Dissertations / Theses on the topic 'Eukaryotic gene'

Die Aufklärung der Mechanismen zur Kontrolle der Genexpression ist eines der wichtigsten Probleme der modernen Molekularbiologie. Detaillierte experimentelle Untersuchungen sind enorm aufwändig aufgrund der komplexen und kombinatorischen Wechselbeziehungen der beteiligten Moleküle. Infolgedessen sind bioinformatische Methoden unverzichtbar. Diese Dissertation stellt drei Methoden vor, die die Vorhersage der regulatorischen Elementen der Gentranskription verbessern. Der erste Ansatz findet Bindungsstellen, die von den Transkriptionsfaktoren erkannt werden. Dieser sucht statistisch überrepräsentierte kurze Motive in einer Menge von Promotersequenzen und wird erfolgreich auf das Genom der Bäckerhefe angewandt. Die Analyse der Genregulation in höheren Eukaryoten benötigt jedoch fortgeschrittenere Techniken. In verschiedenen Datenbanken liegen Hunderte von Profilen vor, die von den Transkriptionsfaktoren erkannt werden. Die Ähnlichkeit zwischen ihnen resultiert in mehrfachen Vorhersagen einer einzigen Bindestelle, was im nachhinein korrigiert werden muss. Es wird eine Methode vorgestellt, die eine Möglichkeit zur Reduktion der Anzahl von Profilen bietet, indem sie die Ähnlichkeiten zwischen ihnen identifiziert. Die komplexe Natur der Wechselbeziehung zwischen den Transkriptionsfaktoren macht jedoch die Vorhersage von Bindestellen schwierig. Auch mit einer Verringerung der zu suchenden Profile sind die Resultate der Vorhersagen noch immer stark fehlerbehafted. Die Zuhilfenahme der unabhängigen Informationsressourcen reduziert die Häufigkeit der Falschprognosen. Die dritte beschriebene Methode schlägt einen neuen Ansatz vor, die die Gen-Anotation mit der Regulierung von multiplen Transkriptionsfaktoren und den von ihnen erkannten Bindestellen assoziiert. Der Nutzen dieser Methode wird anhand von verschiedenen wohlbekannten Sätzen von Transkriptionsfaktoren demonstriert.<br>Understanding the mechanisms which control gene expression is one of the fundamental problems of molecular biology. Detailed experimental studies of regulation are laborious due to the complex and combinatorial nature of interactions among involved molecules. Therefore, computational techniques are used to suggest candidate mechanisms for further investigation. This thesis presents three methods improving the predictions of regulation of gene transcription. The first approach finds binding sites recognized by a transcription factor based on statistical over-representation of short motifs in a set of promoter sequences. A succesful application of this method to several gene families of yeast is shown. More advanced techniques are needed for the analysis of gene regulation in higher eukaryotes. Hundreds of profiles recognized by transcription factors are provided by libraries. Dependencies between them result in multiple predictions of the same binding sites which need later to be filtered out. The second method presented here offers a way to reduce the number of profiles by identifying similarities between them. Still, the complex nature of interaction between transcription factors makes reliable predictions of binding sites difficult. Exploiting independent sources of information reduces the false predictions rate. The third method proposes a novel approach associating gene annotations with regulation of multiple transcription factors and binding sites recognized by them. The utility of the method is demonstrated on several well-known sets of transcription factors. RNA interference provides a way of efficient down-regulation of gene expression. Difficulties in predicting efficient siRNA sequences motivated the development of a library containing siRNA sequences and related experimental details described in the literature. This library, presented in the last chapter, is publicly available at http://www.human-sirna-database.net

Using the Gillespie algorithm, the export of the mRNA molecules from their transcription site to the nuclear pore complex is simulated. The effect of various structures in the nu- cleus on the efficiency of export is discussed. The results show that having some of the space filled by chromatin near the mRNA synthesis site shortens the transport time. Next, the complete eukaryotic gene expression including transcription, splicing, mRNA export, translation, and mRNA degradation is modeled using delay stochastic simulation. This allows for the study of stochastic effects during the process and on the protein production rate patterns. Various protein production patterns can be produced by adjusting the poly-A tail length of the mRNA and the promoter efficiency of the gene. After that, the opposing effects of the chromatin density on the seeking time of the transcription factors for the promoter and the exit time of the mRNA product are discussed.<br>xi, 102 leaves ; 28 cm

We studied ectopic gene conversions, i.e., gene conversions between duplicated genes located at different chromosomal positions, in eukaryotic genomes. In the first part we examined the factors affecting ectopic gene conversions in the human genome and compared their characteristics to those observed in other eukaryotic and prokaryotic species. In the second part, we examined the effect that ectopic conversions have on the GC-content of the duplicated genes found in yeast and Arabidopsis genomes. Using Stanley Sawyer's method implemented in his GENCONV program, we identified and characterized the ectopic gene conversions of the human genome. The human gene families containing 3 or more members contained 483 pairs of converted genes. The average length of conversions is 371+/-752 (+/- standard deviation) nucleotides long with the smallest conversions being 10 nucleotides long and the largest 6011 nucleotides long. Larger gene conversions are found between sequences that are more similar and the frequency of intra-chromosomal gene conversion increases as the distance between genes decreases. Pairs of intra-chromosomal genes sharing the same transcriptional orientation convert more often than intra-chromosomal genes in opposite transcriptional orientation. The excess of conversions in the 3'-end suggest incomplete cDNA molecules are often involved in gene conversions with chromosomal gene copies. Allelic recombination has previously been shown to increase the GC-content of the sequences of a wide variety of eukaryotic species. Ectopic recombination between clustered tandemly repeated genes has also been shown to increase their GC-content. Here we show that gene conversions between the dispersed genes found in the duplicated regions of the yeast and Arabidopsis genomes also increases their GC-content when these genes are more than 88% similar.

