This study aimed to identify the genetic basis of the Aspergillus nidulans hypB5 mutant phenotype. A. nidulans is a filamentous fungus that is widely used as a cell biological and molecular genetic model system. Its hyphae grow by localized polar secretion, producing tubular cells. A. nidulans hypercellular strains define five unlinked genes, hypA1-hypE2, which cause hyphal morphogenesis defects at 42°C. hypA is orthologous to Saccharomyces TRS120, which mediates Golgi transit and is widely conserved. The hypB5 restrictive phenotype resembles hypA1: wide hyphae, short basal cells and small nuclei. Like hypA1, shifting hypB5 mutants from 28°C to 42°C causes cessation of tip growth but isotropic expansion of basal cells. A hypA1, hypB5 double mutant was impaired for growth at 28°C, suggesting these genes have related roles, but neither was epistatic at 37°C so they function in different pathways. The A. nidulans pRG3-AMA1 genomic library was used to clone hypB5 complementing DNA by phenotype rescue, and subcloned to a 5 kb KpnI fragment, pYY2. pYY2 was disrupted and sequenced by Tn1000 insertional mutagenesis. The pYY2 sequence is 4975 bp and encodes a putative Sec7 domain which has 81% identity to the Saccharomyces SEC7 domain. The Sec7 domain is highly conserved from yeasts to mammals. Saccharomyces SEC7 encodes a guanine nucleotide exchange factor involved in COPI vesicle formation and Golgi biogenesis. Insertions in the pYY2 non-Sec7 domain coding region complemented hypB5 efficiently, whereas those in the Sec7 domain did not, indicating that the Sec7 domain is sufficient for function. A point mutation was found in the hypB5 strain Sec7 domain, which could explain temperature sensitivity. However, the pYY2 sequence is found on chromosome I whereas hypB maps to chromosome VII. Although the origin and functional role of the point mutation in the hypB5 strain Sec7 protein remains unresolved, it appears that pYY2 contains an extragenic suppressor. Thus hypB likely encodes an element in the COPI vesicle assembly pathway.

This study aimed to identify the genetic basis of the Aspergillus nidulans hypB5 mutant phenotype. A. nidulans is a filamentous fungus that is widely used as a cell biological and molecular genetic model system. Its hyphae grow by localized polar secretion, producing tubular cells. A. nidulans hypercellular strains define five unlinked genes, hypA1-hypE2, which cause hyphal morphogenesis defects at 42°C. hypA is orthologous to Saccharomyces TRS120, which mediates Golgi transit and is widely conserved. The hypB5 restrictive phenotype resembles hypA1: wide hyphae, short basal cells and small nuclei. Like hypA1, shifting hypB5 mutants from 28°C to 42°C causes cessation of tip growth but isotropic expansion of basal cells. A hypA1, hypB5 double mutant was impaired for growth at 28°C, suggesting these genes have related roles, but neither was epistatic at 37°C so they function in different pathways. The A. nidulans pRG3-AMA1 genomic library was used to clone hypB5 complementing DNA by phenotype rescue, and subcloned to a 5 kb KpnI fragment, pYY2. pYY2 was disrupted and sequenced by Tn1000 insertional mutagenesis. The pYY2 sequence is 4975 bp and encodes a putative Sec7 domain which has 81% identity to the Saccharomyces SEC7 domain. The Sec7 domain is highly conserved from yeasts to mammals. Saccharomyces SEC7 encodes a guanine nucleotide exchange factor involved in COPI vesicle formation and Golgi biogenesis. Insertions in the pYY2 non-Sec7 domain coding region complemented hypB5 efficiently, whereas those in the Sec7 domain did not, indicating that the Sec7 domain is sufficient for function. A point mutation was found in the hypB5 strain Sec7 domain, which could explain temperature sensitivity. However, the pYY2 sequence is found on chromosome I whereas hypB maps to chromosome VII. Although the origin and functional role of the point mutation in the hypB5 strain Sec7 protein remains unresolved, it appears that pYY2 contains an extragenic suppressor. Thus hypB likely encodes an element in the COPI vesicle assembly pathway.

