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When new genes evolve through modification of existing genes, there are often trade-offs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near cognate stop codons and highlight a functional trade-off that we term “collateral toxicity”. We suggest that this type of trade-off may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary trade-offs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds not only to alterations in the demand for RF2 activity, but also for RF1 activity.

A quantitative real-time polymerase chain reaction (PCR) based on the omp P6 gene was developed to detect Haemophilus influenzae. Its specificity was determined by analysis of 29 strains of 11 different Haemophilus spp. and was compared with PCR assays having other target genes: rnpB, 16S rRNA, and bexA. The method was evaluated on nasopharyngeal aspirates from 166 adult patients with community-acquired pneumonia. When 10⁴ DNA copies/mL was used as cutoff limit for the method, P6 PCR had a sensitivity of 97.5% and a specificity of 96.0% compared with the culture. Of 20 culture-negative but P6 PCR-positive cases, 18 were confirmed by fucK PCR as H. influenzae. Five (5.9%) of 84 nasopharyngeal aspirates from adult controls tested PCR positive. We conclude that the P6 real-time PCR is both sensitive and specific for identification of H. influenzae in respiratory secretions. Quantification facilitates discrimination between disease-causing H. influenzae strains and commensal colonization.

Argonautes are conserved RNA-binding proteins that can regulate gene expression post transcriptionally through a process known as RNA interference (RNAi). This is done through the use of small RNAs, e.g. sRNAs that act as a guide for the argonautes, allowing for sequence-specific binding to the target site. This interaction has been studied in many organisms, one of which is the model organism Dictyostelium discoideum. D. discoideum is an amoeba that has been used extensively in genetic experiments due to its unique lifestyle, and ease of use. Being a eukaryotic, unicellular organism, it proves to be a great tool for the study of regulatory systems in eukaryotes, allowing us to study this argonaute-sRNA interaction in detail. By analysing which RNAs bind to the argonautes, we can better understand which genes these proteins regulate and what role RNAi has in the organisms as a whole. 

In this study, I investigate three of the five argonautes found in D. discoideum, namely agnA, agnC and agnE. By transforming FLAG-tagged versions of these genes into the amoeba, I successfully express two of these modified proteins in D. discoideum and verified expression by using antibodies designed specifically to recognise the FLAG-tags. This opens up the possibility for the characterisation of the argonaute proteins to better understand their role and function in the regulation of genes. 

Time-resolved cryogenic electron microscopy (cryo-EM) is a powerful technique for capturingtransient molecular and viral structures during conformational changes. This method providesunique and potentially critical insights into the transient states of biomolecules, which can beinvaluable for drug development. Additionally, it offers glimpses into the pathology of viruses asthey interact with their immediate environment. In this project, a plunge freezer initially designedfor a spray-and-plunge approach in time-resolved cryo-EM [24] was modified to implement aflash-and-freeze system [3]. This modified system was tested on two different viruses: influenzaH3N2 type A and Chaetoceros tenuissimus DNA virus type II. The primary objective was tovisualize an intermediate state during the membrane fusion process between influenza A andliposomes with endosomal characteristics. Although no intermediate state was captured forinfluenza, the activation and pH reduction were successfully achieved. The study on Chaetocerostenuissimus DNA virus type II focused on potential conformational changes due to a drop in pH,rather than capturing intermediate states in a time-resolved manner. Future experiments withmore precise control over blotting, delivered intensity, and the concentration of the cagedcompound are expected to facilitate the capture and analysis of intermediate states.

The proposal that enzymatic catalysis is due to conformational fluctuations has been previously promoted by means of indirect considerations. However, recent works have focused on cases where the relevant motions have components toward distinct conformational regions, whose population could be manipulated by mutations. In particular, a recent work has claimed to provide direct experimental evidence for a dynamical contribution to catalysis in dihydrofolate reductase, where blocking a relevant conformational coordinate was related to the suppression of the motion toward the occluded conformation. The present work utilizes computer simulations to elucidate the true molecular basis for the experimentally observed effect. We start by reproducing the trend in the measured change in catalysis upon mutations (which was assumed to arise as a result of a "dynamical knockout" caused by the mutations). This analysis is performed by calculating the change in the corresponding activation barriers without the need to invoke dynamical effects. We then generate the catalytic landscape of the enzyme and demonstrate that motions in the conformational space do not help drive catalysis. We also discuss the role of flexibility and conformational dynamics in catalysis, once again demonstrating that their role is negligible and that the largest contribution to catalysis arises from electrostatic preorganization. Finally, we point out that the changes in the reaction potential surface modify the reorganization free energy (which includes entropic effects), and such changes in the surface also alter the corresponding motion. However, this motion is never the reason for catalysis, but rather simply a reflection of the shape of the reaction potential surface.

