title: Comparative and Functional Analysis of Gene Expression in Ophiostoma Species

authors: Robson, Lisa Marie

subjects: Ophiostoma

abstract: Ophiostoma floccosum and Ophiostoma piliferum are polymorphic ascomycete fungi found throughout the world. Both species are important economically as they are known to colonise timber and cause discoloration of wood thus reducing its aesthetic value and subsequently price. Albino variants of the two species, in particular O. piliferum, are used as biological control agents to prevent sapstaining and have been used commercially for the past 15 years to reduce pitch/wood extractives in paper manufacturing. Other members of the genus include the plant pathogens O. novo-ulmi and O. clavigerum, known to have a severe effect on forest health and economy around the world. O. floccosum and O. piliferum have been demonstrated in the laboratory to be fermented in large volumes and they are particularly suitable as hosts capable of secreting extracellular recombinant proteins. 

This research aimed to investigate the transcriptome and molecular functioning of Ophiostoma floccosum and compare this to transcriptomic data available for Ophiostoma piliferum and other Ophiostoma species, O. novo-ulmi, O. clavigerum and O. piceae. This research contributes to the development of O. floccosum and O. piliferum as hosts for protein expression and advances the knowledge of gene expression and molecular functioning in this genus.

To gain insight into the molecular functioning of O. floccosum, an expressed sequence tag (EST) collection from yeast-like growth (blastospores) was created during early phase growth.  A total of 1207 EST sequences with an average length of 713 bp were identified. Clustering and assembly of the high-quality EST data set resulted in the identification of 598 unique putative transcripts (UPTs). Functional classification of these UPTs, using both homology searching and ab-initio methods, indicated that the majority of protein transcripts produced were involved in metabolism and cell proliferation. Up-regulation of mitochondrial transcripts involved in respiration and the presence of transcripts homologous to enzymes involved in the tri-carboxylic acid cycle indicated that aerobic respiration was likely the preferred method of ATP production in O. floccosum blastospores. However, the putative identification of genes encoding alcohol dehydrogenases within O. floccosum ESTs and the presence of homologues in other Ophiostoma species would suggest that these Ophiostoma species are also likely to be capable of metabolic functioning under anaerobic conditions.

To identify homologous genes between Ophiostoma species, the O. floccosum EST data set was compared to 20,783 ESTs from other Ophiostoma species including O. piliferum, O. novo-ulmi, O. clavigerum and O. piceae. All UPTs identified within each of the datasets were aligned resulting in the identification of 347 clusters containing EST sequences from more than one Ophiostoma species. Six were identified that had homologues in all of the datasets excluding O. piceae. Three of the six homologous UPTs were predicted to function in core metabolism with two of the UPTs identified as encoding enzymes used in the glycolysis pathway and one encoding a 60S ribosomal protein. The other three homologous UPTs were thought to have a functional role in protein fate and were putatively identified as being a superoxide dismutase, heat-shock protein and a structural alpha-B chain tubulin gene. Of the 347 clusters, 86 of these contained transcripts identified in the O. floccosum EST datasets, and of these 86, only 10 fragments did not align with any significant homology to other fungal sequences contained in the NCBI non redundant database, indicating that the majority these transcripts are conserved in other fungal species.  

Predicted genes within the Ophiostoma EST datasets were also investigated to determine codon usage and to identify the presence of genes predicted to encode proteases. Both are important factors in recombinant protein expression. Protease production can severely inhibit the production of recombinant protein in fungal hosts. Based on sequence homology to known proteases, putative proteases were identified in all of the Ophiostoma species investigated with the exception of O. piceae. Homologues for all six peptidase groups were identified including a possible glutamic acid protease and proportionally high numbers of serine and metallo-protease homologues. This research constitutes the first reported findings of putative peptidases in the aspartic, cysteine, glutamic and threonine peptidase families in Ophiostoma species. 

