Characterization and expression of the pepN gene encoding a general aminopeptidase from Lactobacillus helveticus

Abstract The putative ribosome binding sites preceding 32 of Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H genes were compared. A highly conserved consensus sequence for the ribosome binding sites of LL-H genes was inferred, GAAAGGAG. This study included the characterization of the last nucleotides of the 3'-end of the 16S rRNA molecule from L. delbrueckii subsp. lactis and its comparison to the ribosome binding site consensus sequence.     

Assembly and characterization of canine heart endothelial nitric oxide synthase cDNA and 5'-flanking sequence by homology (RT-)PCR cloning.

A broad spectrum of cardiovascular diseases is studied in canine animal models, in which dysfunction or dysregulation of the endothelial nitric oxide synthase (ecNOS) is of pivotal pathogenetic importance. To provide the tools for subsequent molecular analyses of ecNOS structure or function and to identify putative regulatory factors we isolated and characterized the canine heart ecNOS cDNA and putative regulatory (promoter) sequences. The complete coding sequence, 5'- plus part of 3'-untranslated regions (UTR) of ecNOS cDNA, and part of the 5'-flanking sequence (putative promoter region) were identified by homology (RT-)PCR cloning using canine heart total RNA or genomic DNA. Primer sequences were derived from bovine/human ecNOS cDNAs or genes. An ecNOS sequence contig of 5138 nucleotides length was established containing an open reading frame of 3618 nucleotides (1206 amino acids predicting a 133-kDa protein) and 253 bp 3'-UTR (distal to TGA codon)/1267 bp proximal to ATG codon (containing 5'-UTR and 5'-flanking sequences = putative promoter region). Comparison to human, bovine, murine, or porcine ecNOS sequences at the nucleotide or amino acid level yielded between 86 and 91% or 83 and 84% homologies, respectively. The canine ecNOS 5'-flanking sequence (putative promoter region) revealed stretches of homology up to 86% as compared to the human sequence containing a cluster of binding sites for several regulatory elements. The homology (RT-)PCR cloning strategy is presented as an alternative to common library cloning approaches. The obtained canine ecNOS sequence might serve to further analyze the structure, regulated function (promoter region consensus sites), and expression of ecNOS in different pathophysiological conditions and in other species (GenBank Accession No. BankIt264069 AF143503).     

Distinct DNA elements contribute to Rap1p affinity for its binding sites

The essential Saccharomyces cerevisiae regulatory protein Rap1 contains two tandem Myb-like DNA binding sub-domains that interact with two defined DNA "hemisites", separated by a trinucleotide linker sequence. We have mapped the thermodynamically defined DNA-binding site of Rap1 by a primer extension method coupled with electrophoretic separation of bound and unbound DNAs. Relative to published consensus sequences, we detect binding interactions that extend 3 bp beyond the 5'-end of the putative DNA-binding site. This new site of interaction is located where the DNA minor groove faces the protein, and may account for the major DNA bending induced by Rap1p that previous studies have mapped to a site immediately upstream of the consensus binding site. In addition, we show that a minimal DNA-binding site made of one single consensus hemisite, preceded or followed by a spacer trinucleotide that interacts with the unstructured protein linker between the two Rap1p DNA binding domains, is able to bind the protein, although at lower affinity. These findings may explain the observed in vivo binding properties of Rap1p at many promoters that lack canonical binding sites.