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In this project I have tried to obtain stable transformants of Drosophila melanogaster flies using electroporation. I have completed approximately 200 tests using different DNA concentrations, voltages and cuvettes, including a novel Petri dish cuvette which I developed and manufactured myself. I also developed new and more efficient procedures of egg collection and egg dechorionation. Although I was not  successful in obtaining true stable transformants, control experiments indicate that electroporation of DNA into embryos could be accomplished under the conditions used. The lack of stable transformants was probably due to failure of the electroporated DNA to integrate into the host genome. The reasons for why the DNA did not integrate was not further investigated in this study.

Quantitative analysis of Drosophila melanogaster gap gene expression data reveals valuable information about the nature and strengths of interactions in the gap gene network. We first explore different models for fitting the spatiotemporal gene expression data of Drosophila gap gene system and validate our results by computational analysis and comparison with the existing literature. A fundamental problem in systems biology is to associate these results with the inherent cause of gene regulation, namely the binding of the transcription factors (TF) to their respective binding sites. In order to relate these expression-based estimates of gap gene regulation with the sequence-based information of TF binding site composition, we also explore two related problems of (i) finding a set of regulatory weights that is proportional to the binding site occupancy matrix of the transcription factors in current literature and (ii) finding a set of position weight matrices of the TFs that produce a new binding site occupancy matrix showing a greater level of proportionality with our regulatory weights. Our solution to the first problem yielded a regulatory weight matrix incapable of explaining the true causes of gene expression profile despite its relative numerical accuracy in predicting the gene expressions. On the other hand, the second optimization problem could be solved up to a reasonable level of accuracy, but further analysis on the result demonstrated that this optimization problem may be under-constrained. We devise a simple regularization strategy that helps us to reduce the under-constrained nature of the problem.

Mechanisms of post-transcriptional control are critical to deploy RNAs and proteins asymmetrically to a discrete region of cytoplasm at the posterior of the Drosophila oocyte and embryo, called the pole plasm and thus allow differentiation of the germline. Research presented in this thesis investigates the post-transcriptional control of Drosophila pole plasm component germ cell-less (gcl ). Maternal gcl activity is required for germ cell specification and gcl RNA and protein accumulate asymmetrically in the pole plasm. gcl RNA, but not Gcl protein, is also detected in somatic regions of the embryo, and ectopic expression of Gcl in the soma causes repression of somatic patterning genes suggesting that gcl RNA is subject to translational control. I find that Gcl is expressed during oogenesis, where its expression is regulated by translational repressor Bruno (Bru). Increased levels of Gcl are observed in the oocyte when Bru is reduced (i.e., in an arrest heterozygote) and Bru overexpression reduces the amount of Gcl. Consistent with this, reduction of the maternal dosage of Bru leads to ectopic Gcl expression in the embryo, which, in turn, causes repression of anterior huckebein RNA expression. Bruno binds directly to the gcl3'UTR in vitro, but surprisingly, this binding is largely independent of a Bruno Response Element (BRE) in the gcl 3'UTR and depends upon a novel site. Furthermore, the gcl BRE-like region is not required to repress Gcl expression during oogenesis or embryogenesis. I concluded that Bru regulates gcl translation in a BRE-independent manner. In addition, I established the role of the gcl 3'UTR in gcl RNA localization and translation using transgenes that replace the endogenous 3'UTR with the alpha-tubulin 3'UTR or place it in tandem to the bicoid 3'UTR. I find that accumulation of gcl RNA in the embryonic pole plasm requires the gcl 3'UTR. Moreover, Gel is restricted to the pole plasm by translational repression mediated by the gcl 3'UTR and a limiting pool of trans-acting translational repressors. The phenotypic consequences of loss of this translational control are relatively mild, suggesting that gcl translation does not require stringent repression in the soma.

Evaluating the evolutionary role of mutations depends on an understanding of their major properties, including their rate of origin, U, and the distribution of their fitness effects, f(s). While substantial effort has been put into measuring these properties, most studies have only examined their distributions in a single context. In nature, spontaneous mutations are likely to experience heterogeneity in genetic and environmental context, and this could lead to variation in both U and f(s). My thesis investigates the changes in U and f(s) with different genetic and environmental factors in Drosophila melanogaster, in order to elucidate the nature of context-associated variation in mutational properties. Examination of condition-dependent variation in DNA repair showed that high and low conditioned individuals differ in the use of alternative repair pathways. This could ultimately lead to variance in their heritable mutation rates. However, the assumption that condition dependence in repair arises solely due to a presumed trade-off between accuracy and the energetic costs associated with different repair pathways is too simplistic. Instead, physiological considerations appear to mediate condition-dependent changes in DNA repair. Measurements of selection on individual mutations across different genetic and environment contexts showed that context-associated changes in mutational fitness effects are common. I found that heterogeneity in fitness effects across different environments result in changes to the overall mean and variance of f(s). This does not, however, seem attributable to the degree of ‘adaptedness’ of a population to a particular environment (a prediction generated by previous theoretical analysis). On the other hand, f(s) appears to be relatively robust to differences among genotypes, with epistasis averaging close to zero. This finding suggests that genetic and environmental perturbations may affect mutations differently. Overall, my thesis represents the most rigorous empirical investigation to date of the conceptual and theoretical predictions regarding the nature of context-dependent heterogeneity in U and f(s) for multicellular eukaryotes.

Transgenic organisms are a valuable tool for studying epigenetics, as they provide significant insight into the evolutionary conservation of epigenetic control sequences, the interacting proteins, and the underlying molecular mechanisms. Paramutation is an epigenetic phenomenon in which the epigenetic status and expression level of one allele is heritably altered after pairing with another. At the b1 locus in maize, a control region consisting of seven 853 bp tandem repeats is required for paramutation. To study the conservation of the epigenetic mechanisms underlying maize b1 paramutation, I created transgenic Drosophila carrying the maize b1 control region flanked by FRT sites and adjacent to the Drosophila white reporter gene. The maize b1 tandem repeats caused epigenetic silencing in Drosophila, as white expression consistently increased following repeat removal. A single copy of the tandem repeat sequence was sufficient to cause silencing, and silencing strength increased as the number of repeats increased. Trans interactions, such as pairing-sensitive silencing, were also observed and appear to require a threshold number of b1 tandem repeats, similar to paramutation in maize. Analysis of transcription from the repeats showed that the b1 tandem repeats are transcribed from both strands in Drosophila, as they are in maize. Bidirectional transcription was found to extend to the regions flanking the repeats, and persisted in “repeats-out” transgenes following repeat removal. However, aberrant transcription was lost when a zero-repeat transgene was moved to a new genomic position, suggesting that it may be due to an epigenetic mark that is retained from the previous silenced state. A search for modifiers of b1 repeat-mediated silencing demonstrated that Polycomb group proteins are involved. Together, these results indicate considerable conservation of an epigenetic silencing process between the plant and animal kingdoms. Genomic imprinting is a related epigenetic process in which parent-specific epigenetic states are inherited and maintained in progeny. The conservation of epigenetic mechanisms was further explored via an in-depth review of the molecular mechanisms underlying genomic imprinting in plants, mammals and insects, and identification of potentially imprinted genes in Drosophila by microarray analysis.