Erickson R P, Erickson J M, Betlach C J, Meistrich M L
J Exp Zool. 1980 Oct;214(1):13-9. doi: 10.1002/jez.1402140103.
Mouse testicular cells labelled in vivo with 3H-uridine for 1 hour were separated into enriched cell populations representing different stages of spermatogenesis by centrifugation in an Elutriator rotor. RNA extracted with phenol and chloroform was sized by electrophoresis on 2.4% acrylamide gels. The percentage of newly synthesized RNA which was not ribosomal RNA (or its precursors) and was not transfer RNA, was higher in early postmeiotic, as compared to late premeiotic, stages. RNA was also extracted from fractionated cells in the presence of guanidinium chloride and the proportion of total 3H-RNA-containing poly(A) sequences was determined by binding to an oligo-(dT)-cellulose column. RNA that bound in 0.5 NaCl was eluted with low salt buffer and reapplied twice more, after heating each time in DMSO to disaggregate any non-poly(A)-containing RNA. The percentage of newly synthesized RNA which contained poly(A) did not decrease in early postmeiotic, as compared to late premeiotic, stages. We suggest that at least some part of the 6-15S 3H-RNA and 3H-poly(A)-containing RNA represents mRNA(s) transcribed postmeiotically in haploid germ cells.
用³H-尿苷在体内标记1小时的小鼠睾丸细胞，通过在淘洗转子中离心，分离成代表精子发生不同阶段的富集细胞群体。用苯酚和氯仿提取的RNA在2.4%丙烯酰胺凝胶上进行电泳以确定大小。与减数分裂前后期相比，减数分裂后早期新合成的非核糖体RNA（或其前体）和非转运RNA的RNA百分比更高。还在氯化胍存在下从分级分离的细胞中提取RNA，并通过与寡聚（dT）-纤维素柱结合来确定含³H-RNA的多聚（A）序列在总RNA中的比例。在0.5 NaCl中结合的RNA用低盐缓冲液洗脱，并在每次于二甲基亚砜中加热以解离任何不含多聚（A）的RNA后再重新应用两次。与减数分裂前后期相比，减数分裂后早期含多聚（A）的新合成RNA的百分比没有下降。我们认为，6 - 15S³H-RNA和含³H-多聚（A）的RNA中至少有一部分代表单倍体生殖细胞减数分裂后转录的mRNA。
