A beta-tubulin gene (benA) is widely used in taxonomy and identification of Aspergillus spp. and other Fungi.Across Aspergillus spp. There is either one (benA) or two beta-tubulin paralogs (benA and tubC). The risk ofcontemporary use of sequences of paralogous genes with non-homologous function in the same phylogeneticanalysis is well known. It is evident that it had happened repeatedly in Aspergillus section Nigri. It is alarmingthat conventional primers for amplification of partial benA sequence can specifically amplify tubC paralog insome species. In this work, both paralogs were characterised in a set of species. The beta-tubulin primers in usewere revised and new, more benA specific primers were designed. Applicability of some markers such as basecomposition, codon usage and length of introns for distinguishing -tubulin paralogs benA and tubC is tested. Alarge study on molecular diversity of 349 isolates of Aspergillus (PCR-fingerprint, sequence data - ITS, benA,rpb2, caM) originating from Czech culture collections and from clinical material is also included. 82 specieswere identified, togetherwith nine tentative new taxa belonging to sections with high economic impact - Nigri,Fumigati or Aspergillus (Eurotium spp.). Five species from Section Aspergillus could be synonymised withexisting taxa. A study...

Dbp2p, a member of the large family of DEAD-box proteins and a yeast homolog of human p68, was shown to interact with Upf1p, an essential component of the nonsense-mediated mRNA decay pathway. Dbp2p:Upf1p interaction occurs within a large conserved region in the middle of Upf1p that is largely distinct from its Nmd2p and Sup35/45p interaction domains. Deletion of DBP2, or point mutations within its highly conserved DEAD-box motifs, increased the abundance of nonsense-containing transcripts, leading us to conclude that Dbp2p also functions in the nonsense-mediated mRNA decay pathway. Dbp2p, like Upf1p, acts before or at decapping, is predominantly cytoplasmic, and associates with polyribosomes. Interestingly, Dbp2p also plays an important role in rRNA processing. In dbp2Δ cells, polyribosome profiles are deficient in free 60S subunits and the mature 25S rRNA is greatly reduced. The ribosome biogenesis phenotype, but not the mRNA decay function, of dbp2Δ cells can be complemented by the human p68 gene. We propose a unifying model in which Dbp2p affects both nonsense-mediated mRNA decay and rRNA processing by altering rRNA structure, allowing specific processing events in one instance and facilitating dissociation of the translation termination complex in the other.

This dissertation reports on the technology of cell-free protein synthesis (CFPS) including 1) stabilized lyophilized cell-free systems and 2) enhanced heterogeneous cell extracts. This work further considers applications of CFPS systems in 1) rapid vaccine development, 2) functional virus-based nanoparticles, 3) site-specific protein immobilization, and 4) expanding the language of biology using unnatural amino acids. CFPS technology is a versatile protein production platform that has many features unavailable in in vivo expression systems. The primary benefit cell-free systems provide is the direct access to the reaction environment, which is no longer hindered by the presence of a cell-wall. The “openness" of the system makes it a compelling candidate for many technologies. One limitation of CFPS is the necessity of freezing for long-term viable storage. We demonstrate that a lyophilized CFPS system is more stable against nonideal storage than traditional CFPS reagents. The Escherichia coli-based CFPS system in this work is limited by the biocatalytic machinery found natively in E. coli. To combat these limitations, exogenous biocatalysts can be expressed during fermentation of cells prepared into extract. We demonstrate that simple adjustments in the fermentation conditions can significantly increase the activity of the heterogeneous extract. Towards virus-based particles and vaccines, we demonstrate that the open nature of CFPS can be utilized for coexpression of virus proteins and self-assembly of virus particles. This technique allows for the rapid production of potential vaccines and novel functional virus-based nanoparticles. Unnatural amino acids expand the effective language of protein biology. Utilizing CFPS as an expression system, we demonstrated that the incorporation of a single specific unnatural amino acid allows for site-specific immobilization, thus stabilizing the protein against elevated temperatures and chemical denaturants. Current unnatural amino acid incorporation technologies are limited to one or few simultaneous incorporations and suffer from low efficiency. This work proposes a system that could potentially allow for upwards of 40 unnatural amino acids to be simultaneously incorporated, effectively tripling the protein code. These projects demonstrate the power and versatility of CFPS technologies while laying the foundation for promising technologies in the field of biotechnology.

