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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.

Notes: Abstract The amplified rRNA genes of amphibian oocytes were used as a model system for the development of an in situ hybridization technique to label nascent transcripts in dispersed chromatin. A biotinylated complementary RNA probe was hybridized to nascent transcripts from dispersed nucleoli, and detected by a two step antibody technique utilizing colloidal gold as an electron dense marker. A specific sequence on the rRNA nascent transcript was labeled in a pattern consistent with its location; however, gene morphology was difficult to analyze following in situ hybridization owing to low sample contrast. Proteins associated with the transcripts were apparently lost during the procedure, leading to decreased electron density of the transcripts. The technique was systematically modified in an attempt to identify conditions that preserved gene morphology adequately for ultrastructural analysis, while simultaneously maintaining sufficient levels of specific labeling.

Notes: Abstract Patterns of gene activity on individual chromatids of polytene chromosomes of Drosophila melanogaster white prepupae were ultrastructurally characterized by electron microscopy. The band-interband structure of salivary gland polytene chromosomes is lost when they are dispersed in a low ionic strength detergent solution. Morphologically similar, active genes in close proximity to one another were seen in dispersed white prepupal chromatin. The arrays of genes almost certainly represent sister copies of the same locus. Although lateral register between gene copies on multiple strands was not maintained, analysis of sister transcriptional units of unknown identity was achieved at the periphery of the chromatin arrays. Juxtaposed genes with divergent transcriptional polarity were prevalent. The morphology, size and transcriptional polarity of multiple copies of short, tandemly organized, RNA polymerase dense, co-expressed gene clusters is reported. One highly transcriptionally active region, designated the white prepupal locus (WPP locus), composed of a co-expressed tandem cluster of ten genes within an approximately 50 kb region was analyzed on six separate chromatids. The transcriptional map suggests that the pattern of gene activity for at least one gene within the cluster may not be identical on all homologous strands. The survey of active polytene genes provides ultrastructural correlation with previous molecular data that demonstrate tight linkage of certain developmentally co-regulated Drosophila genes. Our findings are discussed in relation to Drosophila gene organization, clustering, and regulation of gene expression.

Notes: Abstract The transcription of rRNA genes in Calliphora erythrocephala embryonal and larval fat bodies and guts was analyzed by electron microscopy. A gradual increase in the number of active genes and in the RNA polymerase density was observed in both tissues in the hours preceding hatching. Despite this increase, the rRNA genes at hatching are not fully loaded with RNA polymerase molecules. An additional increase in rRNA synthesis is characteristic of the first two days of larval development. This increase in rRNA synthesis is accomplished by an increase in RNA polymerase density, by activation of previously inactive rRNA genes, and by activation of newly replicated rRNA genes. The rRNA seems to be generated exclusively on genes not interrupted by a 6.1 kb intervening sequence. After the first 2 days, the RNA polymerase densities decline. Synthesis of rRNA seems to be coordinately regulated in these two tissues. — Two classes of tandemly repeated nonribosomal transcription units were detected in the fat bodies. The first class has an estimated DNA length of 3.3 kb and cannot be assigned to any of the known genes. The second class appears to represent very short transcription units with no visible RNP fibrils and is a candidate for 5S or tRNA transcription units.