Gillen C, Gleichmann M, Spreyer P, Müller H W
Department of Neurology, University of Düsseldorf, Germany.
J Neurosci Res. 1995 Oct 1;42(2):159-71. doi: 10.1002/jnr.490420203.
In an attempt to identify genes associated with Wallerian degeneration and peripheral nerve regeneration we have performed differential hybridization screening of a cDNA library from crushed rat sciatic nerve (7 days postlesion) using radioactively labeled cDNA prepared from poly(A)+ RNA of normal vs. crushed nerve. Screening of 5,000 randomly selected colonies yielded 24 distinct clones that were regulated following nerve injury. Fifteen of the differentially expressed sequences could be classified as induced, whereas 9 sequences appeared to be repressed at 1 week postcrush. Sequencing and computer-assisted sequence comparison revealed 3 classes of regulated cDNA clones representing 1) novel gene sequences (8 clones) including 3 transcripts containing a repetitive "brain identifier" (ID) element; 2) identified genes (7 clones) with previously undetected expression in the peripheral nervous system (PNS), such as apolipoprotein D, peripheral myelin protein 22kD (PMP22), SPARC (secreted protein, acidic and rich in cysteine), sulfated glycoprotein SGP-1, apoferritin, decorin, and X16/SRp20; and 3) identified genes (9 clones) with known expression in the PNS including, e.g., the myelin protein P0, gamma-actin, vimentin, alpha-tubulin, chargerin II, and cytochrome c-oxidase subunit I. Northern blot and polymerase chain reaction analyses with RNA from crushed and transected nerve demonstrated that sequences with related function, like the group of myelin genes, cytoskeleton genes, genes involved in RNA processing and translation, in lipid transport or energy metabolism showed closely related temporal patterns of expression during nerve degeneration and regeneration. Finally, we compared the differentially expressed genes identified at 7 days after crush injury (this investigation) with the regulated sequences isolated previously by De Leon et al. (J Neurosci Res 29:437-488, 1991) from a 3 day postcrush sciatic nerve cDNA library.
为了鉴定与沃勒变性和周围神经再生相关的基因，我们利用从正常神经与损伤神经的多聚腺苷酸加尾RNA制备的放射性标记cDNA，对损伤后7天的大鼠坐骨神经cDNA文库进行了差异杂交筛选。对随机挑选的5000个菌落进行筛选，得到了24个在神经损伤后表达受到调控的不同克隆。15个差异表达序列可归类为诱导型，而9个序列在损伤后1周似乎受到抑制。测序和计算机辅助序列比较揭示了3类受调控的cDNA克隆，分别代表：1）新基因序列（8个克隆），包括3个含有重复“脑识别符”（ID）元件的转录本；2）已鉴定的基因（7个克隆），其在周围神经系统（PNS）中以前未检测到表达，如载脂蛋白D、周围髓磷脂蛋白22kD（PMP22）、SPARC（分泌性蛋白质，富含酸性和半胱氨酸）、硫酸化糖蛋白SGP-1、脱铁铁蛋白、核心蛋白聚糖和X16/SRp20；3）已鉴定的基因（9个克隆），其在PNS中具有已知表达，例如髓磷脂蛋白P0、γ-肌动蛋白、波形蛋白、α-微管蛋白、电荷调节蛋白II和细胞色素c氧化酶亚基I。用损伤和横断神经的RNA进行的Northern印迹和聚合酶链反应分析表明，具有相关功能的序列，如髓磷脂基因、细胞骨架基因、参与RNA加工和翻译的基因、脂质转运或能量代谢相关基因，在神经变性和再生过程中表现出密切相关的表达时间模式。最后，我们将损伤后7天鉴定的差异表达基因（本研究）与De Leon等人（《神经科学研究杂志》29:437 - 488，1991）先前从损伤后3天的坐骨神经cDNA文库中分离的受调控序列进行了比较。