The relevance of gas-phase protein structure to its solution structure is of the utmost importance in studying biomolecules by mass spectrometry. D-Amino acid substitutions within a minimal protein. Trp-cage. were used to correlate solution-phase properties as measured by circular dichroism with solution/gas-phase conformational features of protein cations probed via charge state distribution (CSD) in electrospray ionization. and gas-phase features revealed by tandem mass spectrometry (MS/MS). The gas-phase features were additionally supported by force-field molecular dynamics simulations. CD data showed that almost any single-residue D-substitution destroys the most prominent CD feature of the "native" all-L isomer, alpha-helicity. CSD was able to qualitatively assess the degree of compactness of solution-phase molecular structures. CSD results were consistent with the all-L form being the most compact in solution among all studied stereoisomers except for the D-Asn(1) isomer. D-substitutions of the aromatic Y(3), W(6) and Q(5) residues generated the largest deviations in CSD data among single amino acid substitutions. consistent with the critical role of these residues in Trp-cage stability. Electron capture dissociation of the stereoisomer dications gave an indication that some gas-phase structural features of Trp-cage are similar to those in solution. This result is supported by MDS data oil five of the studied stereoisomer dications in the gas-phase. The MDS-derived minimum-energy structures possessed more extensive hydrogen bonding than the solution-phase structure of the native form, deviating from the latter by 3-4 angstrom and were not 'inside-out' compared to native structures. MDS data could be correlated with CD data and even with ECD results. which aided in providing a long-range structural constraint for MDS. The overall conclusion is the general resemblance, despite the difference on the detailed level, of the preferred structures in both phases for the mini protein Trp-cage.

An important mechanism for generation of new genes is by duplication-divergence of existing genes. Duplication-divergence includes several different sub-models, such as subfunctionalization where after accumulation of neutral mutations the original function is distributed between two partially functional and complementary genes, and neofunctionalization where a new function evolves in one of the duplicated copies while the old function is maintained in another copy. The likelihood of these mechanisms depends on the longevity of the duplicated state, which in turn depends on the fitness cost and genetic stability of the duplications. Here, we determined the fitness cost and stability of defined gene duplications/amplifications on a low copy number plasmid. Our experimental results show that the costs of carrying extra gene copies are substantial and that each additional kbp of DNA reduces fitness by approximately 0.15%. Furthermore, gene amplifications are highly unstable and rapidly segregate to lower copy numbers in absence of selection. Mathematical modelling shows that the fitness costs and instability strongly reduces the likelihood of both sub- and neofunctionalization, but that these effects can be off-set by positive selection for novel beneficial functions.

Cryptosporidium is a major cause of diarrheal disease worldwide. In developing countries, this infection is endemic and in children, associated with growth faltering and cognitive function deficits, with the most severe impact on those aged <2 years. Little has been reported about symptoms and risk factors for children in industrialized countries, although the disease incidence is increasing in such regions. In November 2010, a large waterborne outbreak of C. hominis occurred in the city of Ostersund in Sweden. Approximately 27,000 of the 60,000 inhabitants were symptomatic. We aimed to describe duration of symptoms and the risk factors for infection with C. hominis in children aged <15 years in a Western setting. Within 2 months after a boil water advisory, a questionnaire was sent to randomly selected inhabitants of all ages, including 753 children aged <15 years. Those with >= 3 loose stools/day were defined as cases of diarrhoea. The response rate was 70.3%, and 211 children (39.9%) fulfilled the case definition. Mean duration of diarrhoea was 7.5 days (median 6, range 1-80 days). Recurrence, defined as a new episode of diarrhoea after >= 2 days of normal stools, occurred in 52.5% of the cases. Significant risk factors for infection, besides living within the distribution area of the contaminated water plant, included a high level of water consumption, male sex, and a previous history of loose stools. The outbreak was characterized by high attack and recurrence rates, emphasizing the necessity of water surveillance to prevent future outbreaks.

