Browsing by Subject "Molecular cloning"
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Item Cloning and functional characterization of the WdSTUA and WdPACC genes of Wangiella dermatitidis(2007) Wang, Qin, 1970 Nov. 15-; Szaniszlo, Paul J.To study the function of WdStuAp and WdPacCp in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast phase predominance, WdSTUA and WdPACC were cloned, sequenced, disrupted and expressed. WdStuAp was most similar to the APSES proteins of Aspergillus species and its APSES DNA-binding domain was located in its N-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich, yeast maintenance agar medium, YPDA, at 37°C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation and invasive hyphal growth on the nitrogen poor, hyphae-inducing agar medium, PDA, at 25°C. Ectopic expression of WdSTU Arepressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. Expression of WdSTUA in S. cerevisiae induced pseudohyphal growth on the nitrogen poor medium. WdPacCp was also most similar to the PacCp proteins of Aspergillus species. Three zinc finger DNA-binding motifs were at the N-terminus, and the C-terminus had the signaling protease cleavage site. WdPACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation of the coding sequence for about 40 residues upstream of the conserved processing protease cleavage site of WdPacCp affected growth on YPDA, increased sensitivity to Na⁺ stress, decreased growth level at neutral-alkaline pH, and repressed hyphal growth on PDA at 25°C. Truncation of the coding sequence for the conserved signaling protease box of WdPacCp impaired growth and reduced RNA expression of class II chitin synthase gene WdCHS1 at acidic pH, and activated hyphal growth on PDA. My results suggested that WdStuAp and WdPacCp play important roles in yeast-hyphal transitions in W. dermatitidis, and that WdPacCp is particularly important for W. dermatitidis to adapt to different ambient pH conditions.Item Cloning of novel low and high molecular weight heat shock genes in wheat(Texas Tech University, 1998-12) Campbell, Janee L.Heat tolerance is an important objective for wheat improvement in the Southern US Great Plains. It is hypothesized that heat shock proteins play a vital role in the plant's ability to survive high temperature stress. Cloning low and high molecular weight HSPs from a thermotolerant variety of wheat will provide a building block for later studies of their function. Moreover, once significance of a HSP gene in plant thermotolerance is confirmed, beneficial cloned genes from thermotolerant varieties could be used to improve other wheat germplasm.Item Expression of the human 5-HT3A receptor using the baculovirus- and Epstein-Barr virus (EBV)-based expression systems(Texas Tech University, 2000-08) Liu, MinghuaDue to the scarcity of 5-HT3A receptors in native tissue, it is extremely difficult to isolate sufficient quantities of protein to perform structural and biophysical studies on them. High-level heterologous expression systems provide a means of overcoming this limitation. The cDNA ofthe human 5-HT3A receptor with a hexa-hisfidine at its Cterminus was constructed with a PCR cloning strategy and was confirmed with dideoxy sequencing. The function ofthe recombinant 5-HT3A receptor was examined using the Xenopus laevis oocyte expression system and two-electrode voltage-clamp electrophysiological recordings. Application of serotonin to recombinant receptors elicited inward sodium currents. No significant difference was revealed in the EC50S of serotonin in wild-type and 5-HT3A-His6 receptors. The cDNA for the human 5-HT3AHis6 receptor was subcloned into a baculovirus transfer plasmid and expressed in baculovirus-infected Sf9 insect cells. The expression ofthe human 5-HT3A-His6 receptor was assessed with both Western blot analysis and nickel affinity purification. In addition, saturation-binding experiments were carried out with crude membranes from baculovirus-infected insect cells and using affinity-purified receptors with the 5-HT3R antagonist [^H] GR65630. The KItem Molecular characterization of genes expressed by the pupal wing epidermis of the silkmoth Antheraea polyphemus(Texas Tech University, 1989-05) Kumar, Murukambat NNot availableItem Molecular cloning and characterization of cellulose synthase genes expressed during tracheary elements differentiation in cultures of Zinnia elegans(Texas Tech University, 2002-08) Hwang, SangjoonIsolated mesophyll cells of Zinnia elegans induced to differentiate into tracheary elements (TEs) semi-synchronously in culture are a valuable experimental system for research on cellulose synthesis. To explore a possible cellulose synthase gene family that might be specific to secondary wall thickening, a RACE (Rapid Amplification of cDNA Ends) strategy was applied using total RNA from Zinnia cells cultured in differentiation medium for 60 hours. Three cDNA fragments, designated ZeCesAl (AF323039), ZeCesA2 (AF323040), and ZeCesA3 (AF323041), that were mainly expressed during TE differentiation and not during primary wall synthesis were isolated from 3' RACE technique. A cDNA fragment corresponding to the 5' part of ZeCesAl gene was obtained from the 5' RACE technique. A full-length ZeCesAl cDNA sequence was constructed by overlapping the 5' and 3'-RACE fragments. In common with known plant cellulose synthase genes iCesAs), ZeCesAl encodes a predicted membrane protein having conserved motifs and domain structure. Based on amino acid sequence comparisons and phylogenetic analyses, ZeCesAl-3 show a closer relationship to secondary wall-specific CesAs, especially to GhCesAl, AtCesAS, and PtCesA2, than to primary wallspecific CesAs. This indicates that ZeCesAl 3 are orthologs of CesA genes from other species that belong to the secondary wall clade. ZeCesAl-3 represent very similar sequences and define a set of paralogous genes, probably duplicated from the same original gene. Northern blot analyses and tissue printing revealed that ZeCesAl-3 were expressed in a close association with TE differentiation in vitro, and ZeCesAl was expressed in regions with developing vascular bundles in stems and leaves.Item The role of endoderm in vascular patterning(2002) Vokes, Steven Alexander; Krieg, Paul A.; Fischer, Janice AnnAngioblasts, the precursor cells that give rise to the endothelial layer of blood vessels, arise from a purely mesodermal population. Individual angioblasts coalesce to form the primary vascular plexus through a process called vasculogenesis. A number of reports in the literature suggest that signals from the adjacent endoderm are necessary to induce angioblast specification within the mesoderm. We present evidence, using both embryological and molecular techniques, indicating that endoderm is not necessary for the induction of angioblasts. While Xenopus embryos lacking endoderm contain aggregates of angioblasts, these angioblasts fail to assemble into endothelial tubes. Endothelial tube formation can be rescued however, by implantation of endodermal tissue from sibling embryos. Based on these studies in Xenopus, and corroborating experiments using the quail embryo, we conclude that endoderm is not required for angioblast specification, but does provide an inductive signal for vascular assembly. In additional experiments using avian embryos, we demonstrate the molecular identity of this inductive signal, showing that endodermally derived Sonic Hedgehog is both necessary and sufficient to form endothelial tubes from angioblasts in avian embryos. This demonstrates a novel role for hedgehog signaling in vascular development and provides a molecular model for vascular assembly.Item The covalent structure of four salmonellar flagellar filament proteins(Texas Tech University, 1986-05) Wei, Li-naNot available