Pukkila P J
Chromosoma. 1975 Nov 20;53(1):71-89. doi: 10.1007/BF00329391.
The loops which transcribe 5S ribosomal RNA in lampbrush chromosomes of the newt, Notophthalmus (Triturus) viridescens, were identified by hybridizing purified 5S DNA to nascent 5S RNA in situ. The genes which code for 5S RNA were found near the centromeres of chromosomes 1, 2, 6, and 7 by hybridizing iodinated 5S RNA to denatured lampbrush and mitotic chromosomes in situ. These genes and their intervening spacer DNA were isolated from Xenopus laevis using sequential silver-cesium sulfate equilibrium centrifugations. This purified 5S DNA was iodinated and hybridized to non-denatured lampbrush chromosomes in situ, where it bound to nascent 5S RNA on loops at the base of the centromeres of chromosomes 1, 2, 6, and 7. The number of 5S genes present in the haploid chromosome complement of N. viridescens was determined. - The 5S loops were chosen for study, since (1) the synthesis of 5S RNA has been demonstrated during the lampbrush stage, (2) both 5S RNA and 5S DNA could be isolated in pure form, and (3) the localization of the repetitive 5S genes could be verified by conventional in situ hybridization procedures. These methods may be applicable to the identification of other loops, leading to a better understanding of lampbrush chromosome function.
通过将纯化的5S DNA与新生5S RNA进行原位杂交，确定了蝾螈（Notophthalmus (Triturus) viridescens）灯刷染色体中转录5S核糖体RNA的环。通过将碘化5S RNA与变性的灯刷染色体和有丝分裂染色体进行原位杂交，发现编码5S RNA的基因位于1、2、6和7号染色体的着丝粒附近。使用连续的硫酸铯平衡离心法从非洲爪蟾（Xenopus laevis）中分离出这些基因及其间隔区DNA。将这种纯化的5S DNA碘化并与未变性的灯刷染色体进行原位杂交，它在1、2、6和7号染色体着丝粒基部的环上与新生5S RNA结合。测定了绿红东美螈（N. viridescens）单倍体染色体组中5S基因的数量。选择5S环进行研究，原因如下：（1）已证实在灯刷期合成5S RNA；（2）5S RNA和5S DNA都可以以纯形式分离；（3）重复的5S基因的定位可以通过传统的原位杂交程序进行验证。这些方法可能适用于鉴定其他环，从而更好地理解灯刷染色体的功能。