Key to the over-expression of recombinant proteins is the optimisation of codons in a cloned gene to better utilise available tRNA species within the recombinant host. No codon bias was apparent between up-regulated and lower frequency transcripts in O. floccosum, O. piliferum, O. clavigerum and O. novo-ulmi. Codon usage was found to be consistent between these Ophiostoma species. However, a large difference between the codon usage in mitochondrially encoded genes compared to nuclear encoded genes in O. floccosum was indicated. 

To optimise the efficiency of a recombinant expression system, we sought to identify promoters in both O. floccosum and O. piliferum that may be applied to a vector system. Using EST data, the most up-regulated UPTs identified from O. floccosum and O. piliferum ESTs were a putative subunit 4 of the NADH-ubiquinone oxidoreductase protein (NADH-UR4) and a possible heat-shock protein (HSP), respectively. A unique hydrolase gene was also identified by molecular probing of O. floccosum genomic DNA. This putative 96 kd protein, called PLIP-Lg, was predicted to be a mitochondrial A1 phospholipase based on both nucleotide and predicted amino acid sequence structure and homology. These gene sequences were investigated using genome walking methods to further elucidate nucleotide sequences in the 5' and 3' directions. In silico investigation of the 5' promoter region of the genes identified a number of predicted transcription factor binding sites, including possible TATA boxes identified previously in the promoter region of an O. floccosum protein. Additionally, RT-PCR methods were used to compare the expression of these transcripts throughout growth in both the mycelial and blastospore forms. All three predicted genes were found to be transcribed throughout growth in both morphological forms and, thus, the use of their promoters in a vector system would not be limited to one morphology. However, the level of expression in blastospores compared to mycelial growth varied by up to 20 fold.  Therefore, the morphological form of the fungi did influence the level of expression of these genes and is a factor for consideration for future promoter use.

This PhD thesis research provides the first comprehensive investigation into gene expression and the transcriptome of O. floccosum while also providing the first comparative look into similarities between the transcriptomes of several Ophiostoma species. Subsequently, this research adds to the knowledge of metabolic functioning in Ophiostoma species and illustrates the usefulness of EST analysis in determining core molecular functioning within this group. Further to addressing these goals, the research will augment future research into various biotechnological applications for the genus, specifically the development of O. floccosum and O. piliferum as hosts for recombinant protein expression.

text: Comparative and Functional Analysis of Gene Expression in Ophiostoma Species Ophiostoma floccosum and Ophiostoma piliferum are polymorphic ascomycete fungi found throughout the world. Both species are important economically as they are known to colonise timber and cause discoloration of wood thus reducing its aesthetic value and subsequently price. Albino variants of the two species, in particular O. piliferum, are used as biological control agents to prevent sapstaining and have been used commercially for the past 15 years to reduce pitch/wood extractives in paper manufacturing. Other members of the genus include the plant pathogens O. novo-ulmi and O. clavigerum, known to have a severe effect on forest health and economy around the world. O. floccosum and O. piliferum have been demonstrated in the laboratory to be fermented in large volumes and they are particularly suitable as hosts capable of secreting extracellular recombinant proteins. 

This research aimed to investigate the transcriptome and molecular functioning of Ophiostoma floccosum and compare this to transcriptomic data available for Ophiostoma piliferum and other Ophiostoma species, O. novo-ulmi, O. clavigerum and O. piceae. This research contributes to the development of O. floccosum and O. piliferum as hosts for protein expression and advances the knowledge of gene expression and molecular functioning in this genus.

To gain insight into the molecular functioning of O. floccosum, an expressed sequence tag (EST) collection from yeast-like growth (blastospores) was created during early phase growth.  A total of 1207 EST sequences with an average length of 713 bp were identified. Clustering and assembly of the high-quality EST data set resulted in the identification of 598 unique putative transcripts (UPTs). Functional classification of these UPTs, using both homology searching and ab-initio methods, indicated that the majority of protein transcripts produced were involved in metabolism and cell proliferation. Up-regulation of mitochondrial transcripts involved in respiration and the presence of transcripts homologous to enzymes involved in the tri-carboxylic acid cycle indicated that aerobic respiration was likely the preferred method of ATP production in O. floccosum blastospores. However, the putative identification of genes encoding alcohol dehydrogenases within O. floccosum ESTs and the presence of homologues in other Ophiostoma species would suggest that these Ophiostoma species are also likely to be capable of metabolic functioning under anaerobic conditions.

