Nucleotide sequence analysis of the long terminal repeat of integrated bovine leukemia provirus DNA and of adjacent viral and host sequences.

The nucleotide sequence of the 3' long terminal repeat and adjacent viral and host sequences was determined for a bovine leukemia provirus cloned from a bovine tumor. The long terminal repeat was found to comprise 535 nucleotides and to harbor at both ends an imperfect inverted repeat of 7 bases. Promoter-like sequences (Hogness box and CAT box), an mRNA capping site, and a core enhancer-related sequence were tentatively located. No kinship was detected between this bovine leukemia proviral fragment and other retroviral long terminal repeats, including that of human T-cell leukemia virus.     

Increased xylanase production in Streptomyces lividans after replacement of the signal peptide: dependence on box and inverted repeat sequence

The signal peptide of the xylanase A gene of Streptomyces lividans was replaced by the signal sequence of the cellulase A preceded by a 57 nucleotides (nt) upstream sequence. This latter contains a 5 nt inverted repeat (5'-TGGGAACGCTCCCA). The 3'-end of the inverted repeat contains a 5 nt box (TCCCA), which is complementary to the 16S rRNA of S. lividans. The effects on the production of xylanase resulting from deletions in the inverted repeat and from variations in the length of the box are shown. Removal of the inverted repeat and box decreased the xylanase production by 75%. Increasing the complementarity of the box with the 16S rRNA to 17 nt decreased the production by 90%. A reduction in the length of the inverted repeat, and consequently in the box, from 5 to 4 nt decreased the production by 40%. Preserving the 4 nt inverted repeat but lengthening the box from 5 to 6 nt increased the production by 1.5-fold. Finally, removing the inverted repeat but introducing an 8 nt box increased the xylanase production by 1.9-fold which then averaged 2.3 g/l of xylanase. The most efficient boxes contained 6-8 nt and were located between 14 and 19 nt downstream from the first initiation codon.     

A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter

Summary The majority of the activation potential of the Saccharomyces cerevisiae TDH3 gene promoter is contained within nucleotides –676 to –381 (relative to the translation initiation codon). An upstream activation sequence (UAS) in this region has been characterized by in vitro and in vivo assays and demonstrated to be composed of two small, adjacent DNA sequence elements. The essential determinant of this upstream UAS is a general regulatory factor 1 (GRF1) binding site at nucleotides –513 to –501. A synthetic DNA element comprising this sequence, or an analogue in which two of the degenerate nucleotides of the GRF1 site consensus sequence were altered, activated 5 deleted TDH3 and CYC1 promoters. The second DNA element of the UAS is a 7 by sequence which is conserved in the promoters of several yeast genes encoding glycolytic enzymes and occurs at positions –486 to –480 of the TDH3 promoter. This DNA sequence represents a novel promoter element: it contains no UAS activity itself, yet potentiates the activity of a GRF1 UAS. The potentiation of the GRFl UAS by this element occurs when placed upstream from the TATA box of either the TDH3 or CYC1 promoters. The characteristics of this element (termed GPE for GRF1 site potentiator element) indicate that it represents a binding site for a different yeast protein which increases the promoter activation mediated by the GRF1 protein. Site-specific deletion and promoter reconstruction experiments suggest that the entire activation potential of the –676 to –381 region of the TDH3 gene promoter may be accounted for by a combination of the GRF1 site and the GPE.     

Purification and characterization of the DNA-binding protein DnrI, a transcriptional factor of daunorubicin biosynthesis in Streptomyces peucetius

The DnrI protein, essential for the biosynthesis of daunorubicin in Streptomyces peucetius , was purified almost to homogeneity from dnrI expression strains of Escherichia coli and S. peucetius through several steps of chromatography. The proteins purified from both organisms had identical chromatographic and electrophoretic behaviour. Purified His-tagged or native DnrI was used to conduct DNA-binding assays by gel mobility-shift analysis, and the results showed no significant difference in the DNA-binding activity of native or His-tagged proteins. DnrI binds specifically to DNA segments containing the intergenic regions separating the putative dnrG–dpsABCD and dpsEF operons, and the dnrC gene and dnrDKPSQ operon. DNase I footprinting assays indicated that the DNA-binding sites for DnrI extended from upstream of the −10 to −35 regions of the dnrG or dpsE promoters to include about 65 bp of the dnrG – dpsE intergenic region and about 80 bp of the dnrC – dnrD intergenic region. Both binding sites contain imperfect inverted repeat sequences of 6–10 bp with a 5'-TCGAG-3' consensus sequence that was present in 4 out of 10 other promoter regions in the cluster of daunorubicin biosynthesis genes.     

A 45 bp inverted repeat is required for cell cycle regulation of the Escherichia coli nrd operon

Expression of β-galactosidase from a nrd–lacZ fusion was used to determine the role in nrd regulation of an inverted sequence upstream of the promoter. Removal or replacement of a 45 bp inverted repeat with an altered sequence including a 48 bp perfect inverted repeat resulted in a mutant phenotype that was low in nrd expression in an exponentially growing culture and that did not increase during DNA synthesis inhibition. Changing the 22 bp in the upstream half of the inverted repeat resulted in the same phenotype, whereas changing the 22 bp in the downstream half of the inverted repeat decreased nrd expression to a lesser extent in an exponentially growing culture and had only a smaller effect on nrd expression during DNA synthesis inhibition. As other mutants with the phenotype of the upstream inverted repeat mutant were found to lack cell cycle regulation, expression of nrd – lac mRNA produced from a plasmid with this mutation in the nrd–lacZ fusion gene was compared with nrd mRNA produced from the chromosomal nrd gene in a synchronized culture. The results indicated that the upstream half of the nrd inverted repeat contains a cis -acting element essential for nrd cell cycle regulation.