In-depth molecular and phenotypic characterization in a rice insertion line library facilitates gene identification through reverse and forward genetics approaches

We report here the molecular and phenotypic features of a library of 31,562 insertion lines generated in the model japonica cultivar Nipponbare of rice (Oryza sativa L.), called Oryza Tag Line (OTL). Sixteen thousand eight hundred and fourteen T-DNA and 12,410 Tos17 discrete insertion sites have been characterized in these lines. We estimate that 8686 predicted gene intervals--i.e. one-fourth to one-fifth of the estimated rice nontransposable element gene complement--are interrupted by sequence-indexed T-DNA (6563 genes) and/or Tos17 (2755 genes) inserts. Six hundred and forty-three genes are interrupted by both T-DNA and Tos17 inserts. High quality of the sequence indexation of the T2 seed samples was ascertained by several approaches. Field evaluation under agronomic conditions of 27,832 OTL has revealed that 18.2% exhibit at least one morphophysiological alteration in the T1 progeny plants. Screening 10,000 lines for altered response to inoculation by the fungal pathogen Magnaporthe oryzae allowed to observe 71 lines (0.7%) developing spontaneous lesions simulating disease mutants and 43 lines (0.4%) exhibiting an enhanced disease resistance or susceptibility. We show here that at least 3.5% (four of 114) of these alterations are tagged by the mutagens. The presence of allelic series of sequence-indexed mutations in a gene among OTL that exhibit a convergent phenotype clearly increases the chance of establishing a linkage between alterations and inserts. This convergence approach is illustrated by the identification of the rice ortholog of AtPHO2, the disruption of which causes a lesion-mimic phenotype owing to an over-accumulation of phosphate, in nine lines bearing allelic insertions.     

Different quaternary structures of human RECQ1 are associated with its dual enzymatic activity

RecQ helicases are essential for the maintenance of chromosome stability. In addition to DNA unwinding, some RecQ enzymes have an intrinsic DNA strand annealing activity. The function of this dual enzymatic activity and the mechanism that regulates it is, however, unknown. Here, we describe two quaternary forms of the human RECQ1 helicase, higher-order oligomers consistent with pentamers or hexamers, and smaller oligomers consistent with monomers or dimers. Size exclusion chromatography and transmission electron microscopy show that the equilibrium between the two assembly states is affected by single-stranded DNA (ssDNA) and ATP binding, where ATP or ATPγS favors the smaller oligomeric form. Our three-dimensional electron microscopy reconstructions of human RECQ1 reveal a complex cage-like structure of approximately 120 Å × 130 Å with a central pore. This oligomeric structure is stabilized under conditions in which RECQ1 is proficient in strand annealing. In contrast, competition experiments with the ATPase-deficient K119R and E220Q mutants indicate that RECQ1 monomers, or tight binding dimers, are required for DNA unwinding. Collectively, our findings suggest that higher-order oligomers are associated with DNA strand annealing, and lower-order oligomers with DNA unwinding.     

CDK1/cyclin B regulation during oocyte maturation in two closely related lugworm species, Arenicola marina and Arenicola defodiens

The molecular mechanisms underlying oocyte maturation in the annelid polychaetes Arenicola marina and Arenicola defodiens were investigated. In both species, a hitherto unidentified hormone triggers synchronous and rapid transition from prophase to metaphase, a maturation process which can be easily reproduced in vitro. Activation of a roscovitine- and olomoucine-sensitive M-phase-specific histone, H1 kinase, occurs during oocyte maturation. Using affinity chromatography on immobilized p9CKShs1, we purified CDK1 and cyclin B from oocyte extracts prepared from both phases and both species. In prophase, CDK1 is present both as an inactive, but Thr161-phosphorylated monomer, and as an inactive (Tyr15-phosphorylated) heterodimer with cyclin B. Prophase to metaphase transition is associated with complete tyrosine dephosphorylation of the cyclin B-associated CDK1, with phosphorylation of cyclin B, and with dramatic activation of the kinase activity of the CDK1/cyclin B complex. We propose that Arenicola oocytes may provide an ideal model system to investigate the acquisition of the ability of oocytes to be fertilized that occurs as oocyte shift from prophase to metaphase, an important physiological event, probably regulated by active CDK1/cyclin B.     

