Abridged 5S rDNA units in sea beet (Beta vulgaris subsp. maritima)

Amplification by polymerase chain reaction of the 5S rDNA repeat units of Beta vulgaris subsp. maritima resulted in a 350-bp product corresponding to the full-length 5S unit, but also revealed 4 abridged unit classes, each with a deletion that removed most of the spacer and 12–76 bp of the coding sequence. Each abridged type lacks at least 1 of the conserved elements involved in transcription of the 5S gene, and so appear to be nonfunctional. Network analysis revealed that the abridged units are evolving in the same manner as the full-length versions.Key words: 5S rDNA, Beta vulgaris subsp. maritima, network analysis, sea beet.

Genomic organization and evolution of the 5S ribosomal DNA in the ancient fish sturgeon

Ribosomal DNA in sturgeon is informative when analyzed at the molecular level because it bears unique characteristics that are, to a certain extent, ancestral within vertebrates. In this paper, we examine the structure and the molecular evolution of the 5S ribosomal DNA (rDNA) region in 13 sturgeon species, comparing both the 5S ribosomal RNA (rRNA) genes and the non-transcribed spacer (NTS) sequences between the coding regions. We have found that different NTS and 5S gene variants are intermixed in the 5S rDNA arrays of the different sturgeon species and that all variants are ancestral, having been maintained over many millions of years. Using predictive models, we have found similar levels of sequence diversity in the coding regions, as well as in the non-coding region, but fixed interspecific differences are underrepresented for 5S genes. However, contrary to the expectations, we have not found fixed differences between NTS sequences when comparing many pairs of species. Specifically, when they belong to the same phylogeographic clade of the four into which the sturgeon is divided, but fixation of mutations and divergence is found between species belonging to different phylogeographic clades. Our results suggest that the evolution of the two parts of the 5S rDNA region cannot be explained exclusively as the outcome of a balance between mutational, homogenizing (i.e., gene conversion as a predominant force in sturgeon), and selective forces. Rather, they suggest that other factors (i.e., hybridization) might be superimposed over those forces and thus could to some extent be masking their effects.Key words: sturgeon, 5S rDNA, NTS sequence, 5S gene, concerted evolution, sequence homogenization, gene conversion, hybridization.

Rearrangement of chromatin domains during development in Xenopus

A dynamic change in the organization of different gene domains transcribed by RNA polymerase I, II, or III occurs during the progression from quiescent [pre-midblastula transition (pre-MBT)] to active (post-MBT) embryos during Xenopus development. In the rDNA, c-myc, and somatic 5S gene domains, a transition from random to specific anchorage to the nuclear matrix occurs when chromatin domains become active. The keratin gene domain was also randomly associated to the nuclear matrix before MBT, whereas a defined attachment site was found in keratinocytes. In agreement with this specification, ligation-mediated (LM)-PCR genomic footprinting carried out on the subpopulation of 5S domains specifically attached to the matrix reveals the hallmarks of determined chromatin after the midblastula transition. In contrast, the same analysis performed on the total 5S gene population does not reveal specific chromatin organization, validating the use of nuclear matrix fractionation to unveil active chromatin domains. These data provide a means for the determination of active chromosomal territories in the embryo and emphasize the role of nuclear architecture in regulated gene expression during development.

Additional intragenic promoter elements of the Xenopus 5S RNA genes upstream from the TFIIIA-binding site.

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.

Additional intragenic promoter elements of the Xenopus 5S RNA genes upstream from the TFIIIA-binding site

The major promoter element of the Xenopus laevis 5S RNA gene is located within the transcribed region of the gene and forms the binding site for the transcription initiation factor TFIIIA. We report an analysis of deletion and substitution mutations within the coding region of the major oocyte-type 5S gene of X. laevis. Our results differ from those of previous mutagenesis studies conducted on the somatic-type genes of Xenopus borealis and X. laevis. Transcription assays in whole oocyte S-150 extracts, with both oocyte- and somatic-type mutants, revealed additional promoter elements between the start site for transcription and the binding site for TFIIIA. These sequences regulate the efficiency of binding TFIIIC, a transcription factor required by the genes transcribed by RNA polymerase III containing intragenic promoters. Under TFIIIC-limiting conditions, the somatic-type gene had a 10-fold-higher affinity for TFIIIC than did the major oocyte-type 5S gene. One mutation in the oocyte-type gene (nucleotides +33 to +39) reduced TFIIIC affinity and transcriptional activity four- to fivefold. Differences in TFIIIC affinity between oocyte- and somatic-type genes may contribute to the differential transcription of these genes observed during Xenopus embryogenesis.