The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

The identification of specific and reproducible biomarkers is critical for the early diagnosis of cancer, which has a profound effect the survival rate of patients. Comprehensive laboratory and clinical evidence needs to be collected to confirm the accuracy of the biomarkers prior to their clinical use. Heterogenous nuclear ribonucleoproteins (hnRNPs) A2 and B1 have been suggested as biomarkers for cancer since 1988 when hnRNP A2/B1 overexpression was first linked with the occurrence of lung cancer. Later studies established a correlation between the expression levels of these hnRNPs and other cancers, such as breast, pancreatic, and lymphatic tumours. In this study, the expression of hnRNPs A1, A2, A3, and B1 has been investigated in various cancer cell lines. hnRNPs A1 and A3, in addition to A2 and B1, were found to be overexpressed in some cancer types. However, the overexpression of none of the hnRNP A/B proteins was universal, and their upregulation may be limited to a few cell types, suggesting they may be effective biomarkers for a subset of cancers. The upregulation of hnRNP A/B proteins in tumours and cancer cell lines led to the hypothesis that they are involved in the uncontrolled cell growth in cancer. According to our Western blot analysis, expression of the hnRNP proteins, A1, A2, and B1, is dependent on the cell cycle whereas no significant change was detected for hnRNP A3, implying that the former three are needed during certain cell cycle stages. The results, together with the transcription factor analysis of the promoter regions of the HNRPA1, HNRPA2, and HNRPA3 genes, suggest that hnRNPs A1, A2, and A3 may have distinct regulatory machineries and cellular functions although they have high amino acid sequence identity. However, their mRNA levels were unchanged across the cell cycle, suggesting the cell-cycle-dependent expression of hnRNPs A1, A2, and B1 is modulated at the translational level. Previous studies showed higher expression of hnRNPs A1 and A2 in rapidly proliferating cells than in quiescent cells, suggesting a role of these proteins in cell proliferation. Though interruption of hnRNP A1 expression did not result in significant change in the viability of murine CB3 cells, simultaneous suppression of hnRNPs A1 and A2 caused apoptosis in a few cell lines. Consistent with this, suppression of hnRNP A1 or A3 expression in our study in Colo16 squamous cells using RNA interference did not affect cell proliferation, but simultaneous suppression of both caused slow cell proliferation. By contrast, reduction of the hnRNP A2 level alone slowed the proliferation of Colo16 cells. These results suggest that although hnRNPs A1, A2, and A3 share some roles in cell proliferation, each of them may have distinct tasks. This conclusion is supported by the data from the comparative analysis of the downstream targets of hnRNPs A1, A2, and A3, which has shown that these three proteins share a limited number of common downstream proteins. The observed impact on cell proliferation of suppressing hnRNP A2 subfamily proteins is in accord with our finding that the downstream targets of hnRNP A2 are overrepresented by genes involved in proliferation regulation, as shown in microarray and real-time PCR analysis. These include cyclin-dependent kinase (CDK) inhibitors, p21 and p27, and their regulatory proteins, such as Skp2 and Rpn10. Skp2 controls the ubiquitination of p21 and p27, and Rpn10 links them to the 26S proteasome, the complex that degrades these two CDK inhibitors. hnRNP A2 also regulates the transcription of securin and separin, which are essential for sister chromatid separation during late anaphase. In addition, hnRNP A2 can also influence cell proliferation through cell growth factors, including fibroblast, vascular endothelial, transforming, and insulin growth factors. Our gene array and real-time PCR analysis have shown that hnRNP A2 regulates the expression of these factors, their receptors, or associated proteins such as IGFBP7 and TGFBR2. The data presented in this thesis link the overexpression of hnRNP A/B proteins, in particular the A2/B1 subfamily, in cancer with their regulatory roles in cell division and cell proliferation. Our findings provide mechanistic evidence that these proteins may be a driving force for the uncontrolled cell growth in cancer, suggesting that some of hnRNP A/B proteins may be potential therapeutic targets for cancer. However, further studies are needed to obtain a global view of the roles of these proteins in cancer.

