Biology subjects

Ozer, E.

Publications and source records attributed to Ozer, E..

4 recordsLinked to original sources

bioRxiv2021

Diverse tumorigenic consequences of human papillomavirus integration in primary oropharyngeal cancers

Human papillomavirus (HPV) causes 5% of all cancers and frequently integrates into host chromosomes, but the impacts of integration in tumorigenesis remain unclear. Analysis of 105 HPV-positive oropharyngeal cancers by whole genome sequencing detects viral integration in 77%, revealing five statistically significant integration hotspots near genes that regulate epithelial stem cell maintenance (i.e. SOX2, TP63, FGFR, MYC) and immune evasion (i.e. CD274). Somatic hyperamplification is enriched 16-fold near HPV integrants, and the extent of focal host genomic instability increases with local density of HPV integrants. Genes expressed at extreme outlier levels are increased 86-fold within +/- 150 kb of integrants. Across 95% of tumors with integration, host gene transcription is disrupted via intragenic integrants, chimeric transcription, outlier expression, gene breaking and/or de novo expression of noncoding or imprinted genes. We conclude that HPV integration contributes substantively to cancer development by causing extensive disruption of host genome structure and gene expression.

genomics↗

bioRxiv2020

An inside look at a biofilm: Pseudomonas aeruginosa flagella bio-tracking

The opportunistic pathogen, Pseudomonas aeruginosa, a flagellated bacterium, is one of the top model organisms for studying biofilm formation. In order to elucidate the role of the bacteria flagella in biofilm formation, we developed a new tool for flagella bio-tracking. We have site-specifically labeled the bacterial flagella by incorporating an unnatural amino acid into the flagella monomer via genetic code expansion. This enabled us to label and track the bacterial flagella during biofilm maturation. Direct, live imaging revealed for the first-time presence and synthesis of flagella throughout the biofilm lifecycle. To ascertain the possible role of the flagella in the strength of a biofilm we produced a "flagella knockout" strain and compared its biofilm to that of the wild type strain. Results showed a one order of magnitude stronger biofilm structure in the wild type in comparison to the flagella knockout strain. This suggests a newly discovered structural role for bacterial flagella in biofilm structure, possibly acting as a scaffold. Based on our findings we suggest a new model for biofilm maturation dynamic and underscore the importance of direct evidence from within the biofilm.

microbiology↗

bioRxiv2019

Simplified Methodology for a Modular and Genetically Expanded Protein Synthesis in Cell-Free Systems

Genetic code expansion, which enables the site-specific incorporation of unnatural amino acids into proteins, has emerged as a new and powerful tool for protein engineering. Currently, it is mainly utilized inside living cells for a myriad of applications. However, utilization of this technology in a cell-free, reconstituted platform has several advantages over living systems. The common limitations to the employment of these systems are the laborious and complex nature of its preparation and utilization. Herein, we describe a simplified method for the preparation of this system from Escherichia coli cells, which is specifically adapted for the expression of the components needed for cell-free genetic code expansion. In addition, we propose and demonstrate a modular approach to its utilization. By this approach, it is possible to prepare and store different extracts, harboring various translational components, and mix and match them as needed for more than four years retaining its high efficiency. We demonstrate this with the simultaneous incorporation of two different unnatural amino acids into a reporter protein. Finally, we demonstrate the advantage of cell-free systems over living cells for the incorporation of {delta}-thio-boc-lysine into ubiquitin by using the methanosarcina mazei wild-type pyrrolysyl tRNACUA and tRNA-synthetase pair, which can not be achieved in a living cell.

synthetic biology↗

bioRxiv2017

Tuning of recombinant protein expression in Escherichia coli by manipulating transcription, translation initiation rates and incorporation of non-canonical amino acids

Protein synthesis in cells has been thoroughly investigated and characterized over the past 60 years. However, some fundamental issues remain unresolved, including the reasons for genetic code redundancy and codon bias. In this study, we changed the kinetics of the E. coli transcription and translation processes by mutating the promoter and ribosome binding domains and by using genetic code expansion. The results expose a counterintuitive phenomenon, whereby an increase in the initiation rates of transcription and translation lead to a decrease in protein expression. This effect can be rescued by introducing slow translating codons into the beginning of the gene, by shortening gene length or by reducing initiation rates. Based on the results, we developed a biophysical model, which suggests that the density of co-transcriptional translation plays a role in bacterial protein synthesis. These findings indicate how cells use codon bias to tune translation speed and protein synthesis.

synthetic biology↗