Ornithine decarboxylase knockout in Tapesia yallundae abolishes infection plaque formation in vitro but does not reduce virulence toward wheat.

A knockout strain of Tapesia yallundae lacking the single ornithine decarboxylase (ODC) allele has been created by targeted gene replacement. A central region of the ODC gene was isolated by polymerase chain reaction with degenerate oligonucleotides and used to probe a lambda genomic library. The gene was sequenced, and the encoded ODC protein sequence was shown to be similar to those from other fungi. The functionality of the T. yallundae ODC was confirmed by complementation of an Aspergillus nidulans mutant (puA) strain devoid of ODC activity, restoring growth in the absence of exogenous polyamines. Transformation-mediated gene replacement was used to create strains that were auxotrophic for putrescine and lack ODC coding sequences. ODC knockout strains were unable to differentiate infection structures after in vitro induction and showed an abnormal hyphal branching phenotype. Pathogenicity studies on these mutants showed that, surprisingly, they are not reduced in virulence compared with nondisrupted transformants. This suggests that the strains carrying an ODC disruption can obtain sufficient polyamines from the host plant for normal growth and differentiation and, therefore, that fungal ODC may not be a suitable target for fungicides.     

An amdS-lacZ fusion for studying gene regulation in Aspergillus

A translational fusion has been constructed between the amdS gene of Aspergillus nidulans and the lacZ gene of Escherichia coli. Sequencing across the fusion junction confirmed the generation of an in-frame fusion at amino acid 34 of amdS and a novel protein has been detected in transformants carrying the fusion plasmid. Transformants of A. nidulans and Aspergillus niger carrying the fusion plasmid were obtained by co-transformation with a second selectable plasmid. These transformants were readily identified on media containing XGal. The intensity of the reaction on XGal media was indicative of the number of copies of the fusion plasmid carried by the transformants. The growth of highly expressing strains of A. nidulans was inhibited on XGal media. The fusion plasmid was used to develop a two-step gene replacement strategy in which the resident amdS gene was replaced with the fusion gene free of vector sequences. Plate tests and in vitro assays of the beta-galactosidase enzyme confirmed that expression of the fusion gene was regulated by amdS flanking sequences and trans-acting regulatory genes.     

Cotransformation of Aspergillus nidulans: a tool for replacing fungal genes

Summary When a non-selected DNA sequence was added during the transformation of amdS320 deletion strains of Aspergillus nidulans with a vector containing the wild-type amdS gene the AmdS⁺ transformants were cotransformed at a high frequency. Cotransformation of an amdS320, trpC801 double mutant strain showed that both the molar ratio of the two vectors and the concentration of the cotransforming vector affected the cotransformation frequency. The maximum frequency obtained was defined by the gene chosen as selection marker for transformation. Cotransformation was used to induce a gene replacement in A. nidulans. An amdS320 strain was transformed to AmdS⁺ and cotransformed with a DNA fragment containing a fusion between a non-functional A. nidulans trpC gene and the Escherichia coli lacZ gene. Ten AmdS⁺, LacZ⁺ transformants with a Trp^– mutant phenotype were selected. All of these strains could be transformed with a functional copy of the A. nidulans trpC gene, but only two strains yielded TrpC⁺ transformants which, with a low frequency, had a LacZ^– phenotype. These latter transformants had also lost the AmdS⁺ phenotype. Southern blotting analysis of DNA from these transformants confirmed the inactivation of the wild-type trpC gene, but revealed that amdS vector sequences were also involved in the gene replacement events.     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.     

Disruption, replacement, and cosuppression of nitrate assimilation genes in Stagonospora nodorum.

