Controls of the expression of aspA, the aspartyl protease gene from Penicillium roqueforti

The gene (aspA) encoding the extracellular aspartyl protease from Penicillium roqueforti was cloned and characterized. Northern hybridization analyses and β-casein degradation assays revealed that aspA was strongly induced by casein in the medium and efficiently repressed by ammonia. External alkaline pH overrides casein induction, resulting in aspA repression. Cis-acting motifs known to mediate nitrogen and pH regulation of fungal gene expression are present in the aspA promoter and protein-DNA binding experiments showed that mycelial proteins interact with various regions of the promoter. Due to the efficient environmental controls on aspA expression, the promoter of aspA is an attractive candidate for the development of a controllable gene expression system in P. roqueforti.     

Ammonium repression of the nitrite-nitrate ( nasAB ) assimilatory operon of Azotobacter vinelandii is enhanced in mutants expressing the nifO gene at high levels

A number of Tn5 mutants were isolated which were unable to fix nitrogen and showed enhanced ammonium repression of the nitrate/nitrite assimilation genes. They also had reduced nitrate reductase activity under fully inducing conditions. Insertions were localized within the nifB gene, and inability to fix nitrogen was shown to be due to disruption of the nifB gene. However, enhanced ammonium repression proved to be the result of constitutive expression of the downstream nifO gene from an `out' promoter present in Tn5. Our results suggest that molybdenum metabolism might function as a regulatory factor that acts through the nitrate reductase. Received: 4 December 1996 / Accepted: 27 March 1997     

Down regulation of the mercury resistance operon by the most promoter-distal gene merD

Summary The effect of the merD gene on the expression of the mer operon was determined from the rates of accumulation of merA-lacZ fusion protein in the presence and absence of an active merD gene in trans. In the presence of the merD gene, -galactosidase activity was 2- to 4-fold lower. The merD gene was cloned in a T7 promoter expression vector and the MerD protein product was visualized by autoradiography.     

拟南芥AtNHX6基因启动子的克隆及表达分析

为了探明拟南芥内膜反向转运体AtNHX6基因的组织表达模式,从基因组中克隆了AtNHX6基因开放阅读框(ORF)上游侧翼调控区1 922bp序列,并成功构建AtNHX6基因启动子与GUS融合表达载体pCAM-BIA1381-proNHX6-GUS,通过农杆菌花序浸染法转化野生型拟南芥获得T3代纯合转基因拟南芥株系,经PCR检测扩增得到2 187bp目的条带。利用组织染色法鉴定转基因拟南芥的GUS表达模式发现,在子叶、下胚轴和花中GUS活性显著。在这些广泛表达的部位中,微管系统中的表达最为显著,真叶中只有局部检测到GUS表达;在根中GUS在根毛和侧根生长部位表达;在未成熟果荚中只有在果荚顶端和基部存在GUS活性,成熟果荚中只在果柄检测到GUS表达;在花中,雄蕊的花丝和花粉粒及雌蕊的柱头中检测到GUS表达。GUS染色分析结果表明,AtNHX6基因启动子与GUS的融合表达载体成功构建并正常启动GUS基因表达,且AtNHX6基因主要在拟南芥的子叶、下胚轴、根、花、果荚中的微管系统、根毛和侧根生长部位以及花丝、花粉、柱头中表达。     

Identification and characterisation of an RPD3 homologue from maize ( Zea mays L.) that is able to complement an rpd3 null mutant of Saccharomycescerevisiae

In mammals, yeast and Drosophila, the histone deacetylase RPD3 proteins can alter the expression of genes involved in fundamental biological processes by affecting the degree of acetylation of histones and changing chromatin structure. Here we report the isolation of a cDNA sequence encoding an RPD3 homologue from maize, which is able to complement the phenotype of an rpd3 null mutant of the yeast Saccharomyces cerevisiae. The expression of the corresponding gene(s) was assessed in different maize tissues. The number of homologous loci was estimated by Southern hybridisation to be in the range of two to three, and the chromosomal location of one of these loci was determined. Phylogenetic analysis and tests for relative divergence rates, using related RPD3 sequences from different species, were performed, and suggest that different polymorphic forms of RPD3-like proteins that evolve at distinct rates are present in the species considered. Received: 5 December 1997 / Accepted: 16 January 1998     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Analysis of an abscisic acid (ABA)-responsive gene promoter belonging to the Asr gene family from tomato in homologous and heterologous systems

Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the β-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems. Received: 24 March 1997 / Accepted: 26 November 1997     

