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Early embryonic development proceeds without zygotic transcription. Genetic control is executed by maternally inherited mRNAs that are expressed in a temporally regulated manner. To set up the body plan, it is pivotal to both exert translational regulation of maternal mRNAs and to integrate maternal signals that drive cell fate determination. Xenopus nanos1, dead end (dnd) and VegT are maternal messages critical for the germline and somatic development. nanos1 and dnd localize to the germ plasm at the vegetal cortex of oocytes, while VegT occupies a cortical region overlapping with, but broader than, that of the germ plasm. In this dissertation, we observed that unlike other mRNAs, synthetic nanos1 RNA translates very poorly if at all after injection into Xenopus oocytes. We find that a RNA secondary structural element immediately downstream of the AUG start site is both necessary and sufficient to prevent ribosome scanning in the absence of a repressor. nanos1 is translated shortly after fertilization, pointing to the existence of a developmentally regulated activator. These observations unravel a new mode of nanos1 regulation at the post-transcriptional level for eukaryotes that is essential for normal development. Further studies showed that co-injection of dnd and nanos1 into oocytes resulted in nanos1 activation. Consistent with Dnd being the activator, Nanos1 expression was attenuated in the absence of dnd activity. Recombinant Dnd interacted directly with nanos1 RNA in vitro and possessed ATPase activity. Our findings suggested that Dnd is the potential activator for nanos1 translation by directly disrupting the repressive structural element. We previously showed that VegT, the maternal transcription factor, is normally inherited by the primordial germ cells (PGCs). VegT is the endoderm determinant and must be silenced in PGCs to preserve the germline. We showed that a classic Pumilio Binding Element (PBE) within the VegT 3’UTR mediated translational repression of a fluorescent reporter in the germline. Accordingly, a direct interaction between Xenopus Pumilio1 RNA binding domain and the VegT PBE was demonstrated in a band shift assay. The Pumilio protein belongs to the Pum-FBF family that functions in translational repression. We show that Pumilio represses VegT in vivo. Our results suggest that the germline stays unresponsive to the somatic determinant VegT in part by Pumilio- and Nanos-mediated translational repression.

The development of genetic transformation systems in insects has revolutionized the field of entomology. Transgenic insects provide tools to identify, isolate and analyze insect genes and to genetically modify insects for the purposes of insect control or disease vector modification. When transformation frequencies are high, multiple transgenic lines can be generated with relative ease. However, in most mosquito species, the results of transformation experiments have been suboptimal. Increased mosquito transformation efficiency is a research priority. Additionally, incorporation of refractory transgenes will not be sufficient to modify natural populations. A gene drive system will be required to allow transgenes to proliferate throughout populations and potentially reach fixation. This study proposes the use of germline-specific regulatory elements to promote confined, regulated transposase expression within the germ tissue. Creation of helper constructs utilizing endogenous promoters will potentially increase genetic transformation frequencies. The generation of lab strains of mosquitoes expressing an endogenous source of transposase within the germline will also serve as a powerful research tool. Endogenous sources of transposase will allow for comparative analysis of integration rates using different donor plasmids. Finally, the generation of autonomous transposable elements will provide a gene drive mechanism to move a tightly-linked refractory gene into a population. Four genes have been identified, cloned, and characterized, revealing expression patterns expected of germline-specific genes. Transcription profiles and in situ hybridization data support these conclusions. Putative cis-acting regulatory elements have been cloned and incorporated into DNA plasmid constructs. These elements are cloned in a manner such that they will regulate fluorescent gene expression. Additionally, similar elements have been cloned upstream of the Mos1 open reading frame, within the inverted terminal repeats of the mariner transposable element, thus creating autonomous elements and a potential gene drive mechanism.

