Nucleotide sequence of DNA controlling expression of genes for maltosaccharide utilization in Streptococcus pneumoniae

An analysis of previous data indicated that four structural genes concerned with maltosaccharide utilization in Streptococcus pneumoniae are organized in two operons that are transcribed in opposite directions from a central control region. This region contains two strong promoters subject to repression by a regulatory gene product in the absence of maltose. The nucleotide sequence of the 554-bp control region DNA and adjacent portions of the malX and malM structural genes was determined. Unique reading frames and initiation codons allowed identification of the oppositely oriented structural genes. Putative ribosome binding sites and −10 and −35 RNA-polymerase-binding sites, as well as AT-rich regions farther upstream, were observed proximal to both the X and M genes. The similarity of these sequences to sites found in Escherichia coli and Bacillus subtilis indicated the conservation of control signals in bacteria, both Gram-negative and Gram-positive. A pair of 17-bp hyphenated repeat sequences in the control region may represent repressor binding sites. Two down promoter mutations, V11 and 69, were shown to be deletions in the control region. The V11 mutation, which affected only the MP operon, deleted the promoter adjacent to the M gene. Mutation 69, which reduced both X and M gene functions, deleted the entire segment between the promoters so that they now overlap at their −35 binding sites. As a consequence of this deletion, the AT-rich regions proximal to the promoters were lost. This suggests that the AT-rich regions are important for promoter strength.     

Convergent evolution of crystallin gene regulation in squid and chicken: The AP-1/ARE connection

Previous experiments have shown that the minimal promoters required for function of the squid SL20-1 and SL11 crystallin genes in transfected rabbit lens epithelial cells contain an overlapping AP-1/antioxidant responsive element (ARE) upstream of the TATA box. This region resembles the PL-1 and PL-2 elements of the chicken B 1-cry stallin promoter which are essential for promoter function in transfected primary chicken lens epithelial cells. Here we demonstrate by site-directed mutagenesis that the AP-1/ARE sequence is essential for activity of the squid SL20-1 and SL11 promoters in transfected embryonic chicken lens cells and fibroblasts. Promoter activity was higher in transfected lens cells than in fibroblasts. Electrophoretic mobility shift and DNase protection experiments demonstrated the formation of numerous complexes between nuclear proteins of the embryonic chicken lens and the AP-1/ARE sequences of the squid SL20-1 and SL11 crystallin promoters. One of these complexes comigrated and cross-competed with that formed with the PL-1 element of the chicken B1-crystallin promoter. This complex formed with nuclear extracts from the lens, heart, brain, and skeletal muscle of embryonic chickens and was eliminated by competition with a consensus AP-1 sequence. The nonfunctional mutant AP-1/ ARE sequences did not compete for complex formation. These data raise the intriguing possibility that entirely different, nonhomologous crystallin genes of the chicken and squid have convergently evolved a similar cis-acting regulatory element (AP-1/ARE) for high expression in the lens. Correspondence to: S. I. Tomarev     

Cloning and identification of the promoter of the tobacco Sar8.2b gene,a gene involved in systemic acquired resistance

Expression of the Sar8.2 gene family is induced by salicylic acid (SA) in tobacco during induction of systemic acquired resistance. Expression of Sar8.2b, one member of this 12-member family, was detected as early as 12 h after treatment with SA and was maximal 36 h after SA treatment. In NahG transgenic tobacco plants, benzothiadiazole and dichloroisonicotinic acid induced expression of Sar8.2b but SA did not, suggesting that expression of the Sar8.2b gene is SA-dependent. Several putative cis-acting elements were found in the Sar8.2b gene promoter region, including an as-1 element and GT-1 and Dof binding sequences. We constructed a series of progressive deletion mutations in the Sar8.2b promoter region linked to the β-glucuronidase (GUS) coding region and analyzed GUS activities by stable expression in transformants of Arabidopsis thaliana. Deletions between −728 and −927 bp or between −351 and −197 bp of the promoter region resulted in a significant reduction in GUS activity induced by SA treatment as shown in stable transformants of A. thaliana. The −197 bp fragment of the promoter region was found to confer a relatively low level of GUS activity induced by SA treatment in stable expression of transformants in A. thaliana. The results suggest that 927 bp of the Sar8.2b gene promoter confers full promoter activity and that cis-acting elements required for high-level SA-inducible expression of the Sar8.2b gene may exist within the regions −728 to −927 bp and −197 to −351 bp.     

Spatial regulation of SpMTA metallothionein gene expression in sea urchin embryos by a regulatory cassette in intron 1

The SpMTA metallothionein (MT) gene of the sea urchin Strongylocentrotus purpuratus is restricted in its expression to the aboral ectoderm in gastrulae and pluteus larvae. The proximal 1.6 kb of the 5′-flanking region together with the 1.12-kb first intron of the SpMTA gene are sufficient for its correct cell-type specific expression in transgenic embryos. This restricted spatial expression is largely eliminated by deletion of an interior 405-bp region in the intron. Within this region is a 295-bp, genomically repetitive, transposon-like segment (Nemer et al., 1993), containing several sequence motifs highly homologous to posited regulatory elements in the promoters of other genes (Thiebaud et al., 1990). The P3A and P5 sites in this apparent regulatory cassette were shown through competition to bind with relatively high affinities the same nuclear factors, bound by their counterpart sites in the CyIIIa actin promoter.