Browsing by Subject "DNA Gyrase"
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Item Algorithmic Developments for Sequence Analysis, Structure Modeling and Functional Prediction of Proteins(2006-12-20) Qi, Yuan; Grishin, Nick V.Sequence, structure and function, being the three most important properties of proteins, are interrelated through homology relationships. In this post-genome era, we are equipped with abundant sequence information. Homology inference is thus of great practical importance because of its ability to make structural and functional predictions through sequence analysis. In an effort to explore and utilize the protein sequence-structure-function relationships, with homology detection and utilization as the central scheme, this work concentrates on algorithmic development of methods and systems for sequence similarity search, structure modeling and functional prediction purposes, as well as performs structure prediction and classification for specific protein families. Three algorithmic developments are described in this dissertation. First, to facilitate identification of structurally or functionally important interactions between positions in a protein family, a program has been developed to perform positional correlation analysis of multiple sequence alignments using different methods. The program has been shown to be useful to identify functionally important position pairs or networks of correlated positions. Second, to further increase the sensitivity of sequence similarity search methods in terms of homology detection and structure modeling ability, a method has been developed by incorporating predicted secondary structure information with sequence profiles. Evaluation on PFAM-based system shows that this method provides improved structure template detection ability and generates alignment of better quality. Third, in order to systematically assess the structure modeling abilities of different sequence similarity search programs, a comprehensive evaluation system has been developed. This large-scale automatic evaluation system assesses the fold recognition ability and alignment quality of different programs from global and local perspectives using both reference-dependent and reference-independent approaches, which provides an instrument to understand the progress and limitations of the field. Two structure prediction and classification projects using manual analysis and existing tools are also described in this dissertation. First, the structure of C-terminal domain of Gyrase A is predicted through inferred homology relationship with regulator of chromosome condensation (RCC1). This prediction has been validated by experimental data. Second, a hierarchical structure classification of thioredoxin-like fold proteins has been carried out, which promotes understanding of fold definitions and sequence-structure-function relationshipsItem Using Gene Overexpression as a Potential Method to Elucidate Antibiotic Resistance Mechanisms in Mycobacteria(2015-03-31) Bacalao, Maria Alexandra; Gumbo, Tawanda; Van Oers, Nicolai S. C.; Kant, Shashi; Wakeland, Edward K.Mycobacterium Tuberculosis remains a major public health threat, with 9 million new cases and 1.5 million deaths in 2013. Also concerning is the rise of antbiotic resistance, leading to the development of Multiple Drug Resistant (MDR) and Extensively Drug Resistance (XDR) Mycobacterium tuberculosis strains. The development of these strains is multifactorial involving both extrinsic factors and intrinsic factors. Extrinsic factors include treatment noncompliance and a delay in diagnosis with existing drug sensitivity methods. Intrinsic factors include heterogeneity in the bacterial population and localization in cavitations and other areas where antibiotic penetration is difficult. Our study analyzed the potential role of a potential mechanism of antibiotic resistance, overexpression of existing wild type M. tuberculosis genes involved in mycobacterial antibiotic defense. We cloned seven M. tuberculosis wild type genes using PCR and the subcloned those constructs into E. Coli. Further subcloning steps placed these genes into shuttle vectors that allowed for transformation into M tuberculosis and M. smegmatis. The M. Smegmatis mutant overexpressing the GyrA gene was selected for dose-response studies to establish whether GyrA overexpression led to increased fluoroquinolone resistance. Two M. smegmatis GyrA mutant and one wild type strain were incubated with increasing concentration of Moxifloxacin. Initial dose-response studies did not show yield an adequate dose-response curves under the study conditions. However, the MIC was higher for the GyrA mutant strains than for wild type M. smegmatis, showing that these GyrA mutants were likely more resistant than wild type and that the role of gene overexpression in M. tuberculosis antibiotic resistance merits further study.