Development of molecular probes for genome-wide genotyping by restriction landmark genomic scanning (RLGS) method.

利用冷冻保存精液的人工授精技术的最新进展大大提高了经济上优良的上级家畜的产量。然而，具有讽刺意味的是，它帮助神秘的遗传疾病以意想不到的速度在世界范围内传播。因此，需要建立一种有效的全基因组筛查方法来检测家畜的遗传异常。为此，目的是开发一种通过限制性标志基因组扫描（RLGS）方法识别基因组DNA结构变化的系统。在RLGS中，基因组DNA被两种或多种不同种类的限制性内切酶消化，并通过二维凝胶电泳（2D/E）分离。因此，它是可能的，灵敏地确定全球基因组的变化，如点突变的限制性内切酶的识别位点，缺失，易位等。RLGS也应该是有用的，以建立一个特定的有价值的表型和一组多个genes.In这项研究中，我们分析了大鼠基因组DNA的CpG岛使用NotI，甲基化敏感酶RLGS。结果，我们发现大约1%的“RLGS斑点”显示组织特异性分布，表明这些斑点是组织特异性甲基化/去甲基化的。在这些斑点中，我们随后可以克隆几个基因组片段，这些片段似乎在胎盘中脱汞，但在肾脏中没有。对分离的克隆进行序列分析，发现其中一个克隆编码线粒体C3转运蛋白基因，进一步分析发现该基因在胎盘中表达较高，而在肾脏中表达较低。这些结果表明，RLGS是一种有效的方法，鉴定和分离基因组DNA片段的基因组扫描的遗传变化。

Recent advance in the artificial insemination technique by using frozen-preserved semen greatly enhanced a production of economically superior livestock animals. It, however, ironically helped cryptic genetic disorders to spread world wide in an unexpected speed. Thus, it has been needed to establish effective way of genome-wide screening for genetic abnormalities in livestock animals. To this end, it was aimed to develop a system to identify changes in a structure of genomic DNA by a restriction landmark genomic scanning (RLGS) method. In the RLGS, genomic DNA is digested by two or more different kind of restriction enzymes, and separated by the 2-dimensional gel electrophoresis (2D/E). It is, therefore, possible to sensitively identify global genomic changes such as point mutations at the recognition sites of restriction enzymes, deletions, translocations, etc. RLGS should be also useful to establish a linkages between a certain valuable phenotype and a set of multiple genes.In this study, we analyzed CpG islands of rat genomic DNA by RLGS using NotI, a methylation sensitive enzyme. As a result, we found approximately 1% of the "RLGS spots" show tissue-specific distribution, suggesting that these spots are tissue-specifically methylated/demethylated. Of these spots, we could subsequently clone several genomic fragments which appeared to be demerhylated in placenta but not in kidney. Sequence analysis of isolated clones revealed that one of the clones encodes mitochondrial C3 transporter gene which appeared to be relatively highly expressed in the placenta but low in kidney in further analysis. These results showed that the RLGS is an effective way of identification and isolation of genomic DNA fragments by genome-wide scanning for the genetic changes.