We used Stagonospora (Septoria) nodorum to explore gene disruption as a general method of fungicide target validation. Nitrate reductase was chosen as a model target because the gene (NIA1) has been cloned from S. nodorum and disruptants should have a readily detectable phenotype (chlorate resistant and nitrate nonutilizing). We have succeeded in disrupting the NIA1 gene by both integration of an unselected vector during cotransformation and one-step gene replacement. Around 2% of transformants from the cotransformation approach became nitrate nonutilizing and Southern analysis confirmed disruption of the resident NIA1 gene. Half of the transformants with the gene replacement vector showed the nitrate nonutilizing phenotype expected from disruption. However, Southern analyses of 14 of these transformants showed that only 6 contained the expected NIA1 gene replacement. Of the remaining transformants, 6 had integrated multiple copies of the vector elsewhere in their genome and still had a functional nitrate reductase gene. Their inability to utilize nitrate was due to a lack of nitrite reductase activity. How this phenotype arose is not clear, but it might involve cosuppression of the nitrite reductase gene as the vector carried 1. 1 kb of the coding region and the complete 5' region of this gene which is adjacent to NIA1. Mutants of both types retained full pathogenicity in detached leaf assays, thereby invalidating both nitrate and nitrite reductase as fungicide targets.     

Human ornithine decarboxylase(ODC)-encoding gene: cloning and expression in ODC-deficient CHO cells

We have cloned a full-length human ornithine decarboxylase (ODC)-encoding gene from a genomic library of human myeloma cells which overproduce ODC due to a selective gene amplification. Correct expression of the cloned gene was assessed by transfecting it into a Chinese hamster ovary (CHO) cell mutant devoid of ODC activity. Transfection with a 10-kb BamHI DNA fragment of the genomic clone, conferred ODC activity to the recipient cells and relieved them of dependence on exogenous polyamines for growth. A set of 40 transformants was isolated, eight of which were further characterized. The transfected ODC gene appeared to be hypomethylated at the cytosine residues in the sequence CpG. The transfectants were all responsive to serum stimulation, but showed different levels of ODC expression depending on both copy number and integration site of the transfected ODC gene. ODC serum induction in the transfectants was sensitive to cycloheximide and polyamine additions, and the half-life of the enzyme was very short, like that in normal CHO cells. These results suggest that the human ODC gene we transfected contains all the elements needed for normal control of ODC expression.     

Transformation of Aspergillus nidulans with the hygromycin-resistance gene, hph

Aspergillus nidulans strain G191 was transformed to hygromycin resistance using plasmid pDH25, which contains the bacterial hygromycin B phosphotransferase gene (hph) fused to promoter elements of the A. nidulans trpC gene. Southern hybridizations of transformants revealed multiple, integrated copies of the vector. A pleiotropic effect conferring increased hygromycin B sensitivity was found to be associated with the A. nidulans pyrG89 allele. Plasmid pDH25 features a ClaI site immediately preceding the hph start codon thus permitting convenient replacement of the trpC sequences with other eukaryotic promoters.     

Identification of Developmental Regulatory Genes in Aspergillus Nidulans by Overexpression

Overexpression of several Aspergillus nidulans developmental regulatory genes has been shown to cause growth inhibition and development at inappropriate times. We set out to identify previously unknown developmental regulators by constructing a nutritionally inducible A. nidulans expression library containing small, random genomic DNA fragments inserted next to the alcA promoter [ alcA (p) ] in an A. nidulans transformation vector. Among 20,000 transformants containing random alcA (p) genomic DNA fusion constructs, we identified 66 distinct mutant strains in which alcA (p) induction resulted in growth inhibition as well as causing other detectable phenotypic changes. These growth inhibited mutants were divided into 52 FIG (Forced expression Inhibition of Growth) and 14 FAB (Forced expression Activation of brlA) mutants based on whether or not alcA (p) induction resulted in accumulation of mRNA for the developmental regulatory gene brlA. In four FAB mutants, alcA (p) induction not only activated brlA expression but also caused hyphae to differentiate into reduced conidiophores that produced viable spores from the tips as is observed after alcA (p) :: brlA induction. Sequence analyses of the DNA fragments under alcA (p) control in three of these four sporulating strains showed that in two cases developmental activation resulted from overexpression of previously uncharacterized genes, whereas in the third strain, the alcA (p) was fused to brlA. The potential uses for this strategy in identifying genes whose overexpression results in specific phenotypic changes like developmental induction are discussed.     

