Genetics - Theses

Permanent URI for this collection

http://hdl.handle.net/11343/292

Search Results

Now showing 1 - 10 of 20

Molecular and cellular biological studies of the Drosophila melanogaster stoned gene

Smith, Michiko. (University of Melbourne, 1999)

The Drosophila melanogaster stoned locus is an essential neurologically expressed gene, of which temperature-sensitive mutants exhibit a lethal interaction with the shibire locus. This, and subsequent evidence gathered from electrophysiological, localisation and sequence analyses of stoned have indicated a likely role critical to the synaptic vesicle cycle. Taking advantage of the genetic tools available for the dissection of stoned function, the role of stoned in the synaptic vesicle cycle was directly implicated in this study. The stoned transcript has a unique tandemly-arranged dicistronic structure, with both open reading frames (ORFs) separated by a fifty-five nucleotide sequence. Presented in this study, is firstly the identification of two distinct translatory products of the stoned ORFs, StonedA and StonedB. Although both novel proteins, the predicted amino acid sequences have allowed for the progressive identification of domains that have homology to a number of proteins implicated in the synaptic vesicle cycle. StonedA and StonedB were determined to have a direct association with synaptic vesicles by co-immunoprecipitation and sedimentation properties. However, rather than a ubiquitous association with synaptic vesicles, StonedA and StonedB are associated with separate subsets within the entire population. The biochemical localisation of StonedB repartitioned over D. melanogaster shibire background subcellular fractions. The data suggests that StonedA and StonedB are indeed part of the molecular mechanisms in the synaptic vesicle cycle. Perhaps StonedB-tagged synaptic vesicles are involved in the elusive kiss-and-run mechanism of the synaptic vesicle cycle.
Molecular and cellular biological studies of the Drosophila melanogaster stoned gene

Smith, Michiko. (University of Melbourne, 1999)

The Drosophila melanogaster stoned locus is an essential neurologically expressed gene, of which temperature-sensitive mutants exhibit a lethal interaction with the shibire locus. This, and subsequent evidence gathered from electrophysiological, localisation and sequence analyses of stoned have indicated a likely role critical to the synaptic vesicle cycle. Taking advantage of the genetic tools available for the dissection of stoned function, the role of stoned in the synaptic vesicle cycle was directly implicated in this study. The stoned transcript has a unique tandemly-arranged dicistronic structure, with both open reading frames (ORFs) separated by a fifty-five nucleotide sequence. Presented in this study, is firstly the identification of two distinct translatory products of the stoned ORFs, StonedA and StonedB. Although both novel proteins, the predicted amino acid sequences have allowed for the progressive identification of domains that have homology to a number of proteins implicated in the synaptic vesicle cycle. StonedA and StonedB were determined to have a direct association with synaptic vesicles by co-immunoprecipitation and sedimentation properties. However, rather than a ubiquitous association with synaptic vesicles, StonedA and StonedB are associated with separate subsets within the entire population. The biochemical localisation of StonedB repartitioned over D. melanogaster shibire background subcellular fractions. The data suggests that StonedA and StonedB are indeed part of the molecular mechanisms in the synaptic vesicle cycle. Perhaps StonedB-tagged synaptic vesicles are involved in the elusive kiss-and-run mechanism of the synaptic vesicle cycle.
Comparison of amidase genes in Aspergillus species

Sharp, Julie Anne. (University of Melbourne, 1998)

A study was undertaken to identify evolutionally conserved sequences within the acetamidase gene regulatory region across Aspergillus species which may define binding sites for transcriptional control regulatory proteins. Six amdS genes were cloned and characterised from five Aspergillus species and compared to A. nidulans and A. oryzae amdS genes. The amdS gene from A. echinulatus was predicted to be non functional due to a termination codon after amino acid 23. A proposed amdS pseudogene was identified in A. ustus. In this species two genes with amdS homology were identified, one with acetamidase activity and one lacking detectable amidase activity. A comparison of the Aspergillus spp. amdS coding region sequences revealed a very high level of conservation. The hydrolysis of amides by the Aspergillus spp. amdS genes was studied by heterologous gene expression in A. nidulans. Acetamide, propionamide, valeramide and butyramide were found to be substrates for A. ustus and A. unguis AmdS. A comparison of the transcriptional regulatory controls governing expression of Aspergillus spp. amdS and A. nidulans amdS genes was performed by heterologous expression of amdS genes fused to a reporter gene. Only some of the transcriptional control pathways regulating A. nidulans amdS were shown to regulate the different Aspergillus spp. amdS genes. During the course of this study, an amidase gene family was discovered in Aspergillus. The identification, cloning, functional characterisation, regulation and evolution of two members of the amidase gene family, gmdB (encoding general amidase B) and gmdC (encoding general amidase C) are described. Four gmdB genes were studied from four Aspergillus species and one gmdC gene was studied in A. nidulans. Substrates for the GmdB enzymes are valeramide, hexanamide and tyrosine amide, however substrate preferences for amide hydrolysis by the different GmdB enzymes differ between Aspergillus species. Transcriptional regulation of gmdB, determined by heterologous gene expression of reporter constructs in A. nidulans is not conserved between the species. The amidase family of genes comprising amdS, gmdB and gmdC is highly conserved in sequence and gene structure and suggests evolution from a common ancestor.
Comparison of amidase genes in Aspergillus species

