Enzymatic characterization of the recombinant Arabidopsis thaliana nitrilase subfamily encoded by the NIT2/NIT1/NIT3-gene cluster

Three of the nitrilase isoenzymes of Arabidopsis thaliana (L.) Heynh. are located on chromosome III in tandem and these genes (NIT2/NIT1/NIT3 in the 5′→3′ direction) encode highly similar polypeptides. Copy DNAs encompassing the entire coding sequences for all three nitrilases were expressed in Escherichia coli as fusion proteins containing a C-terminal hexahistidine extension. All three nitrilases were obtained as enzymatically active proteins, and their characteristics were determined, including a detailed comparative analysis of their substrate preferences. All three nitrilases converted indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA), albeit, compared to the most effective substrates found, phenylpropionitrile (PPN), allylcyanide, (phenylthio)acetonitrile and (methylthio)acetonitrile, with low affinity and velocity. The preferred substrates are either naturally occurring substrates, which may originate from glucosinolate breakdown, or they are close relatives of these. Thus, a major function of NIT1, NIT2 and NIT3 is assigned to be the conversion to carboxylic acids of nitriles from glucosinolate turnover or degradation. While all nitrilases exhibit a similar pH optimum around neutral, and NIT1 and NIT3 exhibit a similar temperature optimum around 30 °C independent of the substrate analyzed (IAN, PPN), NIT2 showed a remarkably different temperature optimum for IAN (15 °C) and PPN (35–40 °C). A potential role for NIT2 in breaking seed dormancy in A. thaliana by low temperatures (stratification), however, was ruled out, although NIT2 was the predominantly expressed nitrilase isoform in developing embryos and in germinating seeds, as judged from an analysis of β-glucuronidase reporter gene expression under the control of the promoters of the four isogenes. It is possible that NIT2 is involved in supplying IAA during seed development rather than during stratification. Received: 13 May 2000 / Accepted: 14 August 2000     

Arabidopsis mutants resistant to the auxin effects of indole-3-acetonitrile are defective in the nitrilase encoded by the NIT1 gene.

Indole-3-acetonitrile (IAN) is a candidate precursor of the plant growth hormone indole-3-acetic acid (IAA). We demonstrated that IAN has auxinlike effects on Arabidopsis seedlings and that exogenous IAN is converted to IAA in vivo. We isolated mutants with reduced sensitivity to IAN that remained sensitive to IAA. These mutants were recessive and fell into a single complementation group that mapped to chromosome 3, within 0.5 centimorgans of a cluster of three nitrilase-encoding genes, NIT1, NIT2, and NIT3. Each of the three mutants contained a single base change in the coding region of the NIT1 gene, and the expression pattern of NIT1 is consistent with the IAN insensitivity observed in the nit1 mutant alleles. The half-life of IAN and levels of IAA and IAN were unchanged in the nit1 mutant, confirming that Arabidopsis has other functional nitrilases. Overexpressing NIT2 in transgenic Arabidopsis caused increased sensitivity to IAN and faster turnover of exogenous IAN in vivo.     

VdToxD1和VdToxD3调控大丽轮枝菌微菌核的萌发

大丽轮枝菌Verticillium dahliae是一种土传性植物病原真菌,可侵染660多种植物,引发黄萎病。微菌核是大丽轮枝菌在侵染后期形成的特殊休眠结构,可在土壤中存活14年,是黄萎病的主要初侵染来源。本研究在前期大丽轮枝菌萌发与休眠微菌核转录组分析的基础上,选择微菌核休眠状态高表达的VdToxD1和VdToxD3基因进行功能研究。结果发现,VdToxD1和VdToxD3敲除突变体菌株的微菌核萌发率分别为87.3%和86.0%,显著高于野生型菌株的64.0%;负调控微菌核萌发的转录因子VdCrz1与VdToxD1上游的-937 bp到-349 bp中的特定基序结合,与VdToxD3上游的-752 bp到-496 bp中的特定基序结合,结合位点均位于2个基因的启动子区。     

The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons

Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4–3A7 and 3A7–3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3′ end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.     

Cloning of the murine unconventional myosin gene Myo9b and identification of alternative splicing

We report the cloning of a cDNA for the mouse unconventional myosin Myo9b, the orthologue of the rat myr5 and human MYOIXb genes. A full-length spleen cDNA of 7087 bp encoding a protein of 1961 amino acids was isolated. By RT–PCR, we show that Myo9b is expressed in a wide range of tissues, including heart, brain, muscle and inner ear. In addition, we have identified two alternatively spliced exons. Equivalent exons have not been previously reported for either the human or rat homologues. These exons are located in the Myo9b specific actin-binding site insert of the head domain and in the tail region. A third splice form utilizing an alternative reading frame within the 3′UTR is also described. Several polymorphisms within the coding region were identified; of interest is an in-frame 33 bp imperfect duplication within the tail region that was observed only in the C57Bl/6 strain. Myo9b has been previously mapped to mouse chromosome 8 and is a candidate for the mouse mutations myodystrophy and quinky.     

