Genomic imprinting is an epigenetic phenomenon by which a subset of genes is expressed in a parent of origin specific manner. Most of the imprinted genes are located in clusters. Genetic evidences suggest that genes in imprinted clusters are regulated by Imprinting Control Regions (ICRs). To elucidate the mechanisms by which the imprinting is maintained in clusters, we have chosen a well characterized cluster at the distal end of mouse chromosome 7. This cluster contains 15 imprinted genes and they have been shown to be regulated by H19 and Kcnq1 ICRs. The mouse H19 ICR, which is shown to have a chromatin insulator function, is implicated in the regulation of H19 and Igf2 genes by interacting with the CTCF protein. It has been documented that CTCF is also involved in the maintenance of differential methylation at the ICR. In this investigation we demonstrated that CTCF maintained differential methylation is lost when we subjected the ICR containing episomal plasmids to de novo methylation machinery of the human choriocarcinoma cell line, JEG3, suggesting that the H19 ICR looses its methylation privilege property under neoplastic conditions. The Kcnq1 ICR has been implicated in the regulation of 11 imprinted genes. The Kcnq1 ICR is methylated on the active maternal allele but unmethylated on the inactive paternal allele and overlaps an oppositely oriented and paternally expressed gene known as Kcnq1ot1. In this investigation, we documented that the Kcnq1 ICR controls the imprinting of neighboring genes by behaving as a bidirectional silencer and that this function is regulated by antisense RNA Kcnq1ot1. Furthermore, we have documented that duration of antisense transcription plays a critical role in the antisense RNA- mediated silencing. In conclusion, this thesis provides more insights into the complex mechanistic aspects by which ICRs, control imprinting of genes in clusters during development and neoplasia.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Antisense RNA (asRNA) strategies are identified as an effective and specific method for gene down-regulation at the post-transcriptional level. In this study, the major purpose is to find a correlation between the expression level and minimum free energy to enable the design of specific asRNA fragments. The thermodynamics of asRNA and mRNA hybridization were computed based on the fluorescent protein reporter genes. Three different fluorescent proteins (i) green fluorescent protein (GFP), (ii) cyan fluorescent protein (CFP) and (iii) yellow fluorescent protein (YFP) were used as reporters. Each fluorescent protein was cloned into the common pUC19 vector. The asRNA fragments were randomly amplified and the resulted antisense DNA fragments were inserted into the constructed plasmid under the control of an additional inducible plac promoter and terminator. The expression levels of fluorescent reporter protein were determined in real time by plate reader. Different results have been observed according to the fluorescent protein and the antisense fragment sequence. The CFP expression level was decreased by 50 to 78% compared to the control. However, with the GFP, the down-regulation did not exceed 30% for the different constructs used. For certain constructs, the effect was the opposite of expected and the expression level was increased. In addition, the YFP showed a weak signal compared to growth media, therefore the expression level was hard to be defined. Based on these results, a thermodynamic model to describe the relationship between the particular asRNA used and the observed expression level of the fluorescent reporter was developed. The minimum free energy and binding percentage of asRNA-mRNA complex were computed by NUPACK software. The expression level was drawn as a function of the minimum free energy. The results showed a weak correlation, but linear trends were observed for low energy values and low expression levels the CFP gene. The linear aspect is not verified for higher energy values. These findings suggest that the lower the energy is, the more stable is the complex asRNA-mRNA and therefore more reduction of the expression is obtained. Meanwhile, the non-linearity involves that there are other parameters to be investigated to improve the mathematical correlation. This model is expected to offer the chance to "fine-tune" asRNA effectiveness and subsequently modulate gene expression and redirect metabolic pathways toward the desired component. In addition, the investigation of the localization of antisense binding indicates that there are some regions that favors the hybridization and promote hence the down-regulation mechanisms. / Master of Science

Au cours d'une étude sur le contrôle transcriptionnel du gène de l'alpha-foetoprotéine, le laboratoire s'était intéressé aux facteurs de transcription de la famille SP/KLF et S. Scohy avait découvert une séquence définissant un nouveau membre: SP6.<p><p>Afin de déterminer la structure du gène chez la souris, nous avons isolé un fragment génomique contenant la totalité du gène et nous l'avons séquencé. Une analyse informatique de cette séquence, l'isolement d'ESTs ainsi qu'une expérience d'extension d'amorce, nous ont permis d'affirmer que le gène Sp6 murin possède deux exons, générant une protéine de 376 acides aminés à partir d'un ATG repéré au début de l'exon 2.<p>En même temps, des travaux réalisés sur un gène nommé epiprofin ont été publiés (Nakamura et al. 2004). Ce gène s'est avéré correspondre au gène Sp6 car il code pour la même protéine. Les exons 2 sont en effet identiques, seuls les exons 1 diffèrent.<p>Nos études sur l'expression du gène Sp6 ont indiqué qu'elle est ubiquiste mais que c'est durant le développement embryonnaire, et surtout pendant les stades les plus tardifs de celui-ci, qu'il est le plus exprimé. Cette expression se localise surtout au niveau des dents, de l'épithélium olfactif, du cerveau, des bourgeons de membres et des follicules pileux de l'embryon. A l'état adulte, l'expression de Sp6 se réduit fortement dans tous les tissus; seuls les poumons présentent un taux d'expression relativement important.<p>Ce travail a également permis de mettre en évidence l'existence d'un transcrit non-codant issu d'une transcription antisens du locus Sp6 et dont le premier exon inclu la totalité de l'exon 2 du gène Sp6. Nous l'avons appelé Sp6os et avons montré que son expression est absente dans de nombreux tissus et est très faible dans les tissus où on détecte le transcrit. Une comparaison de l'expression des transcrits Sp6 et Sp6os nous a permis d'imaginer un rôle pour Sp6 dans le développement et une possible modulation de son activité, par Sp6os, dans certains tissus.<p><p>Afin de préciser la fonction du locus Sp6, nous l'avons invalidé chez la souris. Les mutants Sp6-/- se sont avérés viables mais présentent des anomalies dans tous les tissus où Sp6 est le plus fortement exprimé. En effet, ils n'ont ni pelage, ni vibrisse et montrent des anomalies des dents, des membres et des poumons. Nous avons également noté une dérégulation importante de l'apoptose (et parfois aussi de la prolifération cellulaire) chez ces souris Sp6-/-. / Doctorat en sciences, Spécialisation biologie moléculaire / info:eu-repo/semantics/nonPublished

