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Transcription and Tyranny in the Nucleolus: The Organization, Activation, Dominance and Repression of Ribosomal RNA Genes

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Transcription and Tyranny in the Nucleolus: The Organization, Activation, Dominance and Repression of Ribosomal RNA Genes

Craig S Pikaard. Arabidopsis Book.

Figures

Figure 1.
Figure 1.
Diagrammatic representations of McClintock's observations that defined a specific locus on maize chromosome 6 as the nucleolus organizer region. A. Chromosomes 6 and 9 and their reciprocal translocation products. In wild type maize, a single nucleolus is associated with chromosome 6 on the distal side of a dark knob of heterochromatin known as a chromomere. A secondary constriction is adjacent to this chromomere at metaphase. A reciprocal translocation resulting from double-strand breaks in both chromosomes 6 and 9 produced chromosomes 96 and 69. Nucleoli are associated with both translocated chromosomes, which suggested to McClintock that the breakage site in chromosome 6 must have occurred within a nucleolar organizer whose genetic information was redundant. B. When together in the same microspore (shown at prophase), the 96 chromosome forms a larger nucleolus than does the 69 chromosome, which suggested to McClintock that the two NORs compete for a limiting substance. The graphics are adapted from McClintock's drawings (McClintock, 1934). This figure is reprinted, with permission from the publisher, from (Pikaard, 2000b).
Figure 2.
Figure 2.
Organization of a generic nucleolus organizer region. NORs consist of long head-to-tail repeats of the genes encoding the precursor of the three largest ribosomal RNAs (18S, 5.8S and 25S). The NOR includes both transcriptionally active rRNA genes, which give rise to the secondary constriction on a metaphase chromosome, and silent rRNA genes which are sometimes packaged into dense heterochromatin (as in maize). At metaphase, the proteinaceous remnant of the nucleolus often remains associated with the NOR and is traversed by the secondary constriction. Within the NOR, each rRNA gene is nearly identical in sequence, though variation in the number of repeated DNA elements in the intergenic spacer is common. Intergenic spacer regions evolve rapidly whereas coding regions are highly conserved. This figure is reprinted, with minor modifications, from (Pikaard, 2000b) with permission from the publisher.
Figure 3.
Figure 3.
Organization of NORs and telomeres at the tips of A. thaliana chromosomes 2 and 4 (in the ecotype La-0). NOR2 and NOR4 are each ∼4 Mbp in size, including ∼350–400 rRNA genes at each locus (Copenhaver and Pikaard, 1996b). The locations of the NORs relative to other molecular markers used to map the NORs in 1996 are shown. Telomeres TEL2N and TEL4N directly abut the first (most distal) rRNA genes of NOR2 and NOR4. Digestion with I-PpoI, an essentially rRNA-gene specific endonuclease, releases the ends of the chromosomes as 8 kb or 13 kb fragments (Copenhaver and Pikaard, 1996a). The telomere repeats join the first rRNA gene of NOR4 downstream of the gene promoter such that this gene is probably inactive. The rRNA genes at both NORs are oriented such that transcription proceeds toward the centromere. Whereas all rRNA genes at NOR2 have relatively long intergenic spacers (blue), long, short (yellow) and intermediate-length (green) variants are present at NOR4 (Copenhaver and Pikaard, 1996b). The rRNA gene length variants are not intermingled, but instead are highly clustered, suggesting local spreading of variants as the mode of gene homogenization.
Figure 4.
Figure 4.
Comparison of eukaryotic rRNA gene intergenic spacers. The spacers of multicellular eukaryotes are typically dominated by one or more classes of repetitive elements. In A. thaliana, X. laevis, mouse and D. melanogaster, these include duplicated promoters known as spacer promoters. Repetitive elements that are located between the gene and spacer promoters are also found in Arabidopsis, Xenopus and mouse. Xenopus 60/81 bp repeats, Arabidopsis Sal repeats and mouse 140 bp repeats share no obvious sequence similarity, yet all display enhancer activity when attached to a Xenopus rRNA gene promoter and injected into Xenopus oocytes (Doelling et al., 1993; Pikaard et al., 1990). Note the very different organization of rRNA gene intergenic spacers in yeast, which lack prominent arrays of repetitive elements and which include a 5S RNA gene, transcribed by RNA polymerase III, positioned in opposite orientation relative to the direction of pol I transcription.
Figure 5.
Figure 5.
Purification of RNA polymerase I holoenzyme activity. A. Scheme for sequential chromatography of RNA polymerase I holoenzyme activity from broccoli (Brassica oleracea) using DEAE-Sepharose CL-6B, Biorex 70, Sephacryl S-300, Mono Q, and double-stranded calf thymus DNA-cellulose. B. Mono Q fractions were tested for their ability to program accurate transcription initiation from a cloned B. oleracea rRNA gene promoter. Accurately initiated transcripts were detected using an S1 nuclease protection assay. C. Mono Q fractions subjected to SDS-polyacrylamide gel electrophoresis and blotting to nitrocellulose were probed with antiserum raised against the last exon of the largest A. thaliana RNA polymerase I subunit (190 kd subunit), or against 24.3 kd and 14 kd RNA polymerase subunits. This figure is reprinted, with modifications, from (Saez-Vasquez and Pikaard, 2000) with permission from the publisher.
Figure 6.
Figure 6.
Molecular analysis of nucleolar dominance in Arabidopsis. A. Flower, leaf and whole-plant phenotypes of Arabidopsis thaliana (left), Arabidopsis arenosa (also known as Cardaminopsis arenosa; right) and their allotetraploid hybrid, Arabidopsis suecica (center). Note the intermediate phenotypes of flower and leaf morphologies in A. suecica. B: The ribosomal RNA genes from A. thaliana and A. arenosa are both present in similar abundance in A. suecica. Genomic DNA of A. thaliana (lane 2), A. suecica (lane 3) or A. arenosa (lane 4) was subjected to PCR using one primer corresponding to a region just upstream of the promoter and a second primer corresponding to the beginning of the 18S rRNA coding region. A control reaction in lane 5 lacked template DNA. Bacteriophage lambda DNA cleaved with Hind III served as size markers in lane 1. C: Only A. arenosa ribosomal RNA genes are transcribed in A. suecica, as shown using the S1 nuclease protection assay (compare lanes 5 and 8). Equal aliquots of A. thaliana, A. arenosa or A. suecica RNA were analyzed with A. arenosa (lanes 3–5) or A. thaliana (lanes 6–8)-specific probes that detect rRNA gene transcripts initiated from the correct start sites (+1) of the respective gene promoters. Dideoxynucleotide sequencing reactions served as size markers in lanes 1 and 2. This figure is reprinted from (Pikaard, 1999) with permission from the publisher.

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