Wang Yongping, Xu Zhe, Pierce James C, Guo Ximing
Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, 6959 Miller Avenue, Port Norris, NJ, 08349, USA.
Mar Biotechnol (NY). 2005 May-Jun;7(3):207-14. doi: 10.1007/s10126-004-0051-y. Epub 2005 Jun 8.
Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.
染色体识别是基因组研究中的关键步骤，而迄今为止在牡蛎中尚无法实现这一点。我们利用荧光原位杂交（FISH）技术，检测了用于美洲牡蛎（Crassostrea virginica）染色体识别的噬菌体P1克隆。通过缺口平移法，用洋地黄毒苷-11-dUTP标记P1克隆。用异硫氰酸荧光素标记的抗洋地黄毒苷抗体检测杂交情况，并通过两层抗体进行放大。当使用Cot-1 DNA作为针对重复序列的封闭剂时，所检测的21个P1克隆中有9个产生了清晰且一致的FISH信号。核型分析和共杂交将这9个P1克隆明确地定位到了7条染色体上。其余3条染色体可通过大小和臂比进行区分。对9个P1克隆中的5个进行了两端测序，提供了可用于整合连锁图谱和细胞遗传图谱的序列标签位点。其中一个序列是骨形态发生蛋白1b型受体的一部分，该受体属于转化生长因子超家族，定位于2号染色体长臂的端粒区域。这项研究表明，诸如P1这样的大插入片段克隆作为染色体特异性FISH探针以及用于牡蛎基因定位是有用的。