Sharp, Julie Anne. (University of Melbourne, 1998)

A study was undertaken to identify evolutionally conserved sequences within the acetamidase gene regulatory region across Aspergillus species which may define binding sites for transcriptional control regulatory proteins. Six amdS genes were cloned and characterised from five Aspergillus species and compared to A. nidulans and A. oryzae amdS genes. The amdS gene from A. echinulatus was predicted to be non functional due to a termination codon after amino acid 23. A proposed amdS pseudogene was identified in A. ustus. In this species two genes with amdS homology were identified, one with acetamidase activity and one lacking detectable amidase activity. A comparison of the Aspergillus spp. amdS coding region sequences revealed a very high level of conservation. The hydrolysis of amides by the Aspergillus spp. amdS genes was studied by heterologous gene expression in A. nidulans. Acetamide, propionamide, valeramide and butyramide were found to be substrates for A. ustus and A. unguis AmdS. A comparison of the transcriptional regulatory controls governing expression of Aspergillus spp. amdS and A. nidulans amdS genes was performed by heterologous expression of amdS genes fused to a reporter gene. Only some of the transcriptional control pathways regulating A. nidulans amdS were shown to regulate the different Aspergillus spp. amdS genes. During the course of this study, an amidase gene family was discovered in Aspergillus. The identification, cloning, functional characterisation, regulation and evolution of two members of the amidase gene family, gmdB (encoding general amidase B) and gmdC (encoding general amidase C) are described. Four gmdB genes were studied from four Aspergillus species and one gmdC gene was studied in A. nidulans. Substrates for the GmdB enzymes are valeramide, hexanamide and tyrosine amide, however substrate preferences for amide hydrolysis by the different GmdB enzymes differ between Aspergillus species. Transcriptional regulation of gmdB, determined by heterologous gene expression of reporter constructs in A. nidulans is not conserved between the species. The amidase family of genes comprising amdS, gmdB and gmdC is highly conserved in sequence and gene structure and suggests evolution from a common ancestor.
Sensitisation of DNA by incorporation of iodine or bromine

D'Cunha, Glenn C. (University of Melbourne, 1997)

