1. The hyphae of three basidiomycetes, <I>Coprinus cinereus</I>, <I>Coriolus versicolor</I> and <I>Gloeophyllum trabeum</I>, were stained with DAPI to ascertain their karyotic state. The process of clamp formation was observed in these fungi. 2. Hyphae and clamp connections were treated with CFW to show areas of chitin synthesis. Clamp connections show the same general pattern of chitin deposition as hyphae. Individual hyphae and clamps were treated with nikkomycin, a drug which inhibits chitin synthesis. Again, clamps behaved in a manner similar to hyphae. 3. Genomic DNA was isolated from <I>C. cinereus</I> and used in polymerase chain reactions to try to identify chitin synthase genes. A product was identified, but it is unclear as to whether this is a true chitin synthase gene. 4. Fungi were treated with the chitinase inhibiting drug, allosamidin. The position of branches and clamps relative to the hyphal apex was altered by this antibiotic indicating apical extension was disrupted. Allosamidin did not affect the specific growth rate of fungi in submerged liquid culture. 5. Fungi were grown under conditions of restricted calcium ion supply. With reduced calcium concentration hyphal branch frequency increased as did clamp frequency. Although the pattern of growth was affected, specific growth rate remained constant in cultures grown in submerged liquid culture. 6. The galvanotropic response of fungi was investigated. The magnitude of the response of hyphae to an electric field was dependent upon field strength, length of exposure to the field, pH and calcium ion concentration.

No description available.

No description available.

Tip growth is a form of cellular expansion characteristic of fungal hyphae and some types of plant cells. Currently there is no unified model that satisfactorily describes this in hyphal species. Traditionally turgor has been considered an essential driving force behind cell expansion. In recent years this hypothesis has been challenged by evidence that in some species tip growth can occur despite the absence of measurable hydrostatic pressure. There are currently two contentious theories of hyphal extension. These are the turgor-driven model and the amoeboid-movement theory. Though the essential mechanism underlying cell growth differs between these theories, the actin cytoskeleton is considered important in both. It has been suggested that both the turgor-driven and amoeboid-like modes of growth could occur depending on the whether the hyphae are growing invasively or non-invasively respectively (Money, 1990). It has also been proposed that both modes may occur within the same mycelium (Garrill, 2000). Two distinct patterns of actin have been identified in the hyphal tips of oomycetes. This has lead to the hypothesis that two different mechanisms of apical extension may be employed by some hyphal organisms. During the course of this thesis, actin deplete zones have been observed in a significantly higher number of invasive compared to non-invasive hyphae of the oomycete Achlya bisexualis. Furthermore the difference between burst pressures was found to be lower in invasive hyphae compared to non-invasive hyphae suggestive of a weaker cell wall. A lack of significant difference in turgor pressures between the invasive and non-invasive hyphae of this organism suggests that the deplete zone and weaker wall plays a functional role in enabling hyphae to penetrate substrate. Fractal analysis of mycelial colonies shows that the variation in agar concentration and therefore substrate solidity has a significant effect on mycelial morphology. This is most likely due to an effect at the cellular level. The results of the experiments carried out during the course of this thesis provide the basis for future work towards elucidating the mechanisms of hyphal extension.

Tip growth is a form of cellular expansion characteristic of fungal hyphae and some types of plant cells. Currently there is no unified model that satisfactorily describes this in hyphal species. Traditionally turgor has been considered an essential driving force behind cell expansion. In recent years this hypothesis has been challenged by evidence that in some species tip growth can occur despite the absence of measurable hydrostatic pressure. There are currently two contentious theories of hyphal extension. These are the turgor-driven model and the amoeboid-movement theory. Though the essential mechanism underlying cell growth differs between these theories, the actin cytoskeleton is considered important in both. It has been suggested that both the turgor-driven and amoeboid-like modes of growth could occur depending on the whether the hyphae are growing invasively or non-invasively respectively (Money, 1990). It has also been proposed that both modes may occur within the same mycelium (Garrill, 2000). Two distinct patterns of actin have been identified in the hyphal tips of oomycetes. This has lead to the hypothesis that two different mechanisms of apical extension may be employed by some hyphal organisms. During the course of this thesis, actin deplete zones have been observed in a significantly higher number of invasive compared to non-invasive hyphae of the oomycete Achlya bisexualis. Furthermore the difference between burst pressures was found to be lower in invasive hyphae compared to non-invasive hyphae suggestive of a weaker cell wall. A lack of significant difference in turgor pressures between the invasive and non-invasive hyphae of this organism suggests that the deplete zone and weaker wall plays a functional role in enabling hyphae to penetrate substrate. Fractal analysis of mycelial colonies shows that the variation in agar concentration and therefore substrate solidity has a significant effect on mycelial morphology. This is most likely due to an effect at the cellular level. The results of the experiments carried out during the course of this thesis provide the basis for future work towards elucidating the mechanisms of hyphal extension.

No description available.

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.

This thesis is an investigation of the exopolysaccharides produced by Lactococcus lactis subsp. Cremoris JFR1 and the hyphal cell wall glucans of Candida albicans. L. lactis is an important organism in the dairy industry for the production of fermented dairy products and the exopolysaccharides have been shown to add textural qualities to the foods. C. albicans is a fungal pathogen responsible for the common yeast infection and many post-surgery complications in hospitals and can grow in both the yeast and hyphae form. Through a series of GC-MS, NMR and chemical degradation experiments three unique polysaccharides are discovered in the L. lactis samples giving a molecular basis to the textural qualities provided by these molecules. Additionally, several unique structural features are discovered on the C. albicans hyphal glucan providing possible explanations for the differing immune responses elicited by the hyphae form of the fungus.

Understanding how hyphal organisms grow and develop is essential in order to manipulate mycelial colonies for purposes such as disease prevention and food production. One aspect of hyphal development that is not well understood is hyphal branching. Hyphal organisms branch as a way of creating new hyphal tips required for the search for nutrients, the acquisition of these nutrients and for hyphal fusion events that facilitate communication of signals within a mycelial colony. This investigation focused on the branching process occurring in the fungus N. crassa and in the oomycete A. bisexualis. An induction technique was developed to study branching in N. crassa involving local application of amino acids towards hyphae. This induced a branch to form along the hypha within the field of view. The use of this technique will enable the study of underlying events occurring internally prior to the visible branching stages. The role of Ca²⁺ hyphal branching was investigated in N. crassa suggesting Ca²⁺ is involved in apical dominance of the hyphal tip. This is based on a dose dependent response of increased branch frequency, decreased colony radius and decreased distance between the hyphal tip and the first branch, to the Ca²⁺ channel inhibitor verapamil. The stretch-activated Ca²⁺ channel inhibitors also had an effect on mycelial morphology. Gd³⁺ resulted in an increased branch frequency and a decreased colony radius and La³⁺ resulted in a decreased colony radius. The local application of verapamil towards N. crassa showed an increase in the number of multiple branches forming. Cytoplasmic Ca²⁺ was imaged in hyphae of A. bisexualis and N. crassa showing a tip-high Ca²⁺ gradient in A. bisexualis and Ca²⁺ sequestered into organelles in N. crassa. The role of F-actin in the process of hyphal branching was investigated using Lifeact N. crassa where F-actin could dynamically be seen at the site of both growing and non-growing hyphal branches. The involvement of F-actin at sites of septa development and associated with suspected vesicles was also observed.