No description available.

<p>This thesis details the development of a kinetic model of translation which takes into account codon usage. The process of translation involves ribosomes decoding a sequence of codons to produce a protein. Codon usage is important in the kinetics of translation since experiments have shown that codons are processed at different rates. Codons which code for the same amino acid appear with unequal frequencies and certain synonymous codons are preferred by high expression genes. The relationship between translational efficiency and codon adaptation is explored in this thesis.</p> <p>We use a simple physics model called the totally asymmetric exclusion process (TASEP) to emulate the action of ribosomes, and the decoding of mRNA in protein elongation. The simple model is parameterized by an initiation rate that determines how quickly new ribosomes are introduced onto the lattice, and the rate of motion for ribosomes associated with a site on the lattice (codon message). Based on bioinformatics studies, we assign codon speeds so that codons preferred by high expression genes are translated more quickly.</p> <p>The model captures important aspects of translation like ribosome collision and codons of different speeds, and simulating it allows us to see details in dynamics which are inaccessible to experiments. TASEP has non-trivial behaviour when codon rates, and the rate of ribosome binding is varied. Slow codons can cause ribosomes to pause and may lead to a queue. We approximated real genes with its average rate, and with its slowest codons to test the salient features of how codons are used on mRNAs. We found that codon selection is important in determining when queues occur, and the ribosome density on genes. The model also shows that highly expressed genes queue later than low expression genes. The simple model gives us general insights into the translational selection of codons, and the important kinetic parameters.</p> / Master of Science (MSc)

In evolution alternative genetic trajectories can potentially lead to similar phenotypic outcomes. However, certain trajectories are preferred over others. These preferences bias the genomes of living organisms and the underlying processes can be observed in ongoing evolution. We have studied a variety of biases that can be found in bacterial chromosomes and determined the selective causes and functional consequences for the cell. We have quantified codon usage bias in highly expressed genes and shown that it is selected to optimise translational speed. We further demonstrated that the resulting differences in decoding speed can be used to regulate gene expression, and that the use of ‘non-optimal’ codons can be detrimental to reading frame maintenance. Biased gene location on the chromosome favours recombination between genes within gene families and leads to co-evolution. We have shown that such recombinational events can protect these gene families from inactivation by mobile genetic elements, and that chromosome organization can be selectively maintained because inversions can lead to the formation of unstable hybrid operons. We have used the development of antibiotic resistance to study how different bacterial lifestyles influence evolutionary trajectories. For this we used two distinct pairs of antibiotics and disease-causing bacteria, namely (i) Mycobacterium tuberculosis that is treated with rifampicin and (ii) Escherichia coli that is treated with ciprofloxacin. We have shown that in the slow-growing Mycobacterium tuberculosis, resistance mutations are selected for high-level resistance. Fitness is initially less important, and over time fitness costs can be ameliorated by compensatory mutations. The need for rapid growth causes the selection of ciprofloxacin resistance in Escherichia coli not only to be selected on the basis of high-level resistance but also on high fitness. Compensatory evolution is therefore not required and is not observed. Taken together, our results show that the evolution of a phenotype is the product of multiple steps and that many factors influence which trajectory is the most likely to occur and be most beneficial. Over time, selection will favour this particular trajectory and lead to biased evolution, affecting genome sequence and organization.

