FLP-Mapping: a universal, cost-effective, and automatable method for gene mapping.

Genetic mapping with DNA sequence polymorphisms allows for map-based positional cloning of mutations at any required resolution. Numerous methods have been worked out to assay single nucleotide polymorphisms (SNPs), the most common type of molecular polymorphisms. However, SNP genotyping requires customized and often costly secondary assays on primary PCR products. Small insertions and deletions (InDels) are a class of polymorphisms that are ubiquitously dispersed in eukaryotic genomes and only about fourfold less frequent than SNPs. InDels can be directly and universally detected as fragment length polymorphisms (FLPs) of primary PCR fragments, thus eliminating the need for an expensive secondary genotyping protocol. Genetic mapping with FLPs is suited for both small-scale and automated high-throughput approaches. Two techniques best suited for either strategy and both offering very high to maximal fragment-size resolution are discussed: Analysis on nondenaturing Elchrom gels and on capillary sequencers. Here, we exemplify FLP-mapping for the model organisms Drosophila melanogaster and Caenorhabditis elegans. FLP-mapping can, however, be easily adapted for any other organism as the molecular biology involved is universal. Furthermore, we introduce FLP mapper, a JAVA-based software for graphical visualization of raw mapping data from spreadsheets. FLP mapper is publicly available at http://bio.mcsolutions.ch.

Wntless, a conserved membrane protein dedicated to the secretion of Wnt proteins from signaling cells.

Cell-cell communication via Wnt signals represents a fundamental means by which animal development and homeostasis are controlled. The identification of components of the Wnt pathway is reaching saturation for the transduction process in receiving cells but is incomplete concerning the events occurring in Wnt-secreting cells. Here, we describe the discovery of a novel Wnt pathway component, Wntless (Wls/Evi), and show that it is required for Wingless-dependent patterning processes in Drosophila, for MOM-2-governed polarization of blastomeres in C. elegans, and for Wnt3a-mediated communication between cultured human cells. In each of these cases, Wls is acting in the Wnt-sending cells to promote the secretion of Wnt proteins. Since loss of Wls function has no effect on other signaling pathways yet appears to impede all the Wnt signals we analyzed, we propose that Wls represents an ancient partner for Wnts dedicated to promoting their secretion into the extracellular milieu.

Drosophila sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways.

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) lead to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. Here, we show that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks.

A universal method for automated gene mapping.

Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost.

A reverse genetic screen in Drosophila using a deletion-inducing mutagen.

We report the use of the cross-linking drug hexamethylphosphoramide (HMPA), which introduces small deletions, as a mutagen suitable for reverse genetics in the model organism Drosophila melanogaster. A compatible mutation-detection method based on resolution of PCR fragment-length polymorphisms on standard DNA sequencers is implemented. As the spectrum of HMPA-induced mutations is similar in a variety of organisms, it should be possible to transfer this mutagenesis and detection procedure to other model systems.

Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2.

Centrosomes are major determinants of mitotic spindle structure, but the mechanisms regulating their behavior remain poorly understood. The spd-2 gene of C. elegans is required for centrosome assembly or "maturation." Here we show that spd-2 encodes a coiled-coil protein that localizes within pericentriolar material (PCM) and in the immediate vicinity of centrioles. During maturation, SPD-2 gradually accumulates at the centrosome in a manner that is partially dependent on Aurora-A kinase and cytoplasmic dynein. Interestingly, SPD-2 interacts genetically with dynein heavy chain and SPD-5, another coiled-coil protein required for centrosome maturation. SPD-2 and SPD-5 are codependent for localization to the PCM, but SPD-2 localizes to centrioles independently of SPD-5. Surprisingly, we also find that SPD-2 is required for centrosome duplication and genetically interacts with ZYG-1, a kinase required for duplication. Thus, we have identified SPD-2 as a factor critical for the two basic functions of the centrosome-microtubule organization and duplication.

Systematic functional analysis of the Caenorhabditis elegans genome using RNAi.

A principal challenge currently facing biologists is how to connect the complete DNA sequence of an organism to its development and behaviour. Large-scale targeted-deletions have been successful in defining gene functions in the single-celled yeast Saccharomyces cerevisiae, but comparable analyses have yet to be performed in an animal. Here we describe the use of RNA interference to inhibit the function of approximately 86% of the 19,427 predicted genes of C. elegans. We identified mutant phenotypes for 1,722 genes, about two-thirds of which were not previously associated with a phenotype. We find that genes of similar functions are clustered in distinct, multi-megabase regions of individual chromosomes; genes in these regions tend to share transcriptional profiles. Our resulting data set and reusable RNAi library of 16,757 bacterial clones will facilitate systematic analyses of the connections among gene sequence, chromosomal location and gene function in C. elegans.