Csiszar K, Entersz I, Trackman P C, Samid D, Boyd C D
Department of Surgery, UMDNJ-Robert Wood Johnson Medical School, New Brunswick 08903, USA.
Mol Biol Rep. 1996;23(2):97-108. doi: 10.1007/BF00424435.
Alterations in the synthesis and activity of lysyl oxidase occur concomitant with developmental changes in collagen and elastin deposition and with the pathogenesis of several acquired and heritable connective tissue disorders. To begin to unravel the mechanisms that control lysyloxidase gene expression, we have previously reported the complete exon-intron structure of the human lysyl oxidase gene. We have now sequenced this entire gene, including all six introns and 4 kb of DNA 5' of exon 1. Analysis of over 13 kb of intervening sequence and 5' flanking sequence revealed a concentration of conserved consensus sequence elements within the first intron and 1 kb immediately 5' of exon 1. Analysis of intron 1 and the 5' flanking domain, using recombinant plasmids containing the chloramphenicol acetyl transferase (CAT) reporter gene, identified functional DNA sequence elements within these non-coding domains responsible for inhibition and up-regulation of CAT activity in primary cultures of human skin fibroblasts, in smooth muscle cells, revertant cells derived from an osteosarcoma cell line and malignant c-Ha-ras-transformed osteosarcoma cells. DNA sequence elements within intron 1, in particular, resulted in a marked increase in CAT reporter activity in cultured fibroblasts, smooth muscle cells and osteosarcoma cells. In c-Ha-ras-transformed osteosarcoma cells, however, no such enhancer activity of intron 1 sequence was observed. Ras-transformed osteosarcoma cells exhibited reduced steady-state levels of lysyl oxidase mRNA that was primarily controlled through reduced transcription of the lysyl oxidase gene. The lack of any up-regulation of CAT activity in these ras-transformed cells by sequence elements within intron 1 suggests a complex interaction between cis-acting domains and trans-acting transcriptional factors in the 5' promoter domain and the first intron of the lysyl oxidase gene.
赖氨酰氧化酶合成与活性的改变与胶原蛋白和弹性蛋白沉积的发育变化以及几种获得性和遗传性结缔组织疾病的发病机制同时发生。为了开始阐明控制赖氨酰氧化酶基因表达的机制，我们之前报道了人类赖氨酰氧化酶基因的完整外显子 - 内含子结构。我们现在已经对该完整基因进行了测序，包括所有六个内含子以及外显子1上游4kb的DNA。对超过13kb的间隔序列和5'侧翼序列的分析揭示了在第一个内含子以及外显子1上游紧邻的1kb范围内保守共有序列元件的集中分布。使用含有氯霉素乙酰转移酶（CAT）报告基因的重组质粒对内含子1和5'侧翼结构域进行分析，确定了这些非编码结构域内的功能性DNA序列元件，它们负责在人皮肤成纤维细胞、平滑肌细胞、源自骨肉瘤细胞系的回复细胞以及恶性c-Ha-ras转化的骨肉瘤细胞的原代培养物中抑制和上调CAT活性。特别是内含子1中的DNA序列元件导致培养中的成纤维细胞、平滑肌细胞和骨肉瘤细胞中CAT报告基因活性显著增加。然而，在c-Ha-ras转化的骨肉瘤细胞中，未观察到内含子1序列的这种增强子活性。Ras转化的骨肉瘤细胞中赖氨酰氧化酶mRNA的稳态水平降低，这主要是通过赖氨酰氧化酶基因转录减少来控制的。内含子1中的序列元件在这些ras转化细胞中未能上调CAT活性，这表明在赖氨酰氧化酶基因的5'启动子结构域和第一个内含子中的顺式作用结构域与反式作用转录因子之间存在复杂的相互作用。