This study has focused on the generation of lesions by UV photolysis of bromodeoxyuridine (BrdU)-substituted synthetic oligonucleotides and the characterisation of resulting cleavage products. Two main approaches were undertaken to generate potential new lesions The first involved the use of BrdU substituted synthetic oligonucleotides in photolysis experiments with UVB as the irradiation source. The second approach utilised BrdU- substituted oligonucleotides that also incorporated a ligand binding site (AA 77). These oligonucleotides were irradiated with UVA in the presence of the DNA binding ligand Hoechst 33258 (and analogues). Lesions produced from both these systems were investigated by analysis of cleavage products generated by photolysis alone or photolysis followed by base treatment. The fragments were characterised upon the basis of their size, termini and structure. The nature of the termini of the fragments on either side of the break, were characterised by end-labelling the oligonucleotides from the 5' or the 3' end. The yields of these products were estimated on the substituted and the complementary strand. This study has shown that at least five different types of lesion produced by photolysis of BrdU substituted oligonucleotides. These lesions have been termed A, B, C, D and E. Furthermore, the pathways that lead to the conversion of these lesions to strand breaks has been postulated. Lesion A is converted to a break directly after photolysis, while lesions B-D inclusive, are all alkali-labile lesions (AL) and therefore require further treatment with 1.0M piperidine to be converted to strand-breaks. Lesions B and C require treatment with mild-base (incubation at 50�C for 10 minutes in 1.0M piperidine) to be converted to breaks while lesions D and E require treatment with stringent base (incubation at 90�C for 30 minutes in 1.0M piperidine) to be converted to breaks. Each lesion has been identified and the resulting products have been characterised. Lesions A induced after photolysis is converted directly to a SSB, frank breakage, that results in an of 8bp product referred to as fragment F. The designation of fragment F is based on its electrophoretic mobility and is further discussed below. Lesion B is converted to a strand break with a resulting product that is indistinguishable from fragment F. The yield of fragment F was therefore found to increase following treatment with mild base. Procedures such as PAGE, DNA sequencing, HPLC and mass spectrometry have been used to purify and identify the structure of fragment F. These studies have shown conclusively that fragment F terminates in a phosphoryl group. Lesion C was identified and shown to produce damage at the same site as lesion C. Furthermore, lesion C was only converted to a strand break only under mild base conditions. The resulting fragment, termed S, was shown to be a bulkier species compared to F, based on its slower electrophoretic mobility determined by PAGE. Characterisation of fragment S has revealed it to be an intermediate species only expressed under mild base conditions; under stringent base conditions fragment S is converted to a phosphoryl terminating species indistinguishable from F. This is reflected by the increase in the yield of fragment F when fragment S is treated with stringent base. A third cryptic lesion (lesion D) was also identified and found to be converted to strand break only when treated with stringent base. The resulting fragment is indistinguishable from fragment F generated directly after photolysis or following treatment with mild base. Stringent base treatment also converts a fourth cryptic lesion to a strand break; the resulting ALL was located on the opposite strand. The damage was produced at the A nucleotide that complements the BrdU. Of all the products identified, this is generated in least abundance. The second part of this investigation involved the UVa photolysis of ligands bound to BrdU-DNA. The products generated from the UVA photolysis of BrdU-DNA in the presence of the ligand were found to be identical to those generated from the UVb photolysis of BrdU-DNA in the preceding section. This suggests that the uracilyl radical is the common mediator of damage in the production of SSBs for both systems studied. In addition, the systematic designing of the oligonucleotides used in conjunction with electron donating ligands (from photoionisation of the ligand) has enabled the generation of data on electron transfer. It has been shown that UVA photolysis of BrdU-DNA in the presence of Hoechst 33258 can efficiently generate SSBs at BrdU sites 2-8bp away from the ligand binding site. The radioprotective properties of two newly synthesised Hoechst analogues were compared to Hoechst 33258 and Hoechst 33342, based on the yields of strand cleavage. Of all the ligands tested, Hoechst 33342 has proven to be the most potent radiosensitiser as reflected by the yields of SSBs produced. Both fragments F and S, generated via the UVB or UVA/Hoechst photolysis of BrdU-DNA, are primarily a result of attack of the 2-carbon on the deoxyribose but argue that other interactions are involved suggesting a mixed cleavage chemistry. Such attack results in a high yield of SSBs on BrdU substituted oligoDNA. The mechanisms that lead to the production of strand break have been postulated. The mixture of fragments generated after photolysis and chemical treatment suggest it is unlikely to be solely the result of 2' chemistry.
The isolation and analysis of the hap genes of Aspergillus nidulans

Papagiannopoulos, Peter. (University of Melbourne, 1996)

The 5' regulatory region of the amdS gene of Aspergillus nidulans, which encodes an acetamidase required for growth on acetamide as a carbon and nitrogen source, contains a CCAAT sequence required for setting the basal level of transcription. Mobility shift assays have identified a factor in A. nidulans nuclear extracts that binds specifically to this CCAAT sequence. In Saccharomyces cerevisiae, the HAP3 and HAP5 genes encode components of a highly conserved multi subunit complex which is able to bind CCAAT sequences. The identification, cloning and sequencing of genes from A. nidulans with homology to HAP3 and HAP5, known as hapC and hapE respectively, is described here. The predicted amino acid sequences of the proteins encoded by the hapC and hapE genes share extensive sequence identity to conserved regions in HAP3 and HAP5 respectively. Furthermore, they both show identity to the histone-fold motif, a motif used widely as a means for protein-protein and DNA- protein interactions. A haploid carrying a hapC deletion has been created and is viable, but grows poorly on all media tested. This null mutant grows especially poorly on acetamide as a sole carbon and nitrogen source, indicating that hapC plays a role in amdS expression. In agreement with this notion, the hapC deletion results in reduced levels of amdS expression, particularly under conditions of carbon limitation. Nuclear extracts prepared from the hapC deletion mutant show no CCAAT specific binding to the amdS or gatA promoter, indicating that hapC encodes a component of the complex binding at this sequence. In the presence of the hapC deletion growth on acetamide and amdS
Characterisation of the facB88 translocation of Aspergillus nidulans