L'incessant augment del nombre de seqüències genòmiques, juntament amb <br/>l'increment del nombre de tècniques experimentals de les que es disposa, <br/>permetrà obtenir el catàleg complet de les funcions cel.lulars de <br/>diferents organismes, incloent-hi la nostra espècie. Aquest catàleg <br/>definirà els fonaments sobre els que es podrà entendre millor com els <br/>organismes funcionen a nivell molecular. Al mateix temps es tindran més <br/>pistes sobre els canvis que estan associats amb les malalties. Per tant, <br/>la seqüència en brut, tal i com s'obté dels projectes de seqüenciació de <br/>genomes, no té cap valor sense les anàlisis i la subsegüent anotació de <br/>les característiques que defineixen aquestes funcions. Aquesta tesi <br/>presenta la nostra contribució en tres aspectes relacionats de <br/>l'anotació dels gens en genomes eucariotes.<br/> <br/>Primer, la comparació a nivell de seqüència entre els genomes humà i de <br/>ratolí es va dur a terme mitjançant un protocol semi-automàtic. El <br/>programa de predicció de gens SGP2 es va desenvolupar a partir <br/>d'elements d'aquest protocol. El concepte al darrera de l'SGP2 és que <br/>les regions de similaritat obtingudes amb el programa TBLASTX, es fan <br/>servir per augmentar la puntuació dels exons predits pel programa <br/>geneid, amb el que s obtenen conjunts d'anotacions més acurats <br/>d'estructures gèniques. SGP2 té una especificitat que és prou gran com <br/>per que es puguin validar experimentalment via RT-PCR. La validació de <br/>llocs d'splicing emprant la tècnica de la RT-PCR és un bon exemple de <br/>com la combinació d'aproximacions computacionals i experimentals <br/>produeix millors resultats que per separat.<br/> <br/>S'ha dut a terme l'anàlisi descriptiva a nivell de seqüència dels llocs <br/>d'splicing obtinguts sobre un conjunt fiable de gens ortòlegs per humà, <br/>ratolí, rata i pollastre. S'han explorat les diferències a nivell de <br/>nucleòtid entre llocs U2 i U12, pel conjunt d'introns ortòlegs que se'n <br/>deriva d'aquests gens. S'ha trobat que els senyals d'splicing ortòlegs <br/>entre humà i rossegadors, així com entre rossegadors, estan més <br/>conservats que els llocs no relacionats. Aquesta conservació addicional <br/>pot ser explicada però a nivell de conservació basal dels introns. <br/>D'altra banda, s'ha detectat més conservació de l'esperada entre llocs <br/>d'splicing ortòlegs entre mamífers i pollastre. Els resultats obtinguts <br/>també indiquen que les classes intròniques U2 i U12 han evolucionat <br/>independentment des de l'ancestre comú dels mamífers i les aus. Tampoc <br/>s'ha trobat cap cas convincent d'interconversió entre aquestes dues <br/>classes en el conjunt d'introns ortòlegs generat, ni cap cas de <br/>substitució entre els subtipus AT-AC i GT-AG d'introns U12. Al contrari, <br/>el pas de GT-AG a GC-AG, i viceversa, en introns U2 no sembla ser inusual.<br/> <br/>Finalment, s'han implementat una sèrie d'eines de visualització per <br/>integrar anotacions obtingudes pels programes de predicció de gens i per <br/>les anàlisis comparatives sobre genomes. Una d'aquestes eines, el <br/>gff2ps, s'ha emprat en la cartografia dels genomes humà, de la mosca del <br/>vinagre i del mosquit de la malària, entre d'altres. El programa <br/>gff2aplot i els filtres associats, han facilitat la tasca d'integrar <br/>anotacions de seqüència amb els resultats d'eines per la cerca <br/>d'homologia, com ara el BLAST. S'ha adaptat també el concepte de <br/>pictograma a l'anàlisi comparativa de llocs d splicing ortòlegs, amb el <br/>desenvolupament del programa compi.<br>El aumento incesante del número de secuencias genómicas, junto con el <br/>incremento del número de técnicas experimentales de las que se dispone, <br/>permitirá la obtención del catálogo completo de las funciones celulares <br/>de los diferentes organismos, incluida nuestra especie. Este catálogo <br/>definirá las bases sobre las que se pueda entender mejor el <br/>funcionamiento de los organismos a nivel molecular. Al mismo tiempo, se <br/>obtendrán más pistas sobre los cambios asociados a enfermedades. Por <br/>tanto, la secuencia en bruto, tal y como se obtiene en los proyectos de <br/>secuenciación masiva, no tiene ningún valor sin los análisis y la <br/>posterior anotación de las características que definen estas funciones. <br/>Esta tesis presenta nuestra contribución a tres aspectos relacionados de <br/>la anotación de los genes en genomas eucariotas.<br/> <br/>Primero, la comparación a nivel de secuencia entre el genoma humano y el <br/>de ratón se llevó a cabo mediante un protocolo semi-automático. El <br/>programa de predicción de genes SGP2 se desarrolló a partir de elementos <br/>de dicho protocolo. El concepto sobre el que se fundamenta el SGP2 es <br/>que las regiones de similaridad obtenidas con el programa TBLASTX, se <br/>utilizan para aumentar la puntuación de los exones predichos por el <br/>programa geneid, con lo que se obtienen conjuntos más precisos de <br/>anotaciones de estructuras génicas. SGP2 tiene una especificidad <br/>suficiente como para validar esas anotaciones experimentalmente vía <br/>RT-PCR. La validación de los sitios de splicing mediante el uso de la <br/>técnica de la RT-PCR es un buen ejemplo de cómo la combinación de <br/>aproximaciones computacionales y experimentales produce mejores <br/>resultados que por separado.<br/> <br/>Se ha llevado a cabo el análisis descriptivo a nivel de secuencia de los <br/>sitios de splicing obtenidos sobre un conjunto fiable de genes ortólogos <br/>para humano, ratón, rata y pollo. Se han explorado las diferencias a <br/>nivel de nucleótido entre sitios U2 y U12 para el conjunto de intrones <br/>ortólogos derivado de esos genes. Se ha visto que las señales de <br/>splicing ortólogas entre humanos y roedores, así como entre roedores, <br/>están más conservadas que las no ortólogas. Esta conservación puede ser <br/>explicada en parte a nivel de conservación basal de los intrones. Por <br/>otro lado, se ha detectado mayor conservación de la esperada entre <br/>sitios de splicing ortólogos entre mamíferos y pollo. Los resultados <br/>obtenidos indican también que las clases intrónicas U2 y U12 han <br/>evolucionado independientemente desde el ancestro común de mamíferos y <br/>aves. Tampoco se ha hallado ningún caso convincente de interconversión <br/>entre estas dos clases en el conjunto de intrones ortólogos generado, ni <br/>ningún caso de substitución entre los subtipos AT-AC y GT-AG en intrones <br/>U12. Por el contrario, el paso de GT-AG a GC-AG, y viceversa, en <br/>intrones U2 no parece ser inusual.<br/> <br/>Finalmente, se han implementado una serie de herramientas de <br/>visualización para integrar anotaciones obtenidas por los programas de <br/>predicción de genes y por los análisis comparativos sobre genomas. Una <br/>de estas herramientas, gff2ps, se ha utilizado para cartografiar los <br/>genomas humano, de la mosca del vinagre y del mosquito de la malaria. El <br/>programa gff2aplot y los filtros asociados, han facilitado la tarea de <br/>integrar anotaciones a nivel de secuencia con los resultados obtenidos <br/>por herramientas de búsqueda de homología, como BLAST. Se ha adaptado <br/>también el concepto de pictograma al análisis comparativo de los sitios <br/>de splicing ortólogos, con el desarrollo del programa compi.<br>The constantly increasing amount of available genome sequences, along <br/>with an increasing number of experimental techniques, will help to <br/>produce the complete catalog of cellular functions for different <br/>organisms, including humans. Such a catalog will define the base from <br/>which we will better understand how organisms work at the molecular <br/>level. At the same time it will shed light on which changes are <br/>associated with disease. Therefore, the raw sequence from genome <br/>sequencing projects is worthless without the complete analysis and <br/>further annotation of the genomic features that define those functions. <br/>This dissertation presents our contribution to three related aspects of <br/>gene annotation on eukaryotic genomes.<br/> <br/>First, a comparison at sequence level of human and mouse genomes was <br/>performed by developing a semi-automatic analysis pipeline. The SGP2 <br/>gene-finding tool was developed from procedures used in this pipeline. <br/>The concept behind SGP2 is that similarity regions obtained by TBLASTX <br/>are used to increase the score of exons predicted by geneid, in order to <br/>produce a more accurate set of gene structures. SGP2 provides a <br/>specificity that is high enough for its predictions to be experimentally <br/>verified by RT-PCR. The RT-PCR validation of predicted splice junctions <br/>also serves as example of how combined computational and experimental <br/>approaches will yield the best results.<br/> <br/>Then, we performed a descriptive analysis at sequence level of the <br/>splice site signals from a reliable set of orthologous genes for human, <br/>mouse, rat and chicken. We have explored the differences at nucleotide <br/>sequence level between U2 and U12 for the set of orthologous introns <br/>derived from those genes. We found that orthologous splice signals <br/>between human and rodents and within rodents are more conserved than <br/>unrelated splice sites. However, additional conservation can be <br/>explained mostly by background intron conservation. Additional <br/>conservation over background is detectable in orthologous mammalian and <br/>chicken splice sites. Our results also indicate that the U2 and U12 <br/>intron classes have evolved independently since the split of mammals and <br/>birds. We found neither convincing case of interconversion between these <br/>two classes in our sets of orthologous introns, nor any single case of <br/>switching between AT-AC and GT-AG subtypes within U12 introns. In <br/>contrast, switching between GT-AG and GC-AG U2 subtypes does not appear <br/>to be unusual.<br/> <br/>Finally, we implemented visualization tools to integrate annotation <br/>features for gene- finding and comparative analyses. One of those tools, <br/>gff2ps, was used to draw the whole genome maps for human, fruitfly and <br/>mosquito. gff2aplot and the accompanying parsers facilitate the task of <br/>integrating sequence annotations with the output of homologybased tools, <br/>like BLAST.We have also adapted the concept of pictograms to the <br/>comparative analysis of orthologous splice sites, by developing compi.

