Mrc1 and Srs2 are major actors in the regulation of spontaneous crossover

In vegetative cells, most recombination intermediates are metabolized without an association with a crossover (CO). The avoidance of COs allows for repair and prevents genomic rearrangements, potentially deleterious if the sequences involved are at ectopic locations. We have designed a system that permits to screen spontaneous intragenic recombination events in Saccharomyces cerevisiae and to investigate the CO outcome in different genetic contexts. We have analyzed the CO outcome in the absence of the Srs2 and Sgs1 helicases, DNA damage checkpoint proteins as well as in a mutant proliferating cell nuclear antigen (PCNA) and found that they all contribute to genome stability. Remarkably high effects on COs are mediated by srs2Delta, mrc1Delta and a pol30-RR mutation in PCNA. Our results support the view that Mrc1 plays a specific role in DNA replication, promoting the Srs2 recruitment to PCNA independently of checkpoint signaling. Srs2 would prevent formation of double Holliday junctions (dHJs) and thus CO formation. Sgs1 also negatively regulates CO formation but through a different process that resolves dHJs to yield non-CO products.     

Novel genetic polymorphisms that further delineate the phylogeny of the Mycobacterium tuberculosis complex

In a previous report, we described a PCR protocol for the differentiation of the various species of the Mycobacterium tuberculosis complex (MTC) on the basis of genomic deletions (R. C. Huard, L. C. de Oliveira Lazzarini, W. R. Butler, D. van Soolingen, and J. L. Ho, J. Clin. Microbiol. 41:1637-1650, 2003). That report also provided a broad cross-comparison of several previously identified, phylogenetically relevant, long-sequence and single-nucleotide polymorphisms (LSPs and SNPs, respectively). In the present companion report, we expand upon the previous work (i) by continuing the evaluation of known MTC phylogenetic markers in a larger collection of tubercle bacilli (n = 125), (ii) by evaluating additional recently reported MTC species-specific and interspecific polymorphisms, and (iii) by describing the identification and distribution of a number of novel LSPs and SNPs. Notably, new genomic deletions were found in various Mycobacterium tuberculosis strains, new species-specific SNPs were identified for "Mycobacterium canettii," Mycobacterium microti, and Mycobacterium pinnipedii, and, for the first time, intraspecific single-nucleotide DNA differences were discovered for the dassie bacillus, the oryx bacillus, and the two Mycobacterium africanum subtype I variants. Surprisingly, coincident polymorphisms linked one M. africanum subtype I genotype with the dassie bacillus and M. microti with M. pinnipedii, thereby suggesting closer evolutionary ties within each pair of species than had been previously thought. Overall, the presented data add to the genetic definitions of several MTC organisms as well as fine-tune current models for the evolutionary history of the MTC.     

Over-expression of a cytosolic isoform of the HbCuZnSOD gene in Hevea brasiliensis changes its response to a water deficit

Hevea brasiliensis is the main commercial source of natural rubber. Reactive oxygen species (ROS) scavenging systems are involved in various biotic and abiotic stresses. Genetic engineering was undertaken to study the strengthening of plant defences by antioxidants. To that end, Hevea transgenic plant lines over-expressing a Hevea brasiliensis cytosolic HbCuZnSOD gene were successfully established and regenerated. Over-expression of the HbCuZnSOD gene was not clearly related to an increase in SOD activity in plant leaves. The impact of HbCuZnSOD gene over-expression in somatic embryogenesis and in plant development are presented and discussed. The water deficit tolerance of two HbCuZnSOD over-expressing lines was evaluated. The physiological parameters of transgenic plantlets subjected to a water deficit suggested that plants from line TS4T8An displayed lower stomatal conductance and a higher proline content. Over-expression of the HbCuZnSOD gene and activation of all ROS-scavenging enzymes also suggested that protection against ROS was more efficient in the TS4T8An transgenic line.     

Functional and structural characterization of α-(1->2) branching sucrase derived from DSR-E glucansucrase

ΔN₁₂₃-glucan-binding domain-catalytic domain 2 (ΔN₁₂₃-GBD-CD2) is a truncated form of the bifunctional glucansucrase DSR-E from Leuconostoc mesenteroides NRRL B-1299. It was constructed by rational truncation of GBD-CD2, which harbors the second catalytic domain of DSR-E. Like GBD-CD2, this variant displays α-(1→2) branching activity when incubated with sucrose as glucosyl donor and (oligo-)dextran as acceptor, transferring glucosyl residues to the acceptor via a ping-pong bi-bi mechanism. This allows the formation of prebiotic molecules containing controlled amounts of α-(1→2) linkages. The crystal structure of the apo α-(1→2) branching sucrase ΔN₁₂₃-GBD-CD2 was solved at 1.90 Å resolution. The protein adopts the unusual U-shape fold organized in five distinct domains, also found in GTF180-ΔN and GTF-SI glucansucrases of glycoside hydrolase family 70. Residues forming subsite −1, involved in binding the glucosyl residue of sucrose and catalysis, are strictly conserved in both GTF180-ΔN and ΔN₁₂₃-GBD-CD2. Subsite +1 analysis revealed three residues (Ala-2249, Gly-2250, and Phe-2214) that are specific to ΔN₁₂₃-GBD-CD2. Mutation of these residues to the corresponding residues found in GTF180-ΔN showed that Ala-2249 and Gly-2250 are not directly involved in substrate binding and regiospecificity. In contrast, mutant F2214N had lost its ability to branch dextran, although it was still active on sucrose alone. Furthermore, three loops belonging to domains A and B at the upper part of the catalytic gorge are also specific to ΔN₁₂₃-GBD-CD2. These distinguishing features are also proposed to be involved in the correct positioning of dextran acceptor molecules allowing the formation of α-(1→2) branches.     

