SPLICING OF MAMU AG MRNA SUGGESTS EXISTENCE OF SOLUBLE MHC CLASS I MOLECULE

MAMU AG mRNA 的剪接表明可溶性 MHC I 类分子的存在

• 批准号: 6116422
• 负责人: THADDEUS G GOLOS
• 金额: $ 6.42万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6116422
• 关键词: Mammalia; biotechnology; endocrine gland /system; genome; hormones; mother /infant health care

OBJECTIVE To determine whether mRNAs are expressed in the rhesus placenta which could encode a soluble MHC class I molecule. RESULTS An unusual feature of both the human placental MHC class I molecule HLA-G and the rhesus molecule Mamu-AG is expression of multiple protein isoforms and splice variants. Splice variants also exist for HLA-G which encode a soluble protein but the presence of a mRNA encoding a soluble rhesus MHC class I molecule has not previously been detected. We cloned a fragment of the Mamu-AG gene from rhesus monkey genomic DNA to define the sequences of intron 4 and 5 and noted that the sequence of intron 4 was highly homologous to that of HLA-G (88% identical), which is similar to exon 4 homology with HLA-A sequences (~91%), but higher than that for intron 5, suggesting that evolutionary drift of intron 4 has been restrained and implying selection for functionally important sequences. We then conducted RT-PCR analyses with placental mRNA using an upstream primer specific for Mamu-AG exon 4, and a downstream primer which would amplify through the 3U-untranslated region and allow diagnostic identification of Mamu-AG transcripts via the premature stop codon homologous to HLA-G. RT-PCR products were amplified from placental RNA, cloned and sequenced. A stop codon (TAG) is present in the exact codon location of the TAA stop codon in the soluble HLA-G reading frame of intron 4. The predicted amino acid sequences of the carboxy-terminal end of the 4th exon and the intron 4 peptides indicate identity with the human sequence at 16/21 residues, and conservative substitutions (e.g., G to S) at the other 5 residues. Further studies cloned and defined the sequences of the other introns of Mamu-AG. FUTURE DIRECTIONS These results suggest that the rhesus monkey will be a model for addressing the physiology of soluble Mamu-AG. We will evaluate 1) the expression of soluble mRNA in the fetal membranes, 2) whether antibodies against soluble HLA-G recognize the rhesus molecule, and 3) rhesus amniotic fluid and fetal serum for the presence of soluble MHC class I molecules. KEY WORDS maternal-fetal immune tolerance, placenta, amniotic fluid, splice variant FUNDING NIH HD 26458, HD34216

目的研究恒河猴体内是否存在mRNAs表达。 胎盘可以编码一种可溶性的MHC-I类分子。结果 人类胎盘MHC I类分子的一个不同寻常的特征 人类白细胞抗原-G和恒河猴分子MAMU-AG是多重表达的 蛋白质异构体和剪接变体。拼接变体也存在于 人类白细胞抗原G编码一种可溶性蛋白，但存在一种 编码可溶性恒河猴MHC I类分子以前从未被 检测到。我们克隆了恒河猴MAMU-AG基因的一个片段 基因组DNA来定义内含子4和5的序列，并注意到 内含子4的序列与人类白细胞抗原G基因高度同源(88% 相同)，与外显子4相似，与人类白细胞抗原-A序列同源 (~91%)，但高于内含子5，表明 内含子4的进化漂移受到抑制并暗示 功能重要序列的选择。然后我们进行了 用上游特异性引物RT-PCR分析胎盘组织中的mRNA 针对MAMU-AG外显子4，以及可扩增出 通过3U-非翻译区并允许诊断识别 通过与MAMU-AG同源的提前终止密码子 人类白细胞抗原G。从胎盘RNA中扩增RT-PCR产物，克隆和 已排序。终止密码子(标签)存在于准确的密码子位置 TAA终止密码子位于内含子4可溶的人类白细胞抗原-G阅读框中。 预测的氨基酸序列的羧基末端 第4外显子和第4内含子多肽表明与人类相同 16/21残基的序列和保守的替换(例如，G到 S)在其他5个残基上。进一步的研究克隆和定义了 MAMU-AG其他内含子的序列。这些是未来的方向 结果表明，恒河猴将是解决 可溶性MAMU-AG的生理特性。我们将对1)表达式求值 胎膜中可溶性mRNA的表达，2)抗体是否针对 可溶性人类白细胞抗原-G识别恒河猴分子；3)恒河猴羊水 体液和胎儿血清中可溶性MHC-I类的检测 分子。关键词母胎免疫耐受、胎盘、 羊水，剪接变异资助，美国国立卫生研究院HD 26458，HD34216