RESUMO
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.
Assuntos
Celulase/biossíntese , Isópteros/metabolismo , Animais , Sequência de Bases , Western Blotting , Celulase/metabolismo , Colorimetria , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Fases de Leitura Aberta , Proteínas Recombinantes/metabolismo , TemperaturaRESUMO
Termites express polyphenism during caste differentiation that is mostly undefined at the molecular level. Using the eastern subterranean termite, Reticulitermes flavipes Kollar, we wanted (1) to test juvenile hormone (JH) model assays for their ability to induce detectable molecular changes in worker termites and (2) to investigate hemolymph proteins and their corresponding genes during JH-induced soldier caste differentiation. Our results illustrate pronounced changes in two hemolymph proteins after JH treatment, as well as differences among several caste phenotypes. Significant increases in the expression of four genes encoding hemolymph proteins, including two vitellogenins and two hexamerins, were observed after JH exposure. These findings are the first to demonstrate such protein and gene expression changes during termite caste differentiation. These results also validate the utility of JH model assays for inducing detectable molecular changes in worker termites that have begun presoldier differentiation.
Assuntos
Regulação da Expressão Gênica/fisiologia , Proteínas de Insetos/análise , Isópteros/fisiologia , Sesquiterpenos/metabolismo , Vitelogeninas/sangue , Animais , Regulação da Expressão Gênica/genética , Hemolinfa , Proteínas de Insetos/genética , Isópteros/genética , Vitelogeninas/genéticaRESUMO
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 single-copy 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.
Assuntos
Chlamydomonas reinhardtii/genética , Inativação Gênica , Animais , Evolução Biológica , Bleomicina/toxicidade , Chlamydomonas reinhardtii/efeitos dos fármacos , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Chlamydomonas reinhardtii/metabolismo , Dano ao DNA , Reparo do DNA , Elementos de DNA Transponíveis/genética , Genes de Plantas , Genes de Protozoários , Metanossulfonato de Metila/toxicidade , Mutação , Supressão Genética , Transcrição GênicaRESUMO
In higher plants, mammals, and filamentous fungi, transcriptional gene silencing is frequently associated with DNA methylation. However, recent evidence suggests that certain transgenes can be inactivated by a methylation independent mechanism. In the unicellular green alga Chlamydomonas reinhardtii, single-copy transgenes are transcriptionally silenced without discernible cytosine methylation of the introduced DNA. We have isolated a Chlamydomonas gene, Mut11, which is required for the transcriptional repression of single-copy transgenes. Mut11 appears to have a global role in gene regulation since it also affects transposon mobilization, cellular growth, and sensitivity to DNA damaging agents. In transient expression assays, a fusion protein between the predicted Mut11 gene product (Mut11p) and E. coli beta-glucuronidase localizes predominantly to the nucleus. Mut11p, a polypeptide of 370 amino acids containing seven WD40 repeats, is highly homologous to proteins of unknown function that are widely distributed among eukaryotes. Mut11p also shows similarity to the C-terminal domain of TUP1, a global transcriptional co-repressor in fungi. Based on these findings we speculate that, in Chlamydomonas, the silencing of certain single-copy transgenes and dispersed transposons integrated into euchromatic regions may occur by a mechanism(s) similar to those involving global transcriptional repressors. Our results also support the existence, in methylation-competent organisms, of a mechanism(s) of transcriptional (trans)gene silencing that is independent of DNA methylation.
Assuntos
Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Animais , Núcleo Celular/metabolismo , Clonagem Molecular , Citoplasma/metabolismo , Inativação Gênica , Genes de Plantas , Genes de Protozoários , Teste de Complementação Genética , Dados de Sequência Molecular , Mutação , Fenótipo , Proteínas de Plantas/química , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Sequências Repetitivas de AminoácidosRESUMO
BACKGROUND: Social insects such as termites express dramatic polyphenism (the occurrence of multiple forms in a species on the basis of differential gene expression) both in association with caste differentiation and between castes after differentiation. We have used cDNA macroarrays to compare gene expression between polyphenic castes and intermediary developmental stages of the termite Reticulitermes flavipes. RESULTS: We identified differentially expressed genes from nine ontogenic categories. Quantitative PCR was used to quantify precise differences in gene expression between castes and between intermediary developmental stages. We found worker and nymph-biased expression of transcripts encoding termite and endosymbiont cellulases; presoldier-biased expression of transcripts encoding the storage/hormone-binding protein vitellogenin; and soldier-biased expression of gene transcripts encoding two transcription/translation factors, two signal transduction factors and four cytoskeletal/muscle proteins. The two transcription/translation factors showed significant homology to the bicaudal and bric-a-brac developmental genes of Drosophila. CONCLUSIONS: Our results show differential expression of regulatory, structural and enzyme-coding genes in association with termite castes and their developmental precursor stages. They also provide the first glimpse into how insect endosymbiont cellulase gene expression can vary in association with the caste of a host. These findings shed light on molecular processes associated with termite biology, polyphenism, caste differentiation and development and highlight potentially interesting variations in developmental themes between termites, other insects, and higher animals.