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

目的研究恒河猴体内是否存在mRNA表达 胎盘，可以编码可溶性MHC I类分子。 结果 人类胎盘MHC I类分子 HLA-G和恒河猴分子Mamu-AG的表达是多个 蛋白质同种型和剪接变体。 剪接变体也存在于 HLA-G编码可溶性蛋白质，但存在mRNA 编码可溶性恒河猴MHC I类分子以前没有被 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，并指出， 内含子4与HLA-G同源性为88 相同），其类似于外显子4与HLA-A序列的同源性 （~91%），但高于内含子5，表明 内含子4的进化漂移受到抑制， 选择功能重要的序列。 然后我们进行了 使用上游引物特异性RT-PCR分析胎盘mRNA Mamu-AG外显子4的下游引物，以及将扩增Mamu-AG外显子4的下游引物。 通过3U非翻译区，并允许诊断识别 Mamu-AG转录物通过提前终止密码子与 HLA-G 从胎盘RNA中扩增RT-PCR产物，克隆并 测序 终止密码子（TAG）存在于精确的密码子位置 在内含子4的可溶性HLA-G阅读框中的TAA终止密码子。 预测的氨基酸序列的羧基末端的末端， 第4外显子和内含子4肽表明与人的同一性 16/21残基处的序列，和保守取代（例如，G至 S）在其他5个残基处。 进一步的研究克隆并定义了 Mamu-AG的其他内含子的序列。 未来的方向这些 结果表明，恒河猴将成为解决 可溶性Mamu-AG的生理学。 我们将评估1）表达式 可溶性mRNA在胎膜，2）是否抗体， 可溶性HLA-G识别恒河猴分子，和3）恒河猴羊膜 液体和胎儿血清中可溶性MHC I类的存在 分子。 关键词母胎免疫耐受，胎盘， 羊水，剪接变体基金NIH HD 26458，HD 34216

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