Bendich A J, Taylor W C
Departments of Botany and Genetics, University of Washington, Seattle, Washington 98195.
Plant Physiol. 1977 Apr;59(4):604-9. doi: 10.1104/pp.59.4.604.
Two fractions of a satellite DNA from the muskmelon (Cucumis melo L.) isolated as a unimodal peak from CsCl gradients, differ in melting properties and complexity as estimated by reassociation kinetics. At 49.8 C, all of the low melting fraction was denatured and all of the high melting fraction was native. There were almost no partially denatured molecules detected in the electron microscope at this temperature. This observation provides direct evidence that the two fractions are not closely linked. We conclude that satellite I, the high t(m), low complexity fraction, exists as a 600-nucleotide sequence in blocks of at least 67 tandem repeats. Since the complexity of the low melting fraction, satellite II, is greater than the size of the molecules in our assay, we can only say that the minimum size of each unit of satellite II is 2.5 x 10(7) daltons. It is unlikely that any spacer sequences are interspersed with either satellite.Sequences homologous to those of satellite I were also shown to be present as a minor fraction on 4900 nucleotide pair fragments with main band DNA density. These long main band fragments probably contain in addition at least two repeated sequence elements unrelated to satellite I since they aggregate (form large network structures) when reassociated. Coaggregation of sheared (3)H-satellite I with long main band DNA could not be attributed to contamination of main band with long satellite DNA. We interpret the results as an observation of a recently created family of tandemly repeating sequences whose members are beginning to be scattered throughout the genome.We discuss how the aggregation technique may be generally useful for assessing linkage between a minor and a major DNA fraction when both fractions may be present in the initial DNA preparation. Applications for the technique include the search for DNA sequences in the nucleus which are homologous with chloroplast DNA and for Agrobacterium tumefaciens DNA in the nuclei of plant cells transformed to the tumor phenotype by the bacterium.
从甜瓜(Cucumis melo L.)中分离出的卫星DNA的两个组分,在CsCl梯度中表现为单峰,通过复性动力学估计,它们在解链特性和复杂度上存在差异。在49.8℃时,所有低熔点组分都已变性,而所有高熔点组分仍保持天然状态。在此温度下,电子显微镜几乎未检测到部分变性的分子。这一观察结果直接证明这两个组分并非紧密相连。我们得出结论,卫星I,即高熔解温度(t(m))、低复杂度的组分,以至少67个串联重复的块形式存在,每个块包含600个核苷酸序列。由于低熔点组分卫星II的复杂度大于我们实验中分子的大小,我们只能说卫星II每个单元的最小大小为2.5×10⁷道尔顿。不太可能有任何间隔序列穿插在任何一个卫星中。与卫星I序列同源的序列也被证明以次要组分形式存在于具有主带DNA密度的4900核苷酸对片段上。这些长的主带片段可能还至少包含两个与卫星I无关的重复序列元件,因为它们在复性时会聚集(形成大的网络结构)。剪切后的(³H)卫星I与长的主带DNA的共聚集不能归因于主带被长卫星DNA污染。我们将这些结果解释为对一个最近产生的串联重复序列家族的观察,其成员开始散布于整个基因组中。我们讨论了聚集技术在评估次要和主要DNA组分之间的连锁关系时可能具有的普遍用途,前提是这两个组分可能都存在于初始DNA制备物中。该技术的应用包括在细胞核中寻找与叶绿体DNA同源的DNA序列,以及在被细菌转化为肿瘤表型的植物细胞核中寻找根癌土壤杆菌DNA。