Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Evidence for Sigma Factor Competition in the Regulation of Alginate Production by Pseudomonas aeruginosa

Alginate overproduction, or mucoidy, plays an important role in the pathogenesis of P. aeruginosa lung infection in cystic fibrosis (CF). Mucoid strains with mucA mutations predominantly populate in chronically-infected patients. However, the mucoid strains can revert to nonmucoidy in vitro through suppressor mutations. We screened a mariner transposon library using CF149, a non-mucoid clinical isolate with a misssense mutation in algU (AlgU(A61V)). The wild type AlgU is a stress-related sigma factor that activates transcription of alginate biosynthesis. Three mucoid mutants were identified with transposon insertions that caused 1) an overexpression of AlgU(A61V), 2) an overexpression of the stringent starvation protein A (SspA), and 3) a reduced expression of the major sigma factor RpoD (σ(70)). Induction of AlgU(A61V) in trans caused conversion to mucoidy in CF149 and PAO1DalgU, suggesting that AlgU(A61V) is functional in activating alginate production. Furthermore, the level of AlgU(A61V) was increased in all three mutants relative to CF149. However, compared to the wild type AlgU, AlgU(A61V) had a reduced activity in promoting alginate production in PAO1ΔalgU. SspA and three other anti-σ(70) orthologues, P. aeruginosa AlgQ, E. coli Rsd, and T4 phage AsiA, all induced mucoidy, suggesting that reducing activity of RpoD is linked to mucoid conversion in CF149. Conversely, RpoD overexpression resulted in suppression of mucoidy in all mucoid strains tested, indicating that sigma factor competition can regulate mucoidy. Additionally, an RpoD-dependent promoter (P(ssrA)) was more active in non-mucoid strains than in isogenic mucoid variants. Altogether, our results indicate that the anti-σ(70) factors can induce conversion to mucoidy in P. aeruginosa CF149 with algU-suppressor mutation via modulation of RpoD

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe