Angerer L, Davidson N, Murphy W, Lynch D, Attardi G
Cell. 1976 Sep;9(1):81-90. doi: 10.1016/0092-8674(76)90054-4.
The 4S RNA genes in HeLa mitochondrial DNA (mtDNA) have been mapped by electron microscopy using the electron-opaque label ferritin. This method is based on the high affinity interaction between the protein, avidin,and biotin. 4S RNA, covalently coupled to biotin, was hybridized to single-stranded mtDNA. The hybrids were then labeled with ferritin-avidin conjugates. The positions of ferritin-labeled 4S RNA genes were determined relative to the rRNA genes on both heavy (H) and light (L) strands of mtDNA. This region was recognized as a duplex segment after hybridization either with rRNA in the case of H strands or with DNA complementary to rRNA in the case of L strands. Our studies suggest that at least nineteen 4S RNA genes are present in the HeLa mitochondrial genome. On the H strand, we have confirmed the nine map positions found in a previous electron microscope mapping study (Wu et al., 1972) and obtained evidence for three additional 4S RNA genes. On the L strand, seven 4S RNA genes have been mapped. The nineteen genes are distributed more or less uniformly around the genome. There is a pair of closely spaced genes, approximately 150 nucleotides apart, on the H strand, and another closely spaced pair on the L strand.
利用电子不透明标记物铁蛋白，通过电子显微镜对海拉细胞线粒体DNA（mtDNA）中的4S RNA基因进行了定位。该方法基于蛋白质抗生物素蛋白与生物素之间的高亲和力相互作用。将与生物素共价偶联的4S RNA与单链mtDNA杂交。然后用铁蛋白 - 抗生物素蛋白偶联物对杂交体进行标记。相对于mtDNA重链（H）和轻链（L）上的rRNA基因，确定了铁蛋白标记的4S RNA基因的位置。在H链的情况下，与rRNA杂交后，该区域被识别为双链片段；在L链的情况下，与rRNA的互补DNA杂交后，该区域被识别为双链片段。我们的研究表明，海拉细胞线粒体基因组中至少存在19个4S RNA基因。在H链上，我们证实了先前电子显微镜定位研究（Wu等人，1972年）中发现的9个图谱位置，并获得了另外3个4S RNA基因的证据。在L链上，已经定位了7个4S RNA基因。这19个基因在基因组周围或多或少均匀分布。在H链上有一对紧密间隔的基因，相距约150个核苷酸，在L链上还有另一对紧密间隔的基因。
