Glauco P. Tocchini-Valentini

A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains

BackgroundThe mouse inbred line C57BL/6J is widely used in mouse genetics and its genome has been incorporated into many genetic reference populations. More recently large initiatives such as the International Knockout Mouse Consortium (IKMC) are using the C57BL/6N mouse strain to generate null alleles for all mouse genes. Hence both strains are now widely used in mouse genetics studies. Here we perform a comprehensive genomic and phenotypic analysis of the two strains to identify differences that may influence their underlying genetic mechanisms.ResultsWe undertake genome sequence comparisons of C57BL/6J and C57BL/6N to identify SNPs, indels and structural variants, with a focus on identifying all coding variants. We annotate 34 SNPs and 2 indels that distinguish C57BL/6J and C57BL/6N coding sequences, as well as 15 structural variants that overlap a gene. In parallel we assess the comparative phenotypes of the two inbred lines utilizing the EMPReSSslim phenotyping pipeline, a broad based assessment encompassing diverse biological systems. We perform additional secondary phenotyping assessments to explore other phenotype domains and to elaborate phenotype differences identified in the primary assessment. We uncover significant phenotypic differences between the two lines, replicated across multiple centers, in a number of physiological, biochemical and behavioral systems.ConclusionsComparison of C57BL/6J and C57BL/6N demonstrates a range of phenotypic differences that have the potential to impact upon penetrance and expressivity of mutational effects in these strains. Moreover, the sequence variants we identify provide a set of candidate genes for the phenotypic differences observed between the two strains.

Reliability, robustness, and reproducibility in mouse behavioral phenotyping: a cross-laboratory study.

Establishing standard operating procedures (SOPs) as tools for the analysis of behavioral phenotypes is fundamental to mouse functional genomics. It is essential that the tests designed provide reliable measures of the process under investigation but most importantly that these are reproducible across both time and laboratories. For this reason, we devised and tested a set of SOPs to investigate mouse behavior. Five research centers were involved across France, Germany, Italy, and the UK in this study, as part of the EUMORPHIA program. All the procedures underwent a cross-validation experimental study to investigate the robustness of the designed protocols. Four inbred reference strains (C57BL/6J, C3HeB/FeJ, BALB/cByJ, 129S2/SvPas), reflecting their use as common background strains in mutagenesis programs, were analyzed to validate these tests. We demonstrate that the operating procedures employed, which includes open field, SHIRPA, grip-strength, rotarod, Y-maze, prepulse inhibition of acoustic startle response, and tail flick tests, generated reproducible results between laboratories for a number of the test output parameters. However, we also identified several uncontrolled variables that constitute confounding factors in behavioral phenotyping. The EUMORPHIA SOPs described here are an important start-point for the ongoing development of increasingly robust phenotyping platforms and their application in large-scale, multicentre mouse phenotyping programs.

High-throughput discovery of novel developmental phenotypes.

Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.

Post-publication sharing of data and tools

Despite existing guidelines on access to data and bioresources, good practice is not widespread. A meeting of mouse researchers in Rome proposes ways to promote a culture of sharing.

In vitro catenation and decatenation of DNA and a novel eucaryotic ATP-dependent topoisomerase

Extracts from X. laevis germinal vesicles interlock duplex DNA circles to form catenanes. The catenation activity requires Mg++ and ATP. Negatively supercoiled or relaxed DNA can be used as substrates for the catenation reaction. Homology between donor and acceptor DNA is not required, since catenanes are formed between DNA molecules with unrelated sequences. In the course of the isolation of the activity responsible for the catenation reaction, we discovered a new ATP-dependent topoisomerase. The fractions containing the novel topoisomerase catenate and decatenate DNA, the ionic strength dictating which of the two opposing reactions will occur.

GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs.

The orphan G protein-coupled receptor 37 (GPR37) is a substrate of parkin; its insoluble aggregates accumulate in brain samples of Parkinsons disease patients. We report here that GPR37 interacts with the dopamine transporter (DAT) and modulates DAT activity. GPR37 and DAT were found colocalized in mouse striatal presynaptic membranes and in transfected cells and their interaction was confirmed by coimmunoprecipitation assays. Gpr37-null mutant mice showed enhanced DAT-mediated dopamine uptake in striatal membrane samples, with a significant increase in the number of plasma membrane DAT molecules. The null mutant mice also exhibited a decrease in cocaine-induced locomotor activity and in catalepsy induced by dopamine receptor antagonists. These results reveal the specific role of GPR37, a putative peptidergic G protein-coupled receptor, in modulating the functional expression of DAT and the behavioral responses to dopaminergic drugs.

