Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Cell and Developmental Biology." Includes bibliographical references (p. 45-53).

Dilantin (DPH) is a common anticonvulsant drug used to prevent seizures. It is known to be a human teratogen causing fetal hydantoin syndrome (FHS). FHS is characterized by multiple developmental and growth related abnormalities and mental retardation. Previous studies demonstrated that DPH slowed growth and division in preimplantation mouse embryos in vivo and in vitro. DHP exposure in utero decreased the crown to rump length and weight of 25-35% of embryos and reduced the rate of endochondral bone conversion from cartilage. In vitro preimplantation mouse embryos treated with DPH at 5, 10 and 20 μg/ml showed a reduction of 25-35% in their development, and block at 2-cell or 3-4-cell stages. These embryos also showed a prolonged DNA synthesis (S) phase during the second cell cycle. Nuclear localization and concentration levels of cyclin A , the S phase cyclin, were also altered in vivo in 2-cell DPH treated embryos compared with NaOH control embryos during G1, S phase and G2 of the first, second and third cell cycles. DPH altered patterns of expression of cyclin A were associated with cell cycle disregulation during preimplantation development. The purpose of the current study was to determine whether DPH also affects the concentration of DNA pol δ catalytic subunit in 2-cell preimplantation mouse embryos at G1 and S phases, thus delaying DNA synthesis and contributing to FHS. Immunofluorescence and confocal microscopy were used as tools to determine relative levels and distribution of DNA pol δ (for consistency with text) in the cytoplasm and the nuclei of DPH and NaOH treated 2-cell embryos at G1 and S phase of the second cell cycle. DPH decreased DNA pol δdelta total embryo and nuclear levels by 43% and 36%, respectively, in G1 compared with NaOH controls. Similarly, nuclear levels of DNA pol δ in DPH embryos in S phase near the G2 transition of the second cell cycle increased to 144% of NaOH control levels; there was not a statistically significant difference between total embryonic levels of late S phase DNA pol δ in DPH and NaOH treated control embryos. The results indicated that DPH affects the levels of DNA pol δduring G1 and S phase near the G2 transition of the second cell cycle in preimplantation mouse embryos. The significant alteration in the levels of DNA pol δ during S phase and its probable consequent altered polymerase activity could contribute to an explanation for the extension of S phase in preimplantation embryos observed by Blosser and Chatot. Even more, the alteration in the levels of DNA pol δ and potentially in its exonuclease activity could lead to an increase in the rate of mismatches and mutations suggesting a likely explanation for some features of FHS. / Department of Biology

Thesis (Ph. D.)--Rutgers University, 2007. / "Graduate Program in Computational Biology and Molecular Biophysics." Includes bibliographical references.

Thesis (PH. D.)--Michigan State University. Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on Sept. 2, 2009) Includes bibliographical references. Also issued in print.

No description available.

No description available.

No description available.

Laboratory-engineered proteins such as high-fidelity DNA polymerases, CRISPR base and primeeditors, and chimeric antigen receptors have transformed our ability to probe and manipulate biological systems. To craft these powerful tools, researchers fuse multiple domains into novel chimeras intended to retain the functional properties of their constituent parts. Although this approach has produced a number of important technologies, its low-throughout nature and high costs thwart efforts to explore complex combinatorial landscapes and limit our grasp on the “rules” governing synthetic protein assembly (e.g., which domains work best together, which domain orders are optimal, benefits of fusing multiple copies of the same domain, etc.). Previous state-of-the-art CRISPR activators, including the tripartite activator, VP64-P65- RTA (VPR) and the Synergistic Activation Mediator (SAM), have established the benefit of combining multiple activation domains (ADs) into a single complex for improved transcriptional modulation. While VPR and SAM have proven relatively successful in both in vitro and in vivo applications, neither activator shows uniform activity across targets and cell types. Furthermore, reports that these tools produce toxicity within cellular systems limit their utility in broad-ranging applications. To probe a vast combinatorial landscape of multi-domain CRISPR activators while bypassing the arduous task of generating each construct one one-by-one, we developed a strategy for constructing large combinatorial libraries of protein variants en masse and used this method to functionally evaluate a library of >15,000 CRISPR activators. Importantly, we conduct our screen on multiple target genes to identify tools with consistent performance across the genome. Our findings bring to light a critical yet often overlooked feature of CRISPR activators: toxicity. This work not only highlights the prevalence of this problem but also elucidates several biological factors that contribute to it. Our observation that many high-performing activators elicited minimal effects on cell fitness challenges the notion that toxicity is an inevitable byproduct of a potent activation – and suggests that this model greatly oversimplifies the nuanced relationship between these traits. We also explored how the biochemical properties of ADs (e.g., hydrophobicity and intrinsic disorder) and their combinatorial interactions drive activator performance. Finally, we identified two potent activators, MHV and MMH, that show enhanced activity across diverse targets and cell types over one of the gold-standard CRISPR activators, SAM. Our results underscore the power of high-throughput techniques for both improving our understanding of complex protein assemblies and identifying more powerful tools.

