OverFlap PCR – a reliable approach for generation of plasmid DNA libraries containing random sequences without template bias.

Over the decades the improvement of naturally occurring proteins and creation of novel ones has been the primary goal for many practical biotechnology researchers and it is widely recognized that randomization of protein sequences coupled to various effect screening methodologies is one of the most powerful techniques for fast, efficient and purposeful approach for acquisition of desired improvements. Over the years considerable advancements have been made in this field, however development of PCR based or template guided methodologies has been hampered by the resulting template sequence bias. In this article we present novel whole plasmid amplification based approach, which we named OverFlap PCR, for randomization of virtually any region of the plasmid DNA, without introduction of mentioned bias.

Cotranscriptional R-loop formation by Mfd involves topological partitioning of DNA

R-loops are nucleic acid hybrids which form when an RNA invades duplex DNA to pair with its template sequence. Although they are implicated in a growing number of gene regulatory processes, their mechanistic origins remain unclear. We here report real-time observations of cotranscriptional R-loop formation at single-molecule resolution and propose a mechanism for their formation. We show that the bacterial Mfd protein can simultaneously interact with both elongating RNA polymerase and upstream DNA, tethering the two together and partitioning the DNA into distinct supercoiled domains. A highly negatively supercoiled domain forms in between Mfd and RNA polymerase, and compensatory positive supercoiling appears in front of the RNA polymerase and behind Mfd. The nascent RNA invades the negatively supercoiled domain and forms a stable R-loop that can drive mutagenesis. This mechanism theoretically enables any protein that simultaneously binds an actively translocating RNA polymerase and upstream DNA to stimulate R-loop formation.

Template plasmid integration in germline genome-edited cattle

We analyzed publicly available whole genome sequencing data from cattle which were germline genome-edited to introduce polledness. Our analysis discovered the unintended heterozygous integration of the plasmid and a second copy of the repair template sequence, at the target site. Our finding underscores the importance of employing screening methods suited to reliably detect the unintended integration of plasmids and multiple template copies.

NETSeq reveals heterogeneous nucleotide incorporation by RNA polymerase I

DNA sequence motifs that affect RNA polymerase transcription elongation are well studied in prokaryotic organisms and contribute directly to regulation of gene expression. Despite significant work on the regulation of eukaryotic transcription, the effect of DNA template sequence on RNA polymerase I (Pol I) transcription elongation remains unknown. In this study, we examined the effects of DNA sequence motifs on Pol I transcription elongation kinetics in vitro and in vivo. Specifically, we characterized how the spy rho-independent terminator motif from Escherichia coli directly affects Saccharomyces cerevisiae Pol I activity, demonstrating evolutionary conservation of sequence-specific effects on transcription. The insight gained from this analysis led to the identification of a homologous sequence in the ribosomal DNA of S. cerevisiae. We then used native elongating transcript sequencing (NETSeq) to determine whether Pol I encounters pause-inducing sequences in vivo. We found hundreds of positions within the ribosomal DNA (rDNA) that reproducibly induce pausing in vivo. We also observed significantly lower Pol I occupancy at G residues in the rDNA, independent of other sequence context, indicating differential nucleotide incorporation rates for Pol I in vivo. These data demonstrate that DNA template sequence elements directly influence Pol I transcription elongation. Furthermore, we have developed the necessary experimental and analytical methods to investigate these perturbations in living cells going forward.

Elucidating the mechanisms and dynamics of drug-mediated inhibition and latency reversal in HIV-1 (HIV-1)