Transcription initiation of eucaryotic transfer RNA Genes

RNA polymerase Ill (Pal Ill), an enzyme found in the nuclei of animals, plants and fungi, has been implicated in the in vivo transcription of 5s rRNA, pretRNA, some small viral RNAs and the cellular RNAs derived from certain middle-repetitive genomic sequences. It is a complex -700 kd protein composed of at least ten distinct subunits. The enzyme is not able to transcribe purified genes with fidelity by itself but requires additional components whose number, nature and modes of action are only just beginning to be characterized. Much more is known about the locations of the DNA signals that permit accurate transcription initiation of some Pol Ill genes. This information has come primarily from a two-step experimental approach. In the first step, DNA is progressively deleted from around and within the gene in question; the deleted sequences are then replaced by heterologous DNA. Alternatively, single-base changes or short deletions are introduced into the gene by in vivo or in vitro mutagenesis. The second step is then to assay the transcriptional effects of these sequence manipulations by microinjection of the mutant genes into the nuclei of frog oocytes or by their incubation in a variety of in vitro transcription systems. We shall compare and contrast the results of such analyses with several Pol Ill genes and discuss, in particular, the structure and function of the DNA signals important for tRNA gene transcription initiation. Pol III Promoters Are lntragenic Deletion analyses have shown that the only DNA sequences essential for transcription of a frog 5s RNA gene are located between residues 50 and 83 (Sakonju et al., Cell 79, 13-25, 1980; Bogenhagen et al., Cell 79, 27-35, 1980). A plausible basis for the mode of action of this intragenic promoter was provided by Engelke et al. (Cell 79, 717-728, 19801, who showed that the same region of the 5s RNA gene binds to a 5S-specific transcription factor. These results suggest that this 37 kd transcription factor, once bound, may interact with a Pol III molecule to position its catalytic sites on the transcription start point of the DNA. The same resection approach has shown that tRNA genes also contain intragenic promoters. Their maximum boundaries have been defined as residues 8 and 62 within the genes encoding the initiator tRNAMe’ (Hofstetter et al., Cell 24, 573-585, 1981) and tRNA% (Galli et al., Nature 294, 626-631, 1981) of X. laevis and the tRNAP”’ of Caenorhabditis elegans (Ciliberto et al., PNAS 79, 1195-l 199, 1982). Unlike their counterparts within 5s genes, however, the essential nucleotides are split into two sequence blocks that are set far apart. These sequences, termed the A and B blocks (Galli et al., op. cit.), have the approximate coordinates 8-19 and 52-62, respectively, by the standard system of numbering tRNA genes. Their locations with respect to the tRNA cloverleaf are indicated in Figure 1. Two lines of evidence in particular support the notion of discontinuous intragenic promoters: chimeric tRNA genes containing the 5’ half of one gene and the 3’ half of another can be transcribed well; and transcription can also occur after the replacement of the central region of tRNA genes with DNA of very different sequence (Ciliberto et al., op. cit.; Galli et al., op. cit.). These central regions do appear to have a spacing function, however, because the efficiency of transcription depends on the length of the replaced DNA, the optimal distance between the A and B blocks being 30-40 bp (Ciliberto et al., PNAS 79, 19211925, 1982). In natural tRNA genes this distance can vary from 31 to >74 bp, the variability being due to the length of the V arm and the presence within certain tRNA genes of an intervening sequence. Although not absolutely required for transcription (Wallace et al., Science 209, 1396-1400, 1980), intervening sequences may still influence the efficiency of this process by expanding the distance between the A and B blocks, thereby diminishing promoter strength. An unusual feature of tRNA gene promoters is thus the great latitude in distance between the A and B blocks. In contrast, the distance between the transcription start point and the A block is less variable (10-I 6 bp). Another intriguing feature of these sequences is their close correlation with the most con-

Coevolution of tRNA intron motifs and tRNA endonuclease architecture in Archaea

Members of the three kingdoms of life contain tRNA genes with introns. The introns in pre-tRNAs of Bacteria are self-splicing, whereas introns in archaeal and eukaryal pre-tRNAs are removed by splicing endonucleases. We have studied the structures of the endonucleases of Archaea and the architecture of the sites recognized in their pre-tRNA substrates. Three endonuclease structures are known in the Archaea: a homotetramer in some Euryarchaea, a homodimer in other Euryarchaea, and a heterotetramer in the Crenarchaeota. The homotetramer cleaves only the canonical bulge–helix–bulge structure in its substrates. Variants of the substrate structure, termed bulge–helix–loops, appear in the pre-tRNAs of the Crenarcheota and Nanoarcheota. These variant structures can be cleaved only by the homodimer or heterotetramer forms of the endonucleases. Thus, the structures of the endonucleases and their substrates appear to have evolved together.

Molecular recognition of amino acids by RNA aptamers: the evolution into an L-tyrosine binder of a dopamine-binding RNA motif.

We report the evolution of an RNA aptamer to change its binding specificity. RNA aptamers that bind the free amino acid tyrosine were in vitro selected from a degenerate pool derived from a previously selected dopamine aptamer. Three independent sequences bind tyrosine in solution, the winner of the selection binding with a dissociation constant of 35 microM. Competitive affinity chromatography with tyrosine-related ligands indicated that the selected aptamers are highly L-stereo selective and also recognize L-tryptophan and L-dopa with similar affinity. The binding site was localized by sequence comparison, analysis of minimal boundaries, and structural probing upon ligand binding. Tyrosine-binding sites are characterized by the presence of both tyrosine (UAU and UAC) and termination (UAG and UAA) triplets.

Sustaining the Data and Bioresource Commons

Globalization of biomedical research requires sustained investment for databases and biorepositories. Development of powerful, high-throughput technologies, together with globalization of scientific research, presents the biomedical research community with unprecedented challenges for the management, archiving, and distribution of data and bioresources (1). We need a social contract between funding agencies and the scientific community to accommodate “bottom-up” integration and “top-down” financing of databases and biorepositories on an international scale.