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    Characterising CG5846 (Peep) in Drosophila melanogaster neural function : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatū, New Zealand
    (Massey University, 2024) Wilson, Sarah Jean
    Histone deacetylase 4 (HDAC4) is a transcriptional regulator that has been implicated in a number of neurodevelopmental and neurodegenerative diseases that are associated with intellectual disability, cognitive defects, and/or memory loss. Both the accumulation of nuclear HDAC4 and its loss-of-function have been linked to these conditions, therefore exploring HDAC4’s role in neuronal function is essential to understand the molecular mechanisms underlying these diseases. In Drosophila, overexpression of HDAC4 results in defects in morphogenesis of axons in the mushroom body, a structure essential for memory formation, as well as long-term memory defects and disruption to the development of the compound eye. The molecular mechanisms underlying these HDAC4-induced phenotypes are currently unknown. RNA-sequencing on fly heads in which HDAC4 was overexpressed has previously been performed and showed few genes were transcriptionally regulated by HDAC4. In addition, an enhancer/suppressor rough eye phenotype screen has also been performed which identified a number of genes that interact genetically in the same molecular pathway as HDAC4. To further investigate the molecular mechanisms underlying HDAC4 dysfunction, an RNA interference (RNAi) based candidate screen for potential HDAC4-interactors was performed, which involved quantification of developmental defects in the mushroom body and eye following RNAi knockdown of each candidate. It was hypothesised that if a phenotype resulting from RNAi knockdown was similar to that induced by HDAC4 overexpression, that candidate may function in similar molecular pathways. A single candidate-interactor was selected (CG5846, named Peep) for further investigation. On overexpression, Peep and HDAC4 co- distribute in nuclei of mushroom body neurons, however no physical interaction was detected. Furthermore, overexpression of Peep did not rescue the HDAC4-induced mushroom body or eye defects. Due to the uncharacterised nature of Peep, a thorough investigation was performed to assess the importance of Peep in survival, longevity, motor function, brain development, courtship learning and memory, and wing development. Peep was observed to be essential for survival of glial cells and for normal mushroom body development, which warrants further investigation. Reduced expression of Peep also resulted in a unique severe necrotic eye phenotype, and through this, Peep was shown to play a potential role in processes involved in regulating mitochondrial and proteasomal function, apoptosis and oxidative stress. These data provide the first documented characterisation of the functional role of Peep in Drosophila development and provide the basis for further investigation into the underlying molecular mechanisms involved in mushroom body and eye development.
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    Population genetics and genomics of a marsupial species : analysis of native and invasive brushtail possum populations (Trichosurus vulpecula) : a dissertation presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Conservation Biology, Massey University, Manawatū Campus, New Zealand
    (Masey University, 2024-04-15) Pattabiraman, Nimeshika
    One of the leading causes of global biodiversity decline is the introduction of invasive pest species that destroy native flora and compete with native fauna for food and other resources. Aotearoa New Zealand is one of the foremost countries in the world that has focussed on eradicating pest species and in particular exotic mammals from the archipelago, which lacks native, terrestrial mammals. The New Zealand Government recently set in train the ambitious task of removing all mustelids, rats, and possums from the terrestrial landscape by the year 2050. Brushtail possums (Trichosurus vulpecula) were introduced to Aotearoa New Zealand from Australia in the mid-1800s, after which they were translocated across the country and have become widespread, destroying indigenous habitat, eating native birds and invertebrates, and spreading bovine TB. Control efforts have seen possum numbers decline in the last two decades from close to 75 million in 2002 to 40 million in 2020. There is, however, a gap in the scientific understanding of possum populations with respect to their genetic composition and population structure across the country, and this knowledge could help us develop effective and dynamic management strategies to eradicate possums on a nationwide scale. In this thesis, I focus on three aspects of population structure and diversity of brushtail possums. First, I investigated a small geographical study area - The Kenepuru Peninsula - where I sought evidence of genetic correlations with geography, time and fur colour. I used two types of genetic markers that target the nuclear and mitochondrial regions of possum DNA with large population samples. In every case, it was determined that the possums comprised one freely interbreeding population at this scale. In particular I demonstrated that colour morphs associated with distinct subspecies in Australia, freely interbreed in New Zealand. I then increased the scale of sampling to include representation of populations across New Zealand and Australia, with the same genetic markers. This threw light on the heterogenous nature of possum diversity in New Zealand, and showed that even after ~110 generations, possums retained genetic separation among spatial groups. Additionally, the data showed evidence of multiple possum lineages across New Zealand that are derived from several Australian populations. High haplotype diversity in New Zealand suggests that the rapidly expanding population has retained novel haplotypes and the data thus far indicated a non-homogenous (metapopulation) distribution of possums without geographical concordance. As the project progressed, I was able to apply high-throughput genotyping-by-sequencing to generate a large genomic dataset. This dataset provided much more detail of the genotypic distribution of possums in Australia and among invasive metapopulations in New Zealand, as well as informing us of the relationship between them. This large, robust database of possum population structure and genetic diversity throughout Aotearoa New Zealand will support future studies in providing informed management decisions to eradicate brushtail possums.
