Cloning of the nitrate reductase gene of Stagonospora (Septoria) nodorum and its use as a selectable marker for targeted transformation

The nitrate reductase gene (NIA1) of the phytopathogenic fungus Stagonospora (Septoria) nodorum has been cloned from a cosmid library by homologous hybridisation with a PCR-generated probe. A 6.7-kb fragment carrying the NIA1 gene was subcloned and partially characterised by restriction mapping. Sequencing of the gene indicated a high degree of homology, both at the nucleotide and amino-acid levels, with nitrate reductase genes of other filamentous fungi. Furthermore, consensus regulatory signals thought to be involved in the control of nitrogen metabolism are present in the 5′ flanking region. The cloned NIA1 gene has been used to develop a gene-transfer system based on nitrate assimilation. Stable nia1 mutants of S. nodorum defective in nitrate reductase were isolated by virtue of their resistance to chlorate. These were transformed back to nitrate utilisation with the wild-type S. nodorum NIA1 gene. Southern analyses revealed that transformation occurred as a result of the integration of transforming DNA into the fungal genome; in all cases examined, integration was targeted to the homologous sequence. Received: 30 March / 9 June 1998     

Characterization of the Aspergillus parasiticus niaD and niiA gene cluster

 The nitrate reductase gene (niaD) and nitrite reductase gene (niiA) of Aspergillus parasiticus are clustered and are divergently transcribed from a 1.6-kb intergenic region (niaD-niiA). The deduced aminoacid sequence of the A. parasiticus nitrate reductase demonstrated a high degree of homology to those of other Aspergillus species, as well as to Leptosphaeria maculans, Fusarium oxysporum, Gibberella fujikuroi and Neurospora crassa, particularly in the cofactor-binding domains for molybdenum, heme and FAD. A portion of the deduced nitrite reductase sequence was homologous to those of A. nidulans and N. crassa. The nucleotide sequences in niaD-niiA of A. parasiticus and of A. oryzae were 95% identical, indicating that these two species are closely related. Several GATA motifs, the recognition sites for the N. crassa positive-acting global regulatory protein NIT2 in nitrogen metabolism, were found in A. parasiticus niaD-niiA. Two copies of the palindrome TCCGCGGA and other partial palindromic sequences similar to the target sites for the pathway specific regulatory proteins, N. crassa NIT4 and A. nidulans NirA, in nitrate assimilation, were also identified. A recombinant protein containing the A. nidulans AreA (the NIT2 equivalent) zinc finger and an adjacent basic region was able to bind to segments of niaD-niiA encompassing the GATA motifs. These results suggest that the catalytic and regulatory mechanisms of nitrate assimilation are well conserved in Aspergillus. Received: 17 November 1995/16 January 1996     

Structural analysis of a hmg-coA-reductase pseudogene: insights into evolutionary processes affecting the hmgr gene family in allotetraploid cotton (Gossypium hirsutum L.)

Structural analysis of hmg-coA reductase (hmgr) genes in the allotetraploid cotton species Gossypium hirsutum L. revealed the first-known existence of a pseudogene, ψhmg5, for this important enzyme. Complete sequencing of the genomic clone hmg5 unveiled several deleterious lesions, resulting in an organization that departed significantly from the linear canonical hmgr gene structure. Although analysis of the 5′ flanking region indicated a promoter-like composition based on comparison with other known plant hmgr genes, the precise loss of intron 3, and putative poly-(A) signals, small poly-(A) tracts, and terminal repeats (TRs) found in the 3′-flanking region are characteristic features of retro-pseudogenes. DNA-blot analysis indicated that a ψhmg5-related subfamily exists within a larger hmgr gene family in cotton. Several mechanisms are proposed to account for the formation of this partially intronless pseudogene, including intragenic homologous-replacement recombination and gene conversion involving a cDNA. Alignments of ψhmg5 with functional cotton homologs also raised interesting possibilities for the formation of `chimeric' gene structures, or differential intragenic mutation rates, as potential evolutionary mechanisms involved in shaping the hmgr gene family in cotton. Received: 28 April / 16 July 1998     

