Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa.
Molecular cloning and analysis of the scon-2 negative regulatory gene of Neurospora crassa.
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粗糙脉孢菌scon-2负调控基因的分子克隆与分析
DOI：
10.1128/mcb.10.10.5207-5214.1990
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发表时间：
1990
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
粗脉脉孢菌的硫调控系统由一组高度调控的结构基因（如编码芳基硫酸酯酶的基因）组成，这些结构基因受到scon*（硫控制剂）负调控基因和cys-3+正调控基因的协调控制。 Inscon-1（以前称为sconC）和scon-2突变体，无论细胞可用的硫水平如何，硫结构基因都会持续表达。通过基于粘粒的基因文库的同胞选择筛选克隆了scon-2+基因。该筛选基于铬酸盐的使用，铬酸盐是一种有毒的硫酸盐类似物，它被转运到在高硫条件下生长的scon-2细胞中，但不会转运到已恢复正常硫调节的细胞中。使用限制性片段长度多态性分析来确认克隆片段映射到正确的染色体位置。在野生型细胞中，Northern (RNA) 印迹分析表明，仅在硫去抑制条件下，2.6-kilobasescon-2+ 转录物才以大量水平存在。动力学分析表明，随着细胞变得硫饥饿，scon-2+mRNA含量增加。此外，只有在去抑制条件下，scon-2+RNA才能在核转录测定中被检测到。 Inscon-1、scon-2+mRNA水平呈组成型。在cys-3调节突变体中，scon-2+转录物水平降低，在scon-2突变体中，在去抑制和抑制条件下都存在cys-3+和ars-1+mRNA。重复诱导的点突变产生的 con-2 突变体在表型上与已知突变体相同。
The sulfur regulatory system ofNeurospora crassais composed of a group of highly regulated structural genes (e.g., the gene encoding arylsulfatase) that are under coordinate control ofscon*(sulfur controller) negative andcys-3+positive regulatory genes. Inscon-1(previously designatedsconC) andscon-2mutants, there is constitutive expression of sulfur structural genes regardless of the sulfur level available to the cells. Thescon-2+gene was cloned by sib selection screening of a cosmid-based gene library. The screening was based on the use of chromate, a toxic sulfate analog, which is transported intoscon-2cells grown on high sulfur but is not transported into cells that have regained normal sulfur regulation. Restriction fragment length polymorphism analysis was used to confirm that the cloned segment mapped to the proper chromosomal location. In wild-type cells, Northern (RNA) blot analysis showed that a 2.6-kilobasescon-2+transcript was present at a substantial level only under sulfur-derepressing conditions. Kinetic analysis showed thatscon-2+mRNA content increased as the cells became sulfur starved. Further,scon-2+RNA was detectable in a nuclear transcription assay only under derepressing conditions. Inscon-1,the levels ofscon-2+mRNA were found to be constitutive. In thecys-3regulatory mutant, there was a reduced level ofscon-2+transcript,cys-3+andars-1+mRNAs were present under both derepressing and repressing conditions in thescon-2mutant. Repeat-induced point mutation-generatedscon-2mutants were identical in phenotype to the known mutant.