Fisher R F, Egelhoff T T, Mulligan J T, Long S R
Department of Biological Sciences, Stanford University, California 94305-5020.
Genes Dev. 1988 Mar;2(3):282-93. doi: 10.1101/gad.2.3.282.
Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa. This induction depends on expression of nodD. Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity. We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R. meliloti extracts. The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts. Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes. In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts. The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box. The interaction of R. meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.
苜蓿中华根瘤菌中的结瘤（nod）基因受其共生宿主植物苜蓿产生的黄酮类信号分子（如木犀草素）转录诱导。这种诱导依赖于nodD的表达。在三个可诱导的nod基因簇nodABC、nodFE和nodH的上游，有一个高度保守的序列，称为“nod框”。其上游序列没有其他明显的相似性。我们发现，当用苜蓿中华根瘤菌提取物处理时，包含所有三个可诱导转录本上游区域的DNA片段显示出改变的电泳迁移率。提取物与这些DNA特异性相互作用的能力与nodD1或nodD3的遗传剂量以及提取物中nodD1或nodD3蛋白（NodD1或NodD3）的存在和浓度相关。使用针对NodD的抗血清构建了一个免疫亲和柱，该柱允许对NodD1进行大量纯化；这种NodD1制剂也显示出与含有可诱导nod基因上游发现的DNA序列的限制性片段特异性结合。此外，NodD特异性抗血清从根瘤菌总细胞提取物中去除了特异性DNA结合活性。总提取物和部分纯化的NodD蛋白与nod启动子序列的相互作用与代表nod框3'端25 bp部分的寡核苷酸具有竞争性。苜蓿中华根瘤菌提取物和NodD1蛋白与nod基因上游区域的相互作用独立于细胞或提取物对黄酮诱导剂的暴露而发生。
