Novel approaches to map small molecule–target interactions

The quest for small molecule perturbators of protein function or a given cellular process lies at the heart of chemical biology and pharmaceutical research. Bioactive compounds need to be extensively characterized in the context of the modulated protein(s) or process(es) in living systems to unravel and confirm their mode of action. A crucial step in this workflow is the identification of the molecular targets for these small molecules, for which a generic methodology is lacking. Herein we summarize recently developed approaches for target identification spurred by advances in omics techniques and chemo- and bioinformatics analysis.     

Phase resetting reduces theta–gamma rhythmic interaction to a one-dimensional map

Gamma and theta oscillations of the hippocampus are known to interact, but the mechanisms underlying such interaction are not well understood. We focus on a previously published computational model of hippocampal activity that shows the gamma rhythms nesting in the theta rhythms, and investigate the dynamical mechanisms underlying that interaction. There are three types of neurons in the model: pyramidal cells, fast-spiking interneurons, and “oriens lacunosum-moelculare” (O-LM cells); the latter is an inhibitory cell whose inhibition has a longer time scale, and which has currents associated with intrinsic theta-rhythm behavior. We identify two main modes of interaction among the slow and the fast rhythms in the model, modulated by the strength of the excitatory synapse on the O-LM cells. Using resets of phases after each pyramidal cell and O-LM spike, we extend the use of the phase transition map (PTM) to encode the stability type of spiking patterns in networks where different frequencies interact. The tailored application of the PTM to the model network measures how the interaction between the shape of the phase response curves and the length of the gamma period determines the number of gamma spikes in theta cycles, and provides an explicit formula for the length of theta intervals in nesting regimes. Using the PTM, we also explain the covariance of the gamma and theta rhythms as drive is changed over some intervals.     

Genes for two homologous G-protein α subunits map to different human chromosomes

Summary Signal transduction across biological membranes is modulated by a family of related GTP-binding proteins termed G proteins. These G proteins have a heterotrimeric structure composed of α, β, and γ subunits. The α subunits of the G proteins bind GTP and appear to determine the biochemical specificity of the protein. We have recently cloned and characterized cDNA encoding two G-protein α subunits, α_i and α_h. The former is a substrate for ADP-ribosylation by pertussis toxin. The protein corresponding to α_h has not yet been identified. These cDNAs encode proteins, which demonstrate 90% sequence identity to one another and also show marked similarity to other G proteins. The present studies were designed to determine whether the genes for these related proteins are clustered on a single human chromosome. Genomic DNA isolated from a panel of mouse-human hybrid cell lines was analyzed by hybridization to cDNAs for α_i and α_h. Based on the distribution patterns of α_i and α_h in cell hybrids, the gene for α_i was assigned to human chromosome 7, and the gene for α_h assigned to chromosome 12. These data suggest that the G-protein gene family may be distributed over at least two human chromosomes.     

A genetic linkage map of the cultivated strawberry (Fragaria × ananassa) and its comparison to the diploid Fragaria reference map

The cultivated strawberry, Fragaria × ananassa, is the most economically-important soft-fruit species, but few practical molecular tools for the purpose of marker assisted selection currently exist. As a precursor to the development of such tools, a genetic linkage map was developed from a F₁ population comprising 174 seedlings derived from a cross between two F. × ananassa cultivars, ‘Redgauntlet’ × ‘Hapil’. The resultant map is composed of 315 molecular markers—218 microsatellites, 11 gene-specific markers and 86 AFLP and RAPD markers—and spans 3,116 cM. In total, 69 linkage group fragments were recovered, more than the 56 linkage groups expected for the cultivated strawberry, however, all fragments contained a transferable marker that could be associated with one of 56 linkage group scaffolds. The female (Redgauntlet) and male (Hapil) linkage maps are composed, respectively of 170 loci in 32 linkage groups covering 1,675.3 cM and 182 loci in 37 linkage groups covering 1,440.7 cM, with 37 markers common to both maps. The maximum number of markers in one linkage group was 15, the minimum was two. All linkage groups resolved contained at least one transferable marker (SSR or gene-specific) that had been mapped on the diploid Fragaria reference map (FV × FB), and therefore all linkage groups could be identified as homologous to one of the seven diploid Fragaria linkage groups. When marker order was compared to the diploid Fragaria reference map, effectively complete colinearity was observed. However, the occurrence of duplicated loci on homologues of linkage groups FG1 and FG6 provided evidence of a putative chromosomal duplication or translocation event in Fragaria. The development of this linkage map will facilitate the study and dissection of QTL associated with traits of economic importance such as disease resistance and fruit quality, and provides a foundation for the development of markers for the purpose of marker assisted breeding and selection in the cultivated strawberry, F. × ananassa.     

A corrected Haldane’s map function to calculate genetic distances from recombination data

The estimation of genetic distances from recombination data has no direct relationship due to the fact that multiple crossovers do not generate recombinant gametes that can be recognized in the progeny. The Haldane’s map function is the most widely used mathematical formulation able to relate the observed recombination frequency with the actual number of crossovers. Here I show that the model in which the Haldane’s correction is based on is not correct, and I present a modified map function that takes into consideration the actual number of recombinant gametes produced in cells in which different number of crossovers have occurred. My correction generates shorter genetic distances than the Haldane’s one.     

Commensal ‘trail of bread crumbs’ provide pathogens with a map to the intestinal landscape

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The chaperone activity of α-synuclein: Utilizing deletion mutants to map its interaction with target proteins

α-Synuclein is the principal component of the Lewy body deposits that are characteristic of Parkinson's disease. In vivo, and under physiological conditions in vitro, α-synuclein aggregates to form amyloid fibrils, a process that is likely to be associated with the development of Parkinson's disease. α-Synuclein also possesses chaperone activity to prevent the precipitation of amorphously aggregating target proteins, as demonstrated in vitro. α-Synuclein is an intrinsically disordered (i.e., unstructured) protein of 140 amino acids in length, and therefore studies on its fragments can be correlated directly to the functional role of these regions in the intact protein. In this study, the fragment containing residues 61-140 [α-syn(61-140)] was observed to be highly amyloidogenic and was as effective a chaperone in vitro as the full-length protein, while the N- and C-terminal fragments α-syn(1-60) and α-syn(96-140) had no intrinsic chaperone activity. Interestingly, full-length fibrillar α-synuclein had greater chaperone activity than nonfibrillar α-synuclein. It is concluded that the amyloidogenic NAC region (residues 61-95) contains the chaperone-binding site which is optimized for target protein binding as a result of its β-sheet formation and/or ordered aggregation by α-synuclein. On the other hand, the first 60 residues of α-synuclein modulate the protein's chaperone-active site, while at the same time protecting α-synuclein from fibrillation. On its own, however, this fragment [α-syn(1-60)] had a tendency to aggregate amorphously. As a result of this study, the functional roles of the various regions of α-synuclein in its chaperone activity have been delineated.     

Casares’ map function: no need for a ‘corrected’ Haldane’s map function

A genetic map function M(d) = RF provides a mapping from the additive genetic distance d to the non-additive recombination fraction RF between a given pair of loci, where the recombination fraction is the proportion of gametes that are recombinant between the two loci. Genetic map functions are needed because in most experiments all we can directly observe are the recombination events. However, since a recombination event is only observed if there are an odd number of crossovers between the two loci, recombination fractions are not additive. One of the most widely used map functions is Haldane’s map function, which is derived under the assumptions of no chiasma and no chromatid interference, and has been in widespread use since 1919. However, Casares recently proposed a ‘corrected’ Haldane’s map function – we show here that this ‘corrected’ map function is not correct due to faulty assumptions and mistakes in its derivation.     

Relevance of atmospheric odours and geomagnetic field to pigeon navigation: What is the “map” basis?

• 1.1. Pigeon homing is highly affected by olfactory deprivation, as can be seen from an almost complete lack of homeward orientation of initial bearings, from widely dispersed recovery sites, and from a strong reduction of homing success which achieves, in inexperienced pigeons displaced over longer distances, a level of zero.
• 2.2. Orientational deficits can be produced not only by various methods to eliminate the sense of smell, but also by elimination of odorous substances from the ambient air.
• 3.3. It is concluded that the observed deficits result from an interruption of an information flow necessary for navigation, and are not due to some general, nonspecific distraction or reduced motivation to home.
• 4.4. The range of olfactory navigation is large (radius up to 500 km and more), but not unlimited. Within this range, the pigeons do not depend on olfactory stimuli perceived during displacement to the release site.
• 5.5. Despite some controversial discussions of the matter, no experimental findings have been published contradicting the above statements and conclusions.
• 6.6. Experimental interference with perception of the geomagnetic field resulted, under sun (and thus with the sun compass capable to operate), in increased scatter of initial bearings, sometimes in slightly reduced homing speeds, but not in disoriented or poorly oriented recovery bearings and not in reduced homing success.
• 7.7. Correlations between initial orientation and spatial as well as temporal variations of the geomagnetic field, as reported by several authors, are unsuited to prove involvement of geomagnetism in the process of site localization.
• 8.8. It is concluded that atmospheric odours are a necessary component of the pigeons' navigational “map” system, whereas the geomagnetic field is not. It seems unlikely that the latter is involved in this system at all, and if it is, its role is expected to be quite subordinate and redundant at best.

     

An original method to quantify and map wetland hydrological heterogeneity: the Moëze marsh (France)

We monitored three easily and inexpensively measured parameters to describe the hydrological patterns of a tidal influenced freshwater marsh, characterized by a very complex channel network. Variations in water elevation, electrical conductivity and turbidity were measured simultaneously at 52 sampling stations during 8 months. A Principal Component Analysis showed two different patterns during the winter discharge phase and the summer inflow phase. A Cluster Analysis was used to group stations and rank them according to their increasing hydrological instability computed through an index (I) discussed in this paper. The use of multivariate analyses in combination with the construction of the instability index I enables one to quantify the hydrological instability on a numerical scale and, to map the hydrological patterns of the marsh. This method is proposed to compare several wetland types from a hydrological point of view and to design sampling network in complicated channel network.Corresponding Author: W. Mitsch     

