Midgley J E
Biochem J. 1969 Nov;115(2):171-81. doi: 10.1042/bj1150171.
Bacillus subtilis 168 messenger RNA was determined by DNA-RNA hybridization techniques, with denatured DNA immobilized upon cellulose nitrate membrane filters. The following results were obtained. (1) Cultures of B. subtilis, growing exponentially in enriched glucose-salts medium at 37 degrees , incorporated [5-(3)H]uracil into both ribosomal and messenger RNA fractions without the kinetic delay expected from the presence of the intracellular nucleotide pools. (2) However short the time of labelling with exogenous labelled uracil (down to 7sec.), 32-36% of the rapidly labelled RNA was messenger RNA and 68-64% was an RNA with the hybridization characteristics of ribosomal RNA. Analysis of the apparent nucleotide base composition of total (32)P-labelled rapidly labelled RNA and the two RNA fractions separated by hybridization at a DNA/RNA ratio 5:1 confirmed this finding. Of the rapidly labelled RNA, 31% readily hybridized with DNA at low DNA/RNA ratios and had an apparent base composition like that of the DNA, whereas 69% was hybridized only at low efficiency at low DNA/RNA ratios and had a composition identical with that of ribosomal RNA. (3) In cultures dividing every 48min. at 37 degrees , kinetic analysis of RNA labelled over a 20min. period showed that the average life-time of messenger RNA was 2.7-3.0min. and that its amount was 3.0% of the total RNA. (4) The hybridization of (3)H-labelled randomly labelled RNA with DNA at a DNA/RNA ratio 5:1 showed that 2.9% of the randomly labelled RNA had the characteristics of messenger RNA. (5) Experiments carried out as described by Pigott & Midgley (1968) indicated that hybridization at low DNA/RNA ratios (5:1) effectively accounted for all the messenger RNA in a given specimen. The efficiency coefficient of RNA hybridization lay within the range of 90-95% input, if an excess of DNA sites was offered for RNA binding. (6) These measurements are compared with other results obtained by different methods, and reasons for any major disagreement are suggested.
利用DNA - RNA杂交技术，以固定在硝酸纤维素膜滤器上的变性DNA为材料，测定了枯草芽孢杆菌168的信使RNA。得到了以下结果。（1）枯草芽孢杆菌培养物在37℃的富含葡萄糖 - 盐的培养基中呈指数生长，将[5 - （³H）]尿嘧啶掺入核糖体RNA和信使RNA组分中，没有出现因细胞内核苷酸池的存在而预期的动力学延迟。（2）无论用外源标记尿嘧啶标记的时间有多短（短至7秒），快速标记的RNA中32 - 36%是信使RNA，68 - 64%是具有核糖体RNA杂交特性的RNA。对以5:1的DNA/RNA比例通过杂交分离的总³²P标记的快速标记RNA和两个RNA组分的表观核苷酸碱基组成进行分析，证实了这一发现。在快速标记的RNA中，31%在低DNA/RNA比例下能与DNA快速杂交，其表观碱基组成与DNA相似，而69%在低DNA/RNA比例下仅能低效杂交，其组成与核糖体RNA相同。（3）在37℃下每48分钟分裂一次的培养物中，对20分钟内标记的RNA进行动力学分析表明，信使RNA的平均寿命为2.7 - 3.0分钟，其含量占总RNA的3.0%。（4）³H标记的随机标记RNA以5:1的DNA/RNA比例与DNA杂交表明，2.9%的随机标记RNA具有信使RNA的特性。（5）按照皮戈特和米德格利（1968年）所述进行的实验表明，在低DNA/RNA比例（5:1）下杂交能有效地检测出给定标本中的所有信使RNA。如果提供过量的DNA位点用于RNA结合，RNA杂交的效率系数在输入量的90 - 95%范围内。（6）将这些测量结果与通过不同方法获得的其他结果进行了比较，并提出了出现任何重大差异的原因。