Rhodopsin, a visual pigment protein found in retinal photoreceptors, mediates vision at low-light levels. Recent studies focusing primarily in human and mouse have challenged the assumption of neutral evolution of synonymous substitutions in mammals. Using recently developed likelihood-based codon models accounting for mutational bias and selection, we find significant evidence for selective constraint on synonymous substitutions in mammalian rhodopsins, and a preference for cytosine at 3rd codon positions. A second project investigated adaptive evolution in rhodopsin, in view of theories of nocturnality in early mammals. We detected a significant acceleration of non-synonymous substitution rates at the origins of therian mammals, and a tendency of synonymous substitutions towards C-ending codons prior to that. These findings suggest an evolutionary scenario in which synonymous substitutions that increase mRNA stability and/or translation efficiency may have preceded adaptive non-synonymous evolution in early mammalian rhodopsins. These findings have important implications for theories of early mammalian nocturnality.

Candida parapsilosis is an opportunistic fungal pathogen of humans causing a variety of infections. Immunocompromised individuals represent the most threatened group of patients. The increasing frequency of infections and occurrence of drug resistant strains are the main reasons for research focused on novel antimycotic compounds. Inhibition of secreted aspartic proteases (Sap) of pathogenic Candida spp. appears to be a potential target of therapeutic intervention. The genome of C. parapsilosis contains at least three genes coding for secreted aspartic proteases, denominated SAPP1-3. Protease Sapp1p has been well biochemically and structurally characterized, whereas Sapp2p and Sapp3p have been given less attention. The first part of the thesis is focused on structural analysis of Sapp1p complexes with selected peptidomimetic inhibitors binding to the active site of the enzyme. In addition, complex of the isoenzyme Sapp2p with the well-known secreted aspartate inhibitor Pepstatin A has been analyzed. The second part is related to the fact that C. parapsilosis belongs to the Candida spp. with the unique ability to translate standard leucine CUG codon mostly as serine. Even though it is a non-conservative substitution of hydrophobic amino acids for a hydrophilic one, this unique ability is maintained for more...

This thesis focuses on the genomic study of symbionts of two different groups of hymenopterans: bees and ants. Both groups of insects have major ecological impact, and investigating their microbiomes increases our understanding of their health, diversity and evolution. The study of the bee gut microbiome, including members of Lactobacillus and Bifidobacterium, revealed genomic processes related to the adaptation to the gut environment, such as the expansion of genes for carbohydrate metabolism and the acquisition of genes for interaction with the host. A broader genomic study of these genera demonstrated that some lineages evolve under strong and opposite substitution biases, leading to extreme GC content values. A comparison of codon usage patterns in these groups revealed ongoing shifts of optimal codons. In a separate study we analysed the genomes of several strains of Lactobacillus kunkeei, which inhabits the honey stomach of bees but is not found in their gut. We observed signatures of genome reduction and suggested candidate genes for host-interaction processes. We discovered a novel type of genome architecture where genes for metabolic functions are located in one half of the genome, whereas genes for information processes are located in the other half. This genome organization was also found in other Lactobacillus species, indicating that it was an ancestral feature that has since been retained. We suggest mechanisms and selective forces that may cause the observed organization, and describe processes leading to its loss in several lineages independently. We also studied the genome of a species of Rhizobiales bacteria found in ants. We discuss its metabolic capabilities and suggest scenarios for how it may affect the ants’ lifestyle. This genome contained a region with homology to the Bartonella gene transfer agent (GTA), which is a domesticated bacteriophage used to transfer bacterial DNA between cells. We propose that its unique behaviour as a specialist GTA, preferentially transferring host-interaction factors, originated from a generalist GTA that transferred random segments of chromosomal DNA. These bioinformatic analyses of previously uncharacterized bacterial lineages have increased our understanding of their physiology and evolution and provided answers to old and new questions in fundamental microbiology.