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    Towards better New Zealand adapted industrial hemp (Cannabis sativa L.) cultivars : a quantitative genetic analysis of key traits and evaluation of genetic diversity : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science at Massey University, Manawatu, New Zealand
    (Massey Library, 2023-09-12) Komahan, Dona Harshani Shanika
    Hemp (Cannabis sativa L.) is emerging as a promising commercial crop in New Zealand. Breeding programmes developing material specifically adapted to the country’s target population of environments are at their early stages of establishment. With 20 approved hemp cultivars, 15 of which are imported, it is vital to prioritize research and development focused on the genetic improvement of hemp to generate cultivars that are well adapted to New Zealand’s crop production environments, offering both economic and agronomic benefits. The primary aim of this thesis is to lay the foundation for future hemp breeding in New Zealand by focusing on three critical initial steps: (1) conducting multi-site trials to evaluate the performance of commercial hemp cultivars based on key traits and characterize the effects of Genotype × Environment interaction (G × E interaction), (2) estimating quantitative genetic parameters and predicting genetic gain for key traits to identify breeding strategies that would improve the efficiency of maximizing genetic gain, and (3) assessing genetic diversity using molecular markers and characterizing available genetic resources. These steps are crucial for developing new cultivars that are both climate-resilient and beneficial to New Zealand’s hemp industry. Multi-site trials were conducted for two years over the 2019/2020 and 2020/2021 growing seasons using six hemp cultivars approved to be grown in New Zealand: CFX-2, CRS-1, Ferimon 12, Katani, Futura 75, and Finola. The 2019/2020 trials were conducted in Palmerston North and Wairarapa, while the 2020/2021 trials were conducted at two nearby sites in Palmerston North with different soil characteristics. Across both multi-site trials, biomass yields extend over a broad range from 1.43 to 28.41 t/ha, and seed yields ranged between 0.018 and 3.78 t/ha. The study’s findings on G × E interactions showed a complex scenario that mostly differed from prior reported research. Although significant (P < 0.05) genotypic variation was observed in most traits, only a few, specifically stand establishment in the 2019/2020 season, along with plant height and stem diameter in the 2020/2021 growing season and biomass yield across both years, showed significant (P < 0.05) G × E interactions, suggesting consistent performance across sites. The traits plant height, stem diameter, biomass yield, seed yield, and thousand seed weight have all revealed high cultivar mean broad sense heritability, indicating potential exploitable underlaying genetic variation that could be used to generate breeding pools for cultivar development. A quantitative genetic analysis was conducted using 50 half-sib families (HS families) focused on six key traits: stem diameter, plant height, number of internodes, seed yield, thousand seed weight, and biomass yield to investigate the magnitude of additive genetic variation within a breeding population. The study also aimed to predict the rate of genetic gain for these traits. Significant (P < 0.05) additive genetic variation was observed among the 50 HS families for traits: seed yield, biomass yield, stem diameter, plant height, and the number of internodes, highlighting each trait’s additive genetic variation available for breeding. Deterministic simulation of the breeding strategies, among HS family selection (AF-HS) and among- and within-family selection (AWF-HS), was conducted to predict genetic gain for these traits. AWF-HS yielded the highest genetic gains compared to AF-HS. The study estimated moderate to strong positive genetic correlations among all traits, generally higher than the phenotypic