Transformation of Botrytis cinerea with the nitrate reductase gene (niaD) shows a high frequency of homologous recombination

The nitrate reductase (niaD) gene was isolated from the phytopathogenic ascomycete Botrytis cinerea using a probe obtained by a polymerase chain reaction (PCR) with degenerate oligonucleotides corresponding to domains conserved among three fungal nitrate reductases. The B. cinerea niaD gene encodes a predicted protein of 907 amino acids and contains no intron. Nitrate reductase-deficient mutants of B. cinerea have been isolated. One of them was transformed with the niaD genes of Fusarium oxysporum f.sp. melonis and B. cinerea. The transformation was always ectopic when the donor DNA originated from F. oxysporum, but there was 80% gene replacement when the donor DNA originated from B. cinerea. Received: 19 February / 8 April 1997     

Characterization of the NRAMP1 (SLC11A1) gene in the horse (Equus caballus L.)

The complete coding cDNA sequence of the horse NRAMP1 (SLC11A1) gene was determined (GenBank accession number AF354445 ). The nucleotide sequence of the horse NRAMP1 gene is similar to sequences of this gene in other species. The gene contains 15 exons whose total length of 1635 bp corresponds to 544 amino acids constituting the resulting putative protein. Hydrophobicity profile analysis of the deduced horse NRAMP1 gene product showed a nearly identical structure with the mouse NRAMP1 protein. The gene was found to be located on the short arm of ECA 6p12–13 by fluorescence in situ hybridization (FISH) analysis. Five allelic variants of the 5′ untranslated region (UTR) were identified at the nucleotide sequence level. PCR‐RFLP polymorphisms for NlaIII, TaqI, MspI and AciI were detected. Four out of five alleles could be detected using TaqI and MspI restriction enzymes. Their haplotype frequencies were different in four genetically distinct horse breeds.     

Analysis of the human pancreatic secretory trypsin inhibitor (PSTI) gene mutations in Japanese patients with chronic pancreatitis

Chronic pancreatitis (CP) is a continuing or relapsing inflammatory disease of the pancreas. Several studies have demonstrated that mutations in the cationic trypsinogen (PRSS1) gene and the cystic fibrosis transmembrane conductance regulator (CFTR) gene are causative of the pathogenesis in a subset of hereditary and/or idiopathic CP cases. Recently, the N34S alteration of the pancreatic secretory trypsin inhibitor (PSTI) gene has been suggested to be closely associated with the pathogenesis of hereditary and/or idiopathic CP. Herein we analyzed genetic alterations of the PSTI gene in 32 unrelated Japanese CP patients who developed juvenile-onset CP or had a family history of CP; 5 patients were found to harbor alterations in this gene. In 3 of these 5 patients, heterozygous N34S alterations were found; this frequency is significantly lower than that in Caucasian patients reported previously. Moreover, a novel homozygous G-to-A transition in the promoter region of PSTI at 215 bp upstream from the translation initiation site (−215G>A) was observed in 2 patients. We further surveyed the −215G>A alteration in 117 normal individuals; none of these individuals harbored this alteration. Our results suggested that the −215G>A alteration, as well as the N34S alteration, is a predisposing factor for CP. Received: November 28, 2000 / Accepted: February 1, 2001     

A color-selectable and site-specific integrative transformation system for gene expression studies in the dematiaceous fungus Wangiella (Exophiala) dermatitidis

To explore potential virulence factors in the dematiaceous (melanized) fungus Wangiella dermatitidis, we established a gene expression system with properties of homologous transformation and color identification. Using a polyketide synthase gene (WdPKS1) fragment for targeting, we found that 52% of transformants became albinos easily distinguishable from nonspecific transformants. Southern analysis confirmed that the integrations were at the WdPKS1 locus, which however did not affect transformant growth. With a heterologous promoter, P-glaA, enhanced expression of lacZ was found at 37 °C. Our results indicated that this system allows the efficient production of isogenic strains for gene function analysis in W. dermatitidis. Received: 12 March / 18 June 1999     

The organization of mitochondrial atp6 gene region in male fertile and CMS lines of pepper (Capsicum annuum L.)