Introduction: RNA viruses are economically important pathogens of fish, and among these viruses, infectious pancreatic necrosis virus (IPNV) is of particular concern for the aquaculture industry, especially for farmed rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar). This non-enveloped aquatic virus, which was first isolated in the UK in 1971, belongs to the family of Birnaviridae and has a bi-segmented dsRNA genome of about 6kb. IPNV is classified in 6 genogroups with correspondence to 10 known serotypes and an additional proposed genogroup of marine aquabirnaviruses (MABV). IPNV causes high mortality in fry and a reduced mortality in adult fish, respectively. Fish, which survive, can become carriers and this can lead to a clinical outbreak by releasing infective material into water or by vertical transmission via oocytes, milt and seminal fluids. Methods: This project aimed at determining the phylogeny and genomic changes of IPNV in Scotland by whole genome sequence analysis of IPNV isolates (diagnostic TCID50 supernatants) spanning 3 decades since 1982, using next generation sequencing technology. Viral RNA of IPNV culture supernatant (CHSE-214 and TO cell culture) was processed for next generation sequencing on an Illumina MiSeq platform. Library preparation was performed using the Nextera XT DNA Library Kit, prior to sequencing according to the manufacturer's MiSeq Reagent Kit v3 (150cycles) protocol. To optimize whole genome next generation sequencing for IPNV, we compared two RNA processing protocols, the Glasgow (GLAP) and the Goettingen protocol (GOEP) with focus on missing terminal nucleotides after a de novo genome assembly. Sequences were used to determine the phylogeny and selection pressure on the genome as well as a possible virus-host adaptation. Results: The results showed that both protocols were able to give full length genomes as well as genomes with missing terminal nucleotides. The phylogenetic analysis of 57 sequenced IPVN isolates shows that 78.95 % of the isolates group within genogroup V, which includes serogroup Sp and 5.26 % within genogroup I which includes serogroup Ja. Segment A of 15.79 % of the isolate grouped within genogroup III, which includes serotype Ca1 and Te but only 7.02 % of the segment B isolates grouped in the genogroup III. The remaining 8.77 % of segment B groups within genogroup II, containing the Ab serotype. Previous research has shown that residue substitutions at positions 217 and 221 in the major capsid protein VP2 have an impact on the virulence of the virus, leading to different virulence types: virulent (T217, A221), low virulence (P217, A221), avirulent (T217, T221) and persistent (P217, T221). Whole genome sequence results show that 58.93 % of the sequenced isolates belong to the persistent, 32.14 % to the low virulent type, only one isolate was of a virulent type and 7.15 % had not virulence assigned amino acid compositions in positions 217 and 221. The selection pressure analysis showed that especially VP2 is experiencing selection pressure in the variable region. In the VP1 protein we see two sites under positive selection pressure within specific motifs. VP5 showed positive selected sites mostly within the truncated region of the protein. Other proteins showed no particular interesting sites of selection. The codon adaptation analysis showed highest adaptation index for VP2. Besides VP5, which had an CAI index below one, therefore showing negative adaptation, other IPNV proteins had an CAI of barely above the value of 1. The dinucleotide abundance, focussing on CpG, showed that CpG is underrepresented in segment A and B. Discussion Phylogenetic analysis of the sequenced IPNV strains shows separate clustering of different genogroups. Genetic reassortment is observed in segment B showing a grouping within genogroup III and II although the segment A of these isolates was grouping exclusively within III. We found that over 50 % of the isolates belong to the persistent and over 30 % to the low virulent type, assuming that due to not sterilising vaccination these types were selected in the vaccinated population. The results from the CAI calculations indicate an adaptation of IPNV to its host. Together with the findings that CpG is underrepresented in IPNV it suggests that this leads to an immune escape. Especially since the selection pressure analysis showed positive selection in VP2 within the virulence determination sites of the protein, indicating that IPNV "tries" to downregulate immune recognition. The prevalence of mostly persistent type of isolates indicates together with the assumption of adaptation and immune escape that IPNV is evolving with the host in order to ensure survival.