Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery and Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli

This dissertation focuses on the interaction of proteins and nucleic acids and their applications. It includes two projects: I: Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery and II. Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli.I: Development of a Novel Bistable DNA Sensor for Anti-HIV Drug Discovery Screening drug compounds targeting HIV-1 NCp7 provide attractive candidates for new anti-retroviral therapeutics because of the highly conserved nature of the zinc fingers in NCp71 in selecting and packaging RNA in the HIV-1 life cycle. The unique 3-segment, reversible switch for high throughput screening (HTS) drug targets will be for the HIV-1 nucleocapsid (NC) protein. The Probe is a natural binding element for the NCp7 protein target or, in our case, an aptamer hairpin with loop sequence, TGTGGT, having a nano-molar affinity (Kd=16nM).2 Toggle is a damaged probe where the target-binding sequences are replaced with other bases. Cover is a mostly complementary strand for the probe and the toggle. A hairpin loop forms around the 5Me-dC-brancher in both the ON and OFF forms. Using Visual OMPTM simulation, the following two potential switch molecules (NM-1 and NM-2) were designed and successfully synthesized using a one-step ligation method with at least 90% purity as judged by mass spectrometry. Then fluorescence measurements using NM-1 and NM-2 with NCp7 protein were analyzed to demonstrate proof-of-principle for 3-segment nucleic acid switches. Increasing [NC] causes a dramatic decrease in CY3 fluorescence. The ON/OFF contrast ratio of NM-1 and NM-2 are 2.6 and 3.2 showing the feasibility of 3-segment switches being used to further modify the design of HTS switches for the HIV-1 NC. II. Re-engineering of Recombinant Cyclin T1-Tat Protein with SUMO Fusion in Escherichia Coli. Due to increasing drug resistance for current antiretroviral therapeutic (ART) treatments, it is important to add drugs targeting other aspects of HIV-1 infection to manage AIDS. Tat (trans-activator of transcription) protein up-regulates the transcription of viral-specific proteins by a factor of 1,0003, which makes Tat a very attractive drug target. High-level expression and purification of recombinant GST-CycT1(249-280)-linker(25aa)-Tat(1-101) fusion protein in Escherichia coli (E. coli) was challenging because CycT1-Tat forms inclusion bodies making it difficult to purify and obtain a high concentration of active CycT1-Tat. A commercially available pET-SUMO cloning vector was introduced for the high-level expression of four CycT1-Tat chimeras, F1-F4. The gene associated with the protein sequence was purchased from IDT and SUMO-CycT1-Tat (F1) fusion plasmid was prepared and transformed in E. coli BL21 (DE3). By inducing with IPTG, His6-SUMO-CycT1-Tat fusion protein was able to promote soluble expression making it more efficient to purify CycT1-Tat (F1) compared to GST fusion tag protein. The molecular weight of purified CycT1-Tat was confirmed with mass spectrometry (MALDI-TOF). For initial characterization of the TAR-CycT1-Tat complex, an Electrophoretic Mobility Shift Assay (EMSA) was carried out with partially purified CycT1-Tat protein using TAR-31 and truncated versions that altered the hairpin loop (TAR-H24) and deleted the bulge loop (TAR-B25). A TAR-CycT1-Tat complex was formed and a band shift was observed in the binding of CycT1-Tat to TAR-31 and a lower affinity complex with TAR-B25, but TAR-H24 did not bind

Pathogenicity And Immunogenicity Of Egg-Adapted Very Virulent Infectious Bursal Disease Virus Isolated In Malaysia

The emergence of very virulent infectious bursal disease virus (vvIBDV) strains caused a devastating economic loss to the chicken industry in Malaysia in 1990. The recurrence of infectious bursal disease (IBD) outbreaks and vaccine failure occurred recently. It is very important to study the pathogenicity, immunogenicity and molecular characteristics of these vvIBDV isolates which are prerequisite before developing the IBD vaccine and testing it in the farms. In this study, UPM 93273 (passage 1 to 48 in chorio-allantoic membrane) was characterized by focusing on the hypervariable region of the VP2 protein. The UPM 93273 isolate was selected among seven other isolates obtained from field outbreaks in layer, broiler and indigenous (village) chickens in various areas in West Malaysia during 1993 to 1994. The sequence of the hv region was obtained following total RNA extraction from the wild type UPM 93273 and its different derivative passages. The hv region was amplified by RT-PCR assay and cloned into pCR-TOPO cloning vector. The right clone harboring the hv fragment was selected by EcoRI restriction enzyme digestion. Finally the cloned fragment was sequenced. The nucleotide and the deduced amino acid sequence alignment and analysis was carried out by using the Bio-Edit software. As a result, markers for very virulent IBDV strain were found in UPM 93273 isolate (wild type to passage 48). The UPM 93273 was found to be very immunogenic but also highly pathogenic. The pathogenicity is reduced during the serial passages in embryonated SPF chickens at passage 31. The passage 31 (P31) of UPM 93273 isolate provided protection against the challenge virus with some degree of bursal atrophy. An effective vaccine depends not only on the full attenuation and genetic stability of the virus strain but also on the route of vaccination. This latter was investigated to determine the immune response of the lymphoid organs against IBDV UPM 94283 strain. Ultrastructural, histopathological and immunohistochemistry studies showed that immunocompetent cells were present in the Harderian gland and were involved in the early and strong immune response against IBDV UPM strain administered via ocular route. Thus, this route can stimulate the lymphoid cells in the Harderian gland, and support the proliferation of lymphoid cells in the bursa before the virus reaches the primary target organs. In addition to this rapid immunogenic reaction, some viruses may drain into the oral cavity when administered via ocular route. As a result, these viruses can stimulate the intestinal lymphoid aggregates along the intestinal tract. As a conclusion, all of the serially passaged UPM 93273 isolate up to passage 48 was found to be still unstable and not sufficiently attenuated indicated by virulence markers which are still conserved. The decrease in the virulence and the pathogenicity seen in the serially passaged strain might be due to other unknown factors. Consequently the risk of reversion to the very virulent state is very high. The current numbers of passages are not sufficient and further passages are necessary in order for the virus to reach a fully attenuated and genetically stable state