The eukaryotic elongation factor 1 alpha 2 (eEF1A2) is a protein normally expressed only in the brain, heart and skeletal muscle. eEF1A2 is likely to be a breast and ovarian cancer oncogene based on its high expression in these malignancies and its in vitro transforming capacity . The goal of my thesis is to understand eEF1A2’s role in oncogenesis. In order to determine if eEF1A2 was a prognostic marker for ovarian cancer, we examined eEF1A2 expression in 500 primary human ovarian tumours. We show that eEF1A2 is highly expressed in approximately 30% of ovarian tumours. In serous cancer, high expression of eEF1A2 was associated with an increased 20-year survival probability. Expression of eEF1A2, in a clear cell carcinoma cell line, SK-OV-3, increased the cells ability to form spheroids in hanging drop culture, enhanced in vitro proliferative capacity, increased stress fiber formations, and reduced cell-cell junction spacing. Expression of eEF1A2 did not alter sensitivity to anoikis, cisplatin, or taxol. In order to examine the role of eEF1A2 in breast cancer, we used a three-dimensional culture system. The ability to disrupt the in vitro morphogenesis of breast cells cultured on reconstituted basement membranes is a common property of breast oncogenes. I found that phosphatidylinositol 4-kinase (PI4KIIIβ), a lipid kinase that phosphorylates phosphatidylinositol (PI) to PI(4)P, disrupts in vitro mammary acinar formation. The PI4KIIIβ protein localizes to the basal surface of acini created by the human MCF10A cells and ectopic expression of PI4KIIIβ induces multi-acinar formation. Expression of the PI4KIIIβ activator, eEF1A2, also causes a multi-acinar phenotype. Ectopic expression of PI4KIIIβ or eEF1A2 alters PI(4)P and PI(4,5)P2 localization, indicating a role for these lipids in acinar development. Therefore, eEF1A2 is highly expressed in ovarian carcinomas and its expression enhances cell growth in vitro. eEF1A2 expression is likely to be a useful ovarian cancer prognostic factor in ovarian patients with serous tumours. Furthermore, PI4KIIIβ and eEF1A2 both have an important role in the disruption of three-dimensional morphogenesis of MCF10A cells. Additionally, PI4KIIIβ and eEF1A2 likely have an important role in mammary neoplasia and development and could be anti-cancer targets.

Leaf discs of Saintpaulia ionantha x confusa hybrids (African Violets) were cultured and transferred between hormonal free medium (MS basal medium) and shoot-inducing medium (SIM) to determine whether there is a window of competence in shoot regeneration. The results showed that cultured cells were not responsive to shoot-inducing signals (i.e. not competent) until 3-5 days after explant isolation but the ability to regenerate shoots was not lost in surviving cells/tissues cultured on basal medium. Light microscopic observations found that first periclinal divisions of epidermal cells occurred at 3-5 days on SIM. Meristemoids were then formed from the derivatives of the original epidermal cells. It is proposed that cellular competence for shoot regeneration is acquired in culture. The pre-competent period consists of a resting phase and a "dedifferentiation" phase in which epidermal cells divide periclinally to form "dedifferentiated" cells which are the true target cells for shoot induction. Mutagenic treatments and propagation of a chimeral African Violet cultivar were carried out to study cell origin of adventitious shoots in African Violet leaf culture. The results suggest that adventitious shoots may originate from either multiple cells or single cells. The multiple cell origin is in contrast to the hypothesis of exclusively single cell origin for adventitious shoots proposed in several studies. Several factors were studied to identify the source of variations in shoot regeneration among individual explants and to define conditions favourable for shoot regeneration in African Violet in vitro culture. These factors include donor plant growth temperature, the presence of light and the quality of light in culture, the role of chlorophyll in cultured tissues, position of explants in leaves, age of explant materials, source and type of explants, wounding and leaf disc orientation on media. It was found that the presence of light in culture, age of leaf explants, source (in vitro cultured vs pot plant) and type (leaf vs petiole) of explants, wounding and leaf disc orientation on media all had a statistically significant effect on shoot production.

Thesis (Ph.D.) in Biochemistry and Molecular Biology--University of Maine, 2009. / Includes vita. Includes bibliographical references (leaves 95-107).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 197-236). Also available in electronic version. Access restricted to campus users.

How tissues and organs develop into their final shape during embryogenesis is a fascinating and long-standing question in developmental biology. Tissue morphogenesis is driven by a variety of events at the cellular level and individual cell shape change is one of the central morphogenetic engines. Thus, it is crucial to understand what signals specify the correct cell behavior in specific groups of cells during development. For my doctoral studies, I have focused on two cell shape change events, apical constriction and cilia assembly. First, we present data demonstrating that Shroom3 is essential for cell shape changes and morphogenesis in the developing vertebrate gut, where Shroom3 transcription requires the Pitx1 transcription factor. We identified a Pitx-responsive regulatory element in the genomic DNA upstream of Shroom3, and showed that Pitx proteins directly activated Shroom3 transcription in Xenopus. Moreover, we showed that ectopic expression of Pitx proteins was sufficient to induce Shroom3-dependent cytoskeletal reorganization and epithelial cell shape change. These data demonstrated new breadth to the requirements for Shroom3 in morphogenesis, and also provided a cell-biological mechanism for Pitx transcription factors action during morphogenesis. Next, we focused on understanding the transcriptional regulation of ciliogenesis. We first showed that Rfx2 transcription factor broadly controlled ciliogenesis, and by RNA- and ChIP-sequencing, we showed that Rfx2 directly regulated a wide range of genes encoding diverse ciliogenic machinery. Finally, in addition to ciliogenesis regulation, a large number of non-ciliary genes in our Rfx2 dataset led us to identify a novel role of Rfx2 in controlling insertion of multi-ciliated cells into the overlying mucociliary epithelium. Moreover, we showed here that Slit2, a target of Rfx2, was involved in multi-ciliated cell movements, possibly through mediating cortical E-cadherin level. This work allowed us to begin building a genetic network controlling multi-ciliated cells in mucociliary epithelium. Together, we showed a transcriptional regulation of apical constriction driving gut morphogenesis and a comprehensive transcriptional network that governs multi-ciliated cell development. / text

