Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.
Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.
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粗糙脉孢菌芳基硫酸酯酶结构基因的分子克隆及调控分析。
DOI：
10.1128/mcb.9.9.3630-3637.1989
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发表时间：
1989
影响因子：
5.3
通讯作者：
Paietta,JV
中科院分区：
文献类型：
--
作者：
Paietta,JV
粗粒脉孢菌的Thears-1+基因编码芳基硫酸酯酶。ARS-1+是一组在硫限制条件下表达的高度调控的硫磺相关结构基因，受Cecys-3+和scon+调控基因的协同控制。利用lambda文库，通过染色体走步从qagene簇中克隆了Thears-1+基因。人工神经网络的协同变换。用分离到的lambda克隆和苯菌灵抗性基因突变的crassa ars-1突变体，然后进行芳基硫酸酯酶活性测定，以筛选TARS-1+基因。通过限制性片段长度多态性分析，进一步证实克隆片段定位于TARS-1+基因座。Northern(RNA)印迹分析表明，TARS-1+基因转录得到2.3kb的mRNA。在野生型细胞中，TARS-1+转录本在去硫条件下丰富，而在抑制条件下缺失。时程分析表明，在去硫培养中，ARS-1+信息的出现与芳基硫酸酯酶活力的出现是平行的。此外，在核转录实验中，只有在去抑制条件下才能检测到ars-1+的转录。在不能合成芳基硫酸酯酶(或其他硫控酶)的Acys-3调节突变体中，在抑制或去抑制条件下存在NOARS-1+转录本。在温度敏感的ys-3突变体中，TARS-1+转录本仅在允许的生长温度和硫磺去阻遏下存在。负调控突变体sconc既显示了芳基硫酸酯酶的结构性表达，又显示了ARS-1+信息的含量。
Thears-1+gene ofNeurospora crassaencodes the enzyme arylsulfatase.ars-1+is in a group of highly regulated sulfur-related structural genes that are expressed under conditions of sulfur limitation and are under coordinate control of thecys-3+andscon+regulatory genes. Thears-1+gene was cloned by chromosome walking from theqagene cluster, using a lambda library. Cotransformation of anN. crassa ars-1mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for thears-1+gene. Further confirmation that the cloned segment mapped to thears-1+locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that thears-1+gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, thears-1+transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance ofars-1+message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription ofars-1+was detected only under derepressing conditions in a nuclear transcription assay. In acys-3regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was noars-1+transcript under repressing or derepressing conditions. In a temperature-sensitivecys-3mutant, thears-1+transcript was present only at the permissive growth temperature and under sulfur derepression. A negative regulatory mutant,sconc, displayed both constitutive expression of arylsulfatase enzyme activity and content ofars-1+message.