In maize, a loss-of-function mutation in a MEI2-like gene, terminal ear1 (te1), leads to morphological defects able to be traced back to the shoot apical meristem. One MEI2-like gene has been identified in maize, while six have been identified in rice and nine in Arabidopsis thaliana. In this thesis, a programme of reverse genetic analysis has been designed to investigate if Arabidopsis genes most closely aligned in parsimony trees with TE1, TERMINAL EAR-LIKE 1 (TEL1), TERMINAL EAR-LIKE 2 (TEL2), perform the same function as TE1. The expression pattern of TEL1 and TEL2 genes is restricted to the Shoot Apical Meristem (SAM) and the Root Apical Meristem (RAM) suggesting these genes are important in meristem maintenance or function. Results of the molecular genetic analysis of TEL genes in Arabidopsis support models in which these genes help maintain cells in a pluripotent state. For the first part of the thesis, analysis of lines carrying single knockouts of TEL1 and TEL2 and double knockout lines reveals a slightly accelerated rate of organogenesis, consistent with these genes normally acting to inhibit terminal differentiation pathways. Plants grown on medium containing gibberellic acid and sucrose, at higher than normal concentrations, present a further accelerated rate of organogenesis. As the second part of the thesis, in situ and promoter/reporter GUS fusion analyses indicate TEL1 is preferentially expressed in both the root and shoot apical meristems. Deletion analysis using GFP reporter constructs show that 5' sequences are sufficient to drive quiescent centre (QC) expression in the root while additional sequences are required for central zone (CZ) expression in the SAM. Physiological studies indicate expression of TEL1 in the root is sensitive to the hormones, auxin, gibberellic acid and zeatin, when added at physiological concentrations. To confirm the auxin effect, GFP expression is no longer visible after 12 hours of exposure to auxin transport inhibitors in plants containing GFP under the control of the TEL1 promoter, suggesting, in common with other QC markers, that TEL expression is sensitive to auxin levels. Analysis of mutant plants with altered root patterning suggests QC specific expression of TEL1 requires early acting genes, such as PLETHORA 1 and 2, but does not depend on later acting genes such as SCARECROW or SHORTROOT.

[Abstract not supplied]

Alcohol and tobacco are drugs of abuse which are legal to sell and consume in most western societies. Addiction to these two substances has major social and health implications worldwide. The brain structure known to mediate addictive behaviour is the dopaminergic mesocorticolimbic system. Dopaminegic neurons arise from the ventral tegmental area, project to the nucleus accumbens and interact with the amygdala and the prefrontal cortex. Chronic alcoholism elicits marked damage in the prefrontal cortex with significant loss of neurons and glia. The key components of addiction, tolerance and dependence, are thought to be the result of semipermanent adaptive changes in gene expression. Gene expression profiling of the mesocorticolimbic system from human alcoholics and alcohol-dependent animals has revealed highly region-specific alterations. How these molecular changes result in the development of alcohol dependence in humans is not fully understood. Complicating factors in human alcoholism include a high comorbidity with smoking, socioeconomic factors and the prevalence of underlying psychological pathologies. Gene expression profiling of the prefrontal cortex of six alcoholics and six controls resulted in the identification of functional gene groups sensitive to alcoholism. Mitochondrial function was found down regulated while mRNA levels of genes involved in stress response and cell protection were elevated. These results correlate with the pathology of the prefrontal cortex in chronic alcoholism. Some of the control cases used for gene expression profiling were later identified as chronic smokers, while all of the alcoholics were heavy smokers. To date the heavy co-morbidity of alcoholism with smoking has not been taken into account. Thus the expression of selected genes were investigated by realtime PCR in an extended case set of non-smoking alcoholics, smoking alcoholics, smoking non-alcoholics and non-smoking, non-alcoholics. This study revealed that alcoholism itself had a significant impact on the expression of midkine, the high affinity glial glutamate transporter, member 1 and the tissue inhibitor of the metalloproteinase 3. Heavy smoking itself led to a small but significant elevation of MDK mRNA levels as well as an increase in variation of excitatory amino acid transporter 1 and metalloproteinase inhibitor, member 3 expression. Apolipoprotein D however was induced by chronic smoking but not by alcohol dependence. These results highlight the need of careful case selection in future studies on gene expression in the human alcoholic brain. Peptide antibodies were produced to midkine and a polyclonal antibody against the excitatory amino acid transporter 1 was obtained from a collaborating laboratory. Western blots utilizing these antibodies revealed a marked increase in midkine and excitatory amino acid transporter 1 protein in alcoholics compared to non-smoking and non-drinking controls. In coronal sections of human prefrontal cortex of alcoholics and non-smoking non-drinking controls, immunofluorescence of midkine was obtained from nuclei throughout the layers of the cortex and from the cell bodies of a distinct set of astrocytes in cortical layer II. Double staining with glial fibrillary acidic protein revealed that a portion of midkine-positive nuclei were localised in glial cells. There was no difference in immunostaining of alcohol and control sections with midkine. In summary these results indicate that midkine protein is induced in the prefrontal cortex of the chronic alcoholic. However, this increase in protein may not be strong enough to be visualised by immunohistochemistry. Midkine induction may be reflective of reparative processes in the prefrontal cortex of the chronic alcoholic. Excitatory amino acid transporter 1 staining in non-alcoholic, non-smoking control cases were obtained as a confluent band in cortical layer II and sparsely in deeper cortical layers. Excitatory amino acid transporter 1 immunoreactivity overlapped partially with glial fibrillary acidic protein labelling. In chronic alcoholics, excitatory amino acid transporter 1 staining in the area between the cortical layer II and VI was significantly increased. At withdrawal, glutamate levels may reach toxic levels in the cortex. The increase in cells expressing excitatory amino acid transporter 1 throughout the cortical layers may indicate a protective measure of this brain region in the chronic alcoholic. Additionally, layer specific expression of midkine and excitatory amino acid transporter 1 in the prefrontal cortex of the healthy individual may implicate a specialised role of these astrocytes.

