Exploiting Nucleotide Composition to Engineer Promoters

The choice of promoter is a critical step in optimizing the efficiency and stability of recombinant protein production in mammalian cell lines. Artificial promoters that provide stable expression across cell lines and can be designed to the desired strength constitute an alternative to the use of viral promoters. Here, we show how the nucleotide characteristics of highly active human promoters can be modelled via the genome-wide frequency distribution of short motifs: by overlapping motifs that occur infrequently in the genome, we constructed contiguous sequence that is rich in GC and CpGs, both features of known promoters, but lacking homology to real promoters. We show that snippets from this sequence, at 100 base pairs or longer, drive gene expression in vitro in a number of mammalian cells, and are thus candidates for use in protein production. We further show that expression is driven by the general transcription factors TFIIB and TFIID, both being ubiquitously present across cell types, which results in less tissue- and species-specific regulation compared to the viral promoter SV40. We lastly found that the strength of a promoter can be tuned up and down by modulating the counts of GC and CpGs in localized regions. These results constitute a “proof-of-concept” for custom-designing promoters that are suitable for biotechnological and medical applications

Genomic Signature of Shifts in Selection in a Subalpine Ant and Its Physiological Adaptations

Understanding how organisms adapt to extreme environments is fundamental and can provide insightful case studies for both evolutionary biology and climate-change biology. Here, we take advantage of the vast diversity of lifestyles in ants to identify genomic signatures of adaptation to extreme habitats such as high altitude. We hypothesized two parallel patterns would occur in a genome adapting to an extreme habitat: 1) strong positive selection on genes related to adaptation and 2) a relaxation of previous purifying selection. We tested this hypothesis by sequencing the high-elevation specialist Tetramorium alpestre and four other phylogenetically related species. In support of our hypothesis, we recorded a strong shift of selective forces in T. alpestre, in particular a stronger magnitude of diversifying and relaxed selection when compared with all other ants. We further disentangled candidate molecular adaptations in both gene expression and protein-coding sequence that were identified by our genome-wide analyses. In particular, we demonstrate that T. alpestre has 1) a higher level of expression for stv and other heat-shock proteins in chill-shock tests and 2) enzymatic enhancement of Hex-T1, a rate-limiting regulatory enzyme that controls the entry of glucose into the glycolytic pathway. Together, our analyses highlight the adaptive molecular changes that support colonization of high-altitude environments

Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Sequences of the snapback and reverse primers.

<p>The snapback tail of 16 bases is shown as underlined; 2 nucleotides mismatch at the 5′end are indicated as lowercase; and mismatch nucleotide as underlined bold (A).</p

Nucleotides sequence alignment of the RPO30 gene of CaPVs highlighting the snapback tail binding site.

<p>The RPO30 gene sequences of 7 CaPVs representing GTPVs, LSDVs and SPPVs were aligned. A sequence of sixteen nucleotides complementary to the snapback tail in GTPV (100% match), as well as the corresponding positions in SPPV and LSDV are shown in the box. Note the targeted single nucleotide mismatches inside the box: T:A between GTPV and SPPV, and T:G between GTPV and LSDV. Conserved nucleotides are shown as dots.</p

Snapback primer genotyping of CaPVs.

<p>The fluorescence melting curve analysis of the PCR products shows two melting peaks for each of the CaPV three genotypes (GTPV, SPPV and LSDV) corresponding to the snapback stem melting peak at lower temperature and the full-length PCR amplicon melting peak at higher temperature (see arrows).</p

Secondary structure of the expected GTPV PCR amplicon.

<p>The Snapback hairpin with a 18-nucleotide stem and a loop of 55 bases is shown. The predictions were done on the Mfold web server <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075971#pone.0075971-Zuker1" target="_blank">[28]</a> using the default parameters of the DNA folding form except for the temperature which was set at 45°C and the salt concentration set at 50 mM.</p

High-Resolution melting curve analysis of CaPVs using the Precision Melt Analysis™ software (BioRad).

<p>A: the normalized melt curve of the full-length amplicon; B: the difference curve of full-length amplicon; C: the normalized melt curve of snapback stem; D: The difference curve of the snapback stem. The species are indicated by the arrows: G = GTPV, S = SPPV and L = LSDV.</p

Calculation of the binding constants <i>k</i>_on, <i>k</i>_off, and <i>K_D</i> for the TFIIB binding assays.

<p>Calculation of the binding constants <i>k</i>_on, <i>k</i>_off, and <i>K_D</i> for the TFIIB binding assays.</p