TOUSLED kinase activity oscillates during the cell cycle and interacts with chromatin regulators

The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism.     

Indene bioconversion by a toluene inducible dioxygenase of Rhodococcus sp. I24

Rhodococcus sp. I24 can oxygenate indene via at least three independent enzyme activities: (i) a naphthalene inducible monooxygenase (ii) a naphthalene inducible dioxygenase, and (iii) a toluene inducible dioxygenase (TID). Pulsed field gel analysis revealed that the I24 strain harbors two megaplasmids of 340 and 50 kb. Rhodococcus sp. KY1, a derivative of the I24 strain, lacks the 340 kb element as well as the TID activity. Southern blotting and sequence analysis of an indigogenic, I24-derived cosmid suggested that an operon encoding a TID resides on the 340 kb element. Expression of the tid operon was induced by toluene but not by naphthalene. In contrast, naphthalene did induce expression of the nid operon, encoding the naphthalene dioxygenase in I24. Cell free protein extracts of Escherichia coli cells expressing tidABCD were used in HPLC-based enzyme assays to characterize the indene bioconversion of TID in vitro. In addition to 1-indenol, indene was transformed to cis-indandiol with an enantiomeric excess of 45.2% of cis-(1S,2R)-indandiol over cis-(1R,2S)-indandiol, as revealed by chiral HPLC analysis. The K_m of TID for indene was 380 M. The enzyme also dioxygenated naphthalene to cis-dihydronaphthalenediol with an activity of 78% compared to the formation of cis-indandiol from indene. The K_m of TID for naphthalene was 28 M. TID converted only trace amounts of toluene to 1,2-dihydro-3-methylcatechol after prolonged incubation time. The results indicate the role of the tid operon in the bioconversion of indene to 1-indenol and cis-(1S,2R)-indandiol by Rhodococcus sp. I24.     

New mouse genetic models for human contraceptive development

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.     

Closed state of both binding domains of homodimeric mGlu receptors is required for full activity

Membrane receptors, key components in signal transduction, often function as dimers. These include some G protein-coupled receptors such as metabotropic glutamate (mGlu) receptors that have large extracellular domains (ECDs) where agonists bind. How agonist binding in dimeric ECDs activates the effector domains remains largely unknown. The structure of the dimeric ECDs of mGlu(1) solved in the presence of agonist revealed two specific conformations in which either one or both protomers are in an agonist-stabilized closed form. Here we examined whether both conformations correspond to an active form of the full-length receptor. Using a system that allows the formation of dimers made of a wild-type and a mutant subunit, we show that the closure of one ECD per dimer is sufficient to activate the receptor, but the closure of both ECDs is required for full activity.     

Seasonal Variation of Antifouling Activities of Marine Algae from the Brittany Coast (France)

The antifouling activity of extracts (aqueous, ethanol, and dichloromethane) of 9 marine macroalgae against bacteria, fungi, diatoms, macroalgal spores, mussel phenoloxidase activity, and barnacle cypris larvae has been investigated in relation to season in bimonthly samples from the Bay of Concarneau (France). Of the extracts tested, 48.2% were active against at least one of the fouling organisms, and of these extracts, 31.2% were seasonally active with a peak of activity in summer corresponding to maximal values for water temperature, light intensity, and fouling pressure, and 17% were active throughout the year. This seasonal activity may be adaptive as it coincides with maximal fouling pressure in the Bay of Concarneau. Dichloromethane extracts of Rhodophyceae were the most active in the antifouling assays.     

Diversity of the archaeal community in 44 anaerobic digesters as determined by single strand conformation polymorphism analysis and 16S rDNA sequencing

