Orthogonal translation enables heterologous ribosome engineering in E. coli

The ribosome represents a promising avenue for synthetic biology, but its complexity and essentiality have hindered significant engineering efforts. Heterologous ribosomes, comprising rRNAs and r-proteins derived from different microorganisms, may offer opportunities for novel translational functions. Such heterologous ribosomes have previously been evaluated in E. coli via complementation of a genomic ribosome deficiency, but this method fails to guide the engineering of refractory ribosomes. Here, we implement orthogonal ribosome binding site (RBS):antiRBS pairs, in which engineered ribosomes are directed to researcher-defined transcripts, to inform requirements for heterologous ribosome functionality. We discover that optimized rRNA processing and supplementation with cognate r-proteins enhances heterologous ribosome function for rRNAs derived from organisms with ≥76.1% 16S rRNA identity to E. coli. Additionally, some heterologous ribosomes undergo reduced subunit exchange with E. coli-derived subunits. Cumulatively, this work provides a general framework for heterologous ribosome engineering in living cells

Human Xq28 Inversion Polymorphism: From Sex Linkage to Genomics - A Genetic Mother Lode

An inversion polymorphism of the filamin and emerin genes at the tip of the long arm of the human X-chromosome serves as the basis of an investigative laboratory in which students learn something new about their own genomes. Long, nearly identical inverted repeats flanking the filamin and emerin genes illustrate how repetitive elements can lead to alterations in genome structure (inversions) through nonallelic homologous recombination. The near identity of the inverted repeats is an example of concerted evolution through gene conversion. While the laboratory in its entirety is designed for college level genetics courses, portions of the laboratory are appropriate for courses at other levels. Because the polymorphism is on the X-chromosome, the laboratory can be used in introductory biology courses to enhance understanding of sex-linkage and to test for Hardy-Weinberg equilibrium in females. More advanced topics, such as chromosome interference, the molecular model for recombination, and inversion heterozygosity suppression of recombination can be explored in upper-level genetics and evolution courses. DNA isolation, restriction digests, ligation, long PCR, and iPCR provide experience with techniques in molecular biology. This investigative laboratory weaves together topics stretching from molecular genetics to cytogenetics and sex-linkage, population genetics and evolutionary genetics

HSP25 interactome

The murine 25-kilodalton heat shock protein, HSP25, is a small, non-ATP dependent chaperone and actin binding protein that can be induced to varying degrees under different stress conditions, including exercise. While HSP25’s role as an actin-binding protein is well established and it is known to act as a general chaperone in vitro, it is unclear what client proteins HSP25 may act upon in the exercise-stressed sarcomere. Additionally, although HSP25 is known to interact with a variety of co-chaperones, its interaction with ATP-dependent heat shock proteins (which are necessary for the unbinding of substrate proteins) has only been established in vitro. In order to establish an “interactome” encompassing HSP25’s binding partners in the sarcomere, a co-immunoprecipitation assay was developed using anti-HSP25 antibody crosslinked to protein A/G magnetic beads. These beads were used to co-immunoprecipitate HSP25 and associated proteins from exercised mouse muscle lysate. Eluted proteins were identified using high pressure liquid chromatography-coupled mass spectrometry (HPLC-MS) and immunoblotting. This showed that HSP25 binds co-chaperones including small heat shock proteins and the ATP-dependent heat shock proteins HSP70 and HSP90. Client proteins were enriched for sarcomeric proteins and metabolic enzymes involved in ATP production. Overall, this work presents a method for the co-immunoprecipitation of HSP25. It also suggests that HSP25 acts in concert with several small heat shock proteins as well as ATP-dependent heat shock proteins in order to facilitate the folding of sarcomeric proteins and metabolic enzymes in muscle

Differences in the Frequency of Resistance to Antiretroviral Drug Classes among Human Immunodeficiency Virus Type 1 Clinical Isolates

Genotypic resistance to all antiretroviral classes was widespread among human immunodeficiency virus type 1 isolates failing therapy. Resistance to nonnucleoside reverse transcriptase inhibitors was found most frequently and resistance to protease inhibitors was found least frequently, most likely due to differences in the number of enzymatic amino acid substitutions leading to resistance to each particular drug class