The molecular mechanism(s) responsible for posttranscriptional gene silencing and RNA interference remain poorly understood. We have cloned a gene ( Mut6 ) from the unicellular green alga Chlamydomonas reinhardtii that is required for the silencing of a transgene and two transposon families. Mut6 encodes a protein that is highly homologous to RNA helicases of the DEAH-box family. This protein is necessary for the degradation of certain aberrant RNAs, such as improperly processed transcripts, which are often produced by transposons and some transgenes.

Array-based genomic studies were conducted with the goal of identifying immature (i.e. nymph) and adult reproductive caste-biased gene expression in the termite Reticulitermes flavipes. Using cDNA macro-arrays, we identified thirty-four nymph-biased genes falling into eight ontogenic categories. Based on gene expression profiles among diverse castes and developmental stages (determined by quantitative PCR), several important trends emerged. These findings highlight the importance of several developmental and survival-based factors among immature and adult termite reproductives, including: vitellogenesis, nutrient storage, juvenile hormone sequestration, ribosomal translational and filtering mechanisms, fatty acid biosynthesis, apoptosis inhibition, and both endogenous and symbiont cellulase-assisted nutrition. These findings are highly significant as they are the first to elucidate the molecular biology underlying termite reproductive caste differentiation and reproductive caste-specific biology. Other gene expression results are in agreement with previous findings that suggest roles for vitellogenin-like haemolymph proteins in soldier caste differentiation.

In the unicellular green alga Chlamydomonas reinhardtii, the epigenetic silencing of transgenes occurs, as in land plants, at both the transcriptional and posttranscriptional levels. In the case of singlecopy transgenes, transcriptional silencing takes place without detectable cytosine methylation of the introduced DNA. We have isolated two mutant strains, Mut-9 and Mut-11, that reactivate expression of a transcriptionally silenced single-copy transgene. These suppressors are deficient in the repression of a DNA transposon and a retrotransposon-like element. In addition, the mutants show enhanced sensitivity to DNA-damaging agents, particularly radiomimetic chemicals inducing DNA double-strand breaks. All of these phenotypes are much more prominent in a double mutant strain. These observations suggest that multiple partly redundant epigenetic mechanisms are involved in the repression of transgenes and transposons in eukaryotes, presumably as components of a system that evolved to preserve genomic stability. Our results also raise the possibility of mechanistic connections between epigenetic transcriptional silencing and DNA double-strand break repair.

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Cell-1 is a host-derived beta-1,4-endoglucanase (Glycohydrolase Family 9 [GHF9]) from the lower termite Reticulitermes flavipes. Here, we report on the heterologous production of Cell-1 using eukaryotic (Baculovirus Expression Vector System; BEVS) and prokaryotic (E. coli) expression systems. The BEVS-expressed enzyme was more readily obtained in solubilized form and more active than the E. coli-expressed enzyme. K(m) and V(max) values for BEVS-expressed Cell-1 against the model substrate CMC were 0.993% w/v and 1.056 micromol/min/mg. Additional characterization studies on the BEVS-expressed enzyme revealed that it possesses activity comparable to the native enzyme, is optimally active around pH 6.5-7.5 and 50-60 degrees C, is inhibited by EDTA, and displays enhanced activity up to 70 degrees C in the presence of CaCl(2). These findings provide a foundation on which to begin subsequent investigations of collaborative digestion by coevolved host and symbiont digestive enzymes from R. flavipes that include GHF7 exoglucanases, GHF1 beta glucosidases, phenol-oxidizing laccases, and others.