Plant cell walls play a central role in cell growth and morphogenesis. All plant cells have a primary wall. The formation of a secondary cell wall is restricted to particular cell types, such as the xylem cells, highly lignified cells that provide support and transport functions to the plant. The mechanisms regulating secondary cell wall biogenesis remain largely unknown. To identify genes involved in such mechanisms, a genetic screen for mutants with altered xylem development in the primary root of Arabidopsis thaliana has been conducted. Three different classes of mutants were identified. They are characterised by increased number of xylem strands (m"), altered timing of protoxylem differentiation (tpx) and ectopic lignification (eh). Initial characterisation of the mutant phenotypes, establishment of different complementation groups and their map position in the Arabidopsis genome has been determined. Mutations in the EL [I locus have been characterised in further detail. The eli l mutants exhibited ectopic lignification of cells throughout the plant that never normally lignify. Xylem cells in elil were misshapen and failed to differentiate into continuous strands, causing a disorganised xylem. elil mutants also exhibited altered cell expansion resulting in a stunted phenotype. Abnormal distribution of cellulose and lignin was observed in elil cell walls. Ultrastructural analysis of elil cell walls using an anti-lignin antibody has revealed that that the ectopic deposition of lignin-like compounds occurs within an altered secondary wall. Furthermore, other previously described cell expansion mutants, such as lit, rswl (at the conditional temperature) and det3, exhibited lignification patterns reminiscent to that of elil mutants. Analysis of the genetic interactions of elil with the lit mutant revealed that ELlI and LIT genes act in independent pathways to control cell expansion. These results, together with the double mutant analysis of eli l with other cell expansion mutants suggested a link between cell growth and differentiation of secondary thickened walls. Map-based cloning placed the ELJ1 gene in a 140-Kb interval on the top arm of Arabidopsis chromosome V. A candidate gene approach was used that identified a gene encoding a cellulose synthase catalytic subunit (CesA), AthCesA-3 as a candidate. Sequence analysis revealed that the AthCesA-3 gene is mutated in two elil alleles sequenced, both mutations leading to amino acid substitutions. Initial complementation experiments of elil plants with the wild type AthCesA-3 gene appeared to restore the wild type phenotype, suggesting that mutations in the AthCesA-3 gene gave rise to the elil phenotypes. These studies represent an important contribution to our understanding of the molecular mechanism of cellulose deposition during cell expansion and secondary cell wall deposition during plant morphogenesis.

No description available.

The growth of an organism involves the proliferation and migration of cells within an extracellular matrix. As a cell surface receptor, the Dag1 gene product dystroglycan links the intracellular cytoskeleton to the extracellular basement membrane in many cells. Thought to act as a structural protein dystroglycan may also participate in signal transduction. This study aims to better understand the role of dystroglycan during kidney morphogenesis. I hypothesised that a lack of dystroglycan in the precursor cells of the kidney could lead to altered kidney growth. Chimaeric mice deficient in dystroglycan were generated to test this hypothesis. A total of 38 chimaeras had genetic contribution and histological analysis performed on their kidneys. Of the chimaeras analysed, only four demonstrated altered kidney morphology. Further histological, immunohistochemical and biochemical studies established whether a link existed between this morphology and a deficiency in dystroglycan. Normal laminar architecture and nephrotic structures of the kidneys suggest that normal kidney organogenesis occurred in the absence of dystroglycan. The pattern and expression level of basement membrane components suggests that normal basement membrane formation also occured in the absence of dystroglycan. Biochemical analysis revealed that although dystroglycan protein levels correlate with the genetic contribution of the chimaeric kidney, it does not correlate with the altered morphology. Ureter blockage causing hydronephrosis can explain the morphology observed. A deficiency of dystroglycan in the ureter may in turn have caused this blockage. These findings suggest that dystroglycan is not necessary for kidney organogenesis, since kidney development occurred normally in all 38 chimaeric animals irrespective of genetic contribution.