Murakami M, Fujitani H
Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Kanagawa, Japan.
Genes Genet Syst. 1998 Feb;73(1):9-20. doi: 10.1266/ggs.73.9.
Repetitive DNA sequences (Hi-b; 209 bp in length) were isolated from the HindIII digests of the genomic DNA of the triploid ginbuna, Carassius auratus langsdorfi. Sequence analyses revealed that the Hi-b repetitive units were comprised of the complete coding regions of 5S rDNA (120 bp in size) and their 5'flanking regions. The sequences of the Hi-b units from the same individual were highly homogeneous. Southern blot hybridization to the Hi-b probe displayed intricate patterns that represented the presence of other repetitive units containing the Hi-b related sequences. A major family of repetitive sequences related to the Hi-b was then obtained by the polymerase chain reaction using asymmetry primers for the 5S coding regions. These 331-bp sequences (AZ5S's) contained 5S pseudogenes as well as the almost entire Hi-b sequences, and seemed to be the true 5S rDNAs. The tandem arrangements of the AZ5S sequences explained most of the complex results of Southern blots. Another class of intriguing repeat units (Hi-b-beta and Hi-b-gamma) were also isolated. Fluorescence in situ hybridization data revealed two major signals on a pair of homologous chromosomes and several minor signals on other chromosomes in the triploid ginbuna, indicating the existence of the 5S related sequences as several separate clusters. The major spots were shared with the tetraploid ginbuna and goldfish, but not with the diploid ginbuna. When the genomic organization of the Hi-b related sequences in other cyprinid fishes was examined, the hybridization patterns of the ginbuna were very similar to those of the goldfish, but were clearly different from those of the gengorobuna. The carp genome showed less complex patterns. Thus, the present 5S rDNA-related sequences could be candidates for phylogenetic molecular markers for the crucian carp.
从三倍体银鲫（Carassius auratus langsdorfi）基因组DNA的HindIII酶切片段中分离出重复DNA序列（Hi-b；长度为209 bp）。序列分析表明，Hi-b重复单元由5S rDNA的完整编码区（大小为120 bp）及其5'侧翼区组成。来自同一个体的Hi-b单元序列高度同源。用Hi-b探针进行Southern杂交显示出复杂的图谱，表明存在其他含有Hi-b相关序列的重复单元。然后，使用针对5S编码区的不对称引物通过聚合酶链反应获得了与Hi-b相关的一个主要重复序列家族。这些331 bp的序列（AZ5S）包含5S假基因以及几乎完整的Hi-b序列，似乎是真正的5S rDNA。AZ5S序列的串联排列解释了Southern杂交的大部分复杂结果。还分离出了另一类有趣的重复单元（Hi-b-beta和Hi-b-gamma）。荧光原位杂交数据显示，在三倍体银鲫的一对同源染色体上有两个主要信号，在其他染色体上有几个次要信号，表明5S相关序列以几个独立的簇存在。主要位点在四倍体银鲫和金鱼中共享，但在二倍体银鲫中没有。当检查其他鲤科鱼类中Hi-b相关序列的基因组组织时，银鲫的杂交模式与金鱼非常相似，但与日本白鲫明显不同。鲤鱼基因组的模式较不复杂。因此，目前的5S rDNA相关序列可能是鲫鱼系统发育分子标记的候选者。
