权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Moleculer Mechanism of (functional) genomic imprinting in humans.

人类（功能）基因组印记的分子机制。

基本信息

批准号：
08457630
负责人：
JINNO Yoshihiro
金额：
$ 4.48万
依托单位：
Nagasaki University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-08457630/
关键词：
genomic imprinting H19 gene cis-acting factor repetitive sequence PEN11B gene atypical imprinted gene 非定形的刷り込み遺伝子インプンリティング

项目摘要

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.

第一年分离的新基因的分子分析是今年的重点课题。(1)顺式作用因子的鉴定i)在新基因第一个外显子上游3.2 kb处发现了一条由五核苷酸串联重复组成的重复序列，与H19下游五聚体重复序列具有同源性。 CCCAG的五核苷酸在重复的375bp序列中重复30次，CTCAG重复9次。与 280 bp H19 重复（其中 CCCAG 和 CCCTG 的五核苷酸分别重复 24 次和 20 次）相比，它更加简并。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) 虽然它在大脑中的两个等位基因中同等表达，肝脏和肾脏，在胎盘中发现了两倍等位基因的表达差异。在 9 例检查的病例中，有 5 例可以确定优先表达等位基因的亲本来源，并且始终为母本。因此，我们认为它表明了非典型印记基因。 iv) 到目前为止，尚未在 5' 上游区域和第二内含子中发现差异甲基化，其中存在另一种重复序列。 v) 我们收集了 7 个三联体胎盘和 2 个四联体胎盘，以鉴定负责多态印记的序列。但是，初步实验表明胎盘不适合此目的，因此我们放弃了这个子项目。(2)反式作用因子的分离和鉴定由于上述工作意外地花费了时间，所以我们这个子项目的启动较晚，现在我们正在努力寻找消减PCR的最佳条件。