An Integrated Strategy for Analyzing the Unique Developmental Programs of Different Myoblast Subtypes

An important but largely unmet challenge in understanding the mechanisms that govern the formation of specific organs is to decipher the complex and dynamic genetic programs exhibited by the diversity of cell types within the tissue of interest. Here, we use an integrated genetic, genomic, and computational strategy to comprehensively determine the molecular identities of distinct myoblast subpopulations within the Drosophila embryonic mesoderm at the time that cell fates are initially specified. A compendium of gene expression profiles was generated for primary mesodermal cells purified by flow cytometry from appropriately staged wild-type embryos and from 12 genotypes in which myogenesis was selectively and predictably perturbed. A statistical meta-analysis of these pooled datasets—based on expected trends in gene expression and on the relative contribution of each genotype to the detection of known muscle genes—provisionally assigned hundreds of differentially expressed genes to particular myoblast subtypes. Whole embryo in situ hybridizations were then used to validate the majority of these predictions, thereby enabling true-positive detection rates to be estimated for the microarray data. This combined analysis reveals that myoblasts exhibit much greater gene expression heterogeneity and overall complexity than was previously appreciated. Moreover, it implicates the involvement of large numbers of uncharacterized, differentially expressed genes in myogenic specification and subsequent morphogenesis. These findings also underscore a requirement for considerable regulatory specificity for generating diverse myoblast identities. Finally, to illustrate how the developmental functions of newly identified myoblast genes can be efficiently surveyed, a rapid RNA interference assay that can be scored in living embryos was developed and applied to selected genes. This integrated strategy for examining embryonic gene expression and function provides a substantially expanded framework for further studies of this model developmental system

Competent Program Evolution, Doctoral Dissertation, December 2006

Heuristic optimization methods are adaptive when they sample problem solutions based on knowledge of the search space gathered from past sampling. Recently, competent evolutionary optimization methods have been developed that adapt via probabilistic modeling of the search space. However, their effectiveness requires the existence of a compact problem decomposition in terms of prespecified solution parameters. How can we use these techniques to effectively and reliably solve program learning problems, given that program spaces will rarely have compact decompositions? One method is to manually build a problem-specific representation that is more tractable than the general space. But can this process be automated? My thesis is that the properties of programs and program spaces can be leveraged as inductive bias to reduce the burden of manual representation-building, leading to competent program evolution. The central contributions of this dissertation are a synthesis of the requirements for competent program evolution, and the design of a procedure, meta-optimizing semantic evolutionary search (MOSES), that meets these requirements. In support of my thesis, experimental results are provided to analyze and verify the effectiveness of MOSES, demonstrating scalability and real-world applicability

Assignment of new roles for malectin-like domains to understand their divergent evolution

Malectin is a highly-conserved animal lectin from the endoplasmic reticulum (ER), with a quality control function in the N-Glycosylation process. It has a β-sandwich core with long loops connecting the β-sheets. Malectin binding-pocket is in the loops region. Several carbohydrate-binding modules (CBMs) discovered in other domains of life that shared sequence homology with the malectin, were classified and grouped as a novel CBM57 family by Carbohydrate-Active Enzymes (CAZy) database. The members of this family are expected to have a highly conserved β-sandwich core, but high variance in the binding-pocket residues. To investigate if the specificity of these modules is the same as the malectin, a bioinformatic analysis was performed with 315 members of the CBM57 family found in CAZy database. Several programs were used to predict the protein architecture and to analyse the conservation of amino acids sequences, especially in the binding-pocket. Based on this analysis, we predict animal CBM57 modules to have the same specificity as malectin. However, bacterial CBM57 modules in bacteria domain are predicted, after highlighting the modules associated with glycoside hydrolases from family 2, to have various specificities, and thus different biological functions. For verifying these assumptions, a total of 7 CBMs (family 57 and homologous) associated with glycoside hydrolases from family 2 and belonging to the human gut microbiome – Bacteroides ovatus and Bacteroides thetaiotaomicron- were chosen for characterization studies. A re-cloning was initially performed for the recombinant DNAs, changing the His-tag position. Afterwards, expression tests were realized, in which 2 CBMs of different bacteria were expressed in soluble form. The production of the proteins was then performed at a larger scale, followed by affinity chromatography purification. By the analysis of the gels, the eluted samples had high purity and were suitable for characterization studies. Glycan microarrays were performed for determining the binding-specificities of the 2 CBM modules. The CBM module from B.thetaiotaomicron revealed high specificity for pectin polysaccharides, possible recognizing α 1-3 linked galacturonic acid and ramnose. For structural characterization by X-ray crystallography, several crystallization trials were performed. Crystals were obtained for the B.thetaiotaomicron CBM module, which diffracted to high resolution. The structure is, yet, to be solved

