Mechanisms of checkpoint kinase Rad53 inactivation after a double-strand break in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, double-strand breaks (DSBs) activate DNA checkpoint pathways that trigger several responses including a strong G(2)/M arrest. We have previously provided evidence that the phosphatases Ptc2 and Ptc3 of the protein phosphatase 2C type are required for DNA checkpoint inactivation after a DSB and probably dephosphorylate the checkpoint kinase Rad53. In this article we have investigated further the interactions between Ptc2 and Rad53. We showed that forkhead-associated domain 1 (FHA1) of Rad53 interacts with a specific threonine of Ptc2, T376, located outside its catalytic domain in a TXXD motif which constitutes an optimal FHA1 binding sequence in vitro. Mutating T376 abolishes Ptc2 interaction with the Rad53 FHA1 domain and results in adaptation and recovery defects following a DSB. We found that Ckb1 and Ckb2, the regulatory subunits of the protein kinase CK2, are necessary for the in vivo interaction between Ptc2 and the Rad53 FHA1 domain, that Ckb1 binds Ptc2 in vitro and that ckb1Delta and ckb2Delta mutants are defective in adaptation and recovery after a DSB. Our data thus strongly suggest that CK2 is the kinase responsible for the in vivo phosphorylation of Ptc2 T376.     

MICROSATELLITE DEVELOPMENT IN RHODOPHYTA USING HIGH‐THROUGHPUT SEQUENCE DATA1

Shotgun genome sequencing is rapidly emerging as the method of choice for the identification of microsatellite loci in nonmodel organisms. However, to the best of our knowledge, this approach has not been applied to marine algae so far. Herein, we report the results of using the 454 next‐generation sequencing (NGS) platform to randomly sample 36.0 and 40.9 Mbp (139,786 and 139,795 reads, respectively) of the genome of two red algae from the northwest Iberian Peninsula [Grateloupia lanceola (J. Agardh) J. Agardh and a still undescribed new member of the family Cruoriaceae]. Using data mining tools, we identified 4,766 and 5,174 perfect microsatellite loci in 4,344 and 4,504 sequences/contigs from G. lanceola and the Cruoriaceae, respectively. After conservative removal of potentially problematic loci (redundant sequences, mobile elements), primer design was possible for 1,371 and 1,366 loci, respectively. A survey of the literature indicates that microsatellite density in our Rhodophyta is at the low end of the values reported for other organisms investigated with the same technology (land plants and animals). A limited number of loci were successfully tested for PCR amplification and polymorphism finding that they may be suitable for population genetic studies. This study demonstrates that random genome sequencing is a rapid, effective alternative to develop useful microsatellite loci in previously unstudied red algae.     

Discovery of novel positive allosteric modulators of the metabotropic glutamate receptor 5 (mGlu5)

Novel in vitro mGlu₅ positive allosteric modulators with good potency, solubility, and low lipophilicity are described. Compounds were identified which did not rely on the phenylacetylene and carbonyl functionalities previously observed to be required for in vitro activity. Investigation of the allosteric binding requirements of a series of dihydroquinolinone analogs led to phenylacetylene azachromanone 4 (EC₅₀ 11.5 nM). Because of risks associated with potential metabolic and toxicological liabilities of the phenylacetylene, this moiety was successfully replaced with a phenoxymethyl group (27; EC₅₀ 156.3 nM). Derivation of a second-generation of mGlu₅ PAMs lacking a ketone carbonyl resulted in azaindoline (33), azabenzimidazole (36), and N-methyl 8-azaoxazine (39) phenylacetylenes. By scoping nitrogen substituents and phenylacetylene replacements in 39, we identified phenoxymethyl 8-azaoxazine 47 (EC₅₀ 50.1 nM) as a potent and soluble mGlu₅ PAM devoid of both undesirable phenylacetylene and carbonyl functionalities.     

Phylogenetic evidence for role-reversals of gender-associated mitochondrial DNA in Mytilus (Bivalvia: Mytilidae)

Distinct gender-associated mitochondrial DNA (mtDNA) lineages (i.e., lineages which are transmitted either through males or through females) have been demonstrated in two families of bivalves, the Mytilidae (marine mussels) and the Unionidae (freshwater mussels), which have been separated for more than 400 Myr. The mode of transmission of these M (for male-transmitted) and F (for female-transmitted) molecules has been referred to as doubly uniparental inheritance (DUI), in contrast to standard maternal inheritance (SMI), which is the norm in animals. A previous study suggested that at least three origins of DUI are required to explain the phylogenetic pattern of M and F lineages in freshwater and marine mussels. Here we present phylogenetic evidence based on partial sequences of the cytochrome c oxidase subunit I gene and the 16S RNA gene that indicates the DUI is a dynamic phenomenon. Specifically, we demonstrate that F lineages in three species of Mytilus mussels, M. edulis, M. trossulus, and M. californianus, have spawned separate lineages which are now associated only with males. This process is referred to as "masculinization" of F mtDNA. By extension, we propose that DUI may be a primitive bivalve character and that periodic masculinization events combined with extinction of previously existing M types effectively reset the time of divergence between conspecific gender-associated mtDNA lineages.      

Combining protein evolution and secondary structure

An evolutionary model that combines protein secondary structure and amino acid replacement is introduced. It allows likelihood analysis of aligned protein sequences and does not require the underlying secondary (or tertiary) structures of these sequences to be known. One component of the model describes the organization of secondary structure along a protein sequence and another specifies the evolutionary process for each category of secondary structure. A database of proteins with known secondary structures is used to estimate model parameters representing these two components. Phylogeny, the third component of the model, can be estimated from the data set of interest. As an example, we employ our model to analyze a set of sucrose synthase sequences. For the evolution of sucrose synthase, a parametric bootstrap approach indicates that our model is statistically preferable to one that ignores secondary structure.