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The Human Immunodeficiency Virus Type-1 (HIV-1) is the etiological agent of Acquired Immunodeficiency Syndrome, a disease that causes the host to succumb to secondary infections. There is currently no cure for HIV-1 infection, but Highly Active Anti-Retroviral Therapy (HAART) can bring the viral load in patients down to undetectable levels in the blood (less than 50 copies/mL). Furthermore, when the minimal limit of detection has been reached and the patient stops HAART, the viral load in the blood increases at an exponential rate due to the reactivation of latent HIV-1 infected cells that evaded HAART. Ongoing efforts focus on the eradication of HIV-1 by the development of potent latency reversing agents (LRAs) that can successfully reactivate latently infected cells, and of antivirals that can effectively inhibit re-establishment of infection post reactivation. This dissertation focuses on the evaluations of 2 classes of HIV-1 drugs, Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs), and LRAs, to better understand the mechanisms by which each drug class inhibits and reactivates HIV-1 replication respectively, to aid in the effort towards the development of antivirals that will lead to HIV-1 eradication. Chapter II describes the inhibitory mechanisms of NNRTIs using biochemical methods, which may further explain the differences in potency among drugs of this class. In addition, we explain how changes in the position of HIV-1 RT in the DNA substrate sequence, and the nucleotide terminating the primer 3'-end have a significant effect on the polymerase properties of the enzyme. We demonstrate that there are NNRTI- and site-dependent differences in the potency of NNRTIs, which is demonstrated by the repositioning, or lack there of, of the primer 3'-end of DNA/DNA substrates from the polymerase active site. This is further supported by the efficiency of dNTP or NRTI incorporation in the presence of NNRTI with multiple DNA/DNA substrates, which are representative of different sites in the template sequence. We also show that there are site-dependent differences in the polymerase properties of RT, which is demonstrated by rate of dNTP incorporation and incorporation efficiency at different sites in the template sequence. Chapter III describes the various effects of different types of LRAs, such as histone deacetylase inhibitors and histone methyltransferase inhibitors, on the dynamics of HIV-1 latency reversal in latent cell lines. Here, we demonstrate the use of branched DNA in situ hybridization in combination with immunocytochemistry to study the kinetics and dynamics of latency reversal in various latent cell lines. This technique is augmented with the use of automated screening using microscopy and flow cytometry to quickly detect different populations of latent and reactivated proviruses in thousands of cells in a short amount of time. Understanding the mechanisms by which a drug affects a biological process is important for establishing drug efficacy. Such information can influence what modifications are added to, or removed from drugs, which can cause a change in drug potency. This dissertation outlines assays used to evaluate the mechanisms of various drugs, and the influence of these drugs on the dynamics of HIV-1 replication. It is our hope that the work presented here will help progress efforts to eradicate HIV-1 infection.

HOMOLOGY MODELING OF SUBCUTANEOUS FILARIASIS DHFR PROTEINS

Objective: A systematic technique for protein modeling offers great assistance in the study of protein function, dynamics, interactions with ligands, other proteins and even in drug discovery and drug design. Subcutaneous filariasis is rare parasitic disease caused by Loa Loa (eye worm) and monosonallastreptoscerca species. Methods: The present study develop three dimensional structure of dihydrofolatereductase present in Loa loa species. For this purpose knowledge based homology modeling is used by using Schrodinger Glide 5.6 software.Results: The procedure involves alignment that maps residues in the query sequence to residues in the template sequence to generate structural model of target, which was further refined and final result validated by using Ramchandran plot.Conclusion: In ramchandran plot majority of the amino acids are in the phi-psi distribution and thedevelop model is reliable and of good quality.

Continuous Motion Classification and Segmentation Based on Improved Dynamic Time Warping Algorithm

The continuous image sequence recognition is more difficult than the single image recognition because the classification of continuous image sequences and the image edge recognition must be very accurate. Hence, a method based on sequence alignment for action segmentation and classification is proposed to reconstruct a template sequence by estimating the mean action of a class category, which calculates the distance between a single image and a template sequence by sparse coding in Dynamic Time Warping. The proposed method, the methods of Kulkarni et al. [Continuous action recognition based on sequence alignment, Int. J. Comput. Vis. pp. 1–26.] and Hoai et al. [Joint segmentation and classification of human actions in video, IEEE Conf. Computer Vision and Pattern Recognition, 2008, pp. 108–119.] are compared on the recognition accuracy of the continuous recognition and isolated recognition, which clearly shows that the proposed method outperforms the other methods. When applied to continuous gesture classification, it not only can recognize the gesture categories more quickly and accurately, but is more realistic in solving continuous action recognition problems in a video than the other existing methods.