viii, 111 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1977

viii, 111 leaves : ill., tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1977

Hybridization studies based on the prototrophic selection technique, involving the use of auxotrophic mutants of strains of all accepted species of the genus Kluyveromyces, are reported. Two main groups of mutually interfertile taxa were established within the genus. The first group comprises Kluyveromyces bulgaricus, Kluyveromyces cicerisporus, Kluyveromyces dobzhanskii, Kluyveromyces drosophilarum, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces phaseolosporus, Kluyveromyces vanudenii and Kluyveromyces wikenii. The second group consists of Kluyveromyces dabzhanskii, Kluyveromyces drosophilarum, Kluyveromyces laotis, Kluyveromyces vanudenii and Kluyveromyces wiokerhamii. Hybrids were also detected in crosses involving Kluyveromyces drosophilarum and Kluyveromyces waltii as well as Kluyveromyces marxianus and Kluyveromyces thermotolerans. In terms of the concept of the biological species and in compliance with the requirements of the International Code of Botanical Nomenclature, taxa which hybridize with Kluyveromyces marxianus and form fertile recombinants at frequencies observed in intraspecific crosses, are accepted as varieties of Kluyveromyces marxianus. Hybridization was observed between Kluyveromyces marxianus var. lactis and the presumed imperfect forms of some Kluyveromyces species, namely Candida kefyr, Candida macedoniensis and Torulopsis sphaerica. Recombination was not detected in crosses involving Kluyveromyces marxianus var. marxianus and representatives of other yeast genera, i.e. Pichia, Saccharomyces, Torulaspora and Zygosaccharomyces. Conclusions regarding the relationship between members of the genus Kluyveromyces, reached on the basis of this investigation are compared with those reported by other workers, who based their investigations on phenotypic characteristics as well as on the determinations of mol % G+C and DNA-DNA homology studies.

The SUC2 locus of Saccharomyces cerevisiae encodes two forms of invertase; a constitutively expressed cytoplasmic enzyme and a glucose-repressible secreted and glycosylated enzyme which is initially produced with an amino-terminal signal peptide. The coding sequence of the SUC2 locus has been placed under the control of the constitutive ADH1 promoter and transcription terminator in a centromere based yeast plasmid vector from which invertase is expressed in a Sue" strain of yeast. Oligonucleotide-directed mutagenesis has been used to create a PstI site in the gene at the point encoding the signal peptide cleavage site. An internal methionine codon, the translation start for the cytoplasmic invertase, has been replaced by a serine codon. Mutants in the signal peptide sequence have been produced by replacing the region of the gene upstream of the PstI site with synthetic oligonucleotide cassettes with mixtures of nucleotides at several positions. The mutants could be divided into three classes based on their ability to secrete invertase. The first class of mutants produced secreted invertase, but in reduced amount. There is no obvious correlation between mutation and phenotype. The second class, represented by mutant 4-55B, also exhibited a reduced level of invertase, but a significant fraction (30%) of the enzyme is intracellular. This mutant had a delay in signal peptide cleavage which retards passage of invertase through the secretory pathway. The third class was defective in secretion. Most were defective in translocation from the cytoplasm to the lumen of the endoplasmic reticulum (ER), and produced enzymatically active, non-glycosylated pre-invertase in the cytoplasm. This class of mutant invertases, when transcribed and translated in vitro, was not processed by canine pancreas signal recognition particle (SRP) and microsomes. Comparison of the sequences of the mutant signal peptides of this non-translocating class identifies amino acids at the extreme amino-terminus as the causative defect. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Please read the abstract in the section 00front of this document / Thesis (PhD)--University of Pretoria, 2005. / Genetics / Unrestricted

Studies of breeding relationships are integrated with the nomenclature of Agrocybe praecox. Type studies of potential synonyms are presented. Nomenclatorial and type studies indicate that Agrocybe molesta has been confused with A. praecox sensu lato. New field and microscopic characteristics are introduced which differentiate between A. molesta and the A. praecox group. A. molesta is redescribed to reduce confusion and a neotype is designated for this taxon. Four sibling species of Agrocybe praecox are identified and neotypes designated for A. praecox and A. gibberosa. A new species, Agrocybe montanus is described. / M.S.

The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.

