Cabrera C V, Lee J J, Ellison J W, Britten R J, Davidson E H
J Mol Biol. 1984 Mar 25;174(1):85-111. doi: 10.1016/0022-2836(84)90366-8.
Complementary DNA clones representing cytoplasmic poly(A) RNAs of sea urchin embryos were hybridized with metabolically labeled cytoplasmic RNA preparations and the rates of appearance and of decay for each transcript species were determined at the blastula-gastrula stage of development. The prevalence of the transcripts chosen for this study ranged, on average, from about one molecule per cell to a few hundred molecules per cell. The embryos were labeled continuously for 18 hours with [3H]guanosine, beginning at 24 hours post-fertilization. The amount of cytoplasmic [3H]poly(A) RNA that hybridized to each cloned sequence was determined and the specific activity of the [3H]GTP pool was measured in the same embryos. Rate constants for the entry of each transcript species into the cytoplasm, and for its decay were extracted from these data. The embryo transcript species identified by the cloned probes displayed a range of stabilities. Half-lives of only a few hours were measured both for a very rare sequence and for a moderately prevalent sequence. Other newly synthesized transcripts, including sequences that first appear during embryonic development, as well as sequences also represented in maternal RNA, are far more stable. We conclude that cytoplasmic RNA turnover rate is a major variable in the determination of the cytoplasmic level of expression of embryo genes. The entry rates of the transcripts into the cytoplasm also varied, from a few molecules per embryo per minute to several hundred, depending on the sequence. By comparing the mass of transcripts of a given sequence in the embryo to the mass of transcripts of that sequence accumulating as a result of new synthesis, the point at which embryo transcription accounts for the major fraction of the cytoplasmic molecules could be estimated. This calculation showed that for some sequences maternal transcripts persist well beyond gastrulation, while other embryo poly(A) RNA species are largely the product of transcription in the embryo nuclei from the blastula stage onwards. There is no single stage at which all maternal transcripts are suddenly replaced by newly synthesized embryo transcripts. Primary transcription rates were measured for two sequences by determining accumulation of label in these RNA species soon after addition of [3H]guanosine to the cultures. Comparing these rates to the cytoplasmic entry rates, we did not detect a significantly greater nuclear transcription of the sequence homologous to the cloned probe.
将代表海胆胚胎细胞质多聚腺苷酸化RNA的互补DNA克隆与经代谢标记的细胞质RNA制剂进行杂交，并在囊胚-原肠胚发育阶段测定每个转录本种类的出现率和衰减率。本研究中所选转录本的丰度平均范围为每个细胞约一个分子到每个细胞几百个分子。从受精后24小时开始，胚胎用[3H]鸟苷连续标记18小时。测定与每个克隆序列杂交的细胞质[3H]多聚腺苷酸化RNA的量，并在同一胚胎中测量[3H]GTP库的比活性。从这些数据中提取每个转录本种类进入细胞质的速率常数及其衰减速率常数。由克隆探针鉴定的胚胎转录本种类表现出一系列稳定性。对于一个非常罕见的序列和一个中等丰度的序列，半衰期仅为几个小时。其他新合成的转录本，包括在胚胎发育过程中首次出现的序列以及母源RNA中也存在的序列，稳定性要高得多。我们得出结论，细胞质RNA周转率是决定胚胎基因细胞质表达水平的一个主要变量。转录本进入细胞质的速率也有所不同，根据序列不同，从每个胚胎每分钟几个分子到几百个分子不等。通过比较胚胎中给定序列的转录本质量与由于新合成而积累的该序列转录本质量，可以估计胚胎转录占细胞质分子主要部分的时间点。该计算表明，对于某些序列，母源转录本在原肠胚形成后仍持续存在，而其他胚胎多聚腺苷酸化RNA种类在很大程度上是从囊胚阶段开始胚胎细胞核中转录的产物。不存在所有母源转录本突然被新合成的胚胎转录本取代的单一阶段。通过在向培养物中添加[3H]鸟苷后不久测定这些RNA种类中标记的积累，测量了两个序列的初级转录速率。将这些速率与细胞质进入速率进行比较，我们没有检测到与克隆探针同源的序列在核转录方面有明显更高的水平。