The diversity of Archaea in anaerobic digesters was characterized by strand conformation polymorphism (SSCP) analysis and the sequencing of 16S rDNA genes. The 44 digesters sampled, located in eight different countries, treated effluents from agriculture, the food processing and petro-chemical industries, pulp and paper plant, breweries, slaughterhouses and municipal waste. All the existing processes were represented among the samples (fixed-film, fluidized bed, stirred-tank, UASB, sequential batch reactor, lagoon). Single strand conformation polymorphism analysis targeting the V3 region of 16S rDNA revealed between four to six distinct archaeal peaks per digester. The diversity of dominant Archaea in the 44 digesters was estimated as 23 different 16S rDNA sequences. Cloning of archaeal 16S rRNA genes from 11 distinct total genomic DNA, screening of clones by SSCP and the sequencing of 170 of them made it possible to characterize these SSCP peaks. All the sequences retrieved were members of the Euryarchaeaota subdomain. Furthermore, most of the sequences retrieved were very close to already known and cultivated strains or to environmental clones. The most frequent archaeal sequences were close to Methanosaeta concilii and to a 16S rDNA clone vadinDC06 located in the Methanobacterium clade (84% and 73% of digesters respectively). The other sequences were members of the Methanobacteriales and the Methanomicrobiales families. Only one sequence was far from any sequence of the database and it could be grouped with several sequences of environmental clones. Each digester harboured between two to nine archaeal sequences with only one of them corresponding to a putative acetate-utilizing species. Furthermore, the process in the digesters appeared to play a part in the distribution of archaeal diversity.     

Structural and functional characterization of pi bulges and other short intrahelical deformations

We data-mined the Protein Data Bank for short intrahelical deformations, including pi bulges. These are defined as a contiguous stretch of intrahelical residues deviating from the standard alpha-helical i-->i-4 hydrogen bonding pattern, bilaterally flanked by at least one alpha-helical turn resulting in a helix kink of less than 40 degrees. We find that such motifs exist in 4.7% of a PDB subset filtered by quality metrics (resolution <2.5 A, R-factor <0.25, sequence identity <35%). These are typically characterized by at least one i-->i-5 main chain hydrogen bond, with energetically favorable main chain dihedral angles, followed by a variable number of main chain carbonyl groups that do not accept intrahelical main chain hydrogen bonds. Their stabilization commonly occurs via hydrogen bonding to water molecules or polar groups. Numerous deformations are implicated in basic yet vital functional roles, commonly as ligand binding site contributors.     

Nucleic acid capture assay,a new method for direct quantitation of nucleic acids

Technologies allowing direct detection of specific RNA/DNA sequences occasionally serve as an alternative to amplification methods for gene expression studies. In these direct methods the hybridization of probes takes place in complex mixtures, thus specificity and sensitivity still limit the use of current technologies. To address these challenges, we developed a new technique called the nucleic acid capture assay, involving a direct multi-capture system. This approach combines a 3′-ethylene glycol scaffolding with the incorporation of 2′-methoxy deoxyribonucleotides in the capture sequences. In our design, all nucleotides other than those complementary to the target mRNA have been replaced by an inert linker, resulting in significant reductions in non-specific binding. We also provide a versatile method to detect the presence of captured targets by using specific labeled probes with alkaline phosphatase-conjugated anti-label antibodies. This direct, flexible and reliable technique for gene expression analysis is well suited for high-throughput screening and has potential for DNA microarray applications.     

UVA-induced cyclobutane pyrimidine dimers form predominantly at thymine-thymine dipyrimidines and correlate with the mutation spectrum in rodent cells

Ligation-mediated PCR was employed to quantify cyclobutane pyrimidine dimer (CPD) formation at nucleotide resolution along exon 2 of the adenine phosphoribosyltransferase (aprt) locus in Chinese hamster ovary (CHO) cells following irradiation with either UVA (340–400 nm), UVB (295–320 nm), UVC (254 nm) or simulated sunlight (SSL; λ > 295 nm). The resulting DNA damage spectrum for each wavelength region was then aligned with the corresponding mutational spectrum generated previously in the same genetic target. The DNA sequence specificities of CPD formation induced by UVC, UVB or SSL were very similar, i.e., in each case the overall relative proportion of this photoproduct forming at TT, TC, CT and CC sites was ~28, ~26, ~16 and ~30%, respectively. Furthermore, a clear correspondence was noted between the precise locations of CPD damage hotspots, and of ‘UV signature’ mutational hotspots consisting primarily of C→T and CC→TT transitions within pyrimidine runs. However, following UVA exposure, in strong contrast to the above situation for UVC, UVB or SSL, CPDs were generated much more frequently at TT sites than at TC, CT or CC sites (57% versus 18, 11 and 14%, respectively). This CPD deposition pattern correlates well with the strikingly high proportion of mutations recovered opposite TT dipyrimidines in UVA- irradiated CHO cells. Our results directly implicate the CPD as a major promutagenic DNA photoproduct induced specifically by UVA in rodent cells.