estimates. The genetic correlation coefficients suggest that genotypes with higher seed and biomass yields can be developed. There was significant (P < 0.05) differences between some HS families and the two commercial check cultivars, Fasamo and Férimon 12. HS families outperforming these commercial checks were identified. A laboratory experiment examined the genetic diversity and the degree of variability within and among six New Zealand-approved hemp cultivars using seven previously developed microsatellite (SSR) markers. STRUCTURE analysis identified two distinct genetic clusters that align with the plant’s reproductive biology (monoecious and dioecious), that further subdivided based on end-use (fibre, seed, fibre and seed, and CBD cultivars). The genetic diversity metrics revealed moderate genetic diversity among the six hemp cultivars. The average observed heterozygosity (Ho) and the expected heterozygosity (He) were 0.44 and 0.50, respectively. The Wright’s fixation index (FIS) varied from 0.26 (Futura 75) to -0.02 (Finola). The Analysis of Molecular Variance (AMOVA) revealed that a large portion of the total genetic diversity was found within individual cultivars. While only 11% of the molecular variation was attributed to differences among the cultivars, 19% was attributed to variations among individuals within each cultivar. Notably, the genetic variation within individual plants (69%) exceeded the variation observed both among individuals and among different cultivars. Additionally, considerable molecular variation was observed between male and female individuals of dioecious hemp cultivars. The genetic diversity metrics of female and male groups indicated that female individuals possess greater variation than their male counterparts. Collectively, results from this Ph.D. study lay the groundwork for future hemp breeding research and development in New Zealand. They provide an initial resource, offering insights into genetic diversity, genotypic variation, and the impact of G × E interactions on key traits evaluated among six introduced offshore cultivars. Furthermore, the estimates of quantitative genetic parameters add value to the existing knowledge base, enhancing the development of hemp cultivars suited to New Zealand crop production environments.
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    Primary metabolic processes as drivers of leaf ageing
    (Springer Nature Switzerland AG, 2021-10) Kanojia A; Shrestha DK; Dijkwel PP
    Ageing in plants is a highly coordinated and complex process that starts with the birth of the plant or plant organ and ends with its death. A vivid manifestation of the final stage of leaf ageing is exemplified by the autumn colours of deciduous trees. Over the past decades, technological advances have allowed plant ageing to be studied on a systems biology level, by means of multi-omics approaches. Here, we review some of these studies and argue that these provide strong support for basic metabolic processes as drivers for ageing. In particular, core cellular processes that control the metabolism of chlorophyll, amino acids, sugars, DNA and reactive oxygen species correlate with leaf ageing. However, while multi-omics studies excel at identifying correlative processes and pathways, molecular genetic approaches can provide proof that such processes and pathways control ageing, by means of knock-out and ectopic expression of predicted regulatory genes. Therefore, we also review historic and current molecular evidence to directly test the hypotheses unveiled by the systems biology approaches. We found that the molecular genetic approaches, by and large, confirm the multi-omics-derived hypotheses with notable exceptions, where there is scant evidence that chlorophyll and DNA metabolism are important drivers of leaf ageing. We present a model that summarises the core cellular processes that drive leaf ageing and propose that developmental processes are tightly linked to primary metabolism to inevitably lead to ageing and death.
