A universal approach to gene expression engineering.

In this study, we provide a universal approach to Gene Expression Engineering (GeneEE) for creating artificial expression systems. GeneEE leads to the generation of artificial 5' regulatory sequences (ARES) consisting of promoters and 5' untranslated regions. The ARES lead to the successful recruitment of RNA polymerase, related sigma factors and ribosomal proteins that result in a wide range of expression levels. We also demonstrate that by engaging native transcription regulators, GeneEE can be used to generate inducible promoters. To showcase the universality of the approach, we demonstrate that 200-nucleotide (nt)-long DNA with random composition can be used to generate functional expression systems in six bacterial species, Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Thermus thermophilus, Streptomyces albus and Streptomyces lividans, and the eukaryote yeast Saccharomyces cerevisiae.

Overexpression of RAD51 Enables PCR-Based Gene Targeting in Lager Yeast.

Lager beer fermentations rely on specific polyploid hybrids between Saccharomyces cerevisiae and Saccharomyces eubayanus falling into the two groups of S. carlsbergensis/Saaz-type and S. pastorianus/Frohberg-type. These strains provide a terroir to lager beer as they have long traditional associations and local selection histories with specific breweries. Lager yeasts share, based on their common origin, several phenotypes. One of them is low transformability, hampering the gene function analyses required for proof-of-concept strain improvements. PCR-based gene targeting is a standard tool for manipulating S. cerevisiae and other ascomycetes. However, low transformability paired with the low efficiency of homologous recombination practically disable targeted gene function analyses in lager yeast strains. For genetic manipulations in lager yeasts, we employed a yeast transformation protocol based on lithium-acetate/PEG incubation combined with electroporation. We first introduced freely replicating CEN/ARS plasmids carrying ScRAD51 driven by a strong heterologous promoter into lager yeast. RAD51 overexpression in the Weihenstephan 34/70 lager yeast was necessary and sufficient in our hands for gene targeting using short-flanking homology regions of 50 bp added to a selection marker by PCR. We successfully targeted two independent loci, ScADE2/YOR128C and ScHSP104/YLL026W, and confirmed correct integration by diagnostic PCR. With these modifications, genetic alterations of lager yeasts can be achieved efficiently and the RAD51-containing episomal plasmid can be removed after successful strain construction.

Implication of human herpesviruses in oncogenesis through immune evasion and supression.

All human herpesviruses (HHVs) have been implicated in immune system evasion and suppression. Moreover, two HHV family members, i.e. EBV and KSHV, are recognised as oncogenic viruses. Our literature review summarises additional examples of possible oncogenic mechanisms that have been attributed to other HHVs. In general, HHVs affect almost every cancer-implicated branch of the immune system, namely tumour-promoting inflammation, immune evasion, and immunosuppression. Some HHVs accomplish these effects by inhibiting apoptotic pathways and by promoting proliferation. Mechanisms related to immunosupression and low grade chronic inflammation could eventually result in the initiation and progression of cancer. In this article we open a discussion on the members of Herpesviridae, their immune evasion and suppression mechanisms, and their possible role in cancer development. We conclude that discerning the mechanisms of interplay between HHV, immune system, and cancer is essential for the development of novel preventative and therapeutic approaches for cancer treatment and prophylaxis.