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首页> 外文期刊>Molecular and Cellular Biology >Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.
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Molecular cloning and regulatory analysis of the arylsulfatase structural gene of Neurospora crassa.

机译：Neurospora crassa的芳基硫酸酯酶结构基因的分子克隆和调控分析。

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摘要

The ars-1+ gene of Neurospora crassa encodes 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 the cys-3+ and scon+ regulatory genes. The ars-1+ gene was cloned by chromosome walking from the qa gene cluster, using a lambda library. Cotransformation of an N. crassa ars-1 mutant with the isolated lambda clones and the benomyl resistance gene, followed by assay for arylsulfatase activity, was used to screen for the ars-1+ gene. Further confirmation that the cloned segment mapped to the ars-1+ locus was obtained by restriction-fragment-length polymorphism analysis. Northern (RNA) blot analysis showed that the ars-1+ gene was transcribed to give an mRNA of 2.3 kilobases. In wild-type cells, the ars-1+ transcript was abundant under sulfur-derepressing conditions but absent under repressing conditions. Time course analysis showed that the appearance of ars-1+ message in sulfur-derepressed cultures paralleled the appearance of arylsulfatase enzyme activity. In addition, transcription of ars-1+ was detected only under derepressing conditions in a nuclear transcription assay. In a cys-3 regulatory mutant that was unable to synthesize arylsulfatase (or other sulfur-controlled enzymes), there was no ars-1+ transcript under repressing or derepressing conditions. In a temperature-sensitive cys-3 mutant, the ars-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 of ars-1+ message.
机译：景天孢菌的ars-1 +基因编码芳基硫酸酯酶。 ars-1 +在一组高度调控的硫相关结构基因中，这些结构基因在硫限制的条件下表达，并在cys-3 +和scon +调控基因的协调控制下。使用lambda文库，通过从qa基因簇中行走的染色体克隆了ars-1 +基因。使用分离的λ克隆和苯菌灵抗性基因共转化N. crassa ars-1突变体，然后测定芳基硫酸酯酶活性，以筛选ars-1 +基因。通过限制性片段长度多态性分析进一步获得了定位到ars-1 +基因座的克隆片段。 Northern（RNA）印迹分析表明，ars-1 +基因被转录后可产生2.3 kb的mRNA。在野生型细胞中，ars-1 +转录物在抑制硫的条件下丰富，但在抑制条件下不存在。时程分析表明，在硫降低的培养物中，ars-1 +信息的出现与芳基硫酸酯酶活性的出现平行。另外，仅在去抑制条件下在核转录测定中检测到ars-1 +的转录。在一个无法合成芳基硫酸酯酶（或其他硫控制的酶）的cys-3调节突变体中，在阻遏或阻遏条件下没有ars-1 +转录物。在一个对温度敏感的cys-3突变体中，ars-1 +转录物仅在允许的生长温度和硫抑制下存在。负调节突变体sconc显示芳基硫酸酯酶活性的组成型表达和ars-1 +信息的含量。

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