Fungal model systems and the elucidation of pathogenicity determinants

Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.Peer reviewedPublisher PD

A functional analysis of random coding sequences in Escherichia coli

Adaptation of organisms to continuously changing environments includes the generation of genic novelty in their genomes through mechanisms such as de novo gene evolution, duplication, fusion, lateral gene transfer, etc. De novo gene evolution is a mechanism, wherein new gene functions can evolve from previously non-coding sequences, which are essentially random stretches of nucleotides. Several studies have explored the role of such random sequences as templates for evolutionary innovation. This included a systematic study, where a library of random coding sequences was expressed in Escherichia coli and differential growth was measured to assess fitness effects of individual sequences. Each random sequence from the library was categorized into negative, positive or neutral based on its change in abundance in the population across time. In this thesis, I analyse the effects of individual clones derived from this screen. In order to study effects of random sequences on the fitness of the host, I cloned representative variants from each category into E. coli strains using a multicopy plasmid vector. In the first part of the thesis, I demonstrate that expression of negative random peptides confers a fitness disadvantage (deleterious) in E. coli, followed by a growth recovery. Upon further investigation, I find that these peptides can elicit a stress response in the host instantaneously upon expression. The highly deleterious phenotype can thus be compensated in the host. In addition, I was able to isolate suppressor-of-phenotype clones. Re-sequencing of the suppressors together with each of the ancestor clones helped identify interaction partners for the deleterious peptides. In the second part, I show two mechanisms that the host uses to adapt to deleterious peptide expression: (a) plasmid copy number control by inactivation of the pcnB gene and (b) expression control through inactivation of the LacI inducer binding domain. In the third part of the thesis, I show that the positive random peptides confer competitive fitness advantage only under stressful conditions, for example, an elevated temperature. In conclusion, I show that random sequences indeed affect fitness of the host possibly through targeting specific genes or proteins. This study provides experimental evidence on how random sequences could serve as drivers of de novo gene evolution

Investigating hookworm genomes by comparative analysis of two Ancylostoma species

Background Hookworms, infecting over one billion people, are the mostly closely related major human parasites to the model nematode Caenorhabditis elegans. Applying genomics techniques to these species, we analyzed 3,840 and 3,149 genes from Ancylostoma caninum and A. ceylanicum. Results Transcripts originated from libraries representing infective L3 larva, stimulated L3, arrested L3, and adults. Most genes are represented in single stages including abundant transcripts like hsp-20 in infective L3 and vit-3 in adults. Over 80% of the genes have homologs in C. elegans, and nearly 30% of these were with observable RNA interference phenotypes. Homologies were identified to nematode-specific and clade V specific gene families. To study the evolution of hookworm genes, 574 A. caninum / A. ceylanicum orthologs were identified, all of which were found to be under purifying selection with distribution ratios of nonsynonymous to synonymous amino acid substitutions similar to that reported for C. elegans / C. briggsae orthologs. The phylogenetic distance between A. caninum and A. ceylanicum is almost identical to that for C. elegans / C. briggsae. Conclusion The genes discovered should substantially accelerate research toward better understanding of the parasites' basic biology as well as new therapies including vaccines and novel anthelmintics