No description available.

Dosage compensation is the equalisation of gene expression from unequal doses of genes. Drosophila males up-regulate transcription from their single X chromosome to equal that from the two female X chromosomes. Five malespecific lethal (msl) genes are required in males, and encode the main agents of the up-regulation. At least these proteins, together with either or both of two noncoding RNAs, form the MSL chromatin-modifying complex. Female-specific translational repression of a key component, msl2, limits the complex to males. The MSL complex binds to the X chromosome at hundreds of distinct loci, acetylates nucleosomes, and de-condenses the chromatin. Together with possibly many co-factors, the transcriptional up-regulation caused by MSL complex appears to counteract repressive factors to achieve an average effect of transcriptional doubling. Here, I have studied the initiation of MSL regulation on the X chromosome with a variety of approaches. In order to study early events, dosage compensation was induced in females with ectopic expression of msl2 from the tetracycline system. However, low background expression without activation prohibited further studies. To identify novel factors that affect dosage compensation, a reporter gene system based on variable eye size was evaluated. The system provided a dose-dependent phenotype, but could not report additional up-regulation by the MSL complex, and was thus unsuitable for the proposed mutational screen. The quantifiable lacZ gene was measured in a strict comparison of expression from an eye-specfic (GMR) or a constitutive (armadillo) promoter. At defined locations on the X chromsome, armadillo-lacZ acquired local compensation, but GMR-lacZ did not. Further modifications upstream of GMR-lacZ increased the response, and confirmed the importance of the promoter in attraction of dosage compensation. To corroborate this with the established importance of genic sequences in MSL attraction, a combinatorial model of attraction is proposed. The relative importance of early or constitutive expression was also tested, by providing GMR-lacZ with extra expression through the tetracycline system. A burst of embryonic expression, and constitutive expression, were both insufficient to increase dosage compensation of the transgene. Finally, the compensation of GMRmediated transgenes was confounded by ‘transvection’ effects of chromosome pairing. This effect may have wider implications on the study of compensation at individual genes.

Hydrolytic enzymes degrade macromolecules into smaller components. These enzymes are important in fungal nutrition and have been implicated in the pathogenicity and virulence of pathogenic fungi towards their hosts. However, it is unknown if hydrolytic enzymes play important roles in mutualistic symbioses. In this study, the function of two different classes of hydrolytic enzymes was examined in the mutualistic symbiosis between the fungal endophyte Epichloë festucae and perennial ryegrass (Lolium perenne cv. Nui). Nine members of a gene family encoding subtilisin-like proteases were identified in E. festucae. The prt2, prt3 and prt5 genes encode putative extracellular proteins belonging to the proteinase K subfamily 1, and prt1 and prt6 encode putative extracellular proteins belonging proteinase K subfamily 2. The prt7 and prt8 genes encoded pyrolysin-like enzymes from subfamilies 1 and 2. The prt4 gene encodes a putative vacuolar protease, while the kex2 gene encodes a putative proprotein convertase. Expression analysis showed that the prt1, prt3, prt5, prt4 and kex2 genes, but not the prt2 gene, were expressed in culture. The prt1 and prt3 genes appeared to be up-regulated in planta compared to culture. The function of prt1 and prt2 in the symbiotum between E. festucae and perennial ryegrass was characterised by expressing these genes under the control of the Aspergillus nidulans gpdA or the E. festucae F11 ltmM promoters. No major differences in hyphal or plant morphology were observed between symbioses containing wild type E. festucae or endophyte strains containing the prt1 or prt2 transgenes. The gcnl gene, which encodes a β-1,6-glucanase, was identified immediately downstream of the prt2 gene. The function of the gcnl gene was characterised by gene replacement and testing the phenotype during growth in culture and in planta. E. festucae ∆gcnl strains grew normally on glucose-containing media. On media containing the β-1,6-glucan pustulan, ∆gcnl strains did not form aerial hyphae or hydrolyse pustulan, which the wild type strain did. This phenotype was partially complemented by growth of the ∆gcnl mutant in close proximity to wild type strains, and fully complemented by insertion of the gcnl gene. This suggests that the gcnl gene encodes the major β-1,6-glucanase activity of E. festucae.