Comparative biology: beyond sequence analysis

Comparative analysis is a fundamental tool in biology. Conservation among species greatly assists the detection and characterization of functional elements, whereas inter-species differences are probably the best indicators of biological adaptation. Traditionally, comparative approaches were applied to the analysis of genomic sequences. With the growing availability of functional genomic data, comparative paradigms are now being extended also to the study of other functional attributes, most notably the gene expression. Here we review recent works applying comparative analysis to large-scale gene expression datasets and discuss the central principles and challenges of such approaches.     

Are Biological Effects of Desert Shrubs More Important than Physical Effects on Soil Microorganisms?

Vegetation cover plays a major role in providing organic matter and in acting as a physical barrier, with both together contributing to the formation of “fertile islands,” which play an active role in prolonging biological activity in desert ecosystems. By undertaking this study, a long-term research, we designed an experiment to separate the two components—the physical and biotic parts of the perennial plants—and to identify the factor that contributes the most to the ecosystem. The study site was located in the northern Negev Desert, Israel, where 50 Hammada scoparia shrubs and 50 artificial plants were randomly marked. Soil samples were collected monthly over 3 years of research at three locations: under the canopy of H. scoparia shrubs, in the vicinity of the artificial plants, and between the shrubs (control). The contribution to microbial activity was measured by evaluation of the microbial community functions in soil. The functional aspects of the microbial community that were measured were CO₂ evolution, microbial biomass, microbial functional diversity, and the physiological profile of the community. The results of this study are presented in two ways: (1) according to the three locations/treatments; and (2) according to the phenological situation of the vegetation (annual and perennial plants) in the research field: the growing phase, the drying process, and the absence of annual plants. The only parameters that were found to affect microbial activity were the contribution of the organic matter of perennial shrubs and the growth of vegetation (annual and perennial) during the growing seasons. The physical component was found to have no effect on soil microbial functional diversity, which elucidates the important contribution of the desert shrub in enhancing biological multiplicity and activity.     

Disentangling signaling gradients generated by equivalent sources

Yeast cells approach a mating partner by polarizing along a gradient of mating pheromones that are secreted by cells of the opposite mating type. The Bar1 protease is secreted by a-cells and, paradoxically, degrades the α-factor pheromones which are produced by cells of the opposite mating type and trigger mating in a-cells. This degradation may assist in the recovery from pheromone signaling but has also been shown to play a positive role in mating. Previous studies suggested that widely diffusing protease can bias the pheromone gradient towards the closest secreting cell. Here, we show that restricting the Bar1 protease to the secreting cell itself, preventing its wide diffusion, facilitates discrimination between equivalent mating partners. This may be mostly relevant during spore germination, where most mating events occur in nature.     

A secreted lipolytic enzyme from Xanthomonas campestris pv. vesicatoria is expressed in planta and contributes to its virulence

A recombinase-based in vivo expression technology (RIVET) approach with Xanthomonas campestris pv. vesicatoria (Xcv) revealed that lipA, annotated as putative secreted lipase, is expressed during the interaction between this pathogen and tomato. Here, the tnpR and uidA reporter genes were used to show that lipA is strongly induced in XVM2 minimal medium and during the early stages of tomato infection by Xcv. A mutant strain impaired in lipA was generated by insertional mutagenesis. This mutant grew in a similar manner to the wild-type in rich medium, but its growth was significantly compromised in a medium containing olive oil as a single carbon source. The lipolytic activity of the extracellular fraction of the lipA mutant was reduced significantly relative to that of the wild-type strain, thus confirming that lipA indeed encodes a functional secreted enzyme with lipolytic activity. A plasmid carrying a wild-type copy of lipA complemented the lipA mutant for extracellular lipolytic activity. Dip inoculation experiments with tomato lines Hawaii 7998 (H7998) and Micro Tom showed that the lipA mutant grew to a lesser extent than the wild-type in tomato leaves. Following leaf syringe infiltrations, the mutant strain induced disease symptoms that were less severe than those induced by the wild-type strain, supporting a significant role of lipA in the pathogenicity of Xcv.     

Activin regulation of the follicle-stimulating hormone beta-subunit gene involves Smads and the TALE homeodomain proteins Pbx1 and Prep1

FSH is critical for normal reproductive function in both males and females. Activin, a member of the TGFbeta family of growth factors, is an important regulator of FSH expression, but little is known about the molecular mechanisms through which it acts. We used transient transfections into the immortalized gonadotrope cell line LbetaT2 to identify three regions (at -973/-962, -167, and -134) of the ovine FSH beta-subunit gene that are required for full activin response. All three regions contain homology to consensus binding sites for Smad proteins, the intracellular mediators of TGFbeta family signaling. Mutation of the distal site reduces activin responsiveness, whereas mutation of either proximal site profoundly disrupts activin regulation of the FSHbeta gene. These sites specifically bind LbetaT2 nuclear proteins in EMSAs, and the -973/-962 site binds Smad4 protein. Interestingly, the protein complex binding to the -134 site contains Smad4 in association with the homeodomain proteins Pbx1 and Prep1. Using glutathione S-transferase interaction assays, we demonstrate that Pbx1 and Prep1 interact with Smads 2 and 3 as well. The two proximal activin response elements are well conserved across species, and Pbx1 and Prep1 proteins bind to the mouse gene in vivo. Furthermore, mutation of either proximal site abrogates activin responsiveness of a mouse FSHbeta reporter gene as well, confirming their functional conservation. Our studies provide a basis for understanding activin regulation of FSHbeta gene expression and identify Pbx1 and Prep1 as Smad partners and novel mediators of activin action.     

In silico screening of a saturated mutation library of tomato

A comprehensive mutant population is a basic resource for exploring gene function. We developed an isogenic tomato 'mutation library' in the genetic background of the inbred variety M82. A total of 13 000 M(2) families, derived from EMS (ethyl methane sulfonate) and fast-neutron mutagenesis, were visually phenotyped in the field and categorized into a morphological catalog that includes 15 primary and 48 secondary categories. Currently, 3417 mutations have been cataloged; among them are most of the previously described phenotypes from the monogenic mutant collection of The Tomato Genetics Resource Center, and over a thousand new mutants, with multiple alleles per locus. The phenotypic database indicates that most mutations fall into more than a single category (pleiotropic), with some organs such as leaves more prone to alterations than others. All data and images can be searched and accessed in the Solanaceae Genome Network (SGN) on a site called 'The Genes That Make Tomatoes' (http://zamir.sgn.cornell.edu/mutants/).     

Predicting and controlling the reactivity of immune cell populations against cancer

Heterogeneous cell populations form an interconnected network that determine their collective output. One example of such a heterogeneous immune population is tumor‐infiltrating lymphocytes (TILs), whose output can be measured in terms of its reactivity against tumors. While the degree of reactivity varies considerably between different TILs, ranging from null to a potent response, the underlying network that governs the reactivity is poorly understood. Here, we asked whether one can predict and even control this reactivity. To address this we measured the subpopulation compositions of 91 TILs surgically removed from 27 metastatic melanoma patients. Despite the large number of subpopulations compositions, we were able to computationally extract a simple set of subpopulation‐based rules that accurately predict the degree of reactivity. This raised the conjecture of whether one could control reactivity of TILs by manipulating their subpopulation composition. Remarkably, by rationally enriching and depleting selected subsets of subpopulations, we were able to restore anti‐tumor reactivity to nonreactive TILs. Altogether, this work describes a general framework for predicting and controlling the output of a cell mixture.