Murphy, Rachael L. (University of Melbourne, 1996)

The positively acting regulatory gene facB of Aspergillus nidulans mediates acetate induction of the amdS (acetamidase) gene and genes required for acetate metabolism (facA, acuD and acuE). facB encodes a gene product with a Zn(II)2Cys6 DNA binding cluster, heptad repeats and potential activation domains, and binds to sequences 5' of amdS, facA, acuD and acuE. facB orthologues from A. oryzae and A. niger have been compared to the A. nidulans facB gene at the level of DNA and predicted protein sequences. Highly conserved regions in the predicted translation products have been identified and discussed in terms of their relevance to protein function. Putative transcription factor binding sites were identified in the 5' non-coding regions of each orthologue. The facB88 reciprocal translocation results in high-level constitutive amdS expression (superactivation) and this is mediated by a chimeric gene formed by the translocation event. This chimeric gene was found to encode the N-terminal half of FacB, including the DNA binding domain, fused to a new gene encoding two C2H2 zinc finger DNA binding motifs. The new gene was designated amdX, the cloning, sequencing and transcriptional analysis of which is reported here. Inactivation of amdX and creation of multicopy strains by transformation revealed that amdX is a minor positive regulator of amdS expression. The DNA binding function of AmdX was examined, using an Escherichia coli-expressed AmdX fusion protein, by gel mobility shift assays and DNase I footprinting. AmdX was shown to bind to two sites in the amdS 5' region which overlap the binding sites for the AmdA and CreA regulatory proteins. The facB88 chimeric gene mediating amdS superactivation was designated facB-amdX. Molecular dissection of facB-amdX showed that both the FacB and AmdX DNA binding domains are required for amdS superactivation and that they contribute to amdS expression in a synergistic manner. Cooperative DNA binding of the FacB and AmdX DNA binding domains to the amdS 5' region is proposed to mediate the superactivation ability of the facB-amdX gene product.
Analysis of the facB gene of Aspergillus nidulans

Todd, Richard B. (University of Melbourne, 1995)

The facB gene of Aspergillus nidulans encodes a transcriptional activator which contains a Zn(II)2Cys6 DNA binding cluster, a putative leucine zipper-like dimerization motif and potential acidic activation domains. facB mediates acetate induction of amdS (acetamidase) and the genes of acetate utilisation (facA, acuD, and acuE). This thesis describes an analysis of the facB gene product using a mutational approach and in vitro DNA binding studies. A facB null mutant was constructed by gene replacement This mutant is unable to grow when acetate is the sole carbon source, indicating that facB is required for utilisation of acetate. Important functional regions of FacB were identified by the localisation of lesions in facB mutant alleles. Regions of FacB likely to be involved in DNA binding, dimerization, transactivation, and a possible structural function in acetate metabolism were identified. The Zn(II)2Cys6 motif was shown to be necessary for facB function by in vitro mutagenesis of specific cysteine residues. A facB allele harbouring this Zn(H)2Cys6 mutation failed to complement for growth on acetate upon transformation into a facB null mutant Transformation of this facB allele into a wildtype strain resulted in decreased growth on acetate and acetamide media. The DNA binding function of FacB was examined in detail. A FacB fusion protein was expressed in Escherichia coli and was shown to bind DNA fragments from facB-regulated promoters in vitro in a sequence-specific manner. Abolition of in vitro binding by an expressed FacB fusion protein containing mutated cysteines in the Zn(II)2Cys6 cluster confirmed that this motif was required for DNA binding activity. Expressed FacB proteins with mutations flanking the Zn(II)2Cys6 motif showed altered DNA binding specificity, implicating these regions in determination of DNA binding site specificity. FacB binding sites were defined by Gel Mobility Shift Assay (GMSA), DNase I and Missing Contact Interference Footprinting analyses. FacB binding sites fall into two dissimilar classes. Mutant binding sites from the amdS promoter, known to modulate expression, were shown to alter FacB binding in vitro.
Sites of recombination and transposition in interspecies hybrid cells

