ADA AND PREMATURE TERMINATION CODONS

腺苷脱氨酶缺乏症(ADA)是一种常染色体隐性遗传性 严重的联合免疫缺陷。这项研究的目的是 ADA缺乏症患者ADA基因表达水平降低的病因研究 并探讨无义突变对ADA基因表达的影响 级别。从EB病毒制备的Poly A+富含mRNA的Northern杂交 来自患者JH的转化细胞系没有发现任何 检测到ADA mRNA，但JH细胞中肌动蛋白mRNA的量 很正常。从Poly A+mRNA中制备的JH‘s ADA基因的序列分析 以下是对已发表序列的修改：(1)氨基 破坏Taq限制的外显子1第8密码子上的酸替换 (2)碱基位置334的错义突变 密码子80，将Lys改为Arg，最后(3)在 外显子10中的1050-54个碱基位置，导致移码和 与成熟ADA蛋白的321位对应的过早终止密码子。 上述突变均经测序证实。 JH.基因组DNA之前只有两个美国反兴奋剂机构 缺乏ADA mRNA的突变；这两个(无关)个体都有 ADA基因启动子区和外显子1缺失 是第一个没有ADA mRNA的ADA缺乏症患者 可通过Northern分析检测到，病因似乎是在谁身上 过早终止密码子的存在。之前出版的作品 其他基因已经表明，无义和移码突变会导致 在过早停止的情况下，密码子可能会对mRNA水平产生不同的影响。我们 建议确定JH的ADA mRNA水平降低是否由于 转录功能障碍、细胞质mRNA不稳定、异核 信使核糖核酸的加工或它们的组合。转录功能的研究 来自JH患者的ADA基因将在2005年进行核奔跑研究 JH EBV转化的细胞系和转染腺病毒的CoS细胞 AdA启动子/cDNA构建。细胞质ADA mRNA的稳定性将是 用~(32)P-UTP或放线菌素进行脉冲追逐实验测量 D.异核mRNA的加工将通过转染CoS进行检测 JH‘s ADA基因相关部分含有5个碱基的细胞 缺失和侧翼内含子区域。定点突变和 TRNA抑制子分析将用于证明5个碱基是否 删除或过早终止密码子是导致 降低ADA基因表达水平。来自这些实验的数据可能会揭示 Ada RNA加工。

Adenosine deaminase deficiency (ADA) is an autosomal recessive form of severe combined immunodeficiency. The purpose of this research is to delineate the etiology of reduced ADA mRNA levels in ADA-deficient patient JH and to explore the effects of nonsense mutations upon ADA mRNA levels. Northern blotting of poly A+ enriched mRNA prepared from an EBV- transformed cell line derived from patient JH did not reveal any detectable ADA mRNA but the amount of actin mRNA in JH's cells was normal. Sequencing of JH's ADA cDNA prepared from poly A+ mRNA revealed the following alterations from the published sequence: (1) an amino acid substitution at codon 8 in exon 1 which destroys a Taq restriction site and changes asp to asn, (2) a missense mutation at nt position 334 codon 80 which changes lys to arg, and finally (3) a 5 bp deletion at nt positions 1050-54 in exon 10 which results in a frameshift and a premature stop codon corresponding to a 321 of the mature ADA protein. The above mutations have been verified by sequencing PCR-amplified genomic DNA from JH. There have been only two previously described ADA mutations which lack ADA mRNA; both of these (unrelated) individuals have a deletion in ADA gene encompassing the promoter region and exon 1. JH is the first ADA-deficient patient in which there is no ADA mRNA detectable by Northern analysis and in whom the etiology appears to be the presence of a premature stop codon. Previously published work for other genes has shown that nonsense and frameshift mutations which result in premature stop codons may have varying effects on mRNA levels. We propose to determine whether JH's reduced ADA mRNA levels are due to transcriptional dysfunction, cytoplasmic mRNA instability, heteronuclear mRNA processing or a combination of these. Transcriptional function of the ADA genes from patient JH will be investigated by nuclear run-ons in both JH EBV-transformed cell lines and also in Cos cells transfected with ADA promoter/cDNA constructs. Cytoplasmic ADA mRNA stability will be measured by pulse-chase experiments with 32P-UTP or by using actinomycin D. Processing of heteronuclear mRNA will be examined by transfecting Cos cells with the relevant portions of JH's ADA gene containing the 5 bp deletion and the flanking intronic regions. Site-directed mutagenesis and tRNA suppressor analysis will be employed to prove whether the 5 bp deletion or the premature termination codons are responsible for the reduced ADA mRNA levels. Data from these experiments may shed light upon ADA RNA processing.