Mizuno S, Andrews C, Macgregor H C
Chromosoma. 1976 Oct 12;58(1):1-31. doi: 10.1007/BF00293437.
Intermediate repetitive sequences of Plethodon cinereus which comprised about 30% of the genomic DNA were isolated and iodinated with 125I. About 5% of the 125I-repetitive fraction hybridized with a large excess of DNA from P. dunni at Cot 20. About half of the 125I-DNA in the hybrids was resistant to extensive digestion with S-1 nuclease. The average molecular size of the S-1 nuclease-resistant fraction was about 100 nucleotide pairs. The melting temperature of the S-1 nuclease-resistant fraction was about 2 degrees lower than that of the corresponding fraction made with P. cinereus DNA. These results are taken to indicate the presence in the genomes of P. cinereus and P. dunni of evolutionarily stable "common" repetitive sequences. The average frequency of repetition of the common repetitive sequences is about 6,000 X in both species. The common repetitive fraction is also present in the genomes of other species of Plethodon, although the general populations of intermediate repetitive sequences are markedly different from one species to another. The cinereus--dunni common repetitive sequences could not be detected in plethodontids belonging to different tribes, nor in more distantly related amphibians. The profiles of binding of the common repetitive sequences to CsCl or CS2SO4-Ag+ density gradient fractions of P. dunni DNA suggested that these sequences consisted of heterogeneous components with respect to base compositions, and that they did not include large amounts of the genes for ribosomal RNA, 5S RNA, 4S RNA, or histone messenger RNA. In situ hybridization of the 3H-labelled intermediate repetitive sequences of P. cinereus to male meiotic chromosomes of the same species gave autoradiographs after an exposure of seven days showing all 14 chromosomes labelled. The pattern of labelling appeared not to be random, but was impossible to analyse on account of the irregular shapes and different degrees of stretching of diplotene and prometaphase chromosomes. In situ hybridization of the same sequences to meiotic chromosomes from P. dunni gave autoradiographs after 60 d exposure in which all chromosomes were labelled. These heterologous in situ hybrids can only have involved the "common" repetitive sequences.
分离出占灰林螈基因组DNA约30%的中度重复序列,并用¹²⁵I进行碘化。在Cot 20时,约5%的¹²⁵I重复片段与大量邓氏林螈的DNA杂交。杂交体中约一半的¹²⁵I - DNA对S - 1核酸酶的广泛消化具有抗性。S - 1核酸酶抗性片段的平均分子大小约为100个核苷酸对。S - 1核酸酶抗性片段的解链温度比用灰林螈DNA制备的相应片段低约2摄氏度。这些结果表明,在灰林螈和邓氏林螈的基因组中存在进化上稳定的“共同”重复序列。在这两个物种中,共同重复序列的平均重复频率约为6000X。共同重复片段也存在于其他林螈物种的基因组中,尽管中度重复序列的总体情况在不同物种之间明显不同。在属于不同族的无肺螈科动物中,以及在亲缘关系更远的两栖动物中,未检测到灰林螈 - 邓氏林螈的共同重复序列。共同重复序列与邓氏林螈DNA的CsCl或Cs₂SO₄ - Ag⁺密度梯度组分的结合图谱表明,这些序列在碱基组成方面由异质成分组成,并且它们不包括大量的核糖体RNA、5S RNA、4S RNA或组蛋白信使RNA基因。用³H标记的灰林螈中度重复序列与同一物种的雄性减数分裂染色体进行原位杂交,曝光7天后得到放射自显影片,显示所有14条染色体均被标记。标记模式似乎不是随机的,但由于双线期和前中期染色体形状不规则且伸展程度不同,无法进行分析。将相同序列与邓氏林螈的减数分裂染色体进行原位杂交,曝光60天后得到放射自显影片,其中所有染色体均被标记。这些异源原位杂交只能涉及“共同”重复序列。