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Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum.

ABSTRACT: The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In many eukaryotic systems this challenge has been addressed through polycistronic expression mediated by 2A viral peptides, permitting efficient, co-regulated gene expression. Here, we screen the most commonly used 2A peptides, porcine teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), and foot-and-mouth disease virus 2A (F2A), for activity in D. discoideum and find that all the screened 2A sequences are effective. However, combining the coding sequences of two proteins into a single transcript leads to notable strain-dependent decreases in expression level, suggesting additional factors regulate gene expression in D. discoideum that merit further investigation. Our results show that P2A is the optimal sequence for polycistronic expression in D. discoideum, opening up new possibilities for genetic engineering in this model system.

SUBMITTER: Zhu X

PROVIDER: S-EPMC9980757 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum.

Zhu Xinwen X Ricci-Tam Chiara C Hager Emily R ER Sgro Allyson E AE

PloS one 20230302 3

The social amoeba Dictyostelium discoideum is a model for a wide range of biological processes including chemotaxis, cell-cell communication, phagocytosis, and development. Interrogating these processes with modern genetic tools often requires the expression of multiple transgenes. While it is possible to transfect multiple transcriptional units, the use of separate promoters and terminators for each gene leads to large plasmid sizes and possible interference between units. In many eukaryotic sy ...[more]

PMID: 36862626

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Project description:BackgroundDictyostelium discoideum, a microbial model for social evolution, is known to distinguish self from non-self and show genotype-dependent behