om92 , a glp-1 enhancer mutation, is an allele of ekl-1.

Germline stem cell proliferation in C. elegans requires activation of the GLP-1/Notch receptor, which is located on the germline plasma membrane and encoded by the glp-1 gene. We previously identified several genes whose products directly or indirectly promote activity of the GLP-1 signaling pathway by finding mutations that enhance the germline phenotype of a glp-1(ts) allele, glp-1(bn18) . Here, we report phenotypic and molecular analysis of a new ekl-1 allele, ekl-1(om92) , that enhances the glp-1(bn18) phenotype. ekl-1(om92) is a 244 bp deletion predicted to generate a frameshift and premature termination codon, yielding a severely truncated protein, suggesting it is a null allele.

Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in E. coli.

Fluorescent proteins related to and derived from green fluorescent protein (GFP) are widely used as tools for investigating a wide range of biological processes. In particular, GFP and its relatives have been used extensively as qualitative reporters of gene expression in many different organisms, but relatively few studies have investigated fluorescent proteins as quantitative reporters of gene expression. GFP has some limitations as a reporter gene, including possible toxicity when expressed at high levels. Therefore, it would be useful if other fluorescent proteins could be identified for use as quantitative reporters. Toward this end, we investigated BFP as a quantitative reporter of promoter activity in E. coli and directly compared it with GFPuv using a set of well-characterized synthetic constitutive promoters. The fluorescence produced in E. coli strains expressing GFPuv or BFP grown on solid medium was quantified using a CCD camera and fluorimetry. GFPuv consistently gave more reliable and statistically significant results than did BFP in all assays. Correspondingly, we found that the signal-to-noise ratio for GFPuv fluorescence is substantially higher than for BFP. We conclude that, under the conditions assessed in this study, GFPuv is superior to BFP as a quantitative reporter of promoter activity in E. coli.

The Molecular Chaperone HSP90 Promotes Notch Signaling in the Germline of Caenorhabditis elegans.

In a genetic screen to identify genes that promote GLP-1/Notch signaling in Caenorhabditis elegans germline stem cells, we found a single mutation, om40, defining a gene called ego-3. ego-3(om40) causes several defects in the soma and the germline, including paralysis during larval development, sterility, delayed proliferation of germline stem cells, and ectopic germline stem cell proliferation. Whole genome sequencing identified om40 as an allele of hsp-90, previously known as daf-21, which encodes the C. elegans ortholog of the cytosolic form of HSP90. This protein is a molecular chaperone with a central position in the protein homeostasis network, which is responsible for proper folding, structural maintenance, and degradation of proteins. In addition to its essential role in cellular function, HSP90 plays an important role in stem cell maintenance and renewal. Complementation analysis using a deletion allele of hsp-90 confirmed that ego-3 is the same gene. hsp-90(om40) is an I→N conservative missense mutation of a highly conserved residue in the middle domain of HSP-90 RNA interference-mediated knockdown of hsp-90 expression partially phenocopied hsp-90(om40), confirming the loss-of-function nature of hsp-90(om40) Furthermore, reduced HSP-90 activity enhanced the effect of reduced function of both the GLP-1 receptor and the downstream LAG-1 transcription factor. Taken together, our results provide the first experimental evidence of an essential role for HSP90 in Notch signaling in development.

Isolation of Caenorhabditis elegans genomic DNA and detection of deletions in the unc-93 gene using PCR.

PCR, genomic DNA isolation, and agarose gel electrophoresis are common molecular biology techniques with a wide range of applications. Therefore, we have developed a series of exercises employing these techniques for an intermediate level undergraduate molecular biology laboratory course. In these exercises, students isolate genomic DNA from the nematode Caenorhabditis elegans and use PCR to detect deletions in the C. elegans unc-93 gene. In advance of the exercises, wild-type and three different unc-93 deletion mutant strains are grown, harvested, and frozen by the instructor. In one approach, students isolate genomic DNA from each strain using a genomic DNA isolation kit and use agarose gel electrophoresis to analyze the DNA and to estimate its concentration. PCRs using primers directed to two different regions of the unc-93 gene are carried out on the genomic DNA from wild-type and mutant strains, and the PCR products are analyzed by agarose gel electrophoresis. Students analyze the gel to determine the approximate location and size of deletions in the three mutant strains. Alternatively, students may lyse single nematodes and carry out PCR in one laboratory session. These exercises should be easily adaptable to detection of well characterized deletions in any organism.

Linkage of genetics and ethics: more crossing over is needed.

Since the development of recombinant DNA technology in the mid 1970s, there has been increasing interest in the ethical, legal, and social implications of genetics and related fields. The web sites of five different organizations (government, academic, and independent not-for-profit) that deal explicitly with genetics and ethics are reviewed here. Some of the sites cover genetics and other issues in bioethics while others cover human genetics exclusively. The target audiences for the sites include medical and scientific professionals, students, and the general public. Among the issues examined are genetic testing, genetic discrimination in employment and health insurance, genetically modified foods, stem cells and DNA patenting. Resources for those interested in legal issues are particularly well-represented on these sites.