Nedelkina S, Jupe S C, Blee K A, Schalk M, Werck-Reichhart D, Bolwell G P
Département de Biologie Moléculaire des Plantes, CNRS-IBMP, Strasbourg, France.
Plant Mol Biol. 1999 Apr;39(6):1079-90. doi: 10.1023/a:1006156216654.
cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.8. It contains the conserved cysteine haem-binding site found in all cytochrome P450s. The protein encoded by this cDNA diverges however from other CYP73As in its N- and C-terminus and in four domains internally, so that overall sequence similarity is in the range 58-66%. Many clones contained an identical intron, which may be associated with a novel regulatory mechanism. Sequence similarity is sufficient for it to be classified as CYP73A15, although it is the least similar member of this family classified so far. The cDNA was expressed in yeast. Successful expression of cinnamate 4-hydroxylase activity required removal of the intron. High-level expression also required modification of the N-terminus to that of CYP73A1. Yeast did not process the intron at all and the leader sequence for A15 was not as compatible as that of A1. The mRNA for CYP73A15 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean but induction was more transient than that of phenylalanine ammonia-lyase (PAL). In contrast, induction in cells undergoing xylogenesis was much more coordinate with PAL. The cloned cDNA may represent a cinnamate 4-hydroxylase isoform, whose expression is more related to differentiation than the responses to stress in which the majority of CYP73As cloned so far are involved.
从激发子处理的悬浮培养细胞中表达的mRNA构建的cDNA文库中，分离出了与其他物种的植物CYP73A直系同源基因在大片段上具有高度序列相似性（>70%）的cDNA。这些克隆似乎编码一个全长1554 bp的开放阅读框，带有一个78 bp的5'-非翻译区和一个140 bp的3'-非翻译区。通过序列相似性确定的开放阅读框编码一种预测分子量为59229、等电点为8.8的蛋白质。它包含在所有细胞色素P450中都存在的保守半胱氨酸血红素结合位点。然而，该cDNA编码的蛋白质在其N端和C端以及内部的四个结构域与其他CYP73A不同，因此总体序列相似性在58 - 66%的范围内。许多克隆含有一个相同的内含子，这可能与一种新的调控机制有关。尽管它是迄今为止该家族中最不相似的成员，但序列相似性足以将其归类为CYP73A15。该cDNA在酵母中表达。肉桂酸4-羟化酶活性的成功表达需要去除内含子。高水平表达还需要将N端修饰为CYP73A1的N端。酵母根本不处理内含子，并且A15的前导序列不如A1的兼容。结果表明，菜豆悬浮培养细胞经激发子处理后，CYP73A15的mRNA会迅速被诱导，但诱导比苯丙氨酸解氨酶（PAL）更短暂。相比之下，在进行木质化的细胞中，诱导与PAL更协调。克隆的cDNA可能代表一种肉桂酸4-羟化酶同工型，其表达与分化的关系比与应激反应的关系更大，而迄今为止克隆的大多数CYP73A都参与应激反应。
