The dual lives of bidirectional promoters

The sequencing of the human genome led to many insights into gene organization and structure. One interesting observation was the high frequency of bidirectional promoters characterized by two protein encoding genes whose promoters are arranged in a divergent or "head-to-head" configuration with less than 2000 base pairs of intervening sequence. Computational estimates published by various groups indicate that nearly 10% of the coding gene promoters are arranged in such a manner and the extent of this bias is a unique feature of mammalian genomes. Moreover, as a class, head-to-head promoters appear to be enriched in specific categories of gene function. Here we review the structure, composition, genomic properties and functional classifications of genes controlled by bidirectional promoters and explore the biological implication of these features. This article is part of a Special Issue entitled: Chromatin in time and space.     

Improved baculovirus vectors expressing barnase using promoters from Cotesia plutellae bracovirus

The goal of this study was to create a novel baculovirus expression system that does not require recombinant virus purification steps. Transfection of insect cells with transfer vectors containing barnase under control of the Cotesia plutellae bracovirus (CpBV) promoters ORF3004 or ORF3005 reduced cell growth. Co-transfection with bApGOZA DNA yielded no recombinant viruses and non-recombinant backgrounds. To further investigate the detrimental effects of barnase on insect cells, two recombinant bacmids harboring the barnase gene under control of the CpBV promoters, namely bAcFast-3004ProBarnase and bAcFast-3005ProBarnase, were constructed. While no viral replication was observed when only the recombinant bacmids were transfected, recombinant viruses were generated when the bacmids were co-transfected with the transfer vector, pAcUWPolh, through substitution of the barnase gene with the native polyhedrin gene by homologous recombination. Moreover, no non-recombinant backgrounds were detected from unpurified recombinant stocks using PCR analysis. These results indicate that CpBV promoters can be used to improve baculovirus expression vectors by means of lethal gene expression under the control of these promoters.     

Modulating ectopic gene expression levels by using retroviral vectors equipped with synthetic promoters

The human cytomegalovirus and elongation factor 1?? promoters are constitutive promoters commonly employed by mammalian expression vectors. These promoters generally produce high levels of expression in many types of cells and tissues. To generate a library of synthetic promoters capable of generating a range of low, intermediate, and high expression levels, the TATA and CAAT box elements of these promoters were mutated. Other promoter variants were also generated by random mutagenesis. Evaluation using plasmid vectors integrated at a single site in the genome revealed that these various synthetic promoters were capable of expression levels spanning a 40-fold range. Retroviral vectors were equipped with the synthetic promoters and evaluated for their ability to reproduce the graded expression demonstrated by plasmid integration. A vector with a self-inactivating long terminal repeat could neither reproduce the full range of expression levels nor produce stable expression. Using a second vector design, the different synthetic promoters enabled stable expression over a broad range of expression levels in different cell lines.