The mitochondrial atp6 gene in male fertile (N) and CMS (S) pepper has previously been compared and was found to be present in two copies (Kim et al. in J Kor Soc Hort Sci 42:121–127 2001). In the current study, these atp6 copies were amplified by an inverse PCR technique, and the coding region as well as the 5′ and 3′ flanking regions were sequenced. The atp6 copies in CMS pepper were detected as one intact gene and one pseudogene, truncated at the 3′ coding region. When the atp6 genes in pepper were compared to other plant species, pepper, potato, and petunia all possessed a sequence of 12 identical amino acids at the 3′ extended region, which was considered a hallmark of the Solanaceae family. Northern blot analysis showed differences in mRNA band patterns between CMS and restorer lines, indicating that atp6 gene is one of the candidates for CMS in pepper. GenBank accession number: DQ126682 (atp6-1 genomic sequence common to fertile and CMS pepper), DQ126681 (Fertile atp6-2 genomic sequence), DQ126680 (CMS pseudo-atp6-2 genomic sequence)     

The expression profile of the <Emphasis Type="Italic">Tuber borchii</Emphasis> nitrite reductase suggests its positive contribution to host plant nitrogen nutrition

Ectomycorrhizal symbiosis is a ubiquitous association between plant roots and numerous fungal species. One of the main aspects of the ectomycorrhizal association are the regulation mechanisms of fungal genes involved in nitrogen acquisition. We report on the genomic organisation of the nitrate gene cluster and functional regulation of tbnir1, the nitrite reductase gene of the ectomycorrhizal ascomycete Tuber borchii. The sequence data demonstrate that clustering also occurs in this ectomycorrhizal fungus. Within the TBNIR1 protein sequence, we identified three functional domains at conserved positions: the FAD box, the NADPH box and the two (Fe/S)-siroheme binding site signatures. We demonstrated that tbnir1 presents an expression pattern comparable to that of nitrate transporter. In fact, we found a strong down-regulation in the presence of primary nitrogen sources and a marked tbnir1 mRNA accumulation following transfer to either nitrate or nitrogen limited conditions. The real-time PCR assays of tbnir1 and nitrate transporter revealed that both nitrate transporter and nitrite reductase expression levels are about 15-fold and 10-fold higher in ectomycorrhizal tissues than in control mycelia, respectively. The results reported herein suggest that the symbiotic fungus Tuber borchii contributes to improving the host plant’s ability to make use of nitrate/nitrite in its nitrogen nutrition.     

Thirteen single-nucleotide polymorphisms (SNPs) in the alcohol dehydrogenase 4 (ADH4) gene locus

The human alcohol dehydrogenase 4 (ADH4) gene encodes the class II ADH4 pi subunit, which contributes to the metabolization of a wide variety of substrates, including ethanol, retinol, other aliphatic alcohols, hydroxysteroids, and lipid peroxidation products. Here we report the results of systematic screening for single-nucleotide polymorphisms (SNPs) in the ADH4 gene by means of direct sequencing combined with a polymerase chain reaction method. A total of 16 genetic variations including 13 SNPs were found; 4 in the 5′ flanking region, 4 in the 5′ untranslated region, and 8 within introns. No variation was found in coding, 3′ untranslated, or 3′ flanking regions. Eight of the 13 SNPs were not reported in the NCBI dbSNP database or any previous publications. Our SNP map presented here should provide tools to evaluate the role of ADH4 in complex genetic diseases and a variety of pharmacogenetic effects. Received: September 26, 2001 / Accepted: October 11, 2001     

Size and location of radish chromosome regions carrying the fertility restorer Rfk1 gene in spring turnip rape