The globin family of genes and proteins has been a recurrent object of study for many decades. This interest has generated a vast amount of knowledge. However it has also created an inconsistent and confusing nomenclature, due to the lack of a systematic approach to naming genes and failure to reflect the phylogenetic relationships among genes of the gene family. To alleviate the problems with the existing system, here we propose a standardized nomenclature for the alpha and beta globin family of genes, based on a phylogenetic analysis of vertebrate alpha and beta globins, and following the Guidelines for Human Gene Nomenclature.

The synthesis of proteins, also known as translation, is a fundamental process in every living organism. The steps in the translation of genetic information to functional proteins have been meticulously studied, mostly using in vitro techniques, yielding a detailed model of their mechanisms. However, the use of minimal cell-free systems allows for the possibility to miss interactions from absent components or that reactions are affected by the buffer composition. The work presented in this thesis opens a way to study the kinetics of complex molecular processes, like protein synthesis, directly inside live bacterial cells in real time. We developed and optimized a method to deliver dye-labeled macromolecules inside live cells and generate a kinetic model of the particle’s interactions based on its diffusion inside the cell.

This method facilitated the study of translation elongation and initiation directly in live cells. Our measurements of reaction times of tRNA in the ribosome, agree with previous reports from in vitro techniques. We further applied the method to examine the effects of three aminoglycoside antibiotics and erythromycin directly in live cells. The aminoglycoside antibiotics slowed-down protein synthesis 2- to 4-fold, while the number of elongation cycles per initiation event decreased significantly. In the case of erythromycin, cells showed a 4-fold slower protein synthesis. Additionally, we measured the kinetics of sequence-specific effects of erythromycin: translational arrest, and peptidyl-tRNA drop-off; these in vivo measurements revealed a complex mechanism of action of the drug, in agreement with models suggested by previous experiments. Additionally, we applied the method to measure the effects, on the kinetics of protein synthesis, caused by modifications in the C-terminal tail of the S13 ribosomal protein. Our measurements showed that specific mutations led to different changes in the occupancy and dwell-time of labeled-tRNA in the ribosome.

To summarize, the present work will guide the reader through the development of a method to study the kinetics of protein synthesis directly in live bacterial cells, as well as its application to characterize the effects of different antibiotics within the complex environment of a living organism.

The spread of antibiotic resistance is turning many of the currently used antibiotics less effective against common infections. To address this public health challenge, it is critical to enhance our understanding of the mechanisms of action of these compounds. Aminoglycoside drugs bind the bacterial ribosome, and decades of results from in vitro biochemical and structural approaches suggest that these drugs disrupt protein synthesis by inhibiting the ribosome's translocation on the messenger RNA, as well as by inducing miscoding errors. So far, however, we have sparse information about the dynamic effects of these compounds on protein synthesis inside the cell. In the present study, we measured the effect of the aminoglycosides apramycin, gentamicin, and paromomycin on ongoing protein synthesis directly in live Escherichia coli cells by tracking the binding of dye-labeled transfer RNAs to ribosomes. Our results suggest that the drugs slow down translation elongation two- to fourfold in general, and the number of elongation cycles per initiation event seems to decrease to the same extent. Hence, our results imply that none of the drugs used in this study cause severe inhibition of translocation.

Single-molecule techniques are ideally poised to characterize complex dynamics but are typically limited to investigating a small number of different samples. However, a large sequence or chemical space often needs to be explored to derive a comprehensive understanding of complex biological processes. Here we describe multiplexed single-molecule characterization at the library scale (MUSCLE), a method that combines single-molecule fluorescence microscopy with next-generation sequencing to enable highly multiplexed observations of complex dynamics. We comprehensively profiled the sequence dependence of DNA hairpin properties and Cas9-induced target DNA unwinding-rewinding dynamics. The ability to explore a large sequence space for Cas9 allowed us to identify a number of target sequences with unexpected behaviors. We envision that MUSCLE will enable the mechanistic exploration of many fundamental biological processes.