A Predictive Computational Model of the Dynamic 3D Interphase Yeast Nucleus

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Highlights? We describe a predictive computational model of dynamic chromosomes in the yeast nucleus ? The model quantitatively recapitulates experimental data on nuclear organization ? The model predicts nuclear reorganization in response to treatment by rapamycin ? Large-scale nuclear organization is dominated by unspecific effects of crowded polymers     

DynPeak: an algorithm for pulse detection and frequency analysis in hormonal time series

The endocrine control of the reproductive function is often studied from the analysis of luteinizing hormone (LH) pulsatile secretion by the pituitary gland. Whereas measurements in the cavernous sinus cumulate anatomical and technical difficulties, LH levels can be easily assessed from jugular blood. However, plasma levels result from a convolution process due to clearance effects when LH enters the general circulation. Simultaneous measurements comparing LH levels in the cavernous sinus and jugular blood have revealed clear differences in the pulse shape, the amplitude and the baseline. Besides, experimental sampling occurs at a relatively low frequency (typically every 10 min) with respect to LH highest frequency release (one pulse per hour) and the resulting LH measurements are noised by both experimental and assay errors. As a result, the pattern of plasma LH may be not so clearly pulsatile. Yet, reliable information on the InterPulse Intervals (IPI) is a prerequisite to study precisely the steroid feedback exerted on the pituitary level. Hence, there is a real need for robust IPI detection algorithms. In this article, we present an algorithm for the monitoring of LH pulse frequency, basing ourselves both on the available endocrinological knowledge on LH pulse (shape and duration with respect to the frequency regime) and synthetic LH data generated by a simple model. We make use of synthetic data to make clear some basic notions underlying our algorithmic choices. We focus on explaining how the process of sampling affects drastically the original pattern of secretion, and especially the amplitude of the detectable pulses. We then describe the algorithm in details and perform it on different sets of both synthetic and experimental LH time series. We further comment on how to diagnose possible outliers from the series of IPIs which is the main output of the algorithm.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Significance of the Identification in the Horn of Africa of an Exceptionally Deep Branching Mycobacterium tuberculosis Clade

Molecular and phylogeographic studies have led to the definition within the Mycobacterium tuberculosis complex (MTBC) of a number of geotypes and ecotypes showing a preferential geographic location or host preference. The MTBC is thought to have emerged in Africa, most likely the Horn of Africa, and to have spread worldwide with human migrations. Under this assumption, there is a possibility that unknown deep branching lineages are present in this region. We genotyped by spoligotyping and multiple locus variable number of tandem repeats (VNTR) analysis (MLVA) 435 MTBC isolates recovered from patients. Four hundred and eleven isolates were collected in the Republic of Djibouti over a 12 year period, with the other 24 isolates originating from neighbouring countries. All major M. tuberculosis lineages were identified, with only two M. africanum and one M. bovis isolates. Upon comparison with typing data of worldwide origin we observed that several isolates showed clustering characteristics compatible with new deep branching. Whole genome sequencing (WGS) of seven isolates and comparison with available WGS data from 38 genomes distributed in the different lineages confirms the identification of ancestral nodes for several clades and most importantly of one new lineage, here referred to as lineage 7. Investigation of specific deletions confirms the novelty of this lineage, and analysis of its precise phylogenetic position indicates that the other three superlineages constituting the MTBC emerged independently but within a relatively short timeframe from the Horn of Africa. The availability of such strains compared to the predominant lineages and sharing very ancient ancestry will open new avenues for identifying some of the genetic factors responsible for the success of the modern lineages. Additional deep branching lineages may be readily and efficiently identified by large-scale MLVA screening of isolates from sub-Saharan African countries followed by WGS analysis of a few selected isolates.     

Comparative study of the photophysical properties of indoprofen photoproducts in relation with their DNA photosensitizing properties.

The photophysical properties of indoprofen photoproducts have been examined in various solvents by absorbance and emission spectroscopies in relation with their photosensitizing properties. The photophysical properties of 2-[4-(1-hydroxy)ethylphenyl]isoindolin-1-one (HOINP) and 2-(4-ethylphenyl)isoindolin-1-one (ETINP) are typical of a singlet excited state when the ones of 2-(4-acetylphenyl)isoindolin-1-one (KINP) are based on its triplet excited state according to previous work. The effect of solvent polarity on the absorption and fluorescence properties of HOINP and ETINP has been investigated as a function of Delta f, the Lippert solvent polarity parameter. A solvatochromic effect, function of the polarity region, has been observed for both photoproducts due to a change in the dipole moment of the compound upon excitation. In low-polarity regions, the excited state dipole moment of HOINP undergoes only a moderate increase (11.5 D) as compared to the dipole moment of the ground state (4.5 D) suggesting that the fluorescence arises from the locally excited state while in high-polarity regions it is strongly increased (42.9 D), which can imply that the emission takes place from a charge transfer state. In the case of ETINP, it would seem that the emitting state is rather a charge transfer state whatever the region is (16.9 and 31.8 D for the calculated excited-state dipole moments in the low and high-polarity regions, respectively). HOINP and ETINP do not produce thymine dimers by photosensitization but induce photooxidative damage via an electron transfer mechanism.