Target-dependent suppression of siRNA production modulates the levels of endogenous siRNAs in the Caenorhabditis elegans germline.

Despite the prominent role of endo-siRNAs in transposon silencing, their expression is not limited to these 'nonself' DNA elements. Transcripts of protein-coding genes ('self' DNA) in some cases also produce endo-siRNAs in yeast, plants and animals. How cells distinguish these two populations of siRNAs to prevent unwanted silencing of active genes in animals is not well understood. To address this question, we inserted various self-gene or gfp fragments into an LTR retrotransposon that produces abundant siRNAs and examined the propensity of these gene fragments to produce ectopic siRNAs in the Caenorhabditis elegans germline. We found that fragments of germline genes are generally protected from production of ectopic siRNAs. This phenomenon, which we termed 'target-directed suppression of siRNA production' (or siRNA suppression), is dependent on the germline expression of target mRNA and requires germline P-granule components. We found that siRNA suppression can also occur in naturally produced endo-siRNAs. We suggest that siRNA suppression plays an important role in regulating siRNA expression and preventing self-genes from aberrant epigenetic silencing. This article has an associated 'The people behind the papers' interview.

A Simple Apparatus for Individual C. elegans Culture.

We miniaturized standard, solid-phase C. elegans culture conditions to produce a system in which many isolated, individual C. elegans can be housed throughout their lives. This system, the "worm corral," is compatible with high-resolution brightfield and fluorescent microscopy, allowing imaging of fluorescent transgenes and morphological phenotypes from hatch until death. These culture devices can be constructed on the benchtop with commercially available reagents and standard laboratory equipment, making this an attainable solution for most labs.

Use of the HPRT gene to study nuclease-induced DNA double-strand break repair.

Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases. For I-SceI, our estimated frequencies of accurate or mutagenic non-homologous end-joining and gene correction by homologous recombination are 4.1, 1.5 and 0.16%, respectively. Unexpectedly, I-SceI and Cas9 induced markedly different DSBR profiles. Also, using an I-SceI-sensitive HPRT minigene, we show that gene correction is more efficient when using long double-stranded DNA than single- or double-stranded oligonucleotides. Finally, using both endogenous HPRT and exogenous reporters, we validate novel cell cycle phase-specific I-SceI derivatives for investigating cell cycle variations in DSBR. The results obtained using these novel approaches provide new insights into template design for gene correction and the relationships between multiple DSBR pathways at a single endogenous disease gene.

Dimerization and comments on the reactivity of homophthalic anhydride.

Homophthalic anhydride (HPA) dimerizes under the influence of base to provide, sequentially, the (3-4')-C-acyl dimer, a pair of chiral diastereomeric bis(lactones), 3-(2-carboxybenzyl)isocoumarin-4-carboxylic acid, and finally, 3-(2-carboxybenzyl)isocoumarin. The structures of the bis(lactones) were misassigned in 1970 based on the (presumed) cis thermal decarboxylative elimination reaction of the lower melting one. The preferred pathway should be trans-anti, however, and crystallographic analysis of one of the bis(lactones) reverses the earlier assignment. The formal cycloaddition reaction of HPA with imines occurs in preference to HPA dimerization; the mechanistic implications of this reactivity difference are discussed.

A set of aspartyl protease-deficient strains for improved expression of heterologous proteins in Kluyveromyces lactis.

Secretion of recombinant proteins is a common strategy for heterologous protein expression using the yeast Kluyveromyces lactis. However, a common problem is degradation of a target recombinant protein by secretory pathway aspartyl proteases. In this study, we identified five putative pfam00026 aspartyl proteases encoded by the K. lactis genome. A set of selectable marker-free protease deletion mutants was constructed in the prototrophic K. lactis GG799 industrial expression strain background using a PCR-based dominant marker recycling method based on the Aspergillus nidulans acetamidase gene (amdS). Each mutant was assessed for its secretion of protease activity, its health and growth characteristics, and its ability to efficiently produce heterologous proteins. In particular, despite having a longer lag phase and slower growth compared with the other mutants, a Δyps1 mutant demonstrated marked improvement in both the yield and the quality of Gaussia princeps luciferase and the human chimeric interferon Hy3, two proteins that experienced significant proteolysis when secreted from the wild-type parent strain.

Identification of new human cadherin genes using a combination of protein motif search and gene finding methods.

We have combined protein motif search and gene finding methods to identify genes encoding proteins containing specific domains. Particularly, we have focused on finding new human genes of the cadherin superfamily proteins, which represent a major group of cell-cell adhesion receptors contributing to embryonic neuronal morphogenesis. Models for three cadherin protein motifs were generated from over 100 already annotated cadherin domains and used to search the complete translated human genome. The genomic sequence regions containing motif "hits" were analyzed by eukaryotic GeneMark.hmm to identify the exon-intron structure of new genes. Three new genes CDH-J, PCDH-J and FAT-J were found. The predicted proteins PCDH-J and FAT-J were classified into protocadherin and FAT-like subfamilies, respectively, based on the number and organization of cadherin domains and presence of subfamily-specific conserved amino acid residues. Expression of FAT-J was shown in almost all tested tissues. The exon-intron organization of CDH-J was experimentally verified by PCR with specifically designed primers and its tissue-specific expression was demonstrated. The described methodology can be applied to discover new genes encoding proteins from families with well-characterized structural and functional domains.