To identify homologous genes between Ophiostoma species, the O. floccosum EST data set was compared to 20,783 ESTs from other Ophiostoma species including O. piliferum, O. novo-ulmi, O. clavigerum and O. piceae. All UPTs identified within each of the datasets were aligned resulting in the identification of 347 clusters containing EST sequences from more than one Ophiostoma species. Six were identified that had homologues in all of the datasets excluding O. piceae. Three of the six homologous UPTs were predicted to function in core metabolism with two of the UPTs identified as encoding enzymes used in the glycolysis pathway and one encoding a 60S ribosomal protein. The other three homologous UPTs were thought to have a functional role in protein fate and were putatively identified as being a superoxide dismutase, heat-shock protein and a structural alpha-B chain tubulin gene. Of the 347 clusters, 86 of these contained transcripts identified in the O. floccosum EST datasets, and of these 86, only 10 fragments did not align with any significant homology to other fungal sequences contained in the NCBI non redundant database, indicating that the majority these transcripts are conserved in other fungal species.  

Predicted genes within the Ophiostoma EST datasets were also investigated to determine codon usage and to identify the presence of genes predicted to encode proteases. Both are important factors in recombinant protein expression. Protease production can severely inhibit the production of recombinant protein in fungal hosts. Based on sequence homology to known proteases, putative proteases were identified in all of the Ophiostoma species investigated with the exception of O. piceae. Homologues for all six peptidase groups were identified including a possible glutamic acid protease and proportionally high numbers of serine and metallo-protease homologues. This research constitutes the first reported findings of putative peptidases in the aspartic, cysteine, glutamic and threonine peptidase families in Ophiostoma species. 

Key to the over-expression of recombinant proteins is the optimisation of codons in a cloned gene to better utilise available tRNA species within the recombinant host. No codon bias was apparent between up-regulated and lower frequency transcripts in O. floccosum, O. piliferum, O. clavigerum and O. novo-ulmi. Codon usage was found to be consistent between these Ophiostoma species. However, a large difference between the codon usage in mitochondrially encoded genes compared to nuclear encoded genes in O. floccosum was indicated. 

To optimise the efficiency of a recombinant expression system, we sought to identify promoters in both O. floccosum and O. piliferum that may be applied to a vector system. Using EST data, the most up-regulated UPTs identified from O. floccosum and O. piliferum ESTs were a putative subunit 4 of the NADH-ubiquinone oxidoreductase protein (NADH-UR4) and a possible heat-shock protein (HSP), respectively. A unique hydrolase gene was also identified by molecular probing of O. floccosum genomic DNA. This putative 96 kd protein, called PLIP-Lg, was predicted to be a mitochondrial A1 phospholipase based on both nucleotide and predicted amino acid sequence structure and homology. These gene sequences were investigated using genome walking methods to further elucidate nucleotide sequences in the 5' and 3' directions. In silico investigation of the 5' promoter region of the genes identified a number of predicted transcription factor binding sites, including possible TATA boxes identified previously in the promoter region of an O. floccosum protein. Additionally, RT-PCR methods were used to compare the expression of these transcripts throughout growth in both the mycelial and blastospore forms. All three predicted genes were found to be transcribed throughout growth in both morphological forms and, thus, the use of their promoters in a vector system would not be limited to one morphology. However, the level of expression in blastospores compared to mycelial growth varied by up to 20 fold.  Therefore, the morphological form of the fungi did influence the level of expression of these genes and is a factor for consideration for future promoter use.

