Komissarov A S, Kuznetsova I S, Podgornaia O I
Genetika. 2010 Sep;46(9):1217-21.
The search for all sequences containing centromeric (CEN) minor satellite (MiSat) or pericentromeric (peri-CEN) mouse major satellite (MaSat) was conducted in the whole genome shotgun (WGS) database. The sequences were checked for the presence of the known dispersed repeats using the Censor software. The presence of tandem repeats was tested using Tandem Repeat Finder (TRF). Monotonous MiSat and MaSat arrays and MaSat to MiSat array transitions were detected. Moreover, two other types of contacts were revealed: (1) MiSat transition to fragments of retroelements LINE and IAP (ERV family, intracisternal A-type particles), mainly to ORF2 and 5'-LTR containing elements; (2) MaSat transition to two tandem repeats with monomers 21 bp and 31 bp in size. The presence of the MiSat/IAP transition could be checked experimentally. The common DNA motif among the IAP fragments close to MiSat was isolated. IAP-specific primers were constructed and the fragments obtained in PCR with LAP and MiSat primers compiled the plasmid vector library. Clone n51 with the maximum length of the possible insertion (approximately no. 800 bp) was selected from the library. FISH on extended chromatin fibers (fiberFISH) carried out on the n51 clone demonstrated that the main signal definitely belonged to CEN. However, the signals on the chromosome arms were also detected that could be due to the partial homology of n51 to the dispersed repeats. The duplicated fiberFISH with MiSat and n51 allowed to measure the distances between the fragments. The previously obtained MS3 sequence has some homology to IAP and CEN localization. Accordingly, the regular associations of MiSat with IAP retroelements were shown in silico and in situ. Together with the published data, the present findings suggest that retroelements or their fragments may be essential components of the normal centromere of higher eukaryotes.
在全基因组鸟枪法(WGS)数据库中搜索包含着丝粒(CEN)小卫星(MiSat)或着丝粒周围(peri-CEN)小鼠主要卫星(MaSat)的所有序列。使用Censor软件检查这些序列中已知分散重复序列的存在情况。使用串联重复序列查找器(TRF)测试串联重复序列的存在情况。检测到单调的MiSat和MaSat阵列以及MaSat到MiSat阵列的转变。此外,还发现了另外两种类型的接触:(1)MiSat转变为逆转录元件LINE和IAP(ERV家族,脑内A 型颗粒)的片段,主要是含有ORF2和5'-LTR的元件;(2)MaSat转变为两种串联重复序列,单体大小分别为21 bp和31 bp。MiSat/IAP转变的存在可以通过实验进行检查。分离出靠近MiSat的IAP片段中的共同DNA基序。构建了IAP特异性引物,用LAP和MiSat引物进行PCR获得的片段组成了质粒载体文库。从文库中选择了插入长度可能最大(约800 bp)的克隆n51。对n51克隆进行的伸展染色质纤维荧光原位杂交(fiberFISH)表明,主要信号肯定属于着丝粒。然而,也检测到染色体臂上的信号,这可能是由于n51与分散重复序列的部分同源性所致。用MiSat和n51进行的重复fiberFISH可以测量片段之间的距离。先前获得的MS3序列与IAP有一些同源性且定位于着丝粒。因此,在计算机模拟和原位实验中均显示了MiSat与IAP逆转录元件的规则关联。与已发表的数据一起,目前的研究结果表明,逆转录元件或其片段可能是高等真核生物正常着丝粒的重要组成部分。