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    Genomic differentiation of brushtail possum (Trichosurus vulpecula) populations : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Zoology at Massey University, Manawatū, New Zealand
    (Massey University, 2023-10-15) Carmelet-Rescan, David
    Toxin resistance, an ecologically functional trait, has emerged as a result of the coevolutionary arms race between plants and herbivores. This adaptive response allows organisms to counteract the detrimental effects of toxins. Such adaptation occurs within three primary ecological contexts: predator resistance, prey resistance, and auto-resistance. In the context of plant-herbivore interactions, the production of toxic secondary metabolites by plants plays a pivotal role, triggering a dynamic arms race with herbivores. This ongoing battle leads to the development of diverse defensive strategies in plants and corresponding counter-adaptations in herbivores. Toxin resistance studies have not only shed light on coevolutionary dynamics but also provided insights into regional adaptations and population fragmentation. The case of sodium fluoroacetate, a potent toxin found in certain plant species, offers a specific example of toxin resistance in brushtail possums (Trichosurus vulpecula). Deepening our understanding of the underlying mechanisms driving toxin resistance becomes particularly interesting in this case because of the notable variation in sodium fluoroacetate resistance observed among subspecies. The brushtail possum (Trichosurus vulpecula) is protected in its natural range of Australia but as an invasive pest in New Zealand populations are controlled by the application of human made sodium fluoroacetate, providing a strong selective force for the potential parallel evolution of toxin resistance. I first focus on the population fragmentation and past connectivity of brushtail possum subspecies in their native range using ecological niche modelling and genetic analysis. My results suggest that Pliocene and Pleistocene climate oscillations played a significant role in shaping the distribution and structure of these widespread marsupials. The findings highlight the limited gene flow between subspecies that would have facilitated their adaptations to regional plant assemblages. I studied gene expression using RNA sequencing to reveal significant differences in transcription levels between adult and juvenile brushtail possums. These findings provide insights into the mechanisms associated with possum development, tissue development, cell cycle, and extracellular matrix. Furthermore, the downregulation of specific genes, such as Cytochrome P450, in juveniles may indicate their role in possums' dietary adaptations. Differential gene expression among subspecies of brushtail possum shed light on genomic differentiation and identified candidate genes involved in regional adaptations involving toxin tolerance. Further analyses comparing subspecies has identified genes under positive selection and enriched biological pathways that may be associated with sodium fluoroacetate resistance. These findings support the idea of genomic divergence in spatial populations and provide hypotheses on the metabolic pathways involved in toxin resistance. Collectively, these studies offer valuable insights into the coevolutionary dynamics, toxin resistance mechanisms, gene expression and selection patterns in brushtail possums (Trichosurus vulpecula) and formulated strong hypotheses related to sodium fluoroacetate resistance. This study presents a reliable method to use transcriptomics to investigate the expression and genomic differences behind observed phenotypic variation within a single species.
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    The molecular ecology of an understudied endemic marine isopod - Isocladus armatus : a thesis presented in partial fulfilment of the requirements for the degree of Master of Natural Science at Massey University, Albany, New Zealand
    (Massey University, 2019) Pearman, William Samuel
    The study of populations and the adaptive significance of traits is a major theme in molecular ecology literature. In this thesis I present three lines of research that contribute to the understanding the molecular ecology of a species of New Zealand endemic marine isopod - Isocladus armatus (family: Sphaeromatidae). The goal of this thesis is to develop and utilize a framework to better understand the genomics of marine isopods from a range of genomic perspectives. The first primary chapter aims to assess two ways of enriching mitochondrial DNA from whole genome DNA, and to assemble this species mitochondrial genome. My research indicates that an atypical mitochondrial genome structure, widespread across Isopoda - but previously thought absent within Sphaeromatidae, is present within I. armatus suggesting that this trait has been maintained for an order of magnitude longer than previous estimates. The second primary chapter aims to describe and understand the genetic structure of populations for 8 locations around New Zealand, to understand connectivity and dispersal for I. armatus. Using a panel of 8,020 loci, I find high gene flow on a small spatial scale, while populations on a larger spatial scale exhibit a pattern of Isolation-By-Distance. Additionally, gene flow over one well known biogeographic barrier was much higher than between any other populations on a similar spatial scale, suggesting this barrier may not exhibit a strong effect on this species. Thus, my research indicates a need to revisit and study the way biogeographic barriers affect species with different life histories. The final primary chapter aims to understand the genetic basis for colour polymorphism in I. armatus, with the intention of understanding the adaptive significance and selective mechanism behind this trait. I use genome wide association approaches with a panel of 20,000 loci to answer these questions. I found that loci associated with Colour Polymorphism exhibited signatures of disruptive selection, contrary to initial hypothesis where I expected balancing selection to main colour polymorphism. I propose that substrate heterogeneity in Isocladus armatus’ habitat results in microhabitats, each of which imposes a selective pressure benefiting a specific morph type. The size of these microhabitats is so small that high levels of interbreeding between these microhabitats, and thus between morphs, results in the maintenance of polymorphism across the population.
