Foot-and-mouth disease (FMD) virus causes a highly contagious, economically important disease of cloven-hoofed animals, including livestock animals such as cattle and swine. In South Africa, the disease is controlled primarily through prophylactic vaccination and strict animal movement control. To control effectively the spread of FMD, it has become increasingly more important to develop diagnostic tests that can differentiate FMDV-infected animals from those that have been vaccinated. Currently, the detection of antibodies to nonstructural proteins, especially the 3ABC nonstructural polypeptide, is considered to be the most reliable method to distinguish virus-infected from vaccinated animals. Towards the development of such a serological test, the primary aim of this investigation was to express the 3ABC nonstructural polypeptide of the SAT2 type FMD virus ZIM/7/83 in both a prokaryotic and eukaryotic expression system in order for the recombinant protein to be used as a diagnostic antigen. The nucleotide sequence of the 3ABC-encoding region of SAT2/ZIM/7/83 was determined, the amino acid sequence deduced and subsequently compared to corresponding sequences of other virus isolates representing all seven FMDV serotypes. Phylogenetic analysis revealed that the 3ABC polypeptide of the SAT serotypes, which are mostly restricted to sub-Saharan Africa, clustered separately from the euroasiatic FMDV serotypes (types A, O, C and Asia1). Amino acid sequence alignments also indicated considerable variation in the 3A, 3B and 3C proteins between the SAT and euroasiatic types located mainly in previously identified epitope-containing regions. These results suggest that the sensitivity and specificity of diagnostic tests based on the 3ABC nonstructural polypeptide of the European FMDV types may be compromised when applied to the African sub-continent. Therefore, a SAT-specific diagnostic assay is required to distinguish virus-infected from vaccinated animals. The 3ABC-encoding region of SAT2/ZIM/7/83 was subsequently expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein using the bacterial expression vector pGEX-2T, and in Spodoptera frugiperda insect cells using the BAC-to-BACTM baculovirus expression system. Although high-level expression of the recombinant GST-3ABC protein was obtained, the GST-3ABC protein was insoluble and could not be purified by glutathione affinity chromatography. Therefore, the recombinant GST-3ABC fusion protein was purified from reverse-stained SDS-polyacrylamide gels and shown to be immunoreactive in Western blot analysis using an FMDV-specific serum. Expression of the 3ABC polypeptide in insect cells infected with a recombinant bacmid yielded soluble recombinant protein, but the level of expression was lower compared to that obtained in E. coli. In addition, Western blot analysis of cell extracts prepared from recombinant bacmid-infected cells revealed the presence of three immunoreactive proteins of 47, 25 and 18 kDa. These correspond with the size of the FMDV proteins 3ABC, 3AB and 3A, respectively, suggesting that the 3C protease was responsible for proteolytic cleavage of the 3ABC polypeptide. Based on the results obtained, the bacmid expression system appears to be more suitable for the production of the 3ABC polypeptide. / Dissertation (MSc)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