Selection of a core set of RILs from Forrest × Williams 82 to develop a framework map in soybean

Soybean BAC-based physical maps provide a useful platform for gene and QTL map-based cloning, EST mapping, marker development, genome sequencing, and comparative genomic research. Soybean physical maps for “Forrest” and “Williams 82” representing the southern and northern US soybean germplasm base, respectively, have been constructed with different fingerprinting methods. These physical maps are complementary for coverage of gaps on the 20 soybean linkage groups. More than 5,000 genetic markers have been anchored onto the Williams 82 physical map, but only a limited number of markers have been anchored to the Forrest physical map. A mapping population of Forrest × Williams 82 made up of 1,025 F₈ recombinant inbred lines (RILs) was used to construct a reference genetic map. A framework map with almost 1,000 genetic markers was constructed using a core set of these RILs. The core set of the population was evaluated with the theoretical population using equality, symmetry and representativeness tests. A high-resolution genetic map will allow integration and utilization of the physical maps to target QTL regions of interest, and to place a larger number of markers into a map in a more efficient way using a core set of RILs.     

NMR relaxation parameters of methyl groups as a tool to map the interfaces of helix–helix interactions in membrane proteins

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.     

Genetic linkage map of a wheat×spelt cross

 We constructed a genetic map of a cross between the Swiss winter wheat (Triticum aestivum L.) variety Forno and the Swiss winter spelt (Triticum spelta L.) variety Oberkulmer. For the linkage analysis,176 polymorphic RFLP probes and nine microsatellites were tested on 204 F₅ recombinant inbred lines (RILs) of Forno×Oberkulmer revealing 242 segregating marker loci. Thirty five percent of these loci showed significant (P>0.05) deviation from a 1 : 1 segregation, and the percentage of Forno alleles ranged from 21% to 83% for individual marker loci. Linkage analysis was performed with the program MAPMAKER using the Haldane mapping function. Using a LOD threshold of 10, we obtained 37 linkage groups. After finding the best order of marker loci within linkage groups by multi-point analysis we assembled the linkage groups into 23 larger units by lowering the LOD threshold. All except one of the 23 new linkage groups could be assigned to physical chromosomes or chromosome arms according to hybridisation patterns of nulli-tetrasomic lines of Chinese Spring and published wheat maps. This resulted in a genetic map comprising 230 marker loci and spanning 2469 cM. Since the analysed population is segregating for a wide range of agronomically important traits, this genetic map is an ideal basis for the identification of quantitative trait loci (QTLs) for these traits. Received: 3 August 1998 / Accepted: 28 November 1998     

Recombination hot spots within the I region of the mouse H-2 complex map to the E β and E α genes

Recombinant mouse strains with crossovers in the I region of the H-2 major histocompatibility complex were examined by restriction fragment analysis for the presence of polymorphic restriction sites within the E and E genes. Nine recombinant mouse strains were shown to have crossed over within a 5 kb DNA segment that contains the large intron between the second and third exons of the E gene. These results are in accord with previous studies mapping a recombination hot spot within this gene. Seven recombinant mouse strains between the p and k haplotypes were shown to have crossed over in a 6 kb segment within the E gene. These results show the existence of a recombination hot spot within the E gene. Comparison of the H-2 haplotypes involved in these two recombination hot spots suggests that a specific DNA sequence in b, s, f, and q haplotypes may act to promote recombination in the E gene and a specific DNA sequence in the p haplotype may act to promote recombination in the E gene.     

An updated ‘Essex’ by ‘Forrest’ linkage map and first composite interval map of QTL underlying six soybean traits

DNA marker maps based on single populations are the basis for gene, loci and genomic analyses. Individual maps can be integrated to produce composite maps with higher marker densities if shared marker orders are consistent. However, estimates of marker order in composite maps must include sets of markers that were not polymorphic in multiple populations. Often some of the pooled markers were not codominant, or were not correctly scored. The soybean composite map was composed of data from five separate populations based on northern US germplasm but does not yet include ‘Essex’ by ‘Forrest’ recombinant inbred line (RIL) population (E × F) or any southern US soybean cultivars. The objectives were, to update the E × F map with codominant markers, to compare marker orders among this map, the Forrest physical map and the composite soybean map and to compare QTL identified by composite interval maps to the earlier interval maps. Two hundred and thirty seven markers were used to construct the core of the E × F map. The majority of marker orders were consistent between the maps. However, 19 putative marker inversions were detected on 12 of 20 linkage groups (LG). Eleven marker distance compressions were also found. The number of inverted markers ranged from 1 to 2 per LG. Thus, marker order inversions may be common in southern compared to northern US germplasm. A total of 61 QTL among 37 measures of six traits were detected by composite interval maps, interval maps and single point analysis. Seventeen of the QTL found in composite intervals had previously been detected among the 29 QTL found in simple interval maps. The genomic locations of the known QTL were more closely delimited. A genome sequencing project to compare Southern and Northern US soybean cultivars would catalog and delimit inverted regions and the associated QTL. Gene introgression in cultivar development programs would be accelerated.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.