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    Landscape genetics for conservation management : brushtail possums (Trichosurus vulpecula) in New Zealand : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Conservation Biology at Massey University, New Zealand
    (Massey University, 2019) Pattabiraman, Nimeshika
    The negative impact of brushtail possums (Trichosurus vulpecula) on New Zealand ecosystems became apparent soon after their introduction from Australia in 1858. Possums not only denude native vegetation but prey on native birds and invertebrates. They also carry bovine tuberculosis (TB) impacting the dairy industry and consequently the New Zealand economy. New Zealand possum populations have spread from several introduction sites and densities have increased. The resulting complex patterns of gene flow influences regional diversity, and potentially the effectiveness of control measures. Currently, ~100 million dollars are spent on 1080 management per year, mostly in response to Tb risk, but there is little information about the migration rates associated with resulting population density fluctuations. To determine whether the potential for intermixing between populations since their introductions could have caused a homogenizing effect on the genetic diversity across New Zealand, I began a detailed population genetic analysis by genotyping possums from 19 locations using nuclear microsatellites and mitochondrial DNA haplotyping from across the country to estimate population structure. Initial introductions of possums from multiple locations resulted in genetic and fur colour diversity but, in comparison to natural Australian populations, it appears that only a subset of genetic variants was brought to New Zealand from Australia. Mitochondrial sequence variation analyses showed overall high haplotype diversity with substantial differences among samples in haplotype frequencies, but with relatively low nucleotide diversity. Similarly, analysis of nuclear markers (microsatellite genotypes with Naïve Bayesian clustering) reveals that while there has been admixture between populations in various locations, indicated by shared genotypes, there are genetically distinct regional populations. Concordance of genetic and geographically distant sampling shows a well-developed population structure of possums across New Zealand. These results are also supported by pairwise Fst comparisons between all pairs of populations; although nearly all populations showed significant differences, there was no signature of isolation by distance as expected from their history of introductions. This study provides a foundation for further research into spatial structure of brushtail possums which will enable the effective targeting of management and is essential for modelling population recovery, disease spread, and potentially the emergence of toxin resistance. Predator-free 2050 is an ambitious objective considering current circumstances. In order to achieve its goals, even for the targeted species, we need to efficiently manage our resources and improve the accuracy of control measures to maintain long-term effects.
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    The transcriptional regulation of maspin : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
    (Massey University, 2004) Hollings, Andrew
    Maspin (mammary serine protease inhibitor) is a tumour suppressing member of the serpin superfamily. Maspin is expressed in normal breast and prostate cells, but reportedly down regulated during progression of cancer in these tissues. Maspin has been shown to inhibit cellular migration and invasion in vitro; while in vivo, maspin has been shown to inhibit tumour growth, metastasis, and angiogenesis. Maspin also plays a role in the sensitisation of cells to induced apoptosis. These functions of maspin are independent of serine protease inhibition; however the cellular mobility function is dependent on an intact reactive site loop. Despite this knowledge, the molecular mechanisms for all reported functions of maspin are currently unknown. Maspin is reported to be transcriptionally regulated: to date Ets, Ap1, and p53 transcription factors have been shown to activate transcription of maspin by binding directly to the promoter. Androgen is reported to be a negative regulator through the binding of the androgen receptor to a hormone response element within the promoter. This hormone response element is also responsible for an increase in maspin expression in response to tamoxifen, an anti-oestrogen drug. Transcriptional regulation of maspin has also been reported to be activated by other molecules, including gamma linolenic acid, manganese containing super-oxide dismutase, and nitric oxide, the mechanisms of regulation by these molecules is unknown. Loss of maspin expression in cancerous cells lines has been attributed to loss of one or more of the activating factors, and aberrant methylation of cytosine residues resulting in chromatin compaction. This study investigated the transcriptional regulation of maspin, with the aim of identifying transcriptional effectors important to the regulation of the gene. Identification of such factors may help identify a pathway in which maspin exerts its tumour suppressor functions. To this end, the maspin promoter was cloned and functional assays carried out. identifying several putative regions of the maspin promoter which may be important for the regulation of the gene. To date, the precise activator/repressor binding sites and the cognate proteins responsible for this regulation are unidentified.