Genome-Wide Gene Expression Profiling of Fertilization Competent Mycelium in Opposite Mating Types in the Heterothallic Fungus Podospora anserina

are the major regulators of fertilization, and this study presents a genome-wide view of their target genes and analyzes their target gene regulation. strains. Of the 167 genes identified, 32 genes were selected for deletion, which resulted in the identification of two genes essential for the sexual cycle. Interspecies comparisons of mating-type target genes revealed significant numbers of orthologous pairs, although transcriptional profiles were not conserved between species.This study represents the first comprehensive genome-wide analysis of mating-type direct and indirect target genes in a heterothallic filamentous fungus. Mating-type transcription factors have many more target genes than are found in yeasts and exert a much greater diversity of regulatory actions on target genes, most of which are not directly related to mating

Ligand discovery and structural-functional analysis of proteins involved in plant cell wall degradation

The plant cell wall is constituted by recalcitrant polysaccharides with diverse sequences that comprise an abundant source of terrestrial biomass. To efficiently degrade plant cell wall polysaccharides, some cellulolytic bacterial organisms, such as Clostridium thermocellum and Ruminococcus flavefaciens, have an extracellular multi-enzyme complex with catalytic and non-catalytic carbohydrate-binding modules (CBMs). CBMs play a crucial role in enhancing the catalytic efficiency of the enzymes by proximity effect, cell attachment or targeting and disruptive function. The Carbohydrate Active enZymes database (CAZy) organizes the identified CBMs by sequence similarity into different families. Deposition of CBM sequences in the CAZy database is continually growing for which characterization and structure-function analysis is required. In this study we aim to characterize the carbohydrate ligand specificities of C. thermocellum ATCC 27405 and R. flavefaciens FD-1 CBMs assigned to different families in the CAZy database. We performed carbohydrate microarray screening analysis for ligand discovery and crystallization screenings aiming to solve the 3D structures of the CBM-ligand complexes by X-ray crystallography. To complement the information provided by these methodologies we also performed ITC (Isothermal Titration Calorimetry), MST (Microscale Thermophoresis) and affinity gel electrophoresis. With the implementation of this approach it was possible to elucidate different carbohydrate binding specificities for biotechnologically relevant CBMs. The results from the initial carbohydrate microarray screening constitute a functional start point to target CBMs for structural-functional analysis of carbohydrate-recognition. C. thermocellum family 50 (CtCBM50) reveals to be a novel chitin binding LysM domain and binding with insoluble chitin and a β-(1-4)-GlcNAc chitin oligosaccharide was identified. R. flavefaciens FD-1 family 62 CBM (RfCBM62) reveals to be highly specific for a pectic polysaccharide for which the structure is being investigated and binding to galacturonan DP4 was observed. In the scope of this thesis, and as the structural characterization was not achieved in due time, the sequence similarity to known structures inspired the attempt to computationally produce similarity models for the two CBMs. The (hypothetical) conservation of the secondary structures revealed some structural features of the proteins under study. An important outcome from this integrative study is the possibility to understand the versatility of plant and fungal saccharide sequences and their recognition by the different CBM families. The different binding patterns observed could reflect adaptive pressures of the microorganisms to their respective ecological niches, translating in divergent evolution of the proteome

Generation and application of genomic tools as important prerequisites for sugar beet genome analyses.

