Studies of transcriptional control in eukaryotes have focused mainly on genetic elements that mediate positive regulation. Many positive regulatory elements and transcription factors have been identified that are selectively able to stimulate transcription of specific genes. In most cases, trans-activators bind directly to cis-acting sequences, localized either in the vicinity of the promoter or in a remote position, and transmit a positive signal to the transcription initiation complex. Although many components involved in this activation are known, the mechanisms underlying transcriptional stimulation remain unclear. The activating proteins that bind DNA directly are generally composed of at least two distinct domains: One is responsible for contact of the protein with the DNA and provides the specificity for the recognition of the target site; the second is involved in the 3Present address:
Critical illness in COVID-19 is an extreme and clinically homogeneous disease phenotype that we have previously shown1 to be highly efficient for discovery of genetic associations2. Despite the advanced stage of illness at presentation, we have shown that host genetics in patients who are critically ill with COVID-19 can identify immunomodulatory therapies with strong beneficial effects in this group3. Here we analyse 24,202 cases of COVID-19 with critical illness comprising a combination of microarray genotype and whole-genome sequencing data from cases of critical illness in the international GenOMICC (11,440 cases) study, combined with other studies recruiting hospitalized patients with a strong focus on severe and critical disease: ISARIC4C (676 cases) and the SCOURGE consortium (5,934 cases). To put these results in the context of existing work, we conduct a meta-analysis of the new GenOMICC genome-wide association study (GWAS) results with previously published data. We find 49 genome-wide significant associations, of which 16 have not been reported previously. To investigate the therapeutic implications of these findings, we infer the structural consequences of protein-coding variants, and combine our GWAS results with gene expression data using a monocyte transcriptome-wide association study (TWAS) model, as well as gene and protein expression using Mendelian randomization. We identify potentially druggable targets in multiple systems, including inflammatory signalling (JAK1), monocyte–macrophage activation and endothelial permeability (PDE4A), immunometabolism (SLC2A5 and AK5), and host factors required for viral entry and replication (TMPRSS2 and RAB2A).
We have devised a novel method for the construction of a DNA affinity matrix and tested its use in the purification of a sequence-specific DNA-binding protein from the yeast Saccharomyces cerevisiae. The matrix was prepared in two steps: first, a palindromic oligonucleotide containing an XhoI cohesive end was covalently linked via its loop to a Sepharose matrix; second, directly to this 'universal' primed Sepharose was ligated a 37-bp oligonucleotide, with XhoI cohesive ends, containing the sequence of the upstream activation sequence 1 (UASI) site of the yeast iso-1-cytochrome c (CYCI) gene. After fractionating a yeast crude extract through DEAEcellulose, heparin ultrogel and Mono Q columns, a single pass through the affinity matrix allowed the purification to apparent homogeneity of the 120-kDa protein factor P, which is responsible for the binding to the UASI site.Commonly used procedures for the attachment of DNA to solid supports are based on the immobilization of polynucleotides via one or more bases to activated matrices [l, 21 or end-attachment through terminal phosphate groups [3-61. Good coupling yields have been reported using the first method; however the use of this protocol is restricted to the immobilization of long DNA fragments since the chemical linkage results in multipoint attachments along the DNA chain.Recently, we described a simple two-step procedure allowing rapid covalent linkage of DNA fragments to a solid support such as Sepharose, via a single specific point [7, 81.
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