Informative Query Answering by Using RDQL for E-Commerce

Information Retrieval Related Pape

Opportunities and Inplementation (A Case Study: Economic Development of Kyaukta Village, Sagaing Township)

The study on economic opportunities is a crucial one not only for economic development but also for all round development of the respective area. In this study, economic opportunities of a village are assessed by means of qualitative method. Due to good location and the given economic opportunities of the study area, people in Kyaukta village know well about different economic opportunities in addition to their traditional farming. By taking an interview to the local residents, the past, present and future economic development pattern could be portrayed systematically. Due to the great efforts of local people in their implementation processes, the different types of economic activities were found within one family. The results show that the economic activities for the individual household could be extracted from the minimum of one in one economic activity to the maximum of five in one household. This situation highlights the greater potentiality of the study area to be developed during the time frame of near future

Recloning of regenerated plantlets from elite oil palm (Elaeis guineensis Jacq.) cv. Tenera

Plant regeneration in oil palm cv. Tenera via somatic embryogenesis was conducted using regenerated plantlets as an explant source. Explants from different positions – apex, middle and basal segments of regenerated plantlets – were cultured in N6 medium supplemented with 100, 120 and 140 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) in the presence of activated charcoal. The production of embryogenic calli was affected by 2,4-D concentration and explant position; 2,4-D 120 mg/L was the most effective (62.53%) in inducing embryogenic calli from the basal segment 5 months after inoculation. After 3 months of culture in embryo maturation medium, an average of 36 ± 8 somatic embryos per embryogenic callus was obtained. When transferred to plant regeneration medium for 3 to 4 months, these somatic embryos differentiated into shoots, with ranges of 6 to 40 and 4 to 32 shoots on the medium with and without 2-isopentyladenine (6-dimethylaminopurine) (2iP), respectively. Plantlets (6 to 8 cm height) with balanced shoots and roots were obtained after 12 to 14 months. Histological analysis confirmed the initiation, development and germination of somatic embryos from explants of regenerated plantlets. Simple sequence repeat (SSR) analysis showed the genetic identity and uniformity between the first and second regenerated plantlets at five SSR loci.Key words: Elaeis guineensis Jacq., genetic identity, regenerated plantlets, somatic embryogenesis, SSR marker

Oil Palm Phytochrome-Interacting Factor4 (PIF4) Gene is Conserved and Highly Expressed During Somatic Embryogenesis

Oil palm is used in food, fuel and cosmetic industries. Tissue culture is the best way to propagate oil palm; unfortunately the somatic embryogenesis during tissue culture takes long time. The molecular mechanism of somatic embryogenesis in oil palm remains unknown. Recent research reported that auxin plays an important role in early and post-embryogenic plant. PHYTOCHROME-INTERACTING FACTOR4 (PIF4) regulates levels of auxin and the expression of key auxin biosynthesis genes. Our research aims to characterize oil palm PIF4 gene. Thus, we cloned EgPIF4, analyzed the domain using bioinformatic and examined the expression of EgPIF4 during somatic embryogenesis at different tissue including callus and somatic embryo stages; globular, torpedo, cotyledon, and plantlet stage using real-time PCR method. The result showed that EgPIF4 gene comprised 1,737 bp with 9 exons, which encode 578 amino acid residuals. It contains a conserved domain called basic helix-loop-helix domain. EgPIF4 has high level of expression at somatic embryogenetic stage specifically globular and torpedo stage suggested that EgPIF4 plays an important role during somatic embryogenesis. The future characterization of EgPIF4 function in oil palm will help to understand somatic embryogenesis process and facilitate the improvement of the oil palm tissue culture