Mathematical modelling of gene regulatory networks is a relatively new area which is playing an important role in theoretical and experimental investigations that seek to open the door to understanding the real mechanisms that take place in living systems. The current thesis concentrates on studying the animal stress-response gene regulatory network by seeking to predict the consequence of environmental hazards caused by chemical mixtures (typical of industrial pollution). Organisms exposed to pollutants display multiple defensive stress responses, which together constitute an interlinked gene network (the Stress-Response Network; SRN). Multiple SRN reporter-gene outputs have been monitored during single and combined chemical exposures in transgenic strains of two invertebrates, Caenorhabditis elegans and Drosophila melanogaster. Reporter expression data from both species have been integrated into mathematical models describing the dynamic behaviour of the SRN and incorporating its known regulatory gene circuits. We describe some mathematical models of several types of different stress response networks, incorporating various methods of activation and inhibition, including formation of complexes and gene regulation (through several known transcription factors). Although the full details of the protein interactions forming these types of circuits are not yet well-known, we seek to include the relevant proteins acting in different cellular compartments. We propose and analyse a number of different models that describe four different stress response gene networks and through a combination of analytical (including stability, bifurcation and asymptotic) and numerical methods, we study these models to gain insight on the effect of several stresses on gene networks. A detailed time-dependent asymptotic analysis is performed for relevant models in order to clarify the roles of the distinct biochemical reactions that make up several important proteins production processes. In two models we were able to verify the theoretical predictions with the corresponding laboratory experimental observations that carried out by my coworkers in Britain and India.

Gene regulatory networks are dynamic and continuously remodelled in response to internal and external stimuli. To understand how these networks alter cellular phenotype in response towards specific challenges, my first project sought to develop a methodology to explore how the strength of genetic interactions changes according to environmental context. Defined as sensitivity-based epistasis, the results obtained using this methodology were compared to those generated under the conventional fitness-based approach. By integrating this information with gene expression profiles and physical interaction datasets, we demonstrate that sensitivity-based epistasis specifically highlights genetic interactions with a dynamic component. Having investigated how an external stimulus regulates network dynamics, we next sought to understand of how genome positioning impacts transcription kinetics. This feat was accomplished by cloning two gene-reporter constructs, representing contrasting promoter architectures, across 128 loci along chromosome III in S.Cerevisiae. By comparing expression and noise measurements for promoters with “covered” and “open” chromatin structures against a stochastic model for eukaryotic gene expression, we demonstrate that while promoter structure regulates burst frequency (the rate of promoter activation), positional effects in turn appear to primarily modulate burst size (the number of mRNA produced per gene activation event). By integrating these datasets with information describing global chromatin structure, we suggest that the acetylation state of chromatin regulates burst size across the genome. Interestingly, this hypothesis is further supported by nicotinamide-mediated inhibition of Sir2 which would appear to modulate burst size globally across the genome.

Thesis (D.Sc.D.)--Boston University, Henry M. Goldman School of Graduate Dentistry, 1992 (Oral Biology).<br>Includes bibliographical references (leaves 70-84).<br>The ALG7 gene encodes the tunicamycin-sensitive, dolichol-P-dependent Nacetylglucosamine- 1-phosphate transferase, GPT, that catalyzes the synthesis of the first dolichollinked sugar, Dol-PP-GlcNAc, in the N-glycosylation pathway. ALG7 has been evQlutionarily conserved and is essential for growth in all eukaryotes. The ALG7 gene expression in yeast is known to be regulated in part by the 3' untranslated regions (UTR) of the ALG7 multiple transcripts at the posttranscriptional level. To examine the regulatory features of the mammalian ALG7 gene, cloning and characterization of the hamster ALG7 mRNAs were undertaken. Polymerase chain reaction (PCR) using a single ALG7 gene-specific primer was performed to clone the cDNAs corresponding to the 3' and 5' ends of the ALG7 mRNAs from the Chinese hamster ovary (CHO) cells. The initial Northern blot analysis using a hamster ALG7 genomic DNA as a probe has shown that in the CHO cells the ALG7 gene is transcribed into three major messages, approximately 1.5, 1.9, and 2.2 kb in size. The 1.9 kb transcripts were cloned and sequenced. There is one consensus polyadenylation signal AAUAAA located 12 nucleotides (nt) upstream to the major poly(A) site. Three additional minor poly(A) sites are located at 18, 21 and 29 nt downstream from the AAUAAA sequence in this 1.9 kb class of mRNAs. [TRUNCATED]

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.<br>This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.<br>Cataloged from student submitted PDF version of thesis.<br>Includes bibliographical references.<br>Regulation of genes is fundamental to all living processes and can be exerted at many sequential steps. We studied several eukaryotic gene regulatory mechanisms with an emphasis on understanding the interplay between regulatory processes on a genome-wide scale. Gene splicing involves the joining of exonic RNA stretches from within a precursor messenger RNA (mRNA). Splicing typically occurs co-transcriptionally as the pre-mRNA is being produced from the DNA. We explored the relationship between the chromatin state of the gene-encoding DNA and the splicing machinery. We found a marked enrichment for nucleosomes at exonic DNA in human T cells, as compared to surrounding introns, an effect mostly explained by the biased nucleotide content of exons. The use of nucleosome positioning information improved splicing simulation models, suggesting nucleosome positioning may help determine cellular splicing patterns. Additionally, we found several histone marks enriched or depleted at exons compared to the background nucleosome levels, indicative of a histone code for splicing. These results connect the chromatin regulation and mRNA splicing processes in a genome-wide fashion. Another pre-mRNA processing step is cleavage and polyadenylation, which determines the 30 end of the mature mRNA. We found that 3P-Seq was able to quantify the levels of 30 end isoforms, in addition to the method's previous use for annotating mRNA 30 ends. Using 3P-Seq and a transcriptional shutoff experiment in mouse fibroblasts, we investigated the e?effect of nuclear alternative 30 end formation on mRNA stability, typically regulated in the cytoplasm. In genes with multiple, tandem 30 untranslated regions (30 UTRs) produced by alternative cleavage and polyadenylation, we found the shorter UTRs were significantly more stable in general than the longer isoforms. This di?difference was in part explained by the loss of cis-regulatory motifs, such as microRNA targets and PUF-binding sites, between the proximal and distal isoforms. Finally, we characterized the small interfering RNAs (siRNAs) produced from heterochromatic, silenced genomic regions in fission yeast. We observed a considerable bias for siRNAs with a 5' U, and used this bias to infer patterns of siRNA biogenesis. Furthermore, comparisons with between wild-type and the Cid14 non-canonical poly(A) polymerase mutant demonstrated that the exosome, the nuclear surveillance and processing complex, is required for RNA homeostasis. In the absence of a fully functional exosome complex, siRNAs are produced to normal exosome targets, including ribosomal and transfer RNAs, indicating these processes may compete for substrates and underscoring the interconnectedness of gene regulatory systems.<br>by Noah Spies.<br>Ph.D.