The development of genetic transformation systems in insects has revolutionized the field of entomology. Transgenic insects provide tools to identify, isolate and analyze insect genes and to genetically modify insects for the purposes of insect control or disease vector modification. When transformation frequencies are high, multiple transgenic lines can be generated with relative ease. However, in most mosquito species, the results of transformation experiments have been suboptimal. Increased mosquito transformation efficiency is a research priority. Additionally, incorporation of refractory transgenes will not be sufficient to modify natural populations. A gene drive system will be required to allow transgenes to proliferate throughout populations and potentially reach fixation. This study proposes the use of germline-specific regulatory elements to promote confined, regulated transposase expression within the germ tissue. Creation of helper constructs utilizing endogenous promoters will potentially increase genetic transformation frequencies. The generation of lab strains of mosquitoes expressing an endogenous source of transposase within the germline will also serve as a powerful research tool. Endogenous sources of transposase will allow for comparative analysis of integration rates using different donor plasmids. Finally, the generation of autonomous transposable elements will provide a gene drive mechanism to move a tightly-linked refractory gene into a population. Four genes have been identified, cloned, and characterized, revealing expression patterns expected of germline-specific genes. Transcription profiles and in situ hybridization data support these conclusions. Putative cis-acting regulatory elements have been cloned and incorporated into DNA plasmid constructs. These elements are cloned in a manner such that they will regulate fluorescent gene expression. Additionally, similar elements have been cloned upstream of the Mos1 open reading frame, within the inverted terminal repeats of the mariner transposable element, thus creating autonomous elements and a potential gene drive mechanism.

Attitudes toward human germline engineering were assessed across three studies. In Pilot Study One, we evaluated participant familiarity ratings for a preliminary item pool consisting of potential targets of germline modification in order to screen out unfamiliar targets. The results were used to screen out 54 of 170 items. In Pilot Study Two, we used participant representativeness ratings to validate categorizations for the remaining items by removing items that were indifferently representative of specific goals (therapeutic or enhancing) and types (physical, cognitive, or personality) of germline modifications. The results also indicated that for many items, the distinction between cognitive and personality characteristics broke down, leading us to collapse these categories into a new type category based on general psychological traits. In Study One, we compared mean approval ratings for potential targets of germline engineering sorted according to the goal of modification (therapeutic or enhancing) and the type of characteristic being targeted (physical or psychological). The results indicated that approval was higher for therapeutic modifications than for enhancing modifications, and higher for modifications targeting physical traits than for those targeting psychological traits. A regression analysis showed that approval correlated positively with knowledge, Big Five Agreeableness, and negatively with perceived risk and female gender. Contrary to expectations, approval did not correlate with psychological essentialism or Big Five Openness. Implications and limitations are discussed. / Thesis (Master, Psychology) -- Queen's University, 2011-09-22 11:27:06.734

In mammals, epigenetic modifications and trans-acting effectors coordinate gene expression during development and impose transcriptional memories that define specific cell lineages and cell-types. Methylation at CpG dinucleotides is an epigenetic mechanism through which transcriptional silencing is established and heritably maintained through development. Functionally, DNA methylation regulates key biological processes such as X-chromosome inactivation, transposon repression and genomic imprinting. However, the extent to which DNA methylation is the primary regulator of single-copy gene expression and the precise mechanism of methylation-dependent silencing remain undetermined. Here, I identify a novel set of germline-specific candidate genes putatively regulated by DNA methylation. Analysis of one candidate gene, Tex19, demonstrates that promoter CpG methylation is the primary and exclusive mechanism for regulating developmental silencing in somatic lineages. Genetic or pharmacological removal of CpG methylation triggers robust de-repression of Tex19 and loss of transcriptional memory. Moreover, Tex19 critically relies on de novo methylation, mediated by Dnmt3b, to impose silencing in differentiating ES cells and somatic cells in vivo from embryonic day (E)7.5. Reporter gene and ChIP analysis demonstrate that Tex19 is strongly activated by general transcription factors and is not marked by repressive histone modifications in somatic lineages, consistent with differential DNA methylation per se being the primary mechanism of regulating expression. Full transcriptional silencing of Tex19 is critically dependent on the methyl-binding protein (MBP) Kaiso, which is only recruited to methylated Tex19 promoter. The reliance on DNA methylation and Kaiso for silencing in somatic cells establishes an epigenetic memory responsible for maintaining expression in germline and pluripotent cell types through successive developmental cycles. This thesis represents the first causal report of lineagespecific promoter DNA methylation directing silencing of an in vivo gene through recruitment of an MBP.

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No description available.