Transformation by integration in Aspergillus nidulans

DNA-mediated genetic transformation of Aspergillus nidulans has been achieved by incubating protoplasts from a strain of A. nidulans carrying a deletion in the acetamidase structural gene with DNA of derivatives of plasmid pBR322 containing the cloned structural gene for acetamidase [Hynes et al., Mol. Cell. Biol. 3 (1983) 1430-1439; p3SR2] in the presence of polyethylene glycol and CaCl2. The highest frequency obtained was 25 transformants per microgram of DNA. No enhancement of the transformation frequency was observed when DNAs of plasmids carrying either a fragment of the A. nidulans ribosomal repeat (p3SR2rr) or a fragment containing a possible A. nidulans mitochondrial origin of replication (p3SR2mo) in addition to the acetamidase gene were used. Both pBR322 and acetamidase gene sequences become integrated into the genome of A. nidulans in transformant strains. Integration events into the residual sequences adjacent to the deletion in the acetamidase gene, and probably (for p3SR2rr and p3SR2mo) into the ribosomal repeat unit are described.     

IL-4 and IL-13 upregulate ornithine decarboxylase expression by PI3K and MAP kinase pathways in vascular smooth muscle cells

Ornithine decarboxylase (ODC) is the first and rate-controlling enzyme in the synthesis of polyamines, which are essential for normal cell growth. We have previously demonstrated that IL-4 and IL-13 can stimulate rat aortic smooth muscle cell (RASMC) proliferation. The objective of this study was to determine whether IL-4 and IL-13 induce cell proliferation by upregulating ODC expression in RASMC. The results revealed that incubation of RASMC with IL-4 and IL-13 for 24 h caused four- to fivefold induction of ODC catalytic activity. The increased ODC catalytic activity was attributed to the increased expression of ODC mRNA. Moreover, these observations were paralleled by increased production of polyamines. We further investigated the signal transduction pathways responsible for ODC induction by IL-4 and IL-13. The data illustrated that PD-98059, a MEK (MAPK kinase) inhibitor, LY-294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, and H-89, a protein kinase A (PKA) inhibitor, substantially decreased the induction of ODC catalytic activity and ODC mRNA expression induced by IL-4 and IL-13, suggesting positive regulation of the ODC gene by ERK, PI3K, and PKA pathways. Interestingly, dexamethasone, a known inhibitor of cell proliferation, completely abrogated the response of RASMC to IL-4 and IL-13. Furthermore, the inhibition of ODC by these inhibitors led to the reduced production of polyamines and decreased DNA synthesis as monitored by [(3)H]thymidine incorporation. Our data indicate that upregulation of ODC by IL-4 and IL-13 might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.     

Antitumor effect of antisense ornithine decarboxylase adenovirus on human lung cancer cells

Ornithine decarboxylase (ODC),the first enzyme of polyamine biosynthesis,was found toincrease in cancer cells,especially lung cancer cells.Some chemotherapeutic agents aimed at decreasingODC gene expression showed inhibitory effects on cancer cells,ha this study,we examined the effects ofadenoviral transduced antisense ODC on lung cancer cells.An adenovirus carrying antisense ODC(rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549.The 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to analyze the effect on cell growth.Expression of ODC and concentrationof polyamines in cells were determined by Western blot analysis and high performance liquid chromatography.Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cellapoptosis.The expression of ODC in A-549 cells was reduced to 54%,and that of three polyamines wasalso decreased through the rAd-ODC/Ex3as treatment.Consequently,cell growth was substantially inhibitedand terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3ascould lead to cell apoptosis,with apoptosis index of 46%.This study suggests that rAd-ODC/Ex3as has anantitumor effect on the human lung cancer cells.     