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    Transformations of grapevine pathogens Eutypa lata and Phaeomoniella chlamydospora : a one year project thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 2005) Duan, Guowen
    A transformation system has been developed for the grapevine pathogenic fungi Eutypa lata and Phaeomoniella chlamydospora using a positive selection system based on the Escherichia coli hygromycin B phosphotransferase gene (hph). The system developed could give large, stable transformants at frequencies between 0.7 and 6.5 transformants per µg of DNA. A second type of colony also grew on the selective media. These were believed to be abortive transformants. The first type of transformants were characterized using classical molecular biological technologies such as PCR and Southern hybridization, and the transformation was shown to be successful. Plasmids (pBCH-gfp and pCT74) containing a gfp reporter gene were also transformed into these two fungal species. Expression of the gfp gene was checked using a fluorescence microscope and gfp-expressing E. lata transformants were inoculated onto the host plants blackcurrant and grapevine. Confocal observation of the movement of fungal mycelia in wood tissues was performed but its interaction with host plant was not established in the time available. Purified gfp-expressing P. chlamydospora transformants were also obtained. A vector containing a fragment of the P. chlamydospora putative toxin gene moxY was constructed and transformed into P. chlamydospora. Putative moxY gene disruption transformants were screened with PCR followed by Southern hybridization. The putative moxY gene disruption transformants were spore purified and further confirmed with Southern hybridization. Whilst both PCR and Southern hybridization confirmed disruption of the moxY gene, clear evidence for the presence of an additional wild type moxY was also seen in the same transformants. This led to the suggestions that either P. chlamydospora is a natural diploid, or that moxY is essential for growth and that selective pressures led to the formation of a wild type: moxY-hph diploid.
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    Regulation of the Aspergillus nidulans cytochrome C gene : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand
    (Massey University, 1997) McGlynn, Linda Joy
    The filamentous fungus Aspergillus nidulans has been genetically and biochemically well-characterised and thus provides an attractive model for studies on the regulation of eukaroytic gene expression. This study was undertaken to investigate the factors affecting respiratory function in A. nidulans. Due to the central role of cytochrome c in oxidative respiration, this study was focused on the cytochrome c gene and primarily upon how oxygen availability affects its expression. The Aspergillus nidulans cytochrome c gene (eyeA) appears to be transcriptionally activated in response to oxygen availability (Raitt et al., 1994). In the yeast S. cerevisiae, oxygen availability activates its cytochrome c genes via a heme-activated protein HAP1, which binds to the promoter region of each gene (Pfiefer et al., 1989). Since heme is only synthesised in the presence of oxygen, activity of the HAP1 protein serves as an intracellular signal of oxygen availability. In the upstream region of the cycA gene, a sequence with homology to the S. cerevisiae HAP1 binding site was present (Raitt, 1992). To determine the significance of the putative HAP1 binding site and the role of other promoter sequences in the A. nidulans cycA gene, a promoter-reporter vector was constructed. However, upon sequencing of the cycA promoter in the reporter vector, a sequencing error was discovered in the published cycA gene by Raitt et al. (1994) which affected the position of the major translational start site. Further examination of the cycA sequence also revealed a possible undetected intron (Intron I). To determine the number of introns in the cycA gene, RT- PCR was performed on cycA RNA. Sequencing of the RT-PCR amplified products showed that the previously undetected intron (Intron I) was present, and that the cycA gene contains three instead of two introns as published by Raitt et al. (1994). Since the published ATG start site was located within Intron I, a new translational start site was proposed. The major consequences of these changes to the cycA gene was that the putative HAP1 site was now located within the coding region of the gene, and therefore could not be a regulatory element. In addition only 247 bp of cycA promoter sequence remained cloned for analysis. To obtain additional promoter sequence, an A. nidulans genomic library was screened with a BamHI cDNA probe containing 224 bp of the 5' region of the cycA gene. Three positive clones were obtained, of which λLM9 and λLM5 were identical, and λLM19 was an overlapping clone with λLM9 and λLM5. Restriction enzyme and Southern blott analysis of the two overlapping cycA clones, revealed that 2.1 kb EcoRI fragments from both clones contained the 5' region. The 2.1 kb EcoRI fragment from λLM9 was cloned into pUC18 and sequenced. The completed upstream sequence of the A. nidulans cytochrome c gene was obtained, and putative regulatory signals including the HAP1 binding site were found, and compared with published promoter sequences which regulate the expression of respiratory-encoding genes from yeast.