The Grey Mouse Lemur Uses Season-Dependent Fat or Protein Sparing Strategies to Face Chronic Food Restriction

During moderate calorie restriction (CR) the heterotherm Microcebus murinus is able to maintain a stable energy balance whatever the season, even if only wintering animals enter into torpor. To understand its energy saving strategies to respond to food shortages, we assessed protein and energy metabolisms associated with wintering torpor expression or summering torpor avoidance. We investigated body composition, whole body protein turnover, and daily energy expenditure (DEE), during a graded (40 and 80%) 35-day CR in short-days (winter; SD40 and SD80, respectively) and long-days (summer; LD40 and LD80, respectively) acclimated animals. LD40 animals showed no change in fat mass (FM) but a 12% fat free mass (FFM) reduction. Protein balance being positive after CR, the FFM loss was early and rapid. The 25% DEE reduction, in LD40 group was mainly explained by FFM changes. LD80 animals showed a steady body mass loss and were excluded from the CR trial at day 22, reaching a survival-threatened body mass. No data were available for this group. SD40 animals significantly decreased their FM level by 21%, but maintained FFM. Protein sparing was achieved through a 35 and 39% decrease in protein synthesis and catabolism (protein turnover), respectively, overall maintaining nitrogen balance. The 21% reduction in energy requirement was explained by the 30% nitrogen flux drop but also by torpor as DEE FFM-adjusted remained 13% lower compared to ad-libitum. SD80 animals were unable to maintain energy and nitrogen balances, losing both FM and FFM. Thus summering mouse lemurs equilibrate energy balance by a rapid loss of active metabolic mass without using torpor, whereas wintering animals spare protein and energy through increased torpor expression. Both strategies have direct fitness implication: 1) to maintain activities at a lower body size during the mating season and 2) to preserve an optimal wintering muscle mass and function.     

Development,characterization, validation,and mapping of SSRs derived from <Emphasis Type="Italic">Theobroma cacao</Emphasis> L.<Emphasis Type="Italic">–Moniliophthora perniciosa</Emphasis> interaction ESTs

In this study, we report results of the detection and analysis of SSR markers derived of cacao–Moniliophthora perniciosa expressed sequence tags (ESTs) in relation to cacao resistance to witches’ broom disease (WBD), and we compare the polymorphism of those ESTs (EST-simple sequence repeat (SSR)) with classical neutral SSR markers. A total of 3,487 ESTs was used in this investigation. SSRs were identified in 430 sequences: 277 from the resistant genotype TSH 1188 and 153 from the susceptible one Catongo, totalizing 505 EST-SSRs with three types of motifs: dinucleotides (72.1%), trinucleotides (27.3%), and tetranucleotides (0.6%). EST-SSRs were classified into 16 main categories; most of the EST-SSRs belonged to “Unknown function” and “No homology” categories (45.82%). A high frequency of SSRs was found in the 5’UTR and in the ORF (about 27%) and a low frequency was observed in the 3’UTR (about 8%). Forty-nine EST-SSR primers were designed and evaluated in 21 cacao accessions, 12 revealed polymorphism, having 47 alleles in total, with an average of 3.92 alleles per locus. On the other hand, the 11 genomic SSR markers revealed a total of 47 alleles, with an average of 5.22 alleles per locus. The association of EST-SSR with the genomic SSR enhanced the analysis of genetic distance among the genotypes. Among the 12 polymorphic EST-SSR markers, two were mapped on the F₂ Sca 6 × ICS 1 population reference for WBD resistance.