Worm Breeder's Gazette 10(3): 80
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We have been analyzing transcriptional activity in embryos by preparing extracts and assaying run-on transcription in the presence of [32P]-UTP (WBG Nov. 1987). We reported that early embryos (<30 cells) are transcriptionally active, having about 50% of the activity of later embryos. We have now further characterized these reactions with regard to linearity, developmental timing of specific gene transcripts, amanitin sensitivity, and comparative rates of incorporation in extracts from different embryonic stages. Incorporation of [32P]-UTP is linear to about 15-20 minutes and plateaus by about 60 minutes. The time course of incorporation does not differ significantly between extracts made from embryos at different stages. Amanitin-sensitive incorporation is consistently 80- 90% of the total, but this has not yet been checked for extracts of the earliest embryos analyzed (99% <30 cells). Early and late run-on transcripts (from extracts of 95% <30-cell embryos and >95%o >30-cell embryos, respectively) have been used to probe Southern blots of a collection of genes with known or predicted patterns of developmental regulation during embryogenesis. Histone, tubulin, and actin genes are already being transcribed in the early extracts, and transcription increases significantly in the late extracts. In contrast, unc-54 and col-1 are expressed in late but not in early extracts. Finally, vit-5 transcripts are not detected in either extract. Thus run-on transcription in vitro appears to follow the expected patterns of developmental regulation, at least for these genes. Further quantitation of incorporation by extracts of embryos at different stages suggests that very early embryos are as active transcriptionally as later embryos on a per nucleus basis. Extracts of early embryos (99% <30 cells) incorporate at least as much [32P]- UTP per nucleus as extracts of post-gastrulation embryos. These incorporation rates at 22 C correspond to 300-900 kb of RNA (or about 100-300 mRNA molecules) per nucleus per min. The variation in this number probably reflects the difficulty of accurately determining concentrations of nuclei rather than reflecting stage-specific variations in incorporation rates. Incorporation in these extracts was also normalized to DNA concentration, determined by fluorimetry, and again no significant stage-specific variation of incorporation rates was found. However, DNA readings were 2-3 fold higher than expected based on nuclear estimates. This is presumably due at least in part to high levels of mitochondrial DNA in embryos. Because of this discrepancy the two methods of normalization give different absolute values for [32P]-UTP incorporation/nucleus. However, by both methods early embryos are as active transcriptionally as later embryos. This conclusion appears to be at odds with those of Hecht et al. ( Dev. Biol. 83:374, 1981), who used in situ hybridization of a [3H]- poly(U) probe to squashes of embryos at different stages to estimate levels of nuclear poly(A)+ mRNA per embryo compared to total cellular poly(A)+ mRNA. These authors reported that nuclear poly(A)+ is first detectable around the 90-cell stage, suggesting that transcription of the embryonic genome begins at this time. However, their data (Fig. 3) also show that the average number of grains per nucleus stays approximately constant from the 100-cell to about the 500-cell stage, consistent with our results. Furthermore the level of about 100 poly( A)+ mRNA molecules per nucleus that can be estimated from their data during this time is roughly consistent with our incorporation levels, assuming a short time for transit of nuclear poly(A)+ molecules to the cytoplasm. Therefore, the principal discrepancy between our results and theirs is simply that we find evidence for equivalent levels of transcription in early and late embryos, whereas they do not. One possible explanation is that in our experiments, despite the evidence presented above for apparently normal control of selected transcripts, the earliest incorporation we observe in extracts is an artifact not representative of in vivo transcription. If our results do reflect the in vivo situation, then the discrepancy most probably results from the substantial differences between the two studies in what is being measured: nucleotide incorporation rates vs levels of accumulated nuclear poly(A) present, respectively. Other explanations could be, for example, that: 1) early transcription does not produce mature mRNA's, or produces only poly(A)- mRNA's; or 2) early transcription produces poly(A)+ mRNA's but their transit time to the cytoplasm is shorter than in later embryos, so that the steady state level of nuclear poly(A)+ is below the level of detection by the methods of Hecht et al. We are attempting to distinguish between the various alternatives by analyzing RNA synthesis in permeablized whole early embryos.