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)

Expression of the psbA gene during photoinhibition and recovery in Synechocystis PCC 6714: inhibition and damage of transcriptional and translational machinery prevent the restoration of photosystem II activity

13 Pags.- 8 Figs.The D1 reaction center protein of the photosystem II complex is very sensitive to light. It is continuously being damaged, degraded and resynthesized. Under high light, photosystem II inactivation is observed. This is because the rate of D1 damage is faster than that of its replacement. This process can be reversed if exposure to high light is not too long. In this work we study the changes that occur in the transcriptional and translational machinery that could lead to irreversible photoinhibition in Synechocystis PCC 6714. In the first minutes of photoinhibition, high light induced an accumulation of psbA mRNA due to an increase in psbA transcription initiation. Although the transcription rate of other photosynthetic genes (e.g. psaE and cpcB-cpcA) declined, the high turnover of the psbA transcript was maintained for a long time. When the light stress was too long, the stability of psbA mRNA increased and the psbA transcription rate appeared to decrease. A high level of psbA mRNA was maintained even though translation no longer occurred and the cells were unable to recover. Experiments to measure newly synthesized D1 incorporation into the thylakoid membranes during recovery in the presence of rifampicin showed that the initiation of transcription was not required for translation of psbA mRNA when photoinhibition was still reversible. Since psbA translation did not depend on the level of psbA transcript or on the initiation of psbA transcription, we propose that damage to the translational machinery also occurred during light stress, leading to the inhibition of D1 synthesis and to irreversible photoinhibition.Peer reviewe

Redox control of psbA expression in cyanobacteria Synechocystis strains

10 Pags.- 7 Figs.The D1 reaction-centre protein of the Photosystem II complex is very sensitive to light. It is continuously damaged, degraded and synthesized. The respective rates of these three processes are regulated by the light intensity. The means by which light regulates the expression of the psbA gene encoding the D1 protein in cyanobacteria is still an open question. Our results demonstrate that photosynthetic electron transport has an important role in psbA expression in Synechocystis cells. Under steady illumination, addition of 3-(3,4-dichlorophenyl)-1,1-dimethyl- urea (DCMU) or 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) induces a transient activation of psbA transcription. Transcription of other photosynthetic genes like psaE and cpcBA, respectively encoding the PSA-E subunit of Photosystem I and the β and α subunits of phycocyanin, one of the components of the phycobilisome, decreases under the same experimental conditions. Prolonged incubation with DCMU (or DCMU + methyl-viologen) results in a progressive decrease of psbA transcription and an increased stability of the transcript. Our data point to a control mechanism that involves two different signals: accumulation of QA− specifically activates psbA transcription, whereas oxidation of the electron transfer chain downstream of photosystem II, most probably the plastoquinone pool and/or the cyt b6f, decreases the expression of psbA and that of other photosynthetic genes like psaE and cpcBA.Peer reviewe