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No description available.

No description available.

The quolls are among the largest of the remaining carnivorous marsupials in the Australasian region, and thus occupy an important ecological niche as top predators and scavengers. All quolls are currently in decline and threatened to some degree yet the application of molecular information to the conservation and management of quolls has been unexplored until now. In this thesis I use two independent and highly variable genetic marker systems, the mitochondrial DNA (mtDNA) control region and nuclear microsatellites, to explore various aspects of conservation genetics relevant to the management of quolls. These aspects include an examination of the phylogenetic or evolutionary relationships among all six species of quolls, an examination of the genetic diversity within populations and the degree of differentiation between populations of the four Australian species of quolls, and the definition of units for conservation within these species. The development of suitable nuclear markers was a vital first step in defining levels of genetic variability and differentiation within and between the different populations and species. These markers proved to be highly variable and provided a wealth of information of relevance to the conservation of these species, and will be extremely useful in further studies. The use of the mtDNA control region for phylogenetic analyses was a novel approach to examining this question in quolls and also proved to be highly informative. Results from these phylogenetic analyses highlight the necessity of 1) examining more than one exemplar of each species, as well as 2) finally bringing some consensus to the question of the evolutionary relationships among quolls. Results show that northern quolls form the earliest split from all other quolls and that western quolls are closely related to the two New Guinean species. Furthermore, there is evidence for distinct lineages within species, corresponding to geographically separate or isolated populations. Levels of genetic variability within populations were examined using the microsatellites developed previously. Genetic variation was significantly higher in western quolls than in any other species. This was surprising given the long term and widespread decline of this species. There were also significant differences between populations within species in the level of genetic variability. Low levels of variability were usually found in small or captive bred populations or populations in severe decline. Genetic differentiation between populations was also explored using microsatellites. Significant differentiation in allele frequency distributions was found between most pairwise population comparisons, indicating that each of these populations forms a separate management unit (MU) for conservation purposes. One notable exception was found among populations of tiger quolls from a highly localized area in the Barrington Tops region of New South Wales. Using microsatellites, these populations were not significantly subdivided and thus appeared to be one MU. Using mtDNA, however, these populations were significantly subdivided and thus should be considered separate MUs. Differences in the way these two genetic markers are inherited (mtDNA is maternally inherited, microsatellites are biparentally inherited) provides a clue as to the social structure and organization of these cryptic nocturnal species. Consequently, the use of different genetic marker systems shows that there is sex-biased migration within this species. Finally, the degree of genetic differentiation observed within tiger quolls does not conform to the currently recognized subspecific categories within this species. The major genetic split occurs between the Tasmanian and mainland populations of tiger quolls, not between Dasyurus maculatus maculatus and D. m. gracilis. Thus, the Tasmanian and mainland populations form two distinct evolutionarily significant units (ESUs) for conservation purposes, and I propose that the Tasmanian populations should be elevated to the subspecific status to account for this.

Integrative and conjugative elements (ICEs) are a newly appreciated group of mobile elements that contribute immensely to bacterial evolution. The symbiosis island ICEMlSym[R7A] of Mesorhizobium loti strain R7A is the largest known mobile ICE (502 kb) and confers the ability to form a nitrogen-fixing symbiosis with plants of the genus Lotus. Transfer of ICEMlSym[R7A] to non-symbiotic bacteria has been demonstrated both in the laboratory and the environment and involves chromosomal integration of ICEMlSym[R7A] adjacent to the phe-tRNA gene in the recipient bacterium. Integration of ICEMlSym[R7A] requires the ICEMlSym[R7A]-encoded protein IntS which likely catalyses recombination between the attachment sites attP and attB, present on excised ICEMlSym[R7A] and phe-tRNA gene respectively. In this study it was shown that intS was required for both excision and integration and was required in both the donor and recipient bacterium for efficient transfer. A minimal attP region required for integration was defined and a repetitive sequence motif that likely represents the IntS-binding sequence identified. intS is expressed from two alternate promoters, a weak promoter on attL (left ICEMlSym[R7A]-chromosome junction) and a strong promoter formed on attP. Analysis of nested deletions of the attP region demonstrated that an unusually large region of attP, corresponding to that required for integration, was required for full gene expression. A novel recombination directionality factor RdfS was identified using bioinformatics. A quantitative PCR assay developed to detect the presence of excised ICEMlSym[R7A] in bacterial populations showed that excision of ICEMlSym[R7A] was almost undetectable in an rdfS mutant. Constitutive expression of rdfS resulted in curing of ICEMlSym[R7A] from R7A, creating a non-symbiotic derivative R7ANS. Bioinformatic analyses of rdfS and associated genes on ICEMlSym[R7A] led to discovery of 26 putative ICEs (ICESym family) in 12 α-proteobacterial species. The ICESym clusters contained homologues of up to 25 ICEMlSym[R7A] proteins, encoding functions involved in mating pore formation, DNA processing and regulation, of which 17 proteins were universally conserved. Introduction of pJR174 containing a copy of the ICEMlSym[R7A]-encoded quorum sensing (QS) gene traR induced excision in 100% of cells and stimulated a 100-fold increase in ICEMlSym[R7A] transfer. Stable maintenance of ICEMlSym[R7A] in these cells required the DNA relaxase-encoding gene rlxS. Introduction of pJR174 also induced a 1000-fold increase in the production of 3-oxo-C6-homoserine lactone and several other acyl homoserine lactones; this induction required traI1. The induction of QS was accompanied by various growth-inhibitory effects, similar to those observed in other rhizobial QS systems. The QS system activated ICEMlSym[R7A] excision through expression of msi172 and msi171, genes that are downstream of QS gene traI2. Expression of the adjacent gene msi170 separately repressed both QS and excision. The expression of msi170 was negatively regulated by Msi169, while msi169 was positively autoregulated. msi169 encodes an Xre family DNA-binding protein and homologues of it were identified on the ICESym clusters and on QS-regulated plasmids, suggesting that they have a conserved role as regulators of transfer- and QS-related genes. Overall, the results suggest that ICEMlSym[R7A] excision and transfer are subject to both population-density and cell-cycle-dependent regulation.