Trypanosoma brucei and related protists are distinguished from all other eukaryotes by an unusual mitochondrial genome known as kinetoplast DNA (kDNA) that is a catenated network composed of minicircles and maxicircles. Replication of this single nucleoid involves a release, replicate, and reattach mechanism for the thousands of catenated minicircles and requires at least three DNA polymerase (POLIB, POLIC and POLID) with similarity to E. coli DNA polymerase I. Like other proofreading replicative DNA polymerases, POLIB has both an annotated polymerase domain and an exonuclease domain. Predictive modelling of POLIB indicates that it has the canonical right hand polymerase structure with a unique and large 369 amino acid insertion within the polymerase domain (thumb region) homologous to E. coli RNase T. The goal of this study was to evaluate whether the polymerase domain is necessary for the essential replicative role of POLIB. To study the structure-function relationship, an RNAi-complementation system was designed to ectopically express POLIB variants in T. brucei that has endogenous POLIB silenced by RNAi.Control experiments expressing an ectopic copy of POLIB wildtype (IBWTPTP) or polymerase domain mutant (IBPol-PTP) in the absence of RNAi did not impact fitness in procyclic cells despite protein levels being 5 - 8.5 fold higher than endogenous POLIB levels. Immunofluorescence detection of the tagged variants indicated homogenous expression of the variants in a population of cells and negligible changes in kDNA morphology. Lastly, Southern blot analyses of cells expressing the IBWTPTP or IBPol-PTP variants indicated no changes in free minicircle species. A dually inducible RNAi complementation system was designed and tested with the IBWTPTP and IBPol-PTP variants. Inductions of POLIB RNAi accompanied by ectopic expression of either variant using the standard 1 mg/ml tetracycline resulted in low protein levels of both variants while knockdown of the endogenous POLIB mRNA was greater than 85%. Increasing the tetracycline concentration to 4 mg/ml improved expression levels of both variants. However, levels of the ectopically expressed variants never exceeded that of endogenous POLIB. Using the 4 mg/ml induction conditions, complementation with IBWTPTP resulted in a partial rescue of the POLIB RNAi phenotype based on fitness curves, quantification of kDNA content and Southern blot analysis of free minicircles. IBWTPTP complementation resulted in gradual increase of IBWTPTP protein levels over the 10 day induction, and a small kDNA phenotype instead of the progressive loss of kDNA normally associated with POLIB RNAi. Additionally, the loss of free minicircles was delayed. Complementation with the IBPol-PTP variant produced more consistent levels of IBPol-PTP protein although still below endogenous POLIB levels. Loss of fitness was similar to POLIB RNAi alone. However, a small kDNA phenotype emerged early after just four days of complementation and persisted for the remainder of the induction. The majority of the IBRNAi + IBPol-PTP population (70%) contained small kDNA compared to the parental POLIB RNAi or IBWTPTP complementation that had only 45% and 50% small kDNA, respectively. Lastly, the pattern of free minicircle loss closely resembled POLIB RNAi alone. Together, these data suggest that the dually inducible system results in a partial rescue with the IBWTPTP variant. Rescue with IBPol-PTP variant results in a noticeably different phenotype from either POLIB RNAi alone or IBWTPTP complementation indicating that the POLIB polymerase domain is likely essential for the in vivo role of POLIB during kDNA replication.

Thesis (M.Tech.: Biotechnology)-Dept. of Biotechnology, Durban University of Technology, 2007 vi, 92 leaves / Interest in xylanases from different microbial sources has increased markedly in the past decade, in part because of the application of these enzymes in a number of industries, the main area being the pulp and paper industry. While conventional methods will continue to be applied to enzyme production from micro-organisms, the application of recombinant DNA techniques is beginning to reveal important information on the molecular basis and this knowledge is now being applied both in the laboratory and commercially. In this study, a directed evolution strategy was used to select an enzyme variant with high thermostability. This study describes the use of error-prone PCR to modify the xylanase gene from Thermomyces lanuginosus DSM 5826, rendering it tolerant to temperatures in excess of 80°C. Mutagenesis comprised of different concentrations of nucleotides and manganese ions. The variants were generated in iterative steps and subsequent screening for the best mutant was evaluated using RBB-xylan agar plates. The optimum temperature for the activity of xylanases amongst all the enzyme variants was 72°C whilst the temperature optimum for the wild type enzyme was 70°C. Long term thermostability screening was therefore carried out at 80°C and 90°C. The screen yielded a variant which had a 38% improvement in thermostability compared to the wild type xylanase from pX3 (the unmutated gene). Successive rounds of error-prone PCR were carried out and in each round the progeny mutant displayed better thermostability than the parent. The most stable variant exhibited 71% residual activity after 90 minutes at 80˚C. Sequence analysis revealed four single amino acid residue changes that possibly enhanced their thermostabilities. This in vitro enzyme evolution technique therefore served as an effective tool in improving the thermostable property of this xylanase which is an important requirement in industry and has considerable potential for many industrial applications.