Expression of exogenously introduced bacterial chloramphenicol acetyltransferase genes in Xenopus laevis embryos before the midblastula transition

Abstract

Previous papers have reported that DNAs exogenously injected into Xenopus laevis fertilized eggs are expressed only at and after the midblastula transition (MBT). We have injected fertilized eggs of Xenopus laevis with circular plasmids that contained bacterial chloramphenicol acetyltransferase (CAT) genes connected to the promoter of viral genes (pSV2CAT and pAd12.E1aCAT) or the Xenopus cardiac actin gene (actin-CAT fusion gene), and examined whether these DNAs are expressed during the stage before the MBT. We found that expression of CAT enzyme can be detected before the MBT when CAT genes connected to viral promoters were injected. The activity was low during the cleavage stage on a per-embryo basis; however, the time course of the accumulation of the CAT enzyme activity roughly paralleled the increase in cell number. Therefore, CAT enzyme activity per cell was constant during cleavage and did not change dramatically before and after the MBT stage. CAT mRNA level, detected by CAT antisense RNA, was roughly proportional to the levels of CAT enzyme. Therefore, we assume that the observed enzyme activity reflects the transcriptional activity of injected CAT genes before and after the MBT. When the actin-CAT fusion gene was injected, however, no enzyme activity was detected until embryos had reached the neurula stage, a stage when endogenous actin genes are first activated. On the basis of these results, we conclude that the concept of an initial transcriptional activation of exogenous genes at amphibian MBT has to be changed. We propose that the expression of polymerase-II-transcribed genes is regulated by the nature of the promoters connected to the genes rather than by changes associated with MBT.

Developmental acquisition of enhancer function requires a unique coactivator activity

“…low but constant rate of gene expression per cell prior to the MBT (Shiokawa et al, 1990). Activation of transcription at CAT assay the MBT can require specific enhancers (Krieg and Melton, About 50 injected embryos were incubated for 44 h, harvested in 1987), analogous to the need for an enhancer to activate 250 mM Tris pH 8.0 at a concentration of 0.5 embryo/µl, lysed by freeze-thawing three times in dry ice/ethanol and 37°C baths, centrifuged promoters in two-cell mouse embryos.…”

A unique role for enhancers is revealed during early mouse development

“…Thus, the S phase of a 2-cell mouse embryo appears equivalent to the 6th cleavage stage in Xenopus, where synthesis of heterogeneous, non-ribosomal mRNA is first detected, and the GZ phase of a 2-cell mouse embryo appears equivalent to the 12th cleavage stage in Xenopus where the major onset of RNA polymerase II and Ill transcription occurs [the midblastula transition (MBT)] (74,75). The activity of promoter/ enhancer sequences injected into Xenopus eggs is generally delayed until the MBT, although they appear to exhibit a low but constant rate of gene expression per cell prior to the MBT (76). Activation of transcription at the MBT can require specific enhancers (77), analogous to the need for an enhancer to activate promoters in 2-cell mouse embryos.…”

Maternally-preset program of apoptosis and caspases involved in execution of the apoptosis at midblastula transition (MBT) but not before in <i>Xenopus laevis</i> embryogenesis

“…As the cytologycal manifestation of the rRNA synthesis, nucleoli start to be formed from the MBT stage [32]. The transcriptional activation during the cleavage stage (pre-MBT stage) is also proved for transcription from exogenously-introduced genes like bacterial CAT (chloramphenicole acetyltransferase) genes [33], although this was once also reported and accepted to be expressed only at and after MBT, but not before [34]. The elongation of the cell cycle after the MBT which is due to the appearance of G 1 and G 2 phases may be regulated by the activation of Xchk1 kinase [28], which is essential in remodelling the cell cycle after MBT [28,35,36].…”