carboxypeptidases (9-13). They have been referred to as "serine enzymes" because the reagents react with a serine residue. ,3-Lactamases ofthe metalloenzyme type have also been identified. From incomplete sequence data it is suggested that this class has a different evolutionary origin from that of the serine penicillinases of known sequence (7).The chromosomally encoded ,B-lactamases of Gram-negative enterobacteria in general are basic proteins with a substrate specificity for cephalosporins (14). One such cephalosporinase is encoded by the ampC gene of E. coli K-12. This gene, which is located at 93.8 min on the E. coli linkage map (15), was isolated from a gene bank containing E. coli chromosomal DNA (16,17). By subcloning, ampC was localized to a 1370-base-pair (bp)-long DNA fragment (17, 18). We have reported (19) a characterization of the regulatory region that precedes ampC. T...

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The ubiquitous Ca(2+)-binding protein calmodulin (CaM) is a key protein in Ca2+ homeostasis and activation of eukaryotic cells. CaM is the molecular link between free Ca2+ in the cell and the inhibition, or activation, of numerous enzymes. Many nuclear functions are under Ca2+/CaM control, and some transcriptional activators are known to be Ca2+ modulated indirectly through Ca2+/CaM-dependent protein kinases. But Ca2+/CaM has not yet been found to directly modulate any transcription factor or other DNA-binding protein. Transcription factors of the basic-helix-loop-helix (bHLH) group are important regulators in numerous systems. Here we report that binding of Ca(2+)-loaded CaM to the bHLH domains of several bHLH proteins directly inhibits their DNA binding. Other bHLH proteins are either less sensitive or resistant. Ca2+ ionophore selectively inhibits transcriptional activation by Ca2+/CaM-sensitive bHLH proteins in vivo, implying that Ca2+ can directly influence transcription through differential CaM inhibition of bHLH domains.