精氨酸代谢调控蛋白ArgR调控嗜热链球菌胞外多糖合成

[目的] 研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖（EPS）合成的调控作用。[方法] 利用大肠杆菌异源表达嗜热链球菌ArgR蛋白,通过尿素变性-复性和Ni²⁺亲和层析纯化。采用凝胶电泳迁移（EMSA）和生物膜层干涉（BLI）分析ArgR和eps基因簇中P_epsA启动子的相互作用和动力学信息。构建过表达和弱化argR基因菌株,利用苯酚-硫酸法测定其合成EPS差异。[结果] 大肠杆菌异源表达的ArgR为包涵体,使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白;EMSA和BLI结果显示ArgR和启动子P_epsA有特异性结合,且结合因解离水平低而稳定;过表达argR基因可显著降低嗜热链球菌EPS合成,而弱化argR基因则提高EPS合成。[结论] 本研究表明ArgR能特异性结合嗜热链球菌eps基因簇启动子,并负调控EPS生物合成。     

Expression of a chromosomally integrated, single-copy GFP gene in Candida albicans , and its use as a reporter of gene regulation

Genetically engineered versions of the GFP gene, which encodes the green fluorescent protein of Aequorea victoria, were placed under the control of the constitutively active Candida albicansACT1 promoter and integrated in single copy into the genome of this pathogenic yeast. Integrative transformants in which one of the two ACT1 alleles had been replaced by a GFP gene exhibited a homogeneous, constitutive fluorescent phenotype. Cells expressing GFP with the wild-type chromophore exhibited very weak fluorescence compared to those GFP proteins with the S65T or S65A, V68L, S72A (GFPmut2) chromophore mutations. Substitution of the CTG codon, which specifies serine instead of leucine in C. albicans, by TTG was absolutely necessary for GFP expression. Although GFP mRNA levels in cells containing a GFP gene with the CTG codon were comparable to those of transformants containing GFP with the TTG substitution, only the latter produced GFP protein, as detected by Western blotting, suggesting that the frequent failure to express heterologous genes in C. albicans is principally due to the non-canonical codon usage. Transformants expressing the modified GFP gene from the promoter of the SAP2 gene, which encodes one of the secreted acid proteinases of C. albicans, showed fluorescence only under conditions which promote proteinase expression, thereby demonstrating the utility of stable, chromosomally integrated GFP reporter genes for the study of gene activation in C. albicans. Received: 27 June 1997 / Accepted: 26 September 1997     

厚藤脱水素基因IpDHN启动子IpDHN-Pro的克隆和调控转录活性分析

为了解厚藤(Ipomoea pes-caprae)脱水素基因IpDHN (GenBank登录号:KX426069)启动子的转录活性和对非生物胁迫和植物激素ABA的响应,通过染色体步移法克隆了IpDHN的上游启动子序列IpDHN-Pro,长度为974 bp。构建IpDHN-Pro调控下GUS转基因载体,转化拟南芥(Arabidopsis thaliana)植株获得IpDHN-Pro::GUS转基因植株并进行GUS染色,验证IpDHN-Pro启动转录活性以及在氯化钠、甘露醇、ABA处理后拟南芥GUS基因表达变化。结果表明,扩增获得的IpDHN-Pro序列包含多个顺式作用元件,包括1个ABRE、3个Myb转录因子结合位点、富含TC的重复序列以及Skn-1基序等。转基因拟南芥GUS染色及qRT-PCR表明该序列可驱动GUS基因在拟南芥稳定表达,且表达受高盐、渗透压及ABA的诱导。这表明IpDHN-Pro是一个盐旱、ABA诱导的启动子序列,可应用于相关的植物抗逆遗传工程研究。     

Effects of the sinR and degU32 (Hy) mutations on the regulation of the aprE gene in Bacillus subtilis

The aprE gene of Bacillus subtilis codes for the serine alkaline protease known as subtilisin. Its expression is regulated by a complex network of activators and repressors that includes the products of hpr, degU and sinR. In order to understand the effect of these gene products on subtilisin expression, strains carrying combinations of the degU32(Hy), hpr2 and sinR null mutations, were constructed. We found that in all the genetic backgrounds tested, the sinR null mutation decreased aprE expression. Also, by measuring alkaline phosphatase synthesis and the formation of heat-resistant spores, as indicators of sporulation, we found that some of the mutant strains showed alterations in the sporulation process. These results suggest that these alterations are partially responsible for some of the observed changes in aprE expression. Received: 12 January 1996 / Accepted: 7 July 1996