This PhD thesis research provides the first comprehensive investigation into gene expression and the transcriptome of O. floccosum while also providing the first comparative look into similarities between the transcriptomes of several Ophiostoma species. Subsequently, this research adds to the knowledge of metabolic functioning in Ophiostoma species and illustrates the usefulness of EST analysis in determining core molecular functioning within this group. Further to addressing these goals, the research will augment future research into various biotechnological applications for the genus, specifically the development of O. floccosum and O. piliferum as hosts for recombinant protein expression.

year: 2008

title: Stratigraphy, chronology, and correlation of the Plio-Pleistocene (c. 2.2-0.8 Ma) Kauroa Ash sequence, western central North Island, New Zealand

authors: Horrocks, Joanna L.

abstract: The Kauroa Ash beds (K-beds) comprise a 12-20 m-thick sequence of extremely weathered, clay-rich (40-95% <4 μm clay) beds of tephra and loess, and associated paleosols. Found in isolated remnants throughout the western central North Island, the sequence comprises 15 defined members, with as many as 44 constituent macroscopic beds. The type site, ‘Woodstock’, near Raglan, is the most comprehensive sequence known, but other sites (e.g. Papakura Creek and Tiritirimatangi Peninsula) contain units not found or poorly defined at Woodstock. 

Field properties as well as magnetic susceptibility measurements and particle-size analysis characterise the facies in the sequence. Field properties (in particular colour, consistence, macrofabric) describe the lithostratigraphy. The sequence contains five interpretive (i.e. genetic) ‘facies’: paleosols, primary tephra, very weathered tephra (possibly composite beds), loess and ‘tephric loess’ beds. At least seven loess beds are (newly) identified in the sequence: K4a, K5, K6ai, K8ai, K8bi, K10a and K14ai.

Mass-specific susceptibility and frequency-dependent susceptibility results partly conform to established models (developed mostly on Chinese loess-paleosol deposits) of susceptibility enhancement in paleosols and depletion in loess. Many parts of the sequence do not appear to conform to this model and the results more closely resemble the inverse relationship found on Alaskan loess-paleosol beds. Frequency-dependent susceptibility is reliable in delineating paleosols by their ‘ultrafine’ ferrimagnetic mineral content, and citrate-bicarbonate-dithionite treatments successfully remove all iron oxides so that remeasurement of susceptibility isolates a strictly ‘pedogenic’, rather than lithogenic, fraction. 

Laser diffraction particle-size analysis shows that the Kauroa Ash beds are texturally reasonably homogenous. They have bimodal particle-size distributions with the most dominant mode at around 11.25 ɸ inferred to be the product of intense and prolonged weathering. Other modes are variously centred on 7-8.5 ɸ and, despite weathering and pedogenesis, have some relationship to the original depositional particle-size distributions because trends between facies (i.e. genetic units) are delineated. Principal components analysis objectively characterises these modes as (Wentworth size classes) ‘very fine clay’ and ‘coarse silt’, although there is no proportional relationship between them, supporting a post-depositional origin for the former mode. 

The chronology of the sequence, previously poorly defined, is greatly improved by a combination of tephrochronologic correlations, fission-track dating, and paleomagnetism. Five zircon fission-track dates provide independent age ‘spikes’ and range from 2.24 ± 0.29 Ma in the basal member, K1, to 1.28 ± 0.11 Ma for the distal ignimbrite unit K12a. Paleomagnetism is invaluable in providing additional age information. The top of the sequence, member K15, is dated as >0.78 Ma (Brunhes-Matuyama boundary) because of its reversed polarity; two episodes of normal polarity are found in beds K14b and K2b and are inferred to represent the Jaramillo (1.07-0.99 Ma) and Olduvai (1.95-1.79 Ma) subchrons, respectively. Beds underlying the Kauroa Ash sequence are also of normal polarity, indicating that they were deposited in the Gauss Chron (>2.6 Ma). 

Identification and correlation of tephras by conventional means (fingerprinting by their lithological or geochemical properties) is impossible in the Kauroa Ash sequence because the beds have no remaining volcanic glass, which has instead been altered to an assemblage of authigenic phases (clays) by weathering and pedogenesis. However, a new technique analysing fresh glass found as melt inclusions in quartz grains is successful in circumventing this problem. Inclusions represent samples of non-degassed magma that became entrapped during phenocryst growth prior to eruption. The glass has remained unaltered because it is hermetically sealed in a chemically resistant phenocryst, which has protected it from weathering processes. Electron microprobe analysis of the glass inclusions yield results which are wholly reasonable for glass (totals ranging from 93-97%; low standard deviations of <1 %), and a number of provisional correlations are established by comparing the major element composition of Kauroa Ash tephra beds with those of proximal deposits. The Kauroa Ash sequence may contain deposits correlated with at least seven major TVZ eruptions, in many cases expanding the known extent of the (distal) deposit and, for the first time answering the question as to the origin of the Kauroa Ash beds. 

