Xia X, Werner D, Popanda O, Thielmann H W
Division of Interaction of Carcinogens with Biological Macromolecules, German Cancer Research Center, Heidelberg.
J Cancer Res Clin Oncol. 1994;120(8):454-64. doi: 10.1007/BF01191798.
Differential hybridization was used to detect repair defects in xeroderma pigmentosum (XP) that are not amenable to current analyses. cDNA libraries were constructed from cytoplasmic RNA of normal and XP fibroblast strains (complementation groups A and D) and analyzed for differential gene expression. More than 40,000 lambda gt10 cDNA clones were differentially screened with in vitro transcripts made from cDNA in the pBluescript vector. Six differential clones were detected in the libraries of the XP group A and D strains which caused stronger or weaker signals when probed with transcripts from XP strains than with those from the normal strains. Two clones coded for mitochondrial genes: mitochondrial 16 S rRNA and ATPase 6L. Overexpression of mitochondrial genes in XP may indicate that functions of the ATP-generating system are impaired since such functions are intensified whenever they become insufficient, for example as a consequence of DNA damage. It is tempting to assume that abnormal mitochondria are one of the causes for the neurological malfunctions in XP. Furthermore, densitometric analysis of Northern blots revealed that mRNA of lactate dehydrogenase, chain M, was less abundant in four XP group A strains (extent of reduction: 70%) and in two XP group D strains (extent of reduction: 58%). Enzyme activity was also diminished. In addition, mRNA of the gene for glyceraldehyde-3-phosphate dehydrogenase was less expressed in the same XP group A and D fibroblast strains investigated (reduction in both complementation groups: 50%). Both glycolytic enzymes have nuclear functions apart from their role in sugar metabolism. Lactate dehydrogenase, chain M, is identical to a helix-destabilizing protein; it is closely associated with chromatin and unfolded DNA, suggesting a role in DNA synthesis and transcription. The 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase is involved in transcription and was shown to be identical to uracil-DNA glycosylase, a base-excision repair enzyme. We presume that the nuclear functions of these glycolytic enzymes may be thwarted in the XP strains investigated and may account for malfunctions in XP, particularly for neurological disturbances.
差异杂交用于检测目前分析方法无法检测的着色性干皮病（XP）中的修复缺陷。从正常和XP成纤维细胞系（互补组A和D）的细胞质RNA构建cDNA文库，并分析差异基因表达。用pBluescript载体中的cDNA制备的体外转录本对40000多个λgt10 cDNA克隆进行差异筛选。在XP组A和D菌株的文库中检测到6个差异克隆，用XP菌株的转录本进行探测时，其信号比用正常菌株的转录本更强或更弱。两个克隆编码线粒体基因：线粒体16S rRNA和ATPase 6L。XP中线粒体基因的过表达可能表明ATP生成系统的功能受损，因为每当功能不足时，例如由于DNA损伤，这些功能就会增强。很容易推测异常线粒体是XP神经功能障碍的原因之一。此外，Northern印迹的光密度分析显示，乳酸脱氢酶M链的mRNA在4个XP组A菌株（减少程度：70%）和2个XP组D菌株（减少程度：58%）中含量较低。酶活性也降低。此外，在相同的XP组A和D成纤维细胞系中，甘油醛-3-磷酸脱氢酶基因的mRNA表达较少（两个互补组均减少：50%）。这两种糖酵解酶除了在糖代谢中的作用外，还具有核功能。乳酸脱氢酶M链与一种解螺旋蛋白相同；它与染色质和未折叠的DNA密切相关，表明在DNA合成和转录中起作用。甘油醛-3-磷酸脱氢酶的37 kDa亚基参与转录，并被证明与尿嘧啶-DNA糖基化酶相同，后者是一种碱基切除修复酶。我们推测，在所研究的XP菌株中，这些糖酵解酶的核功能可能受到阻碍，这可能是XP功能障碍的原因，特别是神经紊乱的原因。
