Andrianopoulos A, Hynes M J
Department of Genetics, University of Melbourne, Parkville, Victoria, Australia.
Mol Cell Biol. 1988 Aug;8(8):3532-41. doi: 10.1128/mcb.8.8.3532-3541.1988.
The positively acting regulatory gene amdR of Aspergillus nidulans coordinately regulates the expression of four unlinked structural genes involved in acetamide (amdS), omega amino acid (gatA and gabA), and lactam (lamA) catabolism. By the use of DNA-mediated transformation of A. nidulans, the amdR regulatory gene was cloned from a genomic cosmid library. Southern blot analysis of DNA from various loss-of-function amdR mutants revealed the presence of four detectable DNA rearrangements, including a deletion, an insertion, and a translocation. No detectable DNA rearrangements were found in several constitutive amdRc mutants. Analysis of the fate of amdR-bearing plasmids in transformants showed that 10 to 20% of the transformation events were homologous integrations or gene conversions, and this phenomenon was exploited in developing a strategy by which amdRc and amdR- alleles can be readily cloned and analyzed. Examination of the transcription of amdR by Northern blot (RNA blot) analysis revealed the presence of two mRNAs (2.7 and 1.8 kilobases) which were constitutively synthesized at a very low level. In addition, amdR transcription did not appear to depend on the presence of a functional amdR product nor was it altered in amdRc mutants. The dosage effects of multiple copies of amdR in transformants were examined, and it was shown that such transformants exhibited stronger growth than did the wild type on acetamide and pyrrolidinone media, indicating increased expression of the amdS and lamA genes, respectively. These results were used to formulate a model for amdR-mediated regulation of gene expression in which the low constitutive level of amdR product sets the upper limits of basal and induced transcription of the structural genes. Multiple copies of 5' sequences from the amdS gene can result in reduced growth on substrates whose utilization is dependent on amdR-controlled genes. This has been attributed to titration of limiting amdR gene product. Strong support for this proposal was obtained by showing that multiple copies of the amdR gene can reverse this phenomenon (antititration).
构巢曲霉的正向作用调节基因amdR协同调节参与乙酰胺（amdS）、ω氨基酸（gatA和gabA）以及内酰胺（lamA）分解代谢的四个非连锁结构基因的表达。通过DNA介导的构巢曲霉转化，从基因组粘粒文库中克隆了amdR调节基因。对各种功能丧失型amdR突变体的DNA进行Southern印迹分析，发现存在四种可检测到的DNA重排，包括缺失、插入和易位。在几个组成型amdRc突变体中未发现可检测到的DNA重排。对转化体中携带amdR的质粒的命运分析表明，10%至20%的转化事件是同源整合或基因转换，并且在开发一种策略时利用了这一现象，通过该策略可以轻松克隆和分析amdRc和amdR-等位基因。通过Northern印迹（RNA印迹）分析检测amdR的转录，发现存在两种mRNA（2.7和1.8千碱基），它们以非常低的水平组成型合成。此外，amdR转录似乎不依赖于功能性amdR产物的存在，在amdRc突变体中也没有改变。研究了转化体中amdR多拷贝的剂量效应，结果表明，这些转化体在乙酰胺和吡咯烷酮培养基上比野生型表现出更强的生长，分别表明amdS和lamA基因的表达增加。这些结果被用于构建一个amdR介导的基因表达调节模型，其中amdR产物的低组成水平设定了结构基因基础转录和诱导转录 的上限。来自amdS基因的5'序列的多拷贝可导致在其利用依赖于amdR控制基因的底物上生长减少。这被归因于对有限的amdR基因产物的滴定。通过表明amdR基因的多拷贝可以逆转这种现象（抗滴定），为该提议获得了有力支持。