Includes bibliographical references. / 137, [113] leaves, [13] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Studies the control of prophage induction in coliphage 186 and the expression of a prophage-encoded function, tum. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1995

<p>Accurate initiation of transcription by RNA polymerase II depends on general transcription factors (GTFs), which include the TATA-binding protein (TBP) and the transcription factors (TF) IIB, IIF, IIE and IIH. In order to reconstitute mouse transcription <i>in vitro</i>, we cloned the genes encoding mouse TFIIB, and both subunits of TFIIE and TFIIF from a mouse cDNA library. TBP and TFIIB were expressed in <i>E.coli</i>, while both subunits of TFIIE and the two subunits of TFIIF were expressed in a baculovirus system. All these factors were purified to > 90% homogeneity. The more complex transcription factors, TFIIH and RNA polymerase II, were purified more than 1000-fold and to near homogeneity, respectively, from tissue cultured mouse ascites cells. We have shown that the purified mouse transcription factors are active in a reconstituted RNA polymerase II <i>in vitro</i> transcription assay. The transcription reaction was inhibited by α-amanitine, and dependent on the addition of all the GTFs.</p><p>Ribonucleotide reductase is a key enzyme in deoxyribonucleotide synthesis. It consists of two subunits, R1 and R2, which are both required for the enzyme activity. Transcription of the R1 and R2 genes is restiricted to the S-phase of the cell cycle, but the mechanisms that control this coordinated expression remain to be identified. We have studied initiation of transcription from the mouse R2 gene using a combination of <i>in vivo</i> reporter gene assays and <i>in vitro</i> transcription assays with crude nuclear extracts or with purified transcription factors. This promoter has an atypical TATA-box and a CCAAT-box that binds the transcription factor NF-Y.</p><p>We found that a mutation in the R2 CCAAT-box had no effect on the transcription level in in vitro transcription assays reconstituted with pure transcription factors. However, it significantly decreased the level of transcription in similar experiments using crude nuclear extract. We also found that the sequence downstream from the R2 transcription start site (5´-UTR) (from +1 to +17 base pairs relative to transcription start site) is essential for initiation of transcription from this promoter. The presence of the wild type 5´-UTR made the R2 TATA-box redundant. On the other hand, the R2 5´-UTR had a repressing effect on transcription from the mouse R2 promoter. This region contains a palindrome sequence that covers 10 base pairs, and it is partially conserved in the human R2 promoter. Gel shift assays and <i>in vitro</i> transcription experiments using antibodies against mouse TAF4 (=TAF135) demonstrate that TAF4 is a component of the protein complex that interacts with this palindrome region, and suggest involvement of this component of the TFIID complex in negative regulation of the R2 promoter.</p><p>The Adenovirus Major Late (AdML) promoter is commonly used as a model for studies of transcription initiation and regulation. It is a TATA-box dependent promoter, which also contains an initiator (Inr) element, a CCAAT-box interacting with transcription factor NF-Y, and an E-box binding the upstream stimulatory factor (USF). Using gel shift assays with recombinant NF-Y, USF, and immunopurified human TFIID, we show that binding of USF1 and NF-Y to DNA is not cooperative and that both factors independently facilitate binding of TFIID to the core promoter. The activation domains of NF-Y are expendable for this effect. Negative cofactor (NC2) comprises two subunits, which have a histone-fold structure similar to NF-Y, and represses transcription through formation of an inhibitory complex with TBP. Using an <i>in vitro</i> transcription system based on crude nuclear extracts, we show that NC2 has a negative effect on transcription in the presence of NF-Y or USF1, indicating that the two activators do not act as antirepressors. <i>In vitro</i> transcription using highly purified transcription factors efficiently reproduces repression of transcription by NC2. However, USF1 was inactive and NF-Y had a repressing effect in this system, which suggests that the activator functions of USF and NF-Y depend on cofactors.</p>

Graduation date: 2000

Graduation date: 1998