Structure and Function of A Novel Nuclear Matrix Protein : N/MAX

从人类多种细胞的cDNA文库中分离出编码超过1870个氨基酸的DNA结合蛋白（hN/MAX）的cDNA克隆。N/MAX是一种新型的核基质蛋白，它优先结合双链DNA中任何一条链的胞苷簇。DNA结合所必需的结构域位于N/MAX的c端附近。带N/MAX抗体的间接免疫荧光显微镜显示HeLa细胞核内纤维颗粒结构染色，未见核核。该染色从m期出现的浓缩染色质中排除。Western blot分析显示，核基质部分富集了250 kDa蛋白。N/MAX与大鼠基质蛋白3共有3种结构域（MH1、MH2和MH3）。MH1是两个核基质蛋白n端一个43个氨基酸的序列。MH2是一个约70个氨基酸的序列，在矩阵3和N/MAX中分别重复2次和3次。该结构域与hnRNP I/L中的RNA结合基序具有高度的同源性。MH3是一个60个氨基酸的序列，位于这两种蛋白的c端。M/MAX在MH1重复序列n端有一个富含精氨酸和丝氨酸（RS）的结构域。在DNA结合必需结构域附近，有9次重复序列与LVTVDEVIEEEDL一致。该结构域可以与钙结合，且在小于0.1 mm的浓度范围内，N/MAX的DNA结合能力受到钙的影响。小鼠等效物（mN/MAX）的cdna克隆与hN/MAX具有高度的同源性，并且延伸到RS结构域N端端的75个氨基酸序列是完全保守的。该结构域具有锌指状序列。小鼠cDNA克隆表明存在两种以上的N/MAX mrna，可能是由N/MAX基因的选择性剪接产生的。

cDNA clones encoding a DNA binding protein (hN/MAX) of more than 1870 amino acide were isolated from cDNA libraries of various human cells. N/MAX is a novel nuclear matrix protein which preferentially binds to cytidine clusters in either strand of double stranded DNA.The domain essential for DNA binding was located near C-terminal of N/MAX.Indirect immunofluorescence microscopy with an antibody against N/MAX showed stain of internal fibrogranular structure but not necleolus of HeLa cell nucleus. This stain was excluded from condensed chromatin appeared during M-phase. Western blot analysis showed a 250 kDa protein enriched in nuclear matrix fraction. N/MAX shares three types of domains (MH1, MH2 and MH3) with rat matrin 3. MH1 is a 43 amino acid sequence at N-terminal of both nuclear matrix proteins. MH2 is ca, 70 amino acid sequence repeated twice and three times in matrin 3 and N/MAX,respectively. This domain has high homology to RNA binding motif found in hnRNP I/L.MH3 is a 60 amino acid sequence located at the C-terminus of both proteins. M/MAX has an arginine and serine rich (RS) domain at N-terminal side of MH1 repeats. Close to the domain essential for DNA binding, there are nine times repeated suquences with consensus of LVTVDEVIEEEDL.This domain can bind to calcium and the DNA binding abillity of N/MAX was affected by calcium at concentration range less than 0.1 mM.cDNA clones of mouse equivalent (mN/MAX) showed high homology to hN/MAX and a 75 amino acid sequence extending to N-terminal side of RS domain was perfectly conserved. This domain had a zinc fingerlike sequence. Mouse cDNA clones suggested the pressence of more than two types of N/MAX mRNAs probably produced by alternative splicing of the N/MAX gene.