Using a generalized-clique hidden Markov model (HMM) as the starting point for a eukaryotic gene finder, the objective here is to strengthen the signal information at the transitions between coding and non-coding (c/nc) regions. This is done by enlarging the primitive hidden states associated with individual base labeling (as exon, intron, or junk) to substrings of primitive hidden states or footprint states. Moreover, the allowed footprint transitions are restricted to those that include either one c/nc transition or none at all. (This effectively imposes a minimum length on exons and the other regions.) These footprint states allow the c/nc transitions to be seen sooner and have their contributions to the gene-structure identification weighted more heavily – yet contributing as such with a natural weighting determined by the HMM model itself according to the training data – rather than via introducing an artificial gain-parameter tuning on major transitions. The selection of the generalized HMM model is interpolated to highest Markov order on emission probabilities, and to highest Markov order (subsequence length) on the footprint states. The former is accomplished via simple count cutoff rules, the latter via an identification of anomalous base statistics near the major transitions using Shannon entropy. Preliminary indications, from applications to the C. elegans genome, are that the sensitivity/specificity (SN/SP) result for both the individual state and full exon predictions are greatly enhanced using the generalized-clique HMM when compared to the standard HMM. Here the standard HMM is represented by the choice of the smallest size of footprint state in the generalized-clique HMM. Even with these improvements, we observe that both extremely long and short exon and intron segments would go undetected without an explicit model of the duration of state. The key contributions of this effort are the full derivation and experimental confirmation of a rudimentary, yet powerful and competitive gene finding method based on a higher order hidden Markov model. With suitable extensions, this method is expected to provide superior gene finding capability – not only in the context of pre-conditioned data sets as in the evaluations cited but also in the wider context of less preconditioned and/or raw genomic data.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009.<br>Committee Chair: Mark Borodovky; Committee Member: Jung H. Choi; Committee Member: King Jordan; Committee Member: Leonid Bunimovich; Committee Member: Yury Chernoff. Part of the SMARTech Electronic Thesis and Dissertation Collection.

This study examined the role of <I>PYKI </I>coding sequences in the expression of <I>PYK1::lacZ </I>gene fusions in <I>Saccharomyces cerevisiae. </I>Further aims were to examine the effects of the vector system upon the mRNA levels from these gene fusions the effect that these gene fusions have upon the yeast cell in general. Analysis of the <I>PYK1::lacZ </I>gene fusions revealed that <I>PYK1 </I>coding sequences were responsible for elevating mRNA levels. This elevation was not due to a single element within the coding region of the <I>PYK1 </I>gene as had been previously proposed (Purvis <I>et al.,</I> 1987a; Lithgow, 1989). Models for the stimulatory action of the <I>PYK1 </I>coding region upon the transcription of the <I>PYK1::lacZ</I> gene fusion were presented. <I>PYKI </I>coding region fragments in the <I>PYK1::lacZ</I> gene fusions stabilized the mRNA, but the data presented here were not consistent with a stability element within the <I>PYK1 </I>coding region. An alternative model was presented whereby the translation rate of the mRNA can influence its decay. The effect of expression of these gene fusions upon the yeast cell in general was monitored by examining the mRNA level of two chromosomal loci, <I>PYK1 </I>and <I>PGK1, </I>and by measuring the generation time. In contrast to previous findings, <I>PYK1 </I>and <I>PGK1 </I>mRNA levels were found not to change and so it was concluded that expression of these gene fusions had no general effect upon transcription or mRNA decay. However expression of these gene fusions did lead to an increase in generation time, and it was proposed that this might be due to a general effect upon translation brought about by a reduction in the intracellular pools of tRNAs for non-preferred codons.

Transcriptional elongation is a crucial step in eukaryotic gene regulation whose mis-regulation leads to cellular pathologies. This makes it quite imperative to aim for a better understanding of the processes regulating transcriptional elongation. An important process promoting the association of RNA Polymerase II (RNAPII) with the coding region of the active gene and hence transcriptional elongation is the monoubiquitination of histone H2B at lysine 123. A complex of an E2 conjugase, Rad6p, and an E3 ligase, Bre1p, is essential for this process. Consistent with the role of histone H2B monoubiquitination in promoting the association of RNAPII with the active gene, this process was found to be impaired in the absence of Rad6p or point mutation of lysine 123 to arginine (H2B-K123R). Intriguingly, the association of RNAPII with the coding region of the active gene was not impaired in the absence of Bre1p, even though Bre1p is essential for histone H2B monoubiquitination. However, deletion of Bre1p’s RING domain that is essential for histone H2B monoubiquitination led to an impaired RNAPII association with the active gene. This observation indicates a role of the non-RING domain of Bre1p in repressing the association of RNAPII with the active gene, resulting in no net decrease in RNAPII occupancy in the absence of Bre1p. Taken together, my results implicated both the stimulatory and repressive roles of the histone H2B ubiquitin ligase Bre1p in regulation of RNAPII association with the coding regions of active genes and hence transcriptional elongation. Interestingly, my work also revealed that for efficient transcriptional elongation by histone H2B monoubiquitination, its optimum level needs to be maintained by a proper balance between Rad6p-Bre1p-mediated ubiquitination and de-ubiquitination (DUB) by the DUB module of SAGA. It was found that Sus1p, a subunit of the DUB module, promotes transcriptional elongation, DNA repair and replication via regulation of histone H2B DUB. In addition to Rad6p- Bre1p and the DUB module, global level of histone H2B monoubiquitination is also critically regulated by Cdk9, a kinase essential for phosphorylation of the serine 2 residue in the C-terminal domain (CTD) of RNAPII, which promotes transcriptional elongation. Apart from serine phosphorylation, proline residues at RNAPII-CTD undergo isomerization by proline isomerases, which also regulate transcription. One of the proline isomerases, Rrd1p, has been previously implicated in transcription in response to rapamycin treatment. Based on this fact and Rrd1p’s known interaction with RNAPII-CTD, we predicted that Rrd1p might regulate transcription independently of rapamycin treatment. In agreement with this hypothesis, our work revealed Rrd1p’s role in facilitating transcription of both rapamycin responsive and non-responsive genes in the absence of rapamycin treatment. Consistently, the absence of Rrd1p led to an impaired nucleosomal disassembly at the active gene, which correlates with the role of Rrd1p in promoting transcription. This is because maintenance of proper nucleosomal dynamics is essential for efficient transcription. It is known that transcriptional elongation is facilitated by the regulation of nucleosomal dynamics via the histone chaperone, FACT. Efficient chromatin reassembly in the wake of elongating RNAPII contributing to the fidelity of transcription is promoted by FACT. Being evolutionarily conserved among eukaryotes, FACT is also known to regulate DNA replication and repair, apart from transcription. Intriguingly, FACT has been found to be upregulated in cancers while its downregulation leads to tumor cell death. However, the mechanism which fine-tunes FACT for normal cellular functions remained unknown. My studies revealed a novel mechanism of regulation of FACT by the ubiquitin-proteasome system in yeast. San1p, an E3 ligase involved in nuclear protein quality control, was found to associate with the active gene and regulate transcriptional elongation through its E3 ligase activity- mediated turnover of Spt16p component of FACT. This regulation was found to maintain optimum level of Spt16p/FACT to engage with the active gene for proper transcriptional elongation, DNA repair and replication. In spite of playing such crucial roles in gene regulation, it was not known how FACT is targeted to the active gene. We discovered that a direct physical interaction between FACT and Cet1p, the mRNA capping enzyme, targets FACT to the active gene independently of Cet1p’s mRNA capping activity. Such targeting of FACT to the active gene leads to the release of promoter proximally paused-RNAPII into transcriptional elongation. However, the progress of RNAPII along the active gene during transcriptional elongation is frequently impeded by various kinds of damages along the underlying template DNA. Even though some of these lesions are co-transcriptionally repaired, it was not known whether the repair of extremely toxic DNA double-strand breaks (DSBs) was coupled to transcription. My results showed that DSBs at the transcriptionally active state of a gene are repaired faster than at the inactive state but such repair was not mediated by a co-transcriptional recruitment of DSB repair factors. This observation is in contrast to other DNA repair pathways such as nucleotide excision repair (NER) where repair factors are co-transcriptionally recruited to the lesion containing DNA. In this regard, we found that an NER factor, Rad14p, co-transcriptionally associates with the active gene in the absence of DNA damage to promote transcription, which unraveled a new role of Rad14p in transcription in addition its established role in NER. In summary, my results provide significant novel insights into the regulation of transcriptional elongation and associated processes leading to better understanding of eukaryotic gene expression.

Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2008.<br>Committee Chair: Jordan, I. King; Committee Member: Borodovsky, Mark; Committee Member: Bunimovich, Leonid; Committee Member: Choi, Jung; Committee Member: McDonald, John.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2010.<br>Cataloged from PDF version of thesis.<br>Includes bibliographical references.<br>Transcription of mRNA appears to occur in random, intermittent bursts in a large variety of organisms. The statistics of mRNA expression can be described by two parameters: the frequency at which bursts occur (burst frequency) and the average number of mRNA produced within each burst (burst size). The mean steady-state abundance of mRNA is the product of the burst size and burst frequency. Although the experimental evidence for bursty gene transcription is abundant, little is known about its origins and consequences. We utilize single-molecule mRNA imaging and simple stochastic kinetic models to probe and understand both the mechanistic details and functional responses of transcriptional bursting in budding yeast. At the molecular level, we show that gene-specific activators can control both burst size and burst frequency by differentially utilizing kinetically distinct promoter elements. We also recognize the importance of activator residence time and nucleosome positioning on bursting. This investigation exemplifies how we can exploit spontaneous fluctuations in gene expression to uncover the molecular mechanisms and kinetic pathways of transcriptional regulation. At the network level, we demonstrate the important phenotypic consequences of transcriptional bursting by showing how noise itself can generate a bimodal, all-or-none gene expression profile that switches spontaneously between the low and high expression states in a transcriptional positive-feedback loop. Such bimodality is a hallmark in decision-making circuitry within metabolic, developmental, and synthetic gene regulatory networks. Importantly, we prove that the bimodal responses observed in our system are not due to deterministic bistability, which is an often-stated necessary condition for allor- none responses in positive-feedback loops. By clarifying a common misconception, this investigation provides unique biological insights into the molecular components, pathways and mechanisms controlling a measured phenotype.<br>by Tsz-Leung To.<br>Ph.D.

Gene finding in eukaryotic genomes is an essential part of a comprehensive approach to modern systems biology. Most methods developed in the past rely on a combination of computational prediction and external information about gene structures from transcript sequences and comparative genomics. In the past, external sequence information consisted of a combination of full-length cDNA and expressed sequence tag (EST) sequences. Much improvement in prediction of genes and gene isoforms is promised by availability of RNA-seq data. However, productive use of RNA-seq for gene prediction has been difficult due to challenges associated with mapping RNA-seq reads which span splice junctions to prevalent splicing noise in the cell. This work addresses this difficulty with the development of methods and implementation of two new pipelines: 1/ a novel pipeline for accurate mapping of RNA-seq reads to compact genomes and 2/ a pipeline for prediction of genes using the RNA-seq spliced alignments in eukaryotic genomes. Machine learning methods are employed in order to overcome errors associated with the process of mapping short RNA-seq reads across introns and using them for determining sequence model parameters for gene prediction. In addition to the development of these new methods, genome annotation work was performed on several plant genome projects.

Eukaryotic gene expression is a highly synchronized cellular process whose nuclear phase is comprised of transcription, and mRNA processing and export. Transcription can be further comprised of transcription initiation, and elongation. Regulation of transcription initiation, transcription elongation, and mRNA processing and export are crucial for normal cellular function, since misregulation of these processes are associated with various diseases including cancer. Many factors or proteins are associated with these cellular processes which are modulated by different regulatory processes to maintain normal cellular function. Ubiquitin-proteasome system (UPS) is one of the recently studied regulatory processes. Over the years, ubiquitin and 26S proteasome have emerged as important regulatory factors in coordination of transcription and coupled mRNA export. However, the mechanisms as to how the ubiquitin and 26S proteasome regulate transcription and coupled mRNA export have not been clearly elucidated. Therefore, my dissertation has focused on understanding the role of UPS in these important cellular processes: transcription initiation, transcription elongation and mRNA export. The results have shown the non-proteolytic role of 19S RP of 26S proteasome in regulation of transcriptional initiation of SAGA and TFIID-dependent PHO84 gene. It was found that 19S RP facilitates both SAGA- and NuA4-TFIID-dependent transcriptional initiations of PHO84 via increased recruitment of the coactivators SAGA and NuA4 HAT, which promote TFIID-independent and -dependent PIC formation in the presence and absence of an essential nutrient, Pi, in the growth media for transcriptional initiation, respectively. Next, our studies have uncovered the role of UPS in regulation of transcriptional elongation. It was found that E3 ubiquitin ligase, San1, mediated UPS regulation of transcription elongation factor, FACT is required for stimulating nucleosomal reassembly at the coding sequence of active genes for proper transcription elongation. We also found the interaction of FACT with another important transcription elongation factor, Paf1C via NTD (N-ter domain) of Cet1p (mRNA capping enzyme) to regulate transcription elongation.Subsequently, our results revealed a novel regulation of Paf1 component of Paf1C by UPS to regulate its abundance for proper cellular function. Transcription of genes could be blocked by DNA damage which can be repaired by transcription-coupled DNA repair (TCR) pathways. SUMOylation, another PTM (Post-translational modifications) like ubiquitination, is implicated in regulation of many DNA repair pathways including TCR, but it is not clearly understood how SUMOylation and associated enzymes are involved in regulation of such pathways. Here, we revealed the distinct role of SUMO ligases Siz1 and Siz2 in response to several DNA damaging agents such as UV, MMS (methyl methanesulfonate), HU (Hydroxyurea) and H2O2 (Hydrogen peroxide). Finally, we have extended our research works to understand the regulatory mechanisms of mRNA export by UPS. We found the interaction of TREX (Transcription/Export) component Sub2 with Mdm30 (F-box protein) for ubiquitination and proteasomal degradation of Sub2 in a transcription-dependent manner to regulate mRNA export. We also found the role CBC (Cap binding complex) in regulation of nuclear mRNA export. Collectively, the results of this study postulate a better understanding of regulation of transcription initiation, transcription elongation, and mRNA export by UPS.

Next-generation sequencing has generated enormous amount of DNA and RNA sequences that potentially carry volumes of genetic information, e.g. protein-coding genes. The thesis is divided into three main parts describing i) GeneMarkS-2, ii) GeneMarkS-T, and iii) MetaGeneTack. In prokaryotic genomes, ab initio gene finders can predict genes with high accuracy. However, the error rate is not negligible and largely species-specific. Most errors in gene prediction are made in genes located in genomic regions with atypical GC composition, e.g. genes in pathogenicity islands. We describe a new algorithm GeneMarkS-2 that uses local GC-specific heuristic models for scoring individual ORFs in the first step of analysis. Predicted atypical genes are retained and serve as ‘external’ evidence in subsequent runs of self-training. GeneMarkS-2 also controls the quality of training process by effectively selecting optimal orders of the Markov chain models as well as duration parameters in the hidden semi-Markov model. GeneMarkS-2 has shown significantly improved accuracy compared with other state-of-the-art gene prediction tools. Massive parallel sequencing of RNA transcripts by the next generation technology (RNA-Seq) provides large amount of RNA reads that can be assembled to full transcriptome. We have developed a new tool, GeneMarkS-T, for ab initio identification of protein-coding regions in RNA transcripts. Unsupervised estimation of parameters of the algorithm makes unnecessary several steps in the conventional gene prediction protocols, most importantly the manually curated preparation of training sets. We have demonstrated that the GeneMarkS-T self-training is robust with respect to the presence of errors in assembled transcripts and the accuracy of GeneMarkS-T in identifying protein-coding regions and, particularly, in predicting gene starts compares favorably to other existing methods. Frameshift prediction (FS) is important for analysis and biological interpretation of metagenomic sequences. Reads in metagenomic samples are prone to sequencing errors. Insertion and deletion errors that change the coding frame impair the accurate identification of protein coding genes. Accurate frameshift prediction requires sufficient amount of data to estimate parameters of species-specific statistical models of protein-coding and non-coding regions. However, this data is not available; all we have is metagenomic sequences of unknown origin. The challenge of ab initio FS detection is, therefore, twofold: (i) to find a way to infer necessary model parameters and (ii) to identify positions of frameshifts (if any). We describe a new tool, MetaGeneTack, which uses a heuristic method to estimate parameters of sequence models used in the FS detection algorithm. It was shown on several test sets that the performance of MetaGeneTack FS detection is comparable or better than the one of earlier developed program FragGeneScan.

