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首页> 外文期刊>The Journal of Experomental Medicine >Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.
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Polymorphic structure of the tumor necrosis factor (TNF) locus: an NcoI polymorphism in the first intron of the human TNF-beta gene correlates with a variant amino acid in position 26 and a reduced level of TNF-beta production.

机译：肿瘤坏死因子（TNF）基因座的多态性结构：人类TNF-β基因第一个内含子中的NcoI多态性与26位变异氨基酸和TNF-β产生水平降低相关。

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Since a dysregulated synthesis of tumor necrosis factor alpha (TNF-alpha) may be involved in the pathogenesis of autoimmune diseases, it was of interest to precisely locate the recently reported NcoI restriction fragment length polymorphism (RFLP) of the TNF-alpha region. However, by mapping of 56.8 kb of overlapping cosmid clones and direct sequencing, we could localize the polymorphic NcoI restriction site within the first intron of the TNF-beta gene and not in the TNF-alpha gene. To study whether regulatory mechanisms are affected by this polymorphism, we analyzed the TNF-alpha/TNF-beta production of phytohemagglutinin-stimulated peripheral blood mononuclear cells of individuals homozygous for the TNF-beta NcoI RFLP by ELISA and concomitant Northern blot analysis. On days 2-4 after stimulation with mitogen, the TNFB*1 allele corresponding to a 5.3-kb NcoI fragment presented with a significantly higher TNF-beta response. A mRNA analysis demonstrated that higher protein levels of TNF-beta correlate also with increased amounts of TNF-beta transcripts. No allelic association was found in respect to TNF-alpha production. To further investigate a possible allelic influence on transcription, we determined the DNA sequence of 2 kb of the 5' portion of our cloned TNFB*2 allele and compared it with the available TNF-beta sequences. By computer-aided recognition motif search of DNA binding factors, we report putative binding sites conserved between mouse and man in the 5' flanking region as well as in intron 1 of the TNF-beta gene, found also in other cytokine promoter sequences. In addition, by polymerase chain reaction amplification and sequencing of 740 bp of the 5' part of TNF-beta of individuals typed homozygously for the NcoI RFLP, we could show that amino acid position 26 is conserved as asparagine in the TNFB*1 and as threonine in the TNFB*2 sequence. A previously reported, EcoRI RFLP in the 3' untranslated region of TNF-beta does not segregate with either of the two alleles. Thus, four TNFB alleles can be defined at the DNA level.
机译：由于肿瘤坏死因子α（TNF-α）的合成失调可能与自身免疫性疾病的发病机理有关，因此精确定位最近报道的TNF-α区域的NcoI限制性片段长度多态性（RFLP）引起人们的兴趣。但是，通过绘制56.8 kb重叠的粘粒克隆并直接测序，我们可以将多态性NcoI限制性酶切位点定位在TNF-beta基因的第一个内含子内，而不是在TNF-alpha基因内。为了研究调节机制是否受此多态性影响，我们通过ELISA和伴随的Northern印迹分析分析了纯合了TNF-beta NcoI RFLP的个体的植物血凝素刺激的外周血单核细胞的TNF-alpha / TNF-beta产生。在用有丝分裂原刺激后的第2-4天，对应于5.3-kb NcoI片段的TNFB * 1等位基因表现出明显更高的TNF-beta反应。 mRNA分析表明，较高的TNF-β蛋白水平也与增加的TNF-β转录物量相关。没有发现关于TNF-α产生的等位基因关联。为了进一步研究可能的等位基因对转录的影响，我们确定了我们克隆的TNFB * 2等位基因5'部分2 kb的DNA序列，并将其与可用的TNF-β序列进行了比较。通过计算机辅助识别主题搜索的DNA结合因子，我们报道了在5'侧翼区域以及TNF-β基因的内含子1中在小鼠和人类之间保守的推定结合位点，也发现在其他细胞因子启动子序列中。此外，通过对NcoI RFLP纯合型个体的TNF-beta的5'部分的740 bp的聚合酶链反应扩增和测序，我们可以证明氨基酸位置26在TNFB * 1中作为天冬酰胺保守，并且在TNFB * 2序列中的苏氨酸。先前报道的TNF-beta 3'非翻译区中的EcoRI RFLP不会与两个等位基因中的一个分离。因此，可以在DNA水平上定义四个TNFB等位基因。

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