An excess of catalytically required motions inhibits the scavenger decapping enzyme.

The scavenger decapping enzyme hydrolyzes the protective 5' cap structure on short mRNA fragments that are generated from the exosomal degradation of mRNAs. From static crystal structures and NMR data, it is apparent that the dimeric enzyme has to undergo large structural changes to bind its substrate in a catalytically competent conformation. Here we studied the yeast enzyme and showed that the associated opening and closing motions can be orders of magnitude faster than the catalytic turnover rate. This excess of motion is induced by the binding of a second ligand to the enzyme, which occurs at high substrate concentrations. We designed a mutant that disrupted the allosteric pathway that links the second binding event to the dynamics and showed that this mutant enzyme is hyperactive. Our data reveal a unique mechanism of substrate inhibition in which motions that are required for catalytic activity also inhibit efficient turnover when they are present in excess.

Use of anionic denaturing detergents to purify insoluble proteins after overexpression.

BACKGROUND: Many proteins form insoluble protein aggregates, called "inclusion bodies", when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification. RESULTS: Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC. CONCLUSION: Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology.

Molecular cloning of a dominant roller mutant and establishment of DNA-mediated transformation in the nematode Pristionchus pacificus.

We report the molecular cloning of a dominant Roller mutant of Pristionchus pacificus, which encodes a cuticle collagen. We use the mutant locus as a marker to develop transgenic technique by generating complex arrays and present flourescent-protein based transcriptional reporter constructs for P. pacificus.

Wnt signaling induces vulva development in the nematode Pristionchus pacificus.

The Caenorhabditis elegans vulva is induced by a member of the epidermal growth factor (EGF) family that is expressed in the gonadal anchor cell, representing a prime example of signaling processes in animal development. Comparative studies indicated that vulva induction has changed rapidly during evolution. However, nothing was known about the molecular mechanisms underlying these differences. By analyzing deletion mutants in five Wnt pathway genes, we show that Wnt signaling induces vulva formation in Pristionchus pacificus. A Ppa-bar-1/beta-catenin deletion is completely vulvaless. Several Wnt ligands and receptors act redundantly in vulva induction, and Ppa-egl-20/Wnt; Ppa-mom-2/Wnt; Ppa-lin-18/Ryk triple mutants are strongly vulvaless. Wnt ligands are differentially expressed in the somatic gonad, the anchor cell, and the posterior body region, respectively. In contrast, previous studies indicated that Ppa-lin-17, one of the Frizzled-type receptors, has a negative role in vulva formation. We found that mutations in Ppa-bar-1 and Ppa-egl-20 suppress the phenotype of Ppa-lin-17. Thus, an unexpected complexity of Wnt signaling is involved in vulva induction and vulva repression in P. pacificus. This study provides the first molecular identification of the inductive vulva signal in a nematode other than Caenorhabditis.

Pristionchus pacificus: A Genetic Model System for the Study of Evolutionary Developmental Biology and the Evolution of Complex Life-History Traits.

INTRODUCTIONPristionchus pacificus is a nematode that has been established as a model system for evolutionary developmental biology. Initially, P. pacificus was used as a convenient nematode with which to compare the processes of vulva and gonad development as well as sex determination to Caenorhabditis elegans, one of the best-studied animal models. P. pacificus shares many features with C. elegans, including a short generation time, its ability to be easily cultured in the laboratory, and self-fertilization as a mode of reproduction. These features allowed forward and reverse genetic tools to be developed for this species. The application of these tools for genetic and molecular analysis of vulva formation revealed substantial differences between P. pacificus and C. elegans. The genome of P. pacificus has recently been sequenced and showed an expansion of protein-coding genes compared with C. elegans. Interestingly, the P. pacificus genome encodes some genes, such as cellulases, that are known to be present only in plant-parasitic nematodes. Many of the putative functions of the predicted genes in the genome are related to the ecology of P. pacificus and other Pristionchus species. Pristionchus nematodes can be isolated from beetles and soil, indicating that the ecology of P. pacificus is strikingly different from that of C. elegans. Generally, Pristionchus species show an unexpected level of species specificity in their beetle associations, providing a unique opportunity to study the genetic and molecular mechanisms underlying the interactions of organisms in the environment. Thus, P. pacificus is not only an established model system for evolutionary developmental biology, but also an emerging model system for the evolution of complex life-history traits.

