Moleculer Mechanism of (functional) genomic imprinting in humans.

对第一年分离到的新基因进行分子分析是近年来的研究热点。(1)顺式作用因子的鉴定(1)在新基因第1外显子上游3.2 kb处发现了一个由五核苷酸串联重复序列组成的重复序列，与H19下游五聚体重复序列具有同源性。在375 bp的重复序列中，CCCAG的五核苷酸重复30次，CTCAG重复9次。与CCCAG和CCCTG的五核苷酸分别重复24次和20次的280 bp H19重复相比，该序列更加简并。ii)在3' UTR中鉴定出BstUI/HhaI RFLP。利用该RFLP分析了该基因表达的等位基因特异性。iii)虽然在脑、肝和肾中这两个等位基因的表达相同，但在胎盘中发现了两倍的等位基因表达差异。在所检测的9例中，有5例可确定优先表达等位基因的亲本来源，且始终为母系。因此，我们认为这是一个非典型的印迹基因。iv)到目前为止，在5'上游区域和第二内含子中尚未发现差异甲基化，其中存在另一种重复序列。v)我们收集了7个三胞胎胎盘和2个四胞胎胎盘，以确定多态性印迹的相关序列。但是，初步实验表明胎盘不适合用于此目的，因此我们放弃了这个子项目。(2)反式作用因子的分离与鉴定由于上述工作的意外耗时，本子项目起步较晚，现正努力寻找减法PCR的最佳条件。

Molecular analeses of a novel gene which was isolated in the first year were a main subject in the current year.(1) Identification of cis-acting factorsi) A repetitive sequence consisting of tandem repeats of pentanucleotides, which showed homology to the H19 downstream pentamer repeats, was found at 3.2 kb upstream of the first exon in the novel gene. Pentanucleotode of CCCAG was reiterated 30 times and CTCAG 9 times in the repetitious 375 bp sequence. It was more degenerate compared to the 280 bp H19 repeat in which pentanucleotides of CCCAG and CCCTG were repeated 24 times and 20 times, respectively.ii) A BstUI/HhaI RFLP was identified in the 3' UTR.Allele-specificity in expression of this gene was analyzed by taking advantage of this RFLP.iii) While it was equally expressed from both alleles in the brain, liver and kidney, two-fold allelic difference in expression was found in the placenta. Parental origin of preferrentially expressed allele could be ascertained in 5 cases among nine examined, and it was consistently maternal. Thus, we considered it to indicate an atypical imprinted gene.iv) So far, differential methylation has not yet identified in the 5' upstream region and second intron in which another kind of repetitive sequence was present.v) We collected 7 triplets placentae and 2 quadruplets placentae in order to identify sequences responsible for polymorphic imprinting. But, preliminary experiments suggested insuitability of using placentae for this pourpose, and therefore we abandoned this subproject.(2) Isolation and identification of trans-acting factorsBeing late in starting this subproject because of unexpectedly taking time in the above work, we are now trying to find the best condition of subtractive PCR.