Thesis (Ph.D.)--University of Missouri-Columbia, 2005.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file viewed on (January 25, 2007) Vita. Includes bibliographical references.

A characteristic feature of gene expression in eukaryotes is the addition of a 5' terminal 7-methylguanine "cap" to nascent pre-messenger RNA (mRNA) in the nucleus. It is the 5'capping process, which proves vital to creating a mature mRNA. The synthesis of an mRNA followed by its capping is a complex undertaking which requires a set of protein factors. The capped mRNA is then exclusively bound by a cap-binding complex (CBC). CBC shields mRNA from exonucleases as well as regulates downstream post-transcriptional events, translational initiation and nonsense mediated mRNA decay (NMD). Any misregulation during capping or in the binding of CBC can lead a number of diseases/disorders. Thus, the process and regulation of capping and CBC binding to mRNA are important fields to study the control of gene expression. Over the years, capping apparatus and CBC have been implicated in post-transcriptional regulation. However, it is not yet known whether CBC plays any role in controlling transcriptional initiation or elongation. Thus, the major research focus in my thesis had been to analyze the role of CBC and capping enzymes in regulation of transcriptional initiation and elongation. The results have revealed the role of CBC in stimulating the formation of pre-initiation complex (PIC) at the promoter in vivo via Mot1p (modifier of transcription). Subsequently, we have demonstrated the roles of CBC in transcription elongation, splicing and nuclear export of mRNA. Interestingly, we find that the capping enzyme, Cet1p, decreases promoter proximal accumulation of RNA polymerase II. These results support that Cet1p promotes the release of paused-RNA polymerase II to get engaged into elongating form for productive transcription. Such function of Cet1p appears to be mediated via the Facilitates chromatin transcription (FACT) complex. We find that FACT is targeted to the active gene by the N-terminal domain of Cet1p independently of its capping activity. In the absence of Cet1p, recruitment of FACT to the active gene is impaired, leading to paused-RNA polymerase II. Collectively, the results of my thesis work provide significant insight on the regulation of gene expression by CBC and capping enzyme, Cet1p.

The initial work focused on the interaction between the general coactivator CBP (SID domain) and members of the p160 family of coactivators (AD1 domain), which is a key step in the activation of transcription by nuclear receptors. The solution structure of the CBP SID / SRC1 AD1 complex described in this thesis shows that the two helical domains are intimately associated, with the first helix in SRC1 AD1 and the first three helices in CBP SID forming a four helix bundle, which is capped by the fourth helix of the AD 1 domain. Comparisons with the structure of the related CBP SID / ACTR AD 1 complex showed that while the CBP SID domain adopts a similar fold in complex with different p160 proteins, the topologies of the AD1 domains are strikingly different, a feature that is likely to contribute to functional specificity of these complexes. The second part of the work described here focused on the interaction between the MA-3 domains of the tumour suppressor Pdcd4 and the translation factor eIF4A, which has been shown to inhibit cap-dependent translation. The C-terminal MA-3 domain (Pdcd4 MA-3C) was shown to consist of three atypical HEAT repeats capped by a final helix. This domain was found to interact with the N-terminal domain of eIF4A through a conserved surface region. The comparison of NMR spectra obtained from Pdcd4 MA-3 C and the tandem MA-3 region strongly suggests that the tandem MA-3 region is composed of two equivalent domains connected by a semi-flexible linker. The high resolution structural information obtained provides important insights into the interactions and functional specificity of the protein complexes studied.