Enhanced gene replacement frequency in KU70 disruption strain of Stagonospora nodorum

To improve the efficiency of gene disruption in Stagonospora nodorum, the putative KU70 gene encoding the Ku70 protein involved in the nonhomologous end-joining double DNA break repair pathway was identified and deleted. The KU70 disruption strain showed no apparent defect in vegetable growth, conidiation and pathogenicity on wheat and barley compared with the wild-type strain. The effect of the absence of KU70 on gene replacement frequency was tested by disruption of TOXA encoding toxin A and LIP2 encoding a putative lipase. Frequency of gene replacement for both genes was dramatically increased in the KU70 disruption strain, compared with the low frequency in the wild-type recipient.     

Functional characterization of a maize purine transporter by expression in Aspergillus nidulans

We have characterized the function of Leaf Permease1 (LPE1), a protein that is necessary for proper chloroplast development in maize, by functional expression in the filamentous fungus Aspergillus nidulans. The choice of this ascomycete was dictated by the similarity of its endogenous purine transporters to LPE1 and by particular genetic and physiological features of purine transport and metabolism in A. nidulans. When Lpe1 was expressed in a purine transport-deficient A. nidulans strain, the capacity for uric acid and xanthine transport was acquired. This capacity was directly dependent on Lpe1 copy number and expression level. Interestingly, overexpression of LPE1 from >10 gene copies resulted in transformants with pleiotropically reduced growth rates on various nitrogen sources and the absolute inability to transport purines. Kinetic analysis established that LPE1 is a high-affinity (K(m) = 30 +/- 2.5 microM), high-capacity transporter specific for the oxidized purines xanthine and uric acid. Competition studies showed that high concentrations of ascorbic acid (>30 mM) competitively inhibit LPE1-mediated purine transport. This work defines the biochemical function of LPE1, a plant representative of a large and ubiquitous transporter family. In addition, A. nidulans is introduced as a novel model system for the cloning and/or functional characterization of transporter genes.     

Identification of the sites of action for regulatory genes controlling the amdS gene of Aspergillus nidulans.

The amdS gene of Aspergillus nidulans, which encodes an acetamidase enzyme, is positively regulated by the trans-acting genes amdR, facB, amdA, and areA. Sequence changes in several cis-acting mutations in the 5' region of the gene which specifically affect amdS regulation were determined. The amdI9 mutation, which results in increased facB-dependent acetate induction, is due to a single-base change at base pair -210 relative to the start point of translation. The amdI93 mutation, which abolishes amdR-dependent omega-amino acid induction, is a deletion of base pairs -181 to -151. The amdI66 mutation, which causes increased gene activation in strains carrying amdA regulatory gene mutations, is a duplication of base pairs -107 to -90. Transformation of A. nidulans can generate transformants containing multiple integrated copies of plasmid sequences. When these plasmids carry a potential binding site for a regulatory gene product, growth on substrates whose catabolism requires genes activated by that regulatory gene can be reduced, apparently because of titration of the regulatory gene product. Introduction of 5' amdS sequences via cotransformation into strains of various genotypes was used to localize sequences apparently involved in binding of the products of the amdR, amdA, and facB genes. The position of these sequences is in agreement with the positions of the specific cis-acting mutations. Consistent with these results, a transformant of A. nidulans derived from a plasmid deleted for sequences upstream from -111 was found to have lost amdR- and facB-mediated control but was still regulated by the amdA gene. In addition, amdS expression in this transformant was still dependent on the areA gene.     