The MAT∝ locus of the yeast Saccharomyces cerevisiae encodes two regulatory proteins responsible for determining the ∝cell type. The MAT∝1 gene encodes ∝1, a positive regulator of ∝cell-specific genes, whereas the MAT∝2 gene encodes a negative regulator of a cell-specific genes (∝2). MAT∝2. (in conjunction with the MATα1 gene) also determines the α/∝ diploid cell type by repressing haploid-specific genes. ∝2 exerts its effect at the transcriptional level in the ∝ cell by binding to a sequence located upstream of α cell-specific genes. The present study undertook to examine, through in vitro genetic manipulation, the structure/function relationship of the MAT∝ regulatory proteins, particularly∝2, in their role as gene regulators. The construction of mutant MAT∝2 genes containing termination codons at various points within the gene, and subsequent transformation of the mutant genes into mat∝2 yeast, indicated that the carboxy-terminal one-third of the gene product was necessary for full repressor activity in the haploid as well as in the diploid. A segment within the carboxy-terminal one-third of ∝2 displays some homology to the higher eukaryote homeo domain as well as to a prokaryotic bihelical DNA-binding structural motif. This region of the gene was subjected to semi-random missense mutagenesis in vitro and the mutant genes were analyzed by transformation into strains containing chimaeric genes that encode β-galactosidase from ∝2 and a1/∝2. repressible promoters. In this manner it was demonstrated that most of those residues in ∝2. which correspond to conserved amino acids in the prokaryotic DNA-binding structure and in the homeo domain are essential for the two repressor activities of ∝2. Several mutations more severely affected the ability of ∝2 to repress α-specific genes than haploid-specific genes. Analysis of the temperature dependence of the activities of some of the mutants was consistent with the existence of a helix-turn-helix structure at this region of the protein. Finally, further analysis of some of these mutants in vitro confirmed that the observed defect correlated with a loss of DNA-binding activity. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008

Carbon catabolite repression in A. nidulans is a regulatory system which allows the organism to utilize the most preferable carbon source by repressing the expression of genes encoding enzymes utilizing alternative carbon sources. A ubiquitination pathway was shown to be one of the key mechanisms which regulate carbon source utilization, when creB was found to encode a deubiquitinating enzyme. Strains containing mutations in creB show loss of repression for some metabolic pathways in carbon catabolite repressing conditions, and also grow very poorly on several sole carbon sources such as quinate and proline, suggesting CreB plays multiple roles in the cell. This work describes the analysis of the interaction of CreB with CreA, and with PrnB and QutD. Various epitope-tagged versions of CreA were expressed in A. nidulans, and an internally located HA-epitope tag was found to allow detection of CreA using Western analysis. A diploid strain was constructed between strains containing HA-tagged CreA and FLAG-tagged CreB. When CreB was immunoprecipitated, HA-tagged CreA was also precipitated in the diploid, indicating that CreA and CreB are present in a complex in vivo. To determine whether CreA is a ubiquitinated protein, a version of CreA that was tagged with both an HA epitope and a His-tag was expressed in A. nidulans, and protein extracts were precipitated with an UbiQapture™-Q matrix. Western analysis was used to show that CreA was present in the precipitate. These findings suggest that CreA is a ubiquitinated protein, and a target of the CreB deubiquitination enzyme. To determine whether the proline permease (PrnB) is a direct substrate of CreB, plasmids to express epitope-tagged versions of PrnB were constructed and introduced into the prnB mutant strain. No tagged protein could be detected by Western analysis, even when these constructs were over-expressed from the gpdA promoter. However, a construct to express an HA epitope tagged version of quinate permease (QutD) fully complemented the qutD mutant strain, and HA-tagged QutD could be easily detected in Western analysis when probed with the anti-HA monoclonal antibody. A diploid strain was made between a complementing transformant and a strain expressing a FLAG-tagged CreB construct. When QutDHA was immunoprecipitated, CreBFLAG was detected in the immunoprecipitate of the diploid. A proportion of QutDHA was also co-precipitated in the diploid when CreBFLAG was immunoprecipitated. Thus, CreB is present in a complex with QutD in vivo. Further results showed that the concentration of QutD in the cell is lower in a creB null mutant background than in the wild-type background, indicating that deubiquitination is required to prevent protein turnover. Northern analysis of mRNA showed that the failure of creB mutant strains to grow on quinate medium was not due to a failure of transcriptional induction of qutD, as the amount of mRNA was not lower in a creB1937 mutant background compared to the wild-type. Furthermore, experiments were undertaken that showed that QutD is a ubiquitinated protein. These findings suggest that quinate permease is regulated through deubiquitination involving the CreB deubiquitination protein in A. nidulans. In addition to the candidate protein approach asking whether CreA is a substrate of CreB, a proteomics approach was also used to identify proteins that interact with CreA. However, no clear interacting proteins were identified using this approach. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008