Genetic and physical maps of a genome are essential tools for structural, functional and applied genomics. Genetic maps allow the detection of quantitative trait loci (QTLs), the characterisation of QTL effects and facilitate marker-assisted selection (MAS). The characterisation of genome structure and analysis of evolution is augmented by physical maps. Whole genome physical maps or ultimately complete genomic sequences, respectively, of a species display frameworks that provide essential information for understanding processes in respect to physiology, morphology, development and genetics. However, comprehensive annotation underpins the values a genome sequence or physical map represents. An important task of genome annotation is the linkage of genetic traits to the genome sequence, which is facilitated by integrated genetic and physical maps. In the context of this study several sugar beet (Beta vulgaris L.) genomic tools were developed and applied for evolutionary studies and linkage analysis. A new technique allowing high-throughput identification and genotyping of genetic markers was developed, utilising representational oligonucleotide microarray analysis (ROMA). We tested the performance of the method in sugar beet as a model for crop plants with little sequence information available. Genomic representations of both parents of a mapping population were hybridised on microarrays containing custom oligonucleotides based on sugar beet bacterial artificial chromosome (BAC) end sequences (BESs) and expressed sequence tags (ESTs). Subsequent analysis identified potential polymorphic oligonucleotides, which were placed on new microarrays used for screening of 184 F2 individuals. Exploiting known co-dominant anchor markers, we obtained 511 new dominant markers distributed over all nine sugar beet linkage groups and calculated genetic maps. Besides the method´s transferability to other species, the obtained genetic markers will be an asset for ordering of sequence contigs in the context of the ongoing sugar beet genome sequencing project. In addition, possible linkage of physical and genetic maps was provided, since genetic markers were based on source sequences, which were also used for construction of a BAC based physical map utilising a hybridisation approach. An example of the hybridisation based approach for physical map construction and its application for synteny studies was demonstrated. Since little is known about synteny between rosids and Caryophyllales so far, we analysed the extent of synteny between the genomic sequences of two BAC clones derived from two different Beta vulgaris haplotypes and rosid genomes. For selection of the two BAC clones we hybridised 30 oligonucleotide probes based on ESTs corresponding to Arabidopsis orthologs on chromosomes 1 and 4 that were presumably co-localised in the reconstructed Arabidopsis pseudo ancestral genome (Blanc et al. 2003) on sugar beet BAC macroarrays comprising two different sugar beet libraries. A total of 27,648 clones were screened per sugar beet library, corresponding to 4.4-fold and 5.5-fold, respectively, sugar beet genome coverage. We obtained four and five positive clones for the probes on average. Two clones, one from each haplotype that were positive with the same five EST probes, were selected and their genomic sequences were determined, annotated and exploited for synteny studies. Furthermore, I constructed and characterised a sugar beet fosmid library from the doubled haploid accession KWS2320 encompassing 115,200 independent clones. The insert size of the fosmid library was determined by pulsed field gel electrophoresis to be 39 kbp on average, thus representing 5.9-fold coverage of the sugar beet genome. Fosmids bear the advantage of narrowly defined size of the clone inserts, thus fosmid end sequences will essentially contribute to the future assembly and ordering of sequence contigs. Since repeats are a major obstacle for successful assembly of plant genome sequences, frequently causing gaps and misassembled contigs, I generated a genomic short-insert library. The short-insert library facilitated repeat identification within the sugar beet genome, which was exemplarily shown for three miniature inverted-repeat transposable element (MITE) families. Altogether this work contributed substantially to a deeper understanding of the genome structure of sugar beet and provided the basis for successful sequencing of the sugar beet genome

The characterisation of the peanut agglutinin an evolved plant lectin, with improved specificity to the Thompson Freidenriech antigen

Includes abstract.Includes bibliographical references.Peanut agglutinin (PNA), a carbohydrate binding protein, is able to recognise and bind a number of distinct carbohydrate structures that have been implicated in a number of disease pathologies in humans. In vitro studies of PNA have previously been shown to have some specificity for the Thomson Freidenriech antigen (T-antigen), found on malignant human cells, and this specificity has made PNA an important target for protein engineering experiments aimed at improving its specificity and affinity. A number of tumour cells are characterised by altered states and patterns of glycosylation on cell surfaces and suitably engineered lectins may be able to recognise tumour specific carbohydrate structures. This study was aimed at carrying out the biophysical characterisation of a set of PNA mutants which showed apparent improvement in specificity for the T-Antigen. Previous studies have aimed to engineer this lectin in order to direct its recognition properties towards the T-antigen and away from lactose, the preliminary binding affinities of these mutants being determined using Surface Plasmon Resonance (SPR). Here a set of PNA mutants were characterised, proteins expressed and purified to determine binding activities to the T-antigen, N-Acetyl-Dlactosamine (LacNAc) and lactose through the use of Protein Micro Array technology as well as Enzyme linked immunosorbant assays (ELISA)