In spring turnip rape (Brassica rapa L. spp. oleifera), the most promising F1 hybrid system would be the Ogu-INRA CMS/Rf system. A Kosena fertility restorer gene Rfk1, homolog of the Ogura restorer gene Rfo, was successfully transferred from oilseed rape into turnip rape and that restored the fertility in female lines carrying Ogura cms. The trait was, however, unstable in subsequent generations. The physical localization of the radish chromosomal region carrying the Rfk1 gene was investigated using genomic in situ hybridization (GISH) and bacterial artificial chromosome–fluorescence in situ hybridization (BAC–FISH) methods. The metaphase chromosomes were hybridized using radish DNA as the genomic probe and BAC64 probe, which is linked with Rfo gene. Both probes showed a signal in the chromosome spreads of the restorer line 4021–2 Rfk of turnip rape but not in the negative control line 4021B. The GISH analyses clearly showed that the turnip rape restorer plants were either monosomic (2n = 2x = 20 + 1R) or disomic (2n = 2x = 20 + 2R) addition lines with one or two copies of a single alien chromosome region originating from radish. In the BAC–FISH analysis, double dot signals were detected in subterminal parts of the radish chromosome arms showing that the fertility restorer gene Rfk1 was located in this additional radish chromosome. Detected disomic addition lines were found to be unstable for turnip rape hybrid production. Using the BAC–FISH analysis, weak signals were sometimes visible in two chromosomes of turnip rape and a homologous region of Rfk1 in chromosome 9 of the B. rapa A genome was verified with BLAST analysis. In the future, this homologous area in A genome could be substituted with radish chromosome area carrying the Rfk1 gene.     

Candida albicans ALS3 and insights into the nature of the ALS gene family

The ALS1 (agglutinin-like sequence) gene of Candida albicans encodes a protein similar to alpha-agglutinin, a cell-surface adhesion glycoprotein of Saccharomyces cerevisiae (Hoyer et al. 1995). A central domain of a tandemly repeated 108-bp sequence is found in the ALS1 coding region. This tandem-repeat motif hybridizes to multiple C. albicans genomic DNA fragments, indicating the possibility of other ALS1-like genes in C. albicans (Hoyer et al. 1995). To determine if these fragments constitute a gene family, tandem-repeat-hybridizing genomic fragments were isolated from a fosmid library by PCR screening using primers based on the consensus tandem-repeat sequence of ALS1 (Hoyer et al. 1995). One group of fosmids, designated ALS3, encodes a gene with 81% identity to ALS1. The sequences of ALS1 and ALS3 are most conserved in the tandem-repeat domain and in the region 5′ of the tandem repeats. Northern-blot analysis using unique probes from the 3′ end of each gene demonstrated that ALS1 expression varies, depending on which C. albicans strain is examined, and that ALS3 is hyphal-specific. Both genes are found in a variety of C. albicans and C. stellatoidea strains examined. The predicted Als1p and Als3p exhibit features suggesting that both are cell-surface glycoproteins. Southern blots probed with conserved sequences from the region 5′ of the tandem repeats suggest that other ALS-like sequences are present in the C. albicans genome and that the ALS family may be larger than originally estimated. Received: 24 December 1997 / 12 March 1998     

Disparate sequence characteristics of the Erysiphe graminis f.sp. hordei glyceraldehyde-3-phosphate dehydrogenase gene

The Erysiphe graminis f.sp. hordei (Egh) glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated and characterized. It contains typical promoter elements and has three introns, one of which is positioned in the 5′ untranslated region of the gene. The deduced amino-acid sequence has 87% similarity to gpd genes from other Ascomycete fungi. This is at the same level as previously estimated among these fungi. Comparison at the DNA level reveal similarities of only around 70%, which is 10% lower than previously reported. In an evolutionary tree based on the sequences from 18 fungal gpd genes, Egh falls into the group of Ascomycetes located at a basal position. The regulatory region of the Egh gpd gene has no homology to corresponding sequences in other filamentous Ascomycetes. Codon usage was determined for the four characterized Egh genes (tub2, Egh7, Egh16 and gpd) and found to be similar for all four genes. The results of the codon-usage analysis suggest that Egh is more flexible than other fungi in the choice of nucleotides at the wobble position. Codon-usage preferences in Egh and barley genes indicate a level of difference which may be exploited to discriminate between fungal and plant genes in sequence mixtures. The Egh gpd promoter appears to be superior to that of the Egh β-tubulin gene (tub2) for driving the E. coli β-glucuronidase (GUS) gene in transformation experiments. Received: 15 November 1996 / 22 February 1997