DNA replication timing is deterministic at the level of chromosomal domains but stochastic at the level of replicons in Xenopus egg extracts.

Replication origins in Xenopus egg extracts are located at apparently random sequences but are activated in clusters that fire at different times during S phase under the control of ATR/ATM kinases. We investigated whether chromosomal domains and single sequences replicate at distinct times during S phase in egg extracts. Replication foci were found to progressively appear during early S phase and foci labelled early in one S phase colocalized with those labelled early in the next S phase. However, the distribution of these two early labels did not coincide between single origins or origin clusters on single DNA fibres. The 4 Mb Xenopus rDNA repeat domain was found to replicate later than the rest of the genome and to have a more nuclease-resistant chromatin structure. Replication initiated more frequently in the transcription unit than in the intergenic spacer. These results suggest for the first time that in this embryonic system, where transcription does not occur, replication timing is deterministic at the scale of large chromatin domains (1-5 Mb) but stochastic at the scale of replicons (10 kb) and replicon clusters (50-100 kb).

Cyclic nucleotides, the photosynthetic apparatus and response to a UV-B stress in the Cyanobacterium Synechocystis sp. PCC 6803.

Cyclic nucleotides cAMP and cGMP are ubiquitous signaling molecules that mediate many adaptative responses in eukaryotic cells. Cyanobacteria present the peculiarity among the prokaryotes of having the two types of cyclic nucleotide. Cellular homeostasis requires both cyclases (adenylyl/guanylyl, for their synthesis) and phosphodiesterases (for their degradation). Fully segregated null mutants have been obtained for the two genes, sll1624 and slr2100, which encode putative cNMP phosphodiesterases. We present physiological evidence that the Synechocystis PCC 6803 open reading frame slr2100 could be a cGMP phosphodiesterase. In addition, we show that Slr2100, but not Sll1624, is required for the adaptation of the cells to a UV-B stress. UV-B radiation has deleterious effects for photosynthetic organisms, in particular on the photosystem II, through damaging the protein structure of the reaction center. Using biophysical and biochemical approaches, it was found that Slr2100 is involved in the signal transduction events which permit the repair of the UV-B-damaged photosystem II. This was confirmed by quantitative reverse transcriptase-PCR analyses. Altogether, the data point to an important role for cGMP in signal transduction and photoacclimation processes during a UV-B stress.

Co-ordinated expression of phycobiliprotein operons in the chromatically adapting cyanobacterium Calothrix PCC 7601: a role for RcaD and RcaG.

In the cyanobacterium Calothrix sp. PCC 7601 the cpc2 operon encoding phycocyanin 2 (PC2) is expressed if red radiations are available. RcaD was previously identified in extracts from red-light-grown cells as an alkaline phosphatase-sensitive protein that binds upstream of the transcription start point (TSP) of the cpc2 operon. In this work, RcaD was purified, and the corresponding gene cloned with a PCR probe obtained using degenerated primers based on RcaD peptide sequences (accession no. AJ319541). Purified RcaD binds to the cpc2 promoter region and also to those of the constitutive cpc1 and apc1 operons that encode phycocyanin 1 and allophycocyanin. Escherichia coli-overexpressed RcaD can bind to the cpc2 promoter region. The rcaD gene is upstream of an open reading frame (ORF) termed rcaG. Co-transcription of both genes was demonstrated by reverse transcription (RT)-PCR experiments, and found to be independent of the light wavelengths. A single TSP was mapped. Sequence features of RcaD and RcaG led us to propose a functional relationship between these two proteins. A rcaD mutant generated by allelic exchange exhibited altered expression of the cpc2, cpeBA, apc1 and cpc1 operons upon green to red-light shifts. RcaD seems to be a co-activator co-ordinating the transcription of the phycobiliprotein operons upon changes in light spectral quality.