Grierson D, Hemleben V
Biochim Biophys Acta. 1977 Apr 4;475(3):424-36. doi: 10.1016/0005-2787(77)90058-2.
The percentage of DNA from the crucifer Matthiola incana coding for different types of RNA was measured by filter saturation hybridisation experiments using RNA labelled in vivo. In addition, the melting curves of the various DNA - RNA hybrids formed and the buoyant densities of the DNA sequences complementary to different types of RNA were measured. 1. The RNA preparations used were 25, 18, and 5 S rRNA and 4 S RNA, purified by gel electrophoresis, and poly(A)-containing RNA purified by oligo-(dT)-cellulose chromatography. The molecular weights of the 25 S and 18 S rRNAs, calculated from the mobility in formamide-acrylamide gels relative to Escherichia coli RNA, are 1.25 - 10(6) and 0.64 - 10(6). The rRNA precursor has a molecular weight of approx. 2.1 - 10(6) and the average molecular weight of the poly(A)-containing RNA from both cotyledons and roots is 4 - 10(5). 2. The percentage of the genome, calculated on the basis of double-stranded DNA, coding for these RNAs and the estimated number of genes per haploid DNA amount are approximately 0.46% and 1100 for 25 S plus 18 S rRNA, 0.032% and 3600 for 5 S rRNA and 0.072% and 13 000 for 4 S RNA. In filter hybridisation experiments very little hybridisation of poly(A)-containing RNA was found. A rapidly-hybridising component is attributed to small amounts of contaminating rRNA. 3. M. incana DNA has a main band at 1.697 g - ml-1 in CsCl and a satellite constituting approximately 3% of the DNA, at 1.708 g - ml-1 - 25 and 18 S rRNA hybridise to DNA with a buoyant density of 1.701--2 g - ml-1. The buoyant density of 5 S DNA is slightly less at 1.700--1 g - ml-1. 4. S RNA hybridises to at least two separate regions, one within the main-band DNA and a second lighter component. None of the RNAs tested hybridised to the satellite DNA. The Tm of the DNA - RNA hybrids in 1 X SSC is 89 degrees C for 25 S rRNA, 85 degrees C for 5 S rRNA and 82 degrees C for 4 S RNA. 4. 5 and 4 S RNA preparations contain fragments which hybridise to sequences complementary to high-molecular-weight rRNA. This spurious hybridisation can be eliminated by competition with unlabelled high-molecular-weight RNA.
采用体内标记RNA的滤膜饱和杂交实验,测定了十字花科植物紫罗兰(Matthiola incana)中编码不同类型RNA的DNA所占的百分比。此外,还测定了所形成的各种DNA-RNA杂交体的解链曲线,以及与不同类型RNA互补的DNA序列的浮力密度。1.所用的RNA制剂有25S、18S和5S rRNA以及4S RNA,通过凝胶电泳进行纯化,含poly(A)的RNA则通过寡聚(dT)-纤维素柱层析进行纯化。根据相对于大肠杆菌RNA在甲酰胺-丙烯酰胺凝胶中的迁移率计算,25S和18S rRNA的分子量分别为1.25×10⁶和0.64×10⁶。rRNA前体的分子量约为2.1×10⁶,来自子叶和根的含poly(A)的RNA的平均分子量为4×10⁵。2.以双链DNA为基础计算,编码这些RNA的基因组百分比以及每单倍体DNA量中估计的基因数,对于25S加18S rRNA分别约为0.46%和1100,对于5S rRNA为0.032%和3600,对于4S RNA为0.072%和13000。在滤膜杂交实验中,发现含poly(A)的RNA很少杂交。一个快速杂交的组分归因于少量污染的rRNA。3.紫罗兰DNA在CsCl中的主峰带密度为1.697 g·ml⁻¹,还有一个约占DNA 3%的卫星带,密度为1.708 g·ml⁻¹。25S和18S rRNA与浮力密度为1.701 - 1.702 g·ml⁻¹的DNA杂交。5S DNA的浮力密度略低,为1.700 - 1.701 g·ml⁻¹。4.4S RNA与至少两个不同区域杂交,一个在主带DNA内,另一个是较轻的组分。所测试的RNA均未与卫星DNA杂交。在1×SSC中,DNA-RNA杂交体的解链温度(Tm)对于25S rRNA为89℃,对于5S rRNA为85℃,对于4S RNA为82℃。5.5S和4S RNA制剂中含有与高分子量rRNA互补序列杂交的片段。这种假杂交可以通过与未标记的高分子量RNA竞争来消除。