Madalena C R G, Gorab E
Instituto de Biociências, Universidade de São Paulo, São Paulo, Brazil.
Insect Mol Biol. 2005 Jun;14(3):255-62. doi: 10.1111/j.1365-2583.2005.00554.x.
The characterization of chromosome end sequences of Rhynchosciara americana was initiated with the screening of a plasmid microlibrary made from a microdissected polytene chromosome tip. A 268 bp insert chosen for analysis hybridized specifically to non-telocentric chromosome ends in which reverse transcriptase had been previously identified. Southern-blot hybridization of R. americana genomic DNA cut with XbaI, the only restriction site identified in the 268 bp probe, showed a ladder composed of multimers of a band in the range of 400 bp indicating a tandem array of the repeat partially represented in the cloned fragment. The complete repeat unit obtained by inverse PCR is 414 bp long, 67% AT-rich, characterized by the restriction sites XbaI and SalI and displays features typical of a nematoceran telomeric satellite. The telomere-like repeat is apparently absent from the chromosomes of two other Rhynchosciara species, R. baschanti and R. milleri. Double staining for satellite hybridization and reverse transcriptase in R. americana suggests that the arrays composed of the telomeric-like satellite do not reach the chromosome ends. This may indicate that telomeric-like features of some nematoceran terminal satellites do not warrant their telomeric position.
美洲大蚊染色体末端序列的特征分析始于对一个由显微切割的多线染色体末端构建的质粒微文库的筛选。选择用于分析的一个268 bp插入片段与非端着丝粒染色体末端特异性杂交,此前在这些末端已鉴定出逆转录酶。用XbaI(在268 bp探针中鉴定出的唯一限制性酶切位点)切割美洲大蚊基因组DNA后的Southern杂交显示,有一个由400 bp左右条带的多聚体组成的梯状条带,表明克隆片段中部分代表的重复序列呈串联排列。通过反向PCR获得的完整重复单元长414 bp,富含67%的AT,以限制性酶切位点XbaI和SalI为特征,具有蚋科端粒卫星的典型特征。另外两种美洲大蚊物种,即巴氏美洲大蚊和米勒美洲大蚊的染色体上显然没有类似端粒的重复序列。美洲大蚊中卫星杂交和逆转录酶的双重染色表明,由类似端粒卫星组成的阵列并未延伸至染色体末端。这可能表明,一些蚋科末端卫星的类似端粒特征并不意味着它们处于端粒位置。