Structure and Comparative Analysis of the rDNA Intergenic Spacer of Brassica rapa. Implications for the Function and Evolution of the Cruciferae Spacer

Abstract

The sequence of the intergenic spacer (IGS) of the Brassica rapa rDNA was determined and compared with those of other Cruciferae species. In the 3012-bp IGS, two segments of mostly unique sequence flank a 1.5-kb region consisting of two tandem arrays of repeats. A putative transcription initiation site (TIS) was identified by sequence comparison, 395 bp downstream from the repeat region. The intercalating segment displays unusual sequence patterns, and modelling of its topology predicts intrinsically bent DNA, with two elements of bending centered at positions -118 and -288 relative to the TIS. Comparative analysis of spacers from Cruciferae, revealed a common organization and high sequence similarity in their 5' and, particularly, 3' regions, whereas the repeat region upstream of TIS diverges rapidly. The conservation of structural elements, including the bent DNA upstream from the TIS, is discussed in light of their possible involvement in the IGS functions and structure of spacers in common ancestors. Examination of the Cruciferae spacers shows that, in addition to unequal crossover and gene conversion, insertional mutagenesis and replication slippage are molecular mechanisms significantly contributing to their evolution.

Organization, differential expression and methylation of rDNA in artificial Solanum allopolyploids

“…Besides, in natural and artificial allopolyploids between Brassica oleracea, B. nigra, and B. rapa no correlation between ND and the size of the IGS or the number of subrepeats were found (Chen and Pikaard, 1997a,b). For Brassica it is difficult to discuss presumptive enhancer function of upstream subrepeats, because in the species studied these elements have different size and belong to different structural classes (Bennett and Smith, 1991;Tremousaygue et al, 1992;Da Rocha and Bertrand, 1995;Bhatia et al, 1996). In contrast to Brassica, all Solanum species studied here possess upstream subrepeats of the same structural types differing only by a few base substitutions.…”

Molecular Organization of the 25S–18S rDNA IGS of Fagus sylvatica and Quercus suber: A Comparative Analysis

“…Exploring the IGS sequences for functional elements and domains we have predicted the putative transcription initiation site (TIS) in both spacers: TCTTT A GGGGGG (position - 5 relative to the initiating A) (Figure 5) through the alignment of our IGSs with the regions of initiation of transcription of other species from the Fagaceae [8], Cucurbitaceae [11], [42], [43], Fabaceae [44], [45], Brassicaceae [9], [13], [46]–[49], Solanaceae [3], [50], [51], as well as three monocots [52]–[54]. We have also found two adjacent CAAT-boxes, which is a common cis -acting element in promoter and enhancer regions (position −156 in Beech and −50 in Cork Oak spacer).…”

“…Fagus sylvatica and Quercus suber ribosomal intergenic spacers showed, like in the white oaks [8], an organization typical of most ribosomal IGS composed by five different regions: sub-repeats (SR), AT-rich, promoter and external transcribed sequences (3′ and 5′-ETS) - with structural features of plant IGS sequences and all functional elements needed for rRNA gene activity [3]–[5], [8], [11], [47], [49], [50], [58], [59].…”

“…Moreover, these differences should be maintained by functional compelling rather than by sequence characteristics of this region. For instance, the AT-rich segment upstream the promoter, that has also been referred in the IGS of other plant species [3]–[6], [8], [11], [13], [49], [50], [58], although diverging in their sequence is rich in stretches of AT base pairs, which motifs represent potential binding protein regions involved in transcription initiation sites [65], in gene regulation [66], [67], and also in the initiation of DNA replication [68], [69]. In addition to these elements, SAR/MAR attachment sites like DNA topoisomerase II recognition sites and TG-dinucleotides, found in both spacers are known to maintain the rDNA in correct position during the interphase [70].…”

“…Interestingly, the region between the TIS and this putative TTS has no similarity, which points to a functional meaning of the duplicated TTS. In fact, termination sites have been referred as normal structures in the IGS of animal and plant species [3], [4], [8], [9], [13], [47], [49], [50], [58], and have been clearly implicated in ribosomal transcription enhancement [91]. The terminator will normally be found in close proximity to the promoter-bound TBP1-complex permitting read-trough enhancement by polymerase recycling [86].…”