Thesis (PhD)--Stellenbosch University, 2008.<br>ENGLISH ABSTRACT: Heterologous gene expression is of considerable interest for the production of proteins of therapeutic and industrial importance. As the nature of recombinant proteins has become more complex and as transformation systems have been established in more species, so the variety of hosts available for expression has increased. Every system available has both advantages and disadvantages. The research presented here highlights the advantages of selecting the most appropriate expression system for different recombinant proteins. Expression of different biocatalytically-relevant enzymes, epoxide hydrolases, halohydrin dehalogenase, laccase and mannanase, in different host systems is undertaken, and expression levels and activity are compared. The development of Yarrowia lipolytica as a whole-cell biocatalyst is described. Y. lipolytica is used for the functional expression of epoxide hydrolases (EHs) and halohydrin dehalogenases. EHs are hydrolytic enzymes that convert epoxides to vicinal diols by ring-opening. Two new fungal EHs from Rhodosporidium toruloides NCYC 3181 and NCYC 3158 (a putative Cryptococcus curvatus strain) were identified and cloned. Additional EHs from different sources, including bacteria, yeasts, fungi and plants, were chosen for expression in Y. lipolytica, in order to determine its suitability as the expression system of choice for the production of EHs. Multi-copy integrants were developed, with the genes under control of the growthphase dependent hp4d promoter. A Saccharomyces cerevisiae strain was developed, expressing the EH from Rhodotorula araucariae1, to compare as a whole-cell biocatalyst with Y. lipolytica. This strain proved to be an exceptionally poor wholecell biocatalyst. All the Y. lipolytica strains developed showed varying levels of activity towards different classes of epoxides. Some strains displayed opposite enantioselectivities, allowing for potential complete conversions of racemic epoxides to the desired enantiomeric product. Halohydrins can be considered direct precursors of epoxides. Halohydrin dehalogenases catalyse the nucleophilic displacement of a halogen ion in halohydrins by a vicinal hydroxyl group, yielding an epoxide, a proton and a halide ion. The HheC gene from Agrobacterium radiobacter AD1, codon-optimised to match the codon usage of Y. lipolytica, was over-expressed in Y. lipolytica by generation of multi-copy integrants, further expanding the use of this organism as a host strain for heterologous production of enzymes. Expression levels were maximised by creating tandem repeats of the introduced HheC gene. The ring-closure activity with 2-chloro- 1-phenylethanol as substrate was demonstrated to be broadly dose-dependent. The b-mannanase gene (man1) from Aspergillus aculeatus MRC11624 was expressed in Y. lipolytica with effective secretion in the presence of its native secretion signal, using the hp4d promoter. The same gene was expressed in Aspergillus niger2 under control of the A. niger glyceraldehyde-3-phosphate dehydrogenase promoter (gpdP) and the Aspergillus awamori glucoamylase terminator (glaT). Following optimisation with copy numbers and culture conditions, maximal activity levels of 26,140 nkat.ml-1 for Y. lipolytica, and 16,596 nkat.ml-1 for A. niger were obtained. Laccases are important enzymes for bioremediation, and the best characterised enzymes are from the fungus Trametes versicolor. The objective of this research was to optimise expression of T. versicolor laccases (lcc1 and lcc2) in A. niger D15 and Pichia pastoris3. The Lcc1 enzyme was less active than Lcc2 in both hosts. P. pastoris secreted 0.4 U.L-1 Lcc1 and 2.8 U.L-1 Lcc2, compared to 2,700 U.L-1 produced by A. niger. The Lcc2 enzyme from recombinant A. niger was subsequently purified and characterised in terms of molecular weight and glycosylation, and compared to the wild-type enzyme purified from T. versicolor. The work presented underscores the requirement for experimentation before finalising the choice of an expression system for the optimal production of the desired protein. Every system available has both advantages and disadvantages, and when considering which system to use for producing a recombinant protein, various factors must be taken into consideration. However, the choice is broad and each decision needs to be made empirically. 1 The construction of the S. cerevisiae epoxide hydrolase production strain was carried out by Dr Neeresh Rohitlall of CSIR Biosciences. The Y. lipolytica epoxide hydrolase strains were constructed by the author. 2 The construction of Man1-producing A. niger strain was done by Dr Shaunita Rose of the University of Stellenbosch. The construction of Y. lipolytica Man1 production strains was done by the author. 3 The expression of T. versicolor laccases in P. pastoris was done by Christina Bohlin of Karlstad University. A niger laccase production strains were created by the author.<br>AFRIKAANSE OPSOMMING: Heteroloë geen uitdrukking is van groot belang vir die produksie van proteïene wat van terapeutiese en industriele belang is. Soos die aard van rekombinante proteïene meer ingewikkeld raak en getransformasie-sisteme vir verskeie spesies gevestig raak, is daar ’n groter verskeidenheid van gashere beskikbaar vir geenuitdrukking. Elke sisteem het beide sy voor- en nadele. Hierdie navorsing beklemtoon die voordele wanneer die mees gepaste uitdrukkingssiteem gekies word. Die uitdrukking van verskeie ensieme van biokatalities belang, epoksiedhidrolases, halohidrien dehalogenase, lakkase en mannanase in verskillende gasheersisteme is onderneem en die uitdrukkingsvlakke en aktiwiteite vergelyk. Die ontwikkeling van Yarrowia lipolytica as ’n heelsel biokatalis word beskryf. Y. lipolytica word gebruik vir die funksionele uitdrukking van epoksiedhidrolases (EHs) en halohidrien dehalogenases. EHs is hidroliseringsensieme wat die epoksiede omskakel na aangrensende diole deur middel van ring-opening. Twee nuwe fungi EHs vanaf Rhodosporidiom toruloides NCYC 3181 en NCYC 3158 (’n moontlike Cryptococcus curvatus) is geïdentifiseer en gekloneer. Verdere EHs van verskillende bronne, insluitend bakterieë, giste, fungi en plante, is gekies vir uitdrukking in Y. lipolytica ten einde sy geskiktheid vir die produksie van EHs te bepaal. Multikopie integrante is ook ontwikkel met gene onder beheer van die groei-fase afhanklike hp4d promotor. ’n Saccharomyces cerevisiae ras is ook ontwikkel vir die uitdrukking van die EH van Rhodotorula araucariae4 sodat dit met Y. lipolytica as ’n heelsel biokatalis vergelyk kan word. Hierdie ras was ‘n buitengewone swak heelsel biokatalis. Al die Y. lipolytica rasse wat ontwikkel is het wisselende aktiwiteitsvlakke teenoor verskillende klasse van epoksiede getoon. Sommige rasse het teenoorgestelde enantio-selektiwiteit getoon en het die potensiaal om rasemiese epoksiede volledig na die gewensde enantiomeriese produk om te skakeling. Halohidriene kan as direkte voorgangers van epoksiede beskou word. Halohidrien dehalogenases kataliseer die nukleofiliese vervanging van ’n halogeen-ioon in halohidriene deur ’n aangrensende hidroksiel groep, wat ’n epoksied, ’n proton en ’n halied-ioon lewer. Die HheC geen van Agrobacterium radiobacter AD1 is kodon– geöptimiseer om te pas by die kodon gebruik van Y. lipolytica en was uitgedruk in Y. lipolytica deur die skep van mulitkopie integrante, ’n verdere verbreeding van die toepaslikheid van die organisme as gasheerras vir die heteroloë produksie van ensieme. Maksimum uitdrukkingsvlakke is bereik deur die skep van opeenvolgende herhalings van die ingevoegde HheC-geen. Daar is ook gewys dat die ring-sluitingsaktiwiteit met 2-chloro-1-pheniel-etanol as substraat meestal dosis-afhanklik is. Die -mannanase geen (man1) van Aspergillus aculeatus MRC11624 is uitgedruk en effektief in Y. lipolytica mbv sy eie uitskeidings sein uitgeskei, met die gebruik van die groei-fase afhanklike hp4d promotor. Dieselfde geen is uitgedruk in Aspergillus niger5 onder beheer van die A. niger gliseraldehied-3-fosfaat dehidrogenase promotor (gpdp) en die Aspergillus awamori glikoamilase termineerder (glaT). Verdere optimisering van kopiegetal en voedingskondisies het gelei tot maksimum aktiwiteitsvlakke van 26,140 nkat.ml-1 vir Y. lipolytica en 16,596 nkat.ml-1 vir A. niger. Lakkases is belangrike ensieme vir bio-remediëring, en die ensieme van die fungus Trametes versicolor is die beste gekarateriseer. Die doelwit van hierdie navorsing was die optimisering van die uitdrukking van T. versicolor lakkases (lcc1 en lcc2) in A. niger en Pichia pastoris6. Die Lcc1 ensiem was minder aktief as Lcc2 in altwee die gashere. P. pastoris het 0.4 U.L-1 Lcc1 en 2.8 U.L-1 Lcc2 onderskeidelik uitgeskei, in vergelyking met 2,700 U.L-1 Lcc2 wat deur A. niger geproduseer is. Die Lcc2 ensiem afkomstig van die rekombinante A. niger is vervolgens gesuiwer en gekarakteriseer met betrekking tot molekulêre massa en glikosilering, en daarna vergelyk met die wilde-tipe ensiem wat deur T. versicolor geproduseer word. Die werk wat hier aangebied word, beklemtoon die vereistes vir eksperimentering voor die finale keuse met betrekking tot ’n gepaste uitdrukkingsisteem gemaak kan word vir die optimale produksie van die gewensde proteïen. Elke sisteem het beide voordele en nadele, en wanneer ’n sisteem oorweeg word is daar verskeie faktore wat in ag geneem moet word. ’n wye verskeidenheid van keuses is beskikbaar en elke besluit moet empiries gemaak word. 4 Die konstruksie van die S. cerevisiae epoksiedhidrolase-produserende ras is deur Dr Neeresh Rohitlall van CSIR Biosciences gedoen. Die Y. lipolytica epoxied hydrolase rasse is deur die outeur gemaak. 5 Die konstruksie van die Man1-produserende A. niger ras is deur Dr Shaunita Rose van die Universiteit van Stellenbosch gemaak. Die Y. lipolytica Man1 ras is gemaak deur die outeur. 6 Die uitdrukking van T. versicolor lakkases in P. pastoris is gedoen deur Christina Bohlin van Karlstad University. Die A niger lakkase produksie ras is geskep deur die outeur.

Thesis (Ph.D.)--Biology, Georgia Institute of Technology, 2009.<br>Committee Chair: Lobachev, Kirill; Committee Co-Chair: Chernoff, Yury; Committee Member: Crouse, Gray; Committee Member: Goodisman, Michael; Committee Member: Streelman, Todd.

How genomes evolve through time and how they change in response to selective pressures remains a key area of research in genomics and evolutionary biology. Genome organization is now known to play a significant role in the regulation of expression patterns with significant clustering according to various parameters of gene expression having been reported in all major taxa. In this thesis I present a comprehensive analysis of sex-biased gene expression in the primate genome and show that there is a significant degree of similarity in sex-biased gene expression among neighbouring genes. Whether this clustering of genes with similar expression profiles is functional or instead the result of transcriptional interference with adjacent genes displaying non-functional but significant similarity in patterns of gene expression has only recently started to be addressed. A recent study suggested that although Drosophila melanogaster exhibits significant similarities in gene expression among neighbouring genes, these clusters are not conserved across evolutionary time in the Drosophila lineage. Chapter three of this thesis presents a comprehensive analysis of the conservation of testis overexpressed gene clusters. I show that, as has been found for other Drosophila expression clusters, testis overexpression clusters are also not conserved throughout evolution. Finally, in chapter 4 I present an analysis of allele frequency changes in the Arabidopsis thaliana genome in experimentally selected lines for early flowering under two different growth conditions resembling winter and spring growth seasons. My results reveal widespread changes in allele frequencies in response to selective pressures with a significant degree of parallel changes for independent lines under selection in similar growth conditions. Importantly, from the point of view of conservation crop efficiency efforts, no significant parallel changes were observed when examining the similarity in allele frequency changes across both growth conditions suggesting that adaptation for a particular trait might be only relevant in specific environmental conditions. Together these observations suggest that allele frequencies change on a global scale in response to selective pressures and that while the observed changes are mirrored across replicas in similar environments this is not the case for lines selected under different growth conditions. Overall, the results I present in this thesis provide valuable insights into how gene order relates to gene expression profiles and its functional relevance as well as presenting evidence for patterns of allele changes in response to selective pressures.