Au cours d'une étude sur le contrôle transcriptionnel du gène de l'alpha-foetoprotéine, le laboratoire s'était intéressé aux facteurs de transcription de la famille SP/KLF et S. Scohy avait découvert une séquence définissant un nouveau membre: SP6. Afin de déterminer la structure du gène chez la souris, nous avons isolé un fragment génomique contenant la totalité du gène et nous l'avons séquencé. Une analyse informatique de cette séquence, l'isolement d'ESTs ainsi qu'une expérience d'extension d'amorce, nous ont permis d'affirmer que le gène Sp6 murin possède deux exons, générant une protéine de 376 acides aminés à partir d'un ATG repéré au début de l'exon 2. En même temps, des travaux réalisés sur un gène nommé epiprofin ont été publiés (Nakamura et al. 2004). Ce gène s'est avéré correspondre au gène Sp6 car il code pour la même protéine. Les exons 2 sont en effet identiques, seuls les exons 1 diffèrent. Nos études sur l'expression du gène Sp6 ont indiqué qu'elle est ubiquiste mais que c'est durant le développement embryonnaire, et surtout pendant les stades les plus tardifs de celui-ci, qu'il est le plus exprimé. Cette expression se localise surtout au niveau des dents, de l'épithélium olfactif, du cerveau, des bourgeons de membres et des follicules pileux de l'embryon. A l'état adulte, l'expression de Sp6 se réduit fortement dans tous les tissus; seuls les poumons présentent un taux d'expression relativement important. Ce travail a également permis de mettre en évidence l'existence d'un transcrit non-codant issu d'une transcription antisens du locus Sp6 et dont le premier exon inclu la totalité de l'exon 2 du gène Sp6. Nous l'avons appelé Sp6os et avons montré que son expression est absente dans de nombreux tissus et est très faible dans les tissus où on détecte le transcrit. Une comparaison de l'expression des transcrits Sp6 et Sp6os nous a permis d'imaginer un rôle pour Sp6 dans le développement et une possible modulation de son activité, par Sp6os, dans certains tissus. Afin de préciser la fonction du locus Sp6, nous l'avons invalidé chez la souris. Les mutants Sp6-/- se sont avérés viables mais présentent des anomalies dans tous les tissus où Sp6 est le plus fortement exprimé. En effet, ils n'ont ni pelage, ni vibrisse et montrent des anomalies des dents, des membres et des poumons. Nous avons également noté une dérégulation importante de l'apoptose (et parfois aussi de la prolifération cellulaire) chez ces souris Sp6-/-.

Thesis (Ph. D.)--University of Virginia, 2008. / Title from title page. Includes bibliographical references. Also available online through Digital Dissertations.

Currently, human norovirus is the leading cause of acute gastroenteritis and accounts for most cases of foodborne illnesses in the United States each year. Due to its tissue culture inefficiency, studies of human norovirus have been crippled for more than forty years.Tulane virus (TV) stands out as a suitable surrogate of human norovirus given its high amino acid identity with human norovirus and its well-established cell culture system. It was first isolated from rhesus macaques (Macaca mulatta) in 2008 and identified as a novel Calicivirusrepresenting a new genus, Recovirus genus (Farkas et al., 2008). However, there are still unanswered questions about its infectious cycle and the essential factors for its infection. In this study, we have obtained a TV variant (the 9-6-17 strain) that has lost the binding ability to the B-type histo-blood group antigen (HBGA), which was proposed to be the receptor of both TV and human norovirus. In the first chapter, we outline how the sequence analysis,structural biology studies, and mutagenesis studies of the 9-6-17 TV strain have shed light on the interaction with its host cell receptor. To investigate the key residues for HBGA binding, we established the full-length infectious clone of the 9-6-17 TV strain. We present a highly selective transformation of serine 367, located in the predicted HBGA binding site, into a lysine residu e. Our results advance the understanding of genetic changes in TV required for adaptation to cell culture environments. Cryo-EM is an awarding winning technique that has been the greatest scientific breakthrough in recent years. It was awarded the Nobel Prize in Chemistry in 2017. Despite the technological advances of the direct electron detector and image processing software, several major roadblocks remain for high-resolution structure determination with cryo-EM. In the later chapters, we explored the most efficient way of using VPP to enhance image contrast, how to tackle the airwater interface problem by encapsulating target protein, how to reach a higher resolution by refining high order parameters, and the helical indexing problem in real space. These technical advances would benefit the whole cryo-EM community by providing convenient tools or insights for future directions.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.

Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing.