著絲點蛋白H 是位於著絲點Kinetochore 內層的結構性蛋白。先前的研究顯
示著絲點蛋白H 有助於著絲點蛋白A 和C 聚集在著絲點上以形成有活性著絲
點。然而，著絲點蛋白H 如何幫助著絲點蛋白A 和C 聚集在著絲點上是值得探
討的課題。我們假設著絲點蛋白H 是藉由連結一特定的著絲點DNA 而聚集著絲
點蛋白A 和C 在著絲點上。為了研究著絲點蛋白H 連結的著絲點DNA，我們利
用抗著絲點蛋白H 抗體所抓取的染色質絲(也就是染色質絲免疫沉澱法)建立了
一個小型染色質絲資料庫，叫做CHIP-H。我們利用細胞外蛋白質-DNA 鍵結分
析法篩選小型染色質絲資料庫CHIP-H 菌株，其中有五個菌株的DNA 出現較強
的連結訊號，為了更加確立菌株DNA 在細胞內著絲點蛋白H 的連結能力，我們
將這五個菌株的DNA 送入細胞中，培養48 小時，之後將細胞固定，用免疫螢
光染色及原位螢光雜交分析，菌株DNA 和細胞內著絲點蛋白連結的能力，結果
顯示這五個菌株DNA 確實具有與著絲點蛋白連結的能力。我們再次利用膠體滯
留結合西方吸漬法更加證實這五個菌株DNA 具有著絲點蛋白H連結的能力。 除
此之外，我們更進一步探討著絲點蛋白H 連結DNA 在整體基因組中的關係，因
此我們利用篩選到的菌株DNA 和已知的著絲點蛋白A 連結之特定的著絲點
DNA 當作探針篩選BAC DNAs，結果有三個BAC DNAs 同時具有著絲點蛋白A
連結之特定的著絲點DNA 和著絲點蛋白H 連結之特定的著絲點DNA 的訊號。
接下來，我們利用有系統地定序法定序這些候選的BAC DNAs，雖然這些候選
的BAC DNAs 含有大量的重複性DNA，比一般的定序需要耗費更多的時間，困
難度也相當大。不過目前我們已完成定序這些候選BAC DNAs 的限制酶片段，
這些片段的序列也做了相互的比對及和基因資料庫的比對，有趣的是，著絲點蛋
白A 連結之特定的著絲點DNA 和著絲點蛋白H 連結之特定的著絲點DNA 都和
先前找到的著絲點衛星DNA 有80%以上的相似度，這樣的證據顯示，著絲點蛋
白H 是藉由連結上著絲點DNA，再幫助著絲點蛋白A 和C 聚集在著絲點DNA
上以形成有活性著絲點。所以我們已完成這一計畫所要探討的主要目的了。 另
外，在執行計畫的三年中，我們已有5 篇論文發表，兩篇已被接受，一篇已送出
接受審查，兩篇論文正在撰寫中。
Centromere protein H (CENP-H) is a constituted protein and located within the
inner layer of Kinetochore. The previous studies showed that the CENP-H
contributes gathering the CENP-A and -C together at the centromere to form active
centromere. However, it is not clear how the CENP-H to help the CENP-A and -C
gather at the centromere. We assumed that the CENP-H is associated with a specific
centromeric DNA to recruit the CENP- A and C on the centromere. To study the
CENP-H associating DNA, we used the anti-CENP- H antibodies to catch down the
CENP-H associating chromatin filament (that is, the chromatin immuno-precipitation)
and then constructed a mini-library, designated as CHIP-H. We obtained five
positive clones by screening this mini-library with multi-well protein-DNA binding
assay. Ex-vivo protein assay and gel-retardation combing western blot assay showed
that the isolated clones have CENP-H binding activity. Furthermore, we screened
the centromeric BAC DNA using these five clones and CENP-A associating DNA as
probes for characterizing the genomic organization of CENP-H associating DNA.
Three positive BAC clones obtained were further sequencing. Because the BAC
DNA clones contain the significant repetitive DNA, it is not easy to complete
sequence BAC DNA by shot-gun sequencing or second-generation sequencing.
Even so, we sequenced three BAC DNAs by the systemically hierarchical sequencing.
We had finished all sequences of subclones. We had compared all sequences of
subclones each other and compared them with the nucleotides collected in GeneBank
of NCBI. The sequences comparison result showed that the CENP-H associating
DNA and CENP-A associating DNA shared more 80% identity to centromeric
satellite II. These evidences indicated that the CENP-H recruited CENP-A and C
through associating centromeric satellite II to form an active centromere. Therefore,
we had achieved the main purpose what we want to explore in this project.
During the term of carrying out this project, we had 5 papers published, two papers
accepted in press, one paper submitted and two papers in preparation.