These correlations, together with an improved chronology, enable the Kauroa Ash sequence to be placed in a regional stratigraphic framework alongside other New Zealand Plio-Pleistocene sequences such as those in the Wanganui Basin, Wairarapa, Cape Kidnappers and Port Waikato. Using paleosols as chrono- and climatostratigraphic entities (correlated to warm periods in global climate), the sequence can also be placed alongside a global reference, the marine oxygen isotope stratigraphy. A further correlation to the Chinese loess-paleosol record suggests that large parts of the Kauroa Ash sequence were deposited in an incremental manner akin to deposition of loess, so that the sequence is not only a record of TVZ volcanism, but also of Plio-Pleistocene paleoclimate.

text: Stratigraphy, chronology, and correlation of the Plio-Pleistocene (c. 2.2-0.8 Ma) Kauroa Ash sequence, western central North Island, New Zealand The Kauroa Ash beds (K-beds) comprise a 12-20 m-thick sequence of extremely weathered, clay-rich (40-95% <4 μm clay) beds of tephra and loess, and associated paleosols. Found in isolated remnants throughout the western central North Island, the sequence comprises 15 defined members, with as many as 44 constituent macroscopic beds. The type site, ‘Woodstock’, near Raglan, is the most comprehensive sequence known, but other sites (e.g. Papakura Creek and Tiritirimatangi Peninsula) contain units not found or poorly defined at Woodstock. 

Field properties as well as magnetic susceptibility measurements and particle-size analysis characterise the facies in the sequence. Field properties (in particular colour, consistence, macrofabric) describe the lithostratigraphy. The sequence contains five interpretive (i.e. genetic) ‘facies’: paleosols, primary tephra, very weathered tephra (possibly composite beds), loess and ‘tephric loess’ beds. At least seven loess beds are (newly) identified in the sequence: K4a, K5, K6ai, K8ai, K8bi, K10a and K14ai.

Mass-specific susceptibility and frequency-dependent susceptibility results partly conform to established models (developed mostly on Chinese loess-paleosol deposits) of susceptibility enhancement in paleosols and depletion in loess. Many parts of the sequence do not appear to conform to this model and the results more closely resemble the inverse relationship found on Alaskan loess-paleosol beds. Frequency-dependent susceptibility is reliable in delineating paleosols by their ‘ultrafine’ ferrimagnetic mineral content, and citrate-bicarbonate-dithionite treatments successfully remove all iron oxides so that remeasurement of susceptibility isolates a strictly ‘pedogenic’, rather than lithogenic, fraction. 

Laser diffraction particle-size analysis shows that the Kauroa Ash beds are texturally reasonably homogenous. They have bimodal particle-size distributions with the most dominant mode at around 11.25 ɸ inferred to be the product of intense and prolonged weathering. Other modes are variously centred on 7-8.5 ɸ and, despite weathering and pedogenesis, have some relationship to the original depositional particle-size distributions because trends between facies (i.e. genetic units) are delineated. Principal components analysis objectively characterises these modes as (Wentworth size classes) ‘very fine clay’ and ‘coarse silt’, although there is no proportional relationship between them, supporting a post-depositional origin for the former mode. 

The chronology of the sequence, previously poorly defined, is greatly improved by a combination of tephrochronologic correlations, fission-track dating, and paleomagnetism. Five zircon fission-track dates provide independent age ‘spikes’ and range from 2.24 ± 0.29 Ma in the basal member, K1, to 1.28 ± 0.11 Ma for the distal ignimbrite unit K12a. Paleomagnetism is invaluable in providing additional age information. The top of the sequence, member K15, is dated as >0.78 Ma (Brunhes-Matuyama boundary) because of its reversed polarity; two episodes of normal polarity are found in beds K14b and K2b and are inferred to represent the Jaramillo (1.07-0.99 Ma) and Olduvai (1.95-1.79 Ma) subchrons, respectively. Beds underlying the Kauroa Ash sequence are also of normal polarity, indicating that they were deposited in the Gauss Chron (>2.6 Ma). 