Non-coding DNA in the human genome is widely studied to investigate its effect on coding DNA and gene expression. Non-coding DNA contains cis-regulatory elements that influence transcription of genes upstream, downstream, or nearby. These regulatory elements have largely been studied as enhancers that promote the transcription of genes. To explore these regulatory elements as silencers, we chose validated bifunctional elements to study their silencing capability and their chromatin markers. We used chromatin immunoprecipitation methods with dCas9 to target these elements using single-guide RNAs (sgRNAs). We experimented with various cloning methods to insert dCas9 into the pUAS vector. We initially planned to use the Gibson Assembly method, but after no success, we tried site-directed mutagenesis and traditional cloning with restriction enzymes. We were able to successfully insert dCas9 into the pUAS vector with traditional cloning, and we were then able to inject the construct into Drosophila melanogaster. We designed sgRNAs to target desired elements of DNA that we chose to study as cis-regulatory elements. The sgRNA sequences were cloned into the pCFD5 vector and injected into another line of flies. The transgenic flies containing the pUAS/dCas9 plasmid will then be crossed with the flies containing the pCFD5/sgRNA to develop offspring that express the target elements and could undergo chromatin pulldown to examine the bifunctional regulation of these DNA elements in cells. Results from a quantitative PCR (qPCR) assay on Drosophila expressing the cloned pUAS vector with dCas9 and a sgRNA for the white gene showed chromatin pulldown efficiency and successful transfection. The Drosophila chromatin targeted by the sgRNAs will be pulled down, solubilized, and then analyzed on a western blot to screen for chromatin modifications, primarily histone modifications. We can then identify chromatin markers associated with elements when they act as silencers in the mesoderm versus when they act as non- mesodermal enhancers. We can also determine if the silencer acts by interacting with a promoter or with an enhancer to repress gene expression. If ENCODE can profile the data found in this project, the chromatin markers can act as a predictive tool for the identification of silencers.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第19972号 / 薬科博第63号 / 新制||薬科||7(附属図書館) / 33068 / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 橋田 充, 教授 佐治 英郎, 教授 髙倉 喜信 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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A gene coding for the cambialistic superoxide dismutase (SOD) was isolated from a LambdaGEM-11 genomic library of <i>Bacteroides fragilis</i>. In order to generate a complete genomic library, <i>B. fragilis</i> genomic DNA was partially digested with the restriction endonuclease Sau3AI and was ligated to cloning vector, LambdaGEM-11. After in vitro packaging, DNA was used to infect <i>E. coli</i> KW 251. The genomic library was finally established in the plaque population. Recombinant phage DNAs containing the SOD gene were detected by a ³²P-labelled synthetic oligonucleotide with 17 bases. The sequence of this oligonucleotide was deduced from the N-terminal amino acid sequence of <i>B. fragilis</i> FeSOD. Two recombinant phage DNAs were selected based on he results of plaque hybridization. Further analysis with restriction mapping and DNA sequencing revealed that only one recombinant phage DNA contained the SOD gene. Southern hybridization and restriction mapping located the SOD gene in the SalI-BamHI fragment (2.1 kb). Sequence analysis identified the orientation and open reading frame (ORF) of the gene. Translation of ORF revealed that SOD consists of 193 amino acid residues. The size of the deduced polypeptide is consistent with the molecular weight of SOD subunit (MW 21,000). The B. fragilis SOD sequence was compared with those of other SODs. The amino acid residues contributing metal ligands, the hydrophobic shell of the active site, and amino acids at the subunit contact are almost fully conserved in B. fragilis SOD. Expression of SalI-BamHI fragment in E. coli SOD double mutant (sodA, sodB), QC1799, produced an active SOD whose activity zymogram was identical to that of purified B. fragilis SOD. In addition, Western analysis of the expressed protein separated on SDS acrylamide gel also displayed a band identical to the subunit of B. fragilis SOD. However, a larger molecular weight band was also detected. This band migrated closely to the subunit of B. fragilis SOD. This larger peptide may be the product of gene translation from an ATG 21 bases upstream of the ATG start codon of B. fragilis gene. The cambialistic feature of SOD gene product was also confirmed from in vitro and in vivo metal substitution. / Ph. D.

Thesis advisor: Jianmin Gao / Protein-protein interactions (PPIs) are essential for all biological functions. Developing peptides that disrupt these PPIs is an avid research effort, as peptides possess several advantages over small molecules and monoclonal antibodies. Peptide phage display is a useful tool in identifying peptides for targeting PPIs. This technology displays up to 10^10 unique polypeptides on the surface of bacteriophage, which after several rounds of panning enriches high affinity peptide sequences towards a target protein. Phage display is classically done on immobilized discrete protein; however, we propose to use this technology to identify peptides ligands for overexpressed oncogenic proteins on live cells in-vitro. This is a more accurate representation of the therapeutic target landscape and resembles how the peptide will interact with the receptor in-vivo. Several groups have explored live cell panning, such as Ruoslahti et al. and Cieslewicz et al., and while they demonstrate the capabilities of in-vitro style phage display, there are areas for improvement. We intend to improve on this previous work by 1. Identifying a peptide ligand against specific receptor/protein, and 2. By incorporating the use of covalent phage libraries to elucidate a high affinity binder. This work will be accomplished using the mammalian epidermal oncogenic cell line, A431, that is known to overexpress epidermal growth factor receptor (EGFR). Epidermal growth factor receptor (EGFR) is responsible for cellular proliferation, survival, differentiation and metastasis, which makes it an attractive target to inhibit oncogenic proliferation. Despite successfully marketed monoclonal antibodies and tyrosine kinase inhibitors, EGFR can mutate and develop resistance as diseases progress; this phenomenon, in addition to the benefits of peptides as therapeutics, are driving factors for pursuing this project. Despite our best efforts using non-covalent phage libraries to identify a viable ligand, screening against EGFR extracellular domain (ECD) has proven to be more difficult than anticipated. We hypothesize that non-covalent phage libraries do not possess any sequences with a high enough binding affinity for this protein, and that the use of covalent libraries will be needed to pull out a positive hit. Due to these findings, we have successfully constructed two phage libraries, a ACX7C and a ACX7C-TEV, where the latter introduced a TEV protease cleavage site on the C’-terminal side of the randomized amino acids suitable for covalent warhead modification and screening. Further, we have begun work on constructing an EGF-displaying phage construct to aid in optimizing a live cell panning protocol. In the future, we plan to evaluate ligand affinity and protein density, as well as determine the optimal covalent warhead/peptide combination for live cell screenings. With this information, we intend to apply this to other oncogenic cell lines, such as MCF-10CA1a, to identify potent peptide ligands for overexpressed oncogenic proteins. / Thesis (MS) — Boston College, 2022. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

published_or_final_version / abstract / Zoology / Doctoral / Doctor of Philosophy

Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.