Crystal structure of human ornithine decarboxylase at 2.1 A resolution: structural insights to antizyme binding

The polyamines spermidine and spermine are ubiquitous and required for cell growth and differentiation in eukaryotes. Ornithine decarboxylase (ODC, EC 4.1.1.17) performs the first step in polyamine biosynthesis, the decarboxylation of ornithine to putrescine. Elevated polyamine levels can lead to down-regulation of ODC activity by enhancing the translation of antizyme mRNA, resulting in subsequent binding of antizyme to ODC monomers which targets ODC for proteolysis by the 26S proteasome. The crystal structure of ornithine decarboxylase from human liver has been determined to 2.1 A resolution by molecular replacement using truncated mouse ODC (Delta425-461) as the search model and refined to a crystallographic R-factor of 21.2% and an R-free value of 28.8%. The human ODC model includes several regions that are disordered in the mouse ODC crystal structure, including one of two C-terminal basal degradation elements that have been demonstrated to independently collaborate with antizyme binding to target ODC for degradation by the 26S proteasome. The crystal structure of human ODC suggests that the C terminus, which contains basal degradation elements necessary for antizyme-induced proteolysis, is not buried by the structural core of homodimeric ODC as previously proposed. Analysis of the solvent-accessible surface area, surface electrostatic potential, and the conservation of primary sequence between human ODC and Trypanosoma brucei ODC provides clues to the identity of potential protein-binding-determinants in the putative antizyme binding element in human ODC.     

Saccharomyces cerevisiae centromere CEN11 does not induce chromosome instability when integrated into the Aspergillus nidulans genome.

We constructed Aspergillus nidulans transformation plasmids containing the A. nidulans argB+ gene and either containing or lacking centromeric DNA from Saccharomyces cerevisiae chromosome XI (CEN11). The plasmids transformed an argB Aspergillus strain to arginine independence at indistinguishable frequencies. Stable haploid transformants were obtained with both plasmids, and strains were identified in which the plasmids had integrated into chromosome III by homologous recombination at the argB locus. Plasmid DNA was recovered from a transformant containing CEN11, and the sequence of the essential portion of CEN11 was determined to be unaltered. The transformants were further characterized by using them to construct heterozygous diploids and then testing the diploids for preferential loss of the plasmid-containing chromosomes. The CEN11 sequence had little or no effect on chromosome stability. Thus, CEN11 does not prevent chromosomal integration of plasmid DNA and probably lacks centromere activity in Aspergillus spp.     

Transformation of Penicillium chrysogenum using dominant selection markers and expression of an Escherichia coli lacZ fusion gene

An industrial Penicillium chrysogenum strain was transformed using two dominant selection markers, namely the bacterial gene for phleomycin resistance (ble) fused to a fungal promoter, and the acetamidase (amdS) gene from Aspergillus nidulans. Transformation frequencies of up to 20 transformants per microgram of DNA were obtained with the ble system. With the amdS marker the frequency was up to 120 transformants. Cotransformation was very efficient when using amdS as a selection marker. The introduction of pAN5-41B, a plasmid carrying the Escherichia coli lacZ gene fused to the strong glyceraldehyde-3-phosphate dehydrogenase gene (gpd) promoter from A. nidulans, resulted in the formation of blue colonies on XGal plates indicating expression of the lacZ fusion gene in P. chrysogenum. A more detailed analysis of expression levels in several transformants showed that up to 6% of the total amount of soluble protein consists of the beta-galactosidase fusion protein.     

Antizyme regulates the degradation of ornithine decarboxylase in fission yeast Schizosaccharomyces pombe. Study in the spe2 knockout strains

The mechanism of the regulatory degradation of ornithine decarboxylase (ODC) by polyamines was studied in fission yeast, Schizosaccharomyces pombe. To regulate cellular spermidine experimentally, we cloned and disrupted S-adenosylmethionine decarboxylase gene (spe2) in S. pombe. The null mutant of spe2 was devoid of spermidine and spermine, accumulated putrescine, and contained a high level of ODC. Addition of spermidine to the culture medium resulted in rapid decrease in the ODC activity caused by the acceleration of ODC degradation, which was dependent on de novo protein synthesis. A fraction of ODC forming an inactive complex concomitantly increased. The accelerated ODC degradation was prevented either by knockout of antizyme gene or by selective inhibitors of proteasome. Thus, unlike budding yeast, mammalian type antizyme-mediated ODC degradation by proteasome is operating in S. pombe.