Identification and correlation of tephras by conventional means (fingerprinting by their lithological or geochemical properties) is impossible in the Kauroa Ash sequence because the beds have no remaining volcanic glass, which has instead been altered to an assemblage of authigenic phases (clays) by weathering and pedogenesis. However, a new technique analysing fresh glass found as melt inclusions in quartz grains is successful in circumventing this problem. Inclusions represent samples of non-degassed magma that became entrapped during phenocryst growth prior to eruption. The glass has remained unaltered because it is hermetically sealed in a chemically resistant phenocryst, which has protected it from weathering processes. Electron microprobe analysis of the glass inclusions yield results which are wholly reasonable for glass (totals ranging from 93-97%; low standard deviations of <1 %), and a number of provisional correlations are established by comparing the major element composition of Kauroa Ash tephra beds with those of proximal deposits. The Kauroa Ash sequence may contain deposits correlated with at least seven major TVZ eruptions, in many cases expanding the known extent of the (distal) deposit and, for the first time answering the question as to the origin of the Kauroa Ash beds. 

These correlations, together with an improved chronology, enable the Kauroa Ash sequence to be placed in a regional stratigraphic framework alongside other New Zealand Plio-Pleistocene sequences such as those in the Wanganui Basin, Wairarapa, Cape Kidnappers and Port Waikato. Using paleosols as chrono- and climatostratigraphic entities (correlated to warm periods in global climate), the sequence can also be placed alongside a global reference, the marine oxygen isotope stratigraphy. A further correlation to the Chinese loess-paleosol record suggests that large parts of the Kauroa Ash sequence were deposited in an incremental manner akin to deposition of loess, so that the sequence is not only a record of TVZ volcanism, but also of Plio-Pleistocene paleoclimate.

year: 2000

title: Are historical perpetual leases on Maori owned land (Native reserves) a basis for currently enforceable fiduciary obligations?

authors: Dimitrov, Dara

subjects: fiduciary obligations

abstract: Colonial perpetual leases over what were once native reserves continue to trouble the New Zealand government. The colonial Public Trust Office was tasked with managing and administering Māori property affairs, with a focus on the administration of the native reserves. However, if the perpetual leases continue to be viewed through the contractual lens, relief for the Māori landowners will not be forthcoming. Therefore, examining the rationale for imposing fiduciary duties upon the colonial Public Trust Office is essential. Where traditional legal fiduciary literature explores the onerous proscriptive duties that bind persons occupying a fiduciary position, this article establishes that the extensive statutory role assigned to colonial Public Trust officers in managing the native reserves for their Māori beneficiaries created a fiduciary relationship. Moreover, this article shows that, when the colonial Public Trust Office exercised its powers, this often led to conflicts of interest with their Māori beneficiaries.

text: Are historical perpetual leases on Maori owned land (Native reserves) a basis for currently enforceable fiduciary obligations? Colonial perpetual leases over what were once native reserves continue to trouble the New Zealand government. The colonial Public Trust Office was tasked with managing and administering Māori property affairs, with a focus on the administration of the native reserves. However, if the perpetual leases continue to be viewed through the contractual lens, relief for the Māori landowners will not be forthcoming. Therefore, examining the rationale for imposing fiduciary duties upon the colonial Public Trust Office is essential. Where traditional legal fiduciary literature explores the onerous proscriptive duties that bind persons occupying a fiduciary position, this article establishes that the extensive statutory role assigned to colonial Public Trust officers in managing the native reserves for their Māori beneficiaries created a fiduciary relationship. Moreover, this article shows that, when the colonial Public Trust Office exercised its powers, this often led to conflicts of interest with their Māori beneficiaries.

year: 2024