Littlejohn, Margaret R. (University of Melbourne, 1995)

This work involved isolating DNA rearrangements from interspecies hybrid cell lines. In an interspecies hybrid cell, a human transposable element which has moved into the hamster chromosome complement can be easily identified as a newly transposed element. The ease of isolation of these elements could make interspecies somatic cell hybrids an effective model system for studying transposition, as well as other DNA rearrangements. Previous cytological studies of interspecies somatic cell hybrids have shown many examples of large scale translocations and rearrangements. Genomic libraries were produced from two human x hamster hybrid cell lines in a ? vector. The human specific and hamster specific probes used to screen the libraries were produced by PCR using repetitive element primers and human or hamster genomic DNA as template. A total of 147,000 clones were screened from the hybrid libraries with the human and hamster specific probes. Fourteen clones were isolated which hybridised to both human and hamster DNA. Restriction maps were determined for each of the ten clones showing the pattern of human and hamster DNA. Of the 14 clones, five were found to contain two breakpoint junctions within the X insert, involving inserts of less than 13 kb. Two of those clones contained hamster DNA that had recombined or integrated into human DNA and three contained human DNA that had recombined or integrated into hamster DNA. Therefore, the recombination events appeared to be occurring in both sets of DNA. The remaining nine clones contained one breakpoint junction. These may contain the breakpoint of a translocation, or may be one side of a larger recombination event which could not be contained within a single X clone. None of the clones studied in detail were found to be cloning artifacts produced during library construction. The number of events actually detected would be a minimum number of those occurring as the screening method used means that human DNA fragments inserting into human DNA and hamster DNA fragments inserting into hamster DNA would not be detected. Also, recombination or integration events involving unique sequences would not be detected because of the use of the repetitive element probes. After close analysis of the clones, none of the four events identified using a human LI element probe were found to be due to de novo transposition events. This suggested that integration of human repeats into hamster DNA appeared more likely to be by illegitimate recombination, rather than by genuine transposition events. There are many previous examples of rearrangements in hybrid cells of divergent species. However, no previous study carried out on hybrid genomes has detected recombination events involving such small fragments of DNA. Users of interspecies somatic cell hybrids need to be aware of events such as these when using clones obtained from hybrids for mapping and sequencing. This study raises the possibility that clones from human chromosomes derived from interspecies somatic cell hybrids could be randomly contaminated with short segments of rodent DNA.
Cloning and characterisation of the regulatory gene, amdA, of Aspergillus nidulans

Lints, Robyn. (University of Melbourne, 1993)

Semi-dominant mutations in the amdA gene lead to elevated expression of the gene encoding acetamidase, amdS. These mutations also cause constitutive expression of the acetate-inducible gene, aciA. In the amdS 5' regulatory region, two cis-acting mutations, amdI66 and amdI666, have been isolated which specifically affect amdA activation of amdS. These mutations affect an 18 bp GA-rich sequence, thought to define the amdA site of action within the amdS promoter region. Similar GA-rich sequences have also been found in the 5' region of aciA. The work presented in this thesis describes the cloning and initial functional characterisation of the amdA gene and three of its mutant alleles. The wild-type amdA gene has been cloned by undertaking a chromosome walk from genes gatA and alcC on linkage group VII and localised by complementation of an amdA loss-of-function mutation. Transcriptional analysis reveals that the gene is expressed at low levels and constitutively under growth conditions which affect expression of amdS and aciA. The gene is predicted to encode an 880 amino acid protein which contains two C2H2 zinc fingers, similar to those found in Xenopus laevis transcription factor TFIIIA, a potential nuclear localisation sequence and two potential transcriptional activation domains. Functional analysis of in vitro-generated deletion mutations and of the semi-dominant mutant allele, amdA7, indicate that the acidic activation domain identified in the protein sequence is functionally significant. Interestingly, the C2H2 zinc finger motifs identified in the protein are similar to those found in the carbon catabolite repressor protein, CreA, which also regulates amdS and recognises sequences which coincide with the proposed site of action for AmdA within the promoter of this structural gene.

Genetics - Theses

Permanent URI for this collection

Filters

Date

Author

Subject

Type

Settings

Sort By

Results per page

Statistics

Citations

Search Results