Structural separation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi-functional protein, p43/AIMP1

AIMP1 (previously known as p43) is first found as a factor associated with a macromolecular tRNA synthetase complex. However, it is also secreted and acts on diverse target cells such as endothelial cells, macrophages, and fibroblasts to control angiogenesis, inflammation, and dermal regeneration, respectively. We previously showed that AIMP1 induces the death of endothelial cell but proliferation of fibroblasts and activates macrophages. In this work, we found that elastase 2-cleaved AIMP1 retained its pro-apoptotic activity to endothelial cells but lost the growth-stimulatory activity to fibroblasts. To determine the functional domains responsible for each activity, we generated several deletion fragments of AIMP1 and compared the activities to the target cells. AIMP1 promoted endothelial cell death and caspase-3 activation through its 101-114 amino acid region, fibroblast proliferation through its 6-46 amino acid region, and endothelial migration through its 114-192 amino acid region as revealed by deletion mapping. Thus, this work revealed that AIMP1 uses different regions for its diverse extracellular activities.     

Comparative genomics reveals expansion of the FLC region in the genus Arabidopsis

Mechanisms of genome evolution are poorly understood although recent genome sequencing is providing the tools to begin to illuminate such mechanisms. Using high-resolution molecular cytogenetic tools, we examined the structural evolution of 790 kb surrounding the evolutionarily important FLC locus of Arabidopsis thaliana in three of its relatives, Arabidopsis halleri, Arabidopsis neglecta and Arabidopsis arenosa. Sequenced BACs from A. thaliana were used as heterologous probes across these species and genome expansion was found in all three species relative to A. thaliana, ranging from 16 to 27%. Expansion was seen along the length of the entire region but molecular analyses revealed no characteristic pattern of either intra- or intergenic expansion among these species. Mapping of BACs on DNA fibers from A. thaliana revealed one possible error, ~14 kb missing from the reported sequence, indicating that for comparative studies it is important to confirm the reference sequence to which comparison will be made.     

A Polymerase Chain Reaction Screening Method for Rapid Detection of Microsatellites in Bacterial Artificial Chromosomes

Standard protocols aimed at identifying subclones of interest from bacterial artificial chromosomes (BACs) include the use of hybridization methods that are time consuming and often require the use of radioactive isotopes. Through our efforts to identify microsatellites in BACs from rainbow trout (Oncorhynchus mykiss) we have developed a nonradioactive polymerase chain reaction (PCR)-based screening technique to select microsatellites containing subclones for marker development. Two BACs were subcloned and screened by PCR using a vector-specific primer and a mix of microsatellite repeat primers. The subclones were then sequenced to evaluate the efficiency of the PCR screening method. Correlation between positive PCR amplification and presence of microsatellites varied between the two BACs (21.9% and 71.4%), but still a sufficient number of subclones were identified to enable design and optimization of microsatellite markers.     

Statistical methods in genetics

In recent years, a very large variety of statistical methodologies, at various levels of complexity, have been put forward to analyse genotype data and detect genetic variations that may be responsible for increasing the susceptibility to disease. This review provides a concise account of a number of selected statistical methods for population-based association mapping, from single-marker tests of association to multi-marker data mining techniques for gene-gene interaction detection.     

Mapping tissue-specific genes correlated with age-dependent changes in protein stability and function

Biophysical measurements indicative of protein stability and function were performed on crude extracts from liver, muscle, and lens of a genetically heterogeneous mouse population. Genetic information was used to search for quantitative trait loci (QTL) that influenced the biophysical traits, with emphasis on phenotypes that previously have been shown to be altered in aged animals. Spectroscopic and enzymatic assays of crude liver and muscle tissue extracts from approximately 600 18-month-old mice, the progeny of (BALB/cJxC57BL/6J)F1 females and (C3H/HeJxDBA/2J)F1 males, were used to measure the susceptibility of a ubiquitous glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), to thermal denaturation. The rate constant for thermal inactivation of GAPDH correlated with markers on chromosome 5 (D5Mit79 and D5Mit251) for muscle lysates and chromosome 15 (D15Mit63 and D15Mit100) for liver tissue. The degree of variability of inactivation rate constants, a measure of the heterogeneity of muscle GAPDH in tissue extracts, was also associated with markers on chromosome 5 (D5Mit79 and D5Mit205). In addition, spectroscopic characteristics of extracted eye lens proteins were evaluated for their susceptibility to photooxidative stress. Absorbance and fluorescence emission characteristics of the lens proteins were mapped to QTL on chromosomes 5 and 15 (D5Mit25 and D15Mit171) while the degree of heterogeneity in photochemical oxidation kinetics was associated with a marker on the chromosome 8 (D8Mit42). Recent work has shown that GAPDH possesses a number of non-glycolytic functions including DNA/RNA binding and regulation of protein expression. Tissue specific differences in GAPDH stability may have significant consequences to these alternate functions during aging.     

Genetic mappings in artificial genomes

This paper concerns processing of genomes of artificial (computer-simulated) organisms. Of special interest is the process of translation of genotypes into phenotypes, and utilizing the mapping information obtained during such translation. If there exists more than one genetic encoding in a single artificial life model, then the translation may also occur between different encodings. The obtained mapping information allows to present genes-phenes relationships visually and interactively to a person, in order to increase understanding of the genotype-tophenotype translation process and genetic encoding properties. As the mapping associates parts of the source sequence with the translated destination, it may be also used to trace genes, phenes, and their relationships during simulated evolution. A mappings composition procedure is formally described, and a simple method of visual mapping presentation is established. Finally, advanced visualizations of gene-phene relationships are demonstrated as practical examples of introduced techniques. These visualizations concern genotypes expressed in various encodings, including an encoding which exhibits polygenic and pleiotropic properties.     

Evaluating markers for selecting a patient's treatment

Selecting the best treatment for a patient's disease may be facilitated by evaluating clinical characteristics or biomarker measurements at diagnosis. We consider how to evaluate the potential impact of such measurements on treatment selection algorithms. For example, magnetic resonance neurographic imaging is potentially useful for deciding whether a patient should be treated surgically for Carpal Tunnel Syndrome or should receive less-invasive conservative therapy. We propose a graphical display, the selection impact (SI) curve that shows the population response rate as a function of treatment selection criteria based on the marker. The curve can be useful for choosing a treatment policy that incorporates information on the patient's marker value exceeding a threshold. The SI curve can be estimated using data from a comparative randomized trial conducted in the population as long as treatment assignment in the trial is independent of the predictive marker. Estimating the SI curve is therefore part of a post hoc analysis to determine whether the marker identifies patients that are more likely to benefit from one treatment over another. Nonparametric and parametric estimates of the SI curve are proposed in this article. Asymptotic distribution theory is used to evaluate the relative efficiencies of the estimators. Simulation studies show that inference is straightforward with realistic sample sizes. We illustrate the SI curve and statistical inference for it with data motivated by an ongoing trial of surgery versus conservative therapy for Carpal Tunnel Syndrome.     

Tyrosine residues modification studied by MALDI-TOF mass spectrometry

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.     

Physical education - new technologies for mapping plant genomes

Locating DNA sequences to specific chromosomal segments is essential for associating genes with phenotypes. It is routinely achieved by segregation analysis using meiotic mapping populations that have also been used to collect phenotypic information. However, meiotic mapping is struggling to cope with the shear volume of sequences emerging from high-throughput (HTP) gene-discovery programs. We describe two approaches, Radiation Hybrid and ‘HAPPY’ mapping, which, in conjunction with meiotic mapping, represent valuable HTP tools in the quest to link genes to phenotypes.