Cloning and identification of the promoter of the tobacco Sar8.2b gene,a gene involved in systemic acquired resistance

Expression of the Sar8.2 gene family is induced by salicylic acid (SA) in tobacco during induction of systemic acquired resistance. Expression of Sar8.2b, one member of this 12-member family, was detected as early as 12 h after treatment with SA and was maximal 36 h after SA treatment. In NahG transgenic tobacco plants, benzothiadiazole and dichloroisonicotinic acid induced expression of Sar8.2b but SA did not, suggesting that expression of the Sar8.2b gene is SA-dependent. Several putative cis-acting elements were found in the Sar8.2b gene promoter region, including an as-1 element and GT-1 and Dof binding sequences. We constructed a series of progressive deletion mutations in the Sar8.2b promoter region linked to the β-glucuronidase (GUS) coding region and analyzed GUS activities by stable expression in transformants of Arabidopsis thaliana. Deletions between −728 and −927 bp or between −351 and −197 bp of the promoter region resulted in a significant reduction in GUS activity induced by SA treatment as shown in stable transformants of A. thaliana. The −197 bp fragment of the promoter region was found to confer a relatively low level of GUS activity induced by SA treatment in stable expression of transformants in A. thaliana. The results suggest that 927 bp of the Sar8.2b gene promoter confers full promoter activity and that cis-acting elements required for high-level SA-inducible expression of the Sar8.2b gene may exist within the regions −728 to −927 bp and −197 to −351 bp.     

The Nitrilase ZmNIT2 converts indole-3-acetonitrile to indole-3-acetic acid

We isolated two nitrilase genes, ZmNIT1 and ZmNIT2, from maize (Zea mays) that share 75% sequence identity on the amino acid level. Despite the relatively high homology to Arabidopsis NIT4, ZmNIT2 shows no activity toward beta-cyano-alanine, the substrate of Arabidopsis NIT4, but instead hydrolyzes indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). ZmNIT2 converts IAN to IAA at least seven to 20 times more efficiently than AtNIT1/2/3. Quantitative real-time polymerase chain reaction revealed the gene expression of both nitrilases in maize kernels where high concentrations of IAA are synthesized tryptophan dependently. Nitrilase protein and endogenous nitrilase activity are present in maize kernels together with the substrate IAN. These results suggest a role for ZmNIT2 in auxin biosynthesis.     

Indole acetonitrile-sensitivity of transgenic tobacco containing Arabidopsis thaliana nitrilase genes

 The Arabidopsis thaliana genome has four nitrilase (nitrile aminohydrolase, EC 3.5.5.1) genes (NIT1 to NIT4). These nitrilases catalyze hydrolysis of indole-3-acetonitrile (IAN) to indole-3-acetic acid (IAA). Growth of A. thaliana is inhibited by IAN probably due to hydrolysis of IAN to IAA, while the tobacco (Nicotiana tabacum) genome has only NIT4 homologs and is resistant to IAN. In this study, we introduced A. thaliana NIT1 to NIT4 into tobacco. Introduction of NIT1, NIT2 or NIT3 into tobacco conferred growth inhibition by IAN. NIT2 transgenic plants were highly sensitive to IAN, and NIT1 and NIT3 transgenic plants were moderately sensitive. On the other hand, NIT4 transgenic plants were less sensitive to IAN, although some morphological changes in the roots were observed as the wild-type tobacco. These findings suggest that the ability of transgenic tobacco to convert IAN to IAA in vivo is markedly different among transgenes of NIT1 to NIT4. Received: 22 November 1999 / Revision received: 28 January 2000 / Accepted: 4 February 2000     

糙皮侧耳脲酶基因的克隆和原核表达分析

尿素是现代农业生产中应用较为广泛的一种氮素化肥,而脲酶（EC 3.5.1.5）是尿素分解利用的关键酶。本文以糙皮侧耳Pleurotus ostreatus栽培菌株New 831为试验材料,探究了糙皮侧耳对尿素的利用情况,结果表明：糙皮侧耳可利用尿素作为唯一氮源,平板培养时尿素最适添加量为20mmol/L;在液体摇瓶培养过程中,培养液中的铵根浓度表现为先急剧升高后缓慢降低。通过对P. ostreatus PC15菌株基因组分析,获得了一个功能注释为脲酶的基因,并克隆获得了其全长基因组DNA（gDNA）和编码区（CDS）片段,命名为Pourease。结果表明：Pourease基因的gDNA和CDS长度分别为3 003bp和2 517bp,由10个外显子和9个内含子组成;Pourease蛋白由838个氨基酸组成,预测分子量为90.03kDa,与SDS-PAGE分析结果相符;Pourease蛋白与细菌、真菌和植物来源的脲酶具有52%-82%的一致性,且含有脲酶保守的镍离子结合位点;与洋刀豆脲酶空间结构类似,Pourease蛋白也以同源三聚体的形式存在。