The Amipurimycin and Miharamycin Biosynthetic Gene Clusters: Unraveling the Origins of 2-Aminopurinyl Peptidyl Nucleoside Antibiotics.

Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds have tripartite structures composed of a core saccharide, a nucleobase, and one or more amino acids. In particular, amipurimycin and the miharamycins are novel 2-aminopurinyl PNAs with complex nine-carbon core saccharides and include the unusual amino acids (-)-cispentacin and N5-hydroxyarginine, respectively. Despite their interesting structures and properties, these PNAs have heretofore eluded biochemical scrutiny. Herein is reported the discovery and initial characterization of the miharamycin gene cluster in Streptomyces miharaensis (mhr) and the amipurimycin gene cluster (amc) in Streptomyces novoguineensis and Streptomyces sp. SN-C1. The gene clusters were identified using a comparative genomics approach, and heterologous expression of the amc cluster as well as gene interruption experiments in the mhr cluster support their role in the biosynthesis of amipurimycin and the miharamycins, respectively. The mhr and amc biosynthetic gene clusters characterized encode enzymes typical of polyketide biosynthesis instead of enzymes commonly associated with PNA biosynthesis, which, along with labeled precursor feeding studies, implies that the core saccharides found in the miharamycins and amipurimycin are partially assembled as polyketides rather than derived solely from carbohydrates. Furthermore, in vitro analysis of Mhr20 and Amc18 established their roles as ATP-grasp ligases involved in the attachment of the pendant amino acids found in these PNAs, and Mhr24 was found to be an unusual hydroxylase involved in the biosynthesis of N5-hydroxyarginine. Finally, analysis of the amc cluster and feeding studies also led to the proposal of a biosynthetic pathway for (-)-cispentacin.

Analysis of Gal4 Expression Patterns in Adult Drosophila Females.

Precise genetic manipulation of specific cell types or tissues to pinpoint gene function requirement is a critical step in studies aimed at unraveling the intricacies of organismal physiology. Drosophila researchers heavily rely on the UAS/Gal4/Gal80 system for tissue-specific manipulations; however, it is often unclear whether the reported Gal4 expression patterns are indeed specific to the tissue of interest such that experimental results are not confounded by secondary sites of Gal4 expression. Here, we surveyed the expression patterns of commonly used Gal4 drivers in adult Drosophila female tissues under optimal conditions and found that multiple drivers have unreported secondary sites of expression beyond their published cell type/tissue expression pattern. These results underscore the importance of thoroughly characterizing Gal4 tools as part of a rigorous experimental design that avoids potential misinterpretation of results as we strive for understanding how the function of a specific gene/pathway in one tissue contributes to whole-body physiology.

RNAi-based screens uncover a potential new role for the orphan neuropeptide receptor Moody in Drosophila female germline stem cell maintenance.

Reproduction is highly sensitive to changes in physiology and the external environment. Neuropeptides are evolutionarily conserved signaling molecules that regulate multiple physiological processes. However, the potential reproductive roles of many neuropeptide signaling pathways remain underexplored. Here, we describe the results of RNAi-based screens in Drosophila melanogaster to identify neuropeptides/neuropeptide receptors with potential roles in oogenesis. The screen read-outs were either the number of eggs laid per female per day over time or fluorescence microscopy analysis of dissected ovaries. We found that the orphan neuropeptide receptor encoded by moody (homologous to mammalian melatonin receptors) is likely required in somatic cells for normal egg production and proper germline stem cell maintenance. However, the egg laying screens had low signal-to-noise ratio and did not lead to the identification of additional candidates. Thus, although egg count assays might be useful for large-scale screens to identify oogenesis regulators that result in dramatic changes in oogenesis, more labor-intensive microscopy-based screen are better applicable for identifying new physiological regulators of oogenesis with more subtle phenotypes.