Le ribosome est un complexe macromoléculaire impliqué dans la synthèse protéique de toutes les cellules vivantes. L’étape d’élongation de cette synthèse est un processus itératif débutant par la sélection au sein du ribosome d’un ARNt aminoacylé suivie par le transfert du peptide du site P- vers le site A- et de la translocation de l’ARNm et de l’ARNt. Le facteur d’élongation 2 (eEF2), qui catalyse la translocation, est l’un des acteurs majeur de cette étape d’élongation chez les eucaryotes. Cependant le mécanisme par lequel eEF2 induit ce processus est encore aujourd’hui inconnu. Dans cette étude structurale, nous présentons la première structure à haute résolution (3.1 Å) du complexe de pré-translocation résolu par cristallographie aux rayons X. La structure obtenue nous a permis d’identifier les différents composants du complexe de translocation et de proposer le rôle de l’His699 et celui de la diphtamide, modification post-traductionnelle d’eEF2, lors du stade de pré-translocation<br>Elongation is the longest stage of protein synthesis that takes place on the ribosome and represents a cycle that begins with an aminoacyl-tRNA selection followed by the catalysis of peptide transfer from the P- to the A-site and mRNA-tRNA translocation. Elongation factor 2 (eEF2) is one of the key player of elongation cycle in eukaryotes that catalyzes translocation of mRNA and tRNA on the ribosome. However the mechanism how eEF2 induces translocation on the ribosome is unknown. Current work investigates the structural aspect of protein synthesis machinery in eukaryotes. In particular we present first high resolution structure of functional pretranslocation complex solved at 3.1 A by X-ray crystallography. The obtained structure allowed us to see several features of translocation complex and to propose the role of His699 and post translational modification of eEF2 diphthamide during at pretranslocation stage

Thesis (Ph. D.)--University of California, Riverside, 2010.<br>Includes abstract. Available via ProQuest Digital Dissertations. Title from first page of PDF file (viewed April 24, 2010). Includes bibliographical references. Also issued in print.

The diagnostic value of extracellular occurring DNA (eoDNA) is limited by our lack of understanding its biological function. eoDNA exists in a number of forms, namely vesicle bound DNA, histone/DNA complexes or nucleosomes and virtosomes. These forms of DNA can also be categorized under the terms circulating DNA, cell free DNA, free DNA and extracellular DNA. The DNA can be released by means of form–specific mechanisms and seem to be governed by cell cycle phases and apoptosis. Active release is supported by evidence of energy dependant release mechanisms and various immunological– and messenger functions. Sequencing has shown that eoDNA sequences present in the nucleome reflects traits and distribution of genome sequences and are regulated by ways of release and/or clearance. eoDNA enables the horizontal transfer of gene sequences from one cell to another, over various distances. The ability of eoDNA to partake in horizontal gene transfer makes it an important facet in the field of epigenetic variation. Clinical implementation of eoDNA diagnostics requires that all of the subgroups of eoDNA be properly investigated. It is suggested that eoDNA is the result of the metabolic fraction of DNA that is released by the cell. Various observations indicate that eoDNA may also be incorporated into the genome of a cell, from where it may affect cell function. Therefore horizontal gene transfer in higher organisms is a real possibility. In this study, variations and increases in eoDNA levels over time correlate with stressors that are subjected to 143B human osteosarcoma cells. It seems viable to assume that a stressor is met by a change in the molecular machinery of a cell, required to neutralise the onset of metabolic instability. This may be done by amplification of necessary cistrons, producing metabolic DNA, that may then be observed after its release as eoDNA. The presence of hydrolysing enzymes gives an updated real time picture of the state of eoDNA. The eogenics hypothesis emanating from this study, suggests that amplification and horizontal transfer of cistrons affect tissue and organ function over long periods of time, in order for an organism to evolve one or more a specialized genomes.<br>Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2011.

Das Cyanobakterium Microcystis aeruginosa PCC 7806 enthält zwei Proteine unbekannter Funktion, welche eine hohe Sequenzähnlichkeit mit Bausteinen des eukaryotischen Aktinzytoskeletts haben. Eines dieser Proteine ist Aktin selbst, das andere ist das Aktinbindeprotein Profilin. Die vorliegende Arbeit enthält eine detaillierte Charakterisierung beider Proteine sowie Vergleiche mit ihren eukaryotischen Verwandten. So inhibiert, im Gegensatz zu Eukaryoten, cyanobakterielles Aktin nicht das Enzym DNaseI. Es bildet jedoch Polymere, die hier mit Phalloidin visualisiert wurden. Konfokale Mikroskopie offenbart klare Unterschiede in den Polymeren, da die cyanobakteriellen eine Länge von 10 µm nicht überschreiten und breiter sind als die zylindrischen, ca. 100 µm langen Filamente eukaryotischen Aktins. Röntgen-Kleinwinkelstreuungsdaten zeigen, dass cyanobakterielle Aktinpolymere in ihrer Form am ehesten einem Band ähneln. Es bestehen auch Unterschiede hinsichtlich des Profilins: während es in Eukaryoten ausschließlich Aktinmonomere bindet, assoziiert cyanobakterielles Profilin mit Aktinfilamenten und vermittelt die Entstehung flächiger Heteropolymere. GFP-Fusionsstudien zeigen, dass die Koexpression von Aktin und Profilin die Bildung eines Hohlraumkompartiments in E.coli nach sich zieht. Ähnliche Gebilde wurden bereits in Microcystis gezeigt und könnten auf die beobachteten Heteropolymere zurückzuführen sein. Diese Arbeit verdeutlicht, dass beide Proteine in einer natürlichen Bakterienpopulation etabliert sind und dort Merkmale tragen, die ihre eukaryotischen Vorläufer nicht zeigen. Folglich könnte die Anpassung an die räumlichen Begrenzungen einer Bakterienzelle, welcher die für die Regulierung der Polymerisation notwendigen Aktinbindeproteine fehlen, die Triebkraft für eine Koevolution von cyanobakteriellem Aktin und Profilin gewesen sein. Dieser Prozess gipfelte möglicherweise in der Entstehung eines neuartigen intrazellulären Gebildes von potentiell struktureller Bedeutung.<br>The cyanobacterium Microcystis aeruginosa PCC 7806 harbors two proteins with unknown functions that were transferred horizontally from eukaryotes and show a high degree of sequence identity with key components of the eukaryotic actin cytoskeleton. One is actin itself; the other is profilin, an actin binding protein. This work presents the detailed characterization of both proteins and comparisons with the eukaryotic archetype. In contrast to bona fide actin, its cyanobacterial counterpart does not inhibit DNaseI. It forms polymers that can be visualized with labeled phalloidin, resembling eukaryotic actin in that respect. However, confocal microscopy reveals key differences between polymers of eukaryotic and cyanobacterial actin. Whereas the former appear as cylindrical filaments about 100 µm in length, the latter are shorter and wider arresting polymerization at 5-10 µm. Structural elucidation by Small-angle X-ray scattering shows that cyanobacterial actin polymers are ribbon-shaped. This work also shows fundamental differences between cyanobacterial and eukaryotic profilin. Most importantly, cyanobacterial profilin binds actin filaments and mediates their assembly into heteropolymeric sheets. GFP labeling experiments show that the co-expression of cyanobacterial profilin and actin results in the formation of large hollow enclosures in E.coli. These structures resemble the shell-like distribution of actin in Microcystis aeruginosa and may be based on the actin/profilin heteropolymers observed in vitro. This work shows that both cyanobacterial proteins are established in a natural bacterial community where they have gained properties unknown from their eukaryotic ancestors. Consequently, the adaptation to the confined space of a bacterial cell devoid of binding proteins usually regulating actin polymerization in eukaryotes may have driven the co-evolution of cyanobacterial actin and profilin, giving rise to an intracellular entity of potential structural relevance.