Isolation of pristionchus nematodes from beetles.

INTRODUCTIONIn this procedure, nematodes disembark from a beetle carcass and feed on Escherichia coli OP50. The nematodes are then monitored for a few days and identified using simple morphological characteristics. This method is rapid, easy, and biased for Pristionchus species.

Assessment of the olfactory response to chemicals or bacteria in pristionchus nematodes.

INTRODUCTIONIn the soil environment, nematodes must rely on a number of host-specific chemical cues in order to find potential beetle hosts. They must also discriminate among different food choices (i.e., bacteria), which is important because if the nematodes concentrate on an unsuitable food source, they may die. To detect the bacteria and host-specific chemicals, nematodes use sensory structures called amphids and phasmids that are located on the head and tail, respectively. The olfactory response of nematodes can be studied in the laboratory using the very simple agar-based assay described here. This assay determines the attractiveness of Pristionchus to a range of beetle-associated compounds and has allowed detailed molecular mechanisms of olfaction to be studied in Pristionchus. It can also be used to examine the food choice of these nematodes when they are faced with a number of bacteria.

HAIRY-like transcription factors and the evolution of the nematode vulva equivalence group.

BACKGROUND: Nematode vulva formation provides a paradigm to study the evolution of pattern formation and cell-fate specification. The Caenorhabditis elegans vulva is generated from three of six equipotent cells that form the so-called vulva equivalence group. During evolution, the size of the vulva equivalence group has changed: Panagrellus redivivus has eight, C. elegans six, and Pristionchus pacificus only three cells that are competent to form vulval tissue. In P. pacificus, programmed cell death of individual vulval precursor cells alters the size of the vulva equivalence group. RESULTS: We have identified the genes controlling this cell-death event and the molecular mechanism of the reduction of the vulva equivalence group. Mutations in Ppa-hairy, a gene that is unknown from C. elegans, result in the survival of two precursor cells, which expands the vulva equivalence group. Mutations in Ppa-groucho cause a similar phenotype. Ppa-HAIRY and Ppa-GROUCHO form a molecular module that represses the Hox gene Ppa-lin-39 and thereby reduces the size of the vulva equivalence group. The C. elegans genome does not encode a similar hairy-like gene, and no typical HAIRY/GROUCHO module exists. CONCLUSIONS: We conclude that the vulva equivalence group in Pristionchus is patterned by a HAIRY/GROUCHO module, which is absent in Caenorhabditis. Thus, changes in the number, structure, and function of nematode hairy-like transcription factors are involved in the evolutionary alteration of this equivalence group.

Genetic flexibility in the convergent evolution of hermaphroditism in Caenorhabditis nematodes.

The self-fertile hermaphrodites of C. elegans and C. briggsae evolved from female ancestors by acquiring limited spermatogenesis. Initiation of C. elegans hermaphrodite spermatogenesis requires germline translational repression of the female-promoting gene tra-2, which allows derepression of the three male-promoting fem genes. Cessation of hermaphrodite spermatogenesis requires fem-3 translational repression. We show that C. briggsae requires neither fem-2 nor fem-3 for hermaphrodite development, and that XO Cb-fem-2/3 animals are transformed into hermaphrodites, not females as in C. elegans. Exhaustive screens for Cb-tra-2 suppressors identified another 75 fem-like mutants, but all are self-fertile hermaphrodites rather than females. Control of hermaphrodite spermatogenesis therefore acts downstream of the fem genes in C. briggsae. The outwardly similar hermaphrodites of C. elegans and C. briggsae thus achieve self-fertility via intervention at different points in the core sex determination pathway. These findings are consistent with convergent evolution of hermaphroditism, which is marked by considerable developmental genetic flexibility.