Notes: [Auszug] The induction of neural differentiation was assayed by the expression of N-CAM RNA, a pan-neural marker (neurons and glia), or of NF-M (neurofilament-M) RNA, a pan-neuronal marker (neurons only), in injected animal caps explanted at early gastrula stage and cultured until siblings reached the ...

Notes: [Auszug] Cell signalling through Frizzled receptors has evolved to considerable complexity within the metazoans. The Frizzled-dependent signalling cascade comprises several branches, whose differential activation depends on specific Wnt ligands, Frizzled receptor isoforms and the cellular context. In ...

Notes: Summary An expression vector was constructed containing the entire bovine papilloma virus (BPV-1) genome and part of the a-actin gene of Xenopus laevis cloned in the antisense orientation into the neomycin resistance gene under the control of the herpes simplex virus (HSV) thymidine kinase (TK) promoter. When this vector is microinjected into X. laevis embryos it replicates extrachromosomally, at least up to the tadpole stage, and a fusion RNA is synthesized after the mid blastula transition (MBT). The expression of the antisense gene results in a morphological abnormality of somites demonstrating that antisense RNA generated by an episomal replicating expression vector can inhibit the expression of a selected gene during early embryogenesis of X. laevis.

Notes: Abstract DNA sequencing and subsequent functional in vitro analysis of the Xenopus laevis rDNA transcription termination has led to the identification of three transcription termination sequence elements: T1, located at the 3′ end of the 28S rDNA; T2, a putative processing site 235 bp downstream of T1; T3, the principal terminator positioned 215 bp upstream of the gene promoter. As demonstrated for nuclear run-off assays, T3 was found to be the main terminator for Xenopus rDNA transcription. These in vitro data are in obvious contradiction to results obtained by electron microscopic (EM) spread preparations from rapidly isolated amplified oocyte nucleoli, i.e., an rDNA chromatin probe thought to represent the in vivo situation, indicative of transcription termination at sites T1-2. However, most interestingly, T3 had-again by the EM method-been identified as the exclusive terminator for NTS spacer transcription units. In order to answer the question of whether read-through transcription of the complete rDNA spacer sequence is obligatory for 40S pre-rRNA in vivo transcription, we analyzed several hundreds of spread rRNA genes from Xenopus oocyte nucleoli in great detail, applying two different spreading procedures, e.g., dispersal of amplified oocyte nucleoli shortly in detergent-free or detergent containing low-salt media prior to the EM spreading technique. Quantitation of EM spreads resulted in the finding that read-through rDNA spacer transcription beyond T1-2 termination sites (i.e., indicative of T3 transcription termination) can be visualized for the in vivo situation at a frequency of less than 3% of rRNA genes analyzed. In order to discriminate whether termination in vivo occurs preferentially at sites T1 or T2, we used the S1 nuclease protection assay and localized the 3′ end of the primary 40S rRNA transcript at site T2. Chromatin spread preparations using amplified amphibian oocyte nucleoli have opened the gate for the present understanding of transcription organization in higher eukaryotes (Miller and Beatty 1969a, b; for review, see Miller 1981). The overall notion from numerous electron microscopic (EM) studies using nucleolar chromatin from a wide range of different eukaryotes was that a general pattern exists for rRNA transcription, namely, a regular alternation of transcribed rDNA segments (‘matrix units’, sensu Miller and Beatty 1969a, b) and non-transcribed rDNA spacer segments (Beyer et al. 1979; Franke et al. 1979; Miller 1981). From the initial studies on, it was assumed that the typical Christmastree pattern of gradually lengthening precursor ribosomal RNA transcripts associated with ribonucleoproteins (pre-rRNP fibrils) starts at the 5′ site of the 40S rDNA sequence and terminates near the 3′ 40S coding site. The results obtained by analyses of fully hydrated spread rRNA genes by video-enhanced light microscopy were in agreement with the EM data (Spring and Trendelenburg 1990). A more direct functional analysis of Xenopus rRNA transcription became possible when the Xenopus rDNA unit had been sequenced and the positions of promoters and terminators determined (Boseley et al. 1979; Moss et al. 1980; Labhart and Reeder 1987a). It was shown that the terminator sequence T1 is positioned at the 3′ end of the 28S rDNA coding region, sequence T2 235 bp downstream of T1, and T3 215 bp upstream of the gene promoter (Trendelenburg 1981, 1982; Labhart and Reeder 1986). To elucidate the function of the regulatory elements of the rDNA spacer so far identified, in most of the more recent assays nuclear run-off experiments were used for rDNA transcript analysis. One of the most striking outcomes of these studies was the finding that in contrast to the in vivo situation (see above), in nuclear run-off assays transcription constitutively passed the T1-2 termination sites, resulting in read-through transcription of the entire rDNA spacer sequence up to transcription termination site, T3, located immediately up-stream of the 5′ 40S main promoter element. It was thus concluded that for both organisms studied, i.e., Xenopus and Drosophila, the non-transcribed spacer rDNA segments should be regarded as an constitutive, integral part of the primary pre-rRNA transcription unit (Labhart and Reeder 1986; Tautz and Dover 1986; Labhart and Reeder 1987b, c). The main argument to discuss the striking discrepancy of run-off analysis with in vivo EM observations was the claim that possibly the association of rDNA spacer-sequence-bound transcription complexes and their RNA polymerase particles downstream of T1-2 terminators might be too unstable to be visualized by the chromatin spreading technique. In the present study the aim was thus to have a closer look at this discrepancy. In order to answer the question of whether read-through transcription of the complete rDNA spacer sequence is also obligatory for the in vivo situation, we reexamined several hundreds of spread rRNA genes from Xenopus oocyte nucleoli to quantitate the percentage of read-through transcription and spacer promoter initiation. To discriminate whether termination in vivo occurs preferentially at sites T2 or T3, we used the S1 nuclease protection assay and localized the 3′ end of the primary 40S rRNA transcript at site T2.