Boretsky Yuriy R, Kapustyak Kostyantyn Y, Fayura Lyubov R, Stasyk Oleh V, Stenchuk Mykola M, Bobak Yaroslav P, Drobot Lyudmyla B, Sibirny Andriy A
Department of Molecular Genetics and Biotechnology Institute of Cell Biology, NAS of Ukraine, Lviv.
FEMS Yeast Res. 2005 Jun;5(9):829-37. doi: 10.1016/j.femsyr.2005.03.007.
It is known for many years that iron represses synthesis of riboflavin (RF) and most of RF-synthesizing enzymes in several yeast species, known as flavinogenic yeasts. However, the mechanism of such repression is not known. We have found that iron represses transcription of RIB1 and RIB7 genes coding for the first and the last enzymes of RF biosynthesis in the model flavinogenic organism Pichia guilliermondii. To decipher molecular mechanisms of iron-dependent repression, isolation and study of the regulatory mutants defective in corresponding regulation is desirable. However, no suitable methods for isolation of such mutants were previously available. We have produced a single-point transition mutation in the RIB1 gene. The corresponding rib1-86 mutant exhibits leaky phenotype and is unable to grow in iron-sufficient minimal medium without exogenous RF. However, it can grow in minimal iron-deficient medium without RF, or in iron-sufficient medium upon introduction of the previously-isolated regulatory mutation rib81, which leads to increase in RF production. Using the rib1-86 mutant as parental strain, a collection of mutants able to grow in iron-sufficient medium without exogenous RF has been isolated. The mutants appeared to be defective in regulation of RF biosynthesis and iron homeostasis and were divided into six new complementation groups. Study of one corresponding mutant, red6, showed derepression of RIB1 mRNA synthesis in iron-sufficient medium.
多年来已知，在几种被称为产黄素酵母的酵母物种中，铁会抑制核黄素（RF）的合成以及大多数RF合成酶的合成。然而，这种抑制的机制尚不清楚。我们发现，在典型的产黄素生物季也蒙毕赤酵母中，铁会抑制编码RF生物合成的第一种和最后一种酶的RIB1和RIB7基因的转录。为了解析铁依赖性抑制的分子机制，分离和研究在相应调控中存在缺陷的调控突变体是很有必要的。然而，以前没有合适的方法来分离这种突变体。我们在RIB1基因中产生了一个单点转换突变。相应的rib1-86突变体表现出渗漏表型，在没有外源RF的铁充足的基本培养基中无法生长。然而，它可以在没有RF的缺铁基本培养基中生长，或者在引入先前分离的调控突变rib81后在铁充足的培养基中生长，这会导致RF产量增加。以rib1-86突变体作为亲本菌株，分离出了一组能够在没有外源RF的铁充足培养基中生长的突变体。这些突变体似乎在RF生物合成和铁稳态的调控方面存在缺陷，并被分为六个新的互补群。对一个相应的突变体red6的研究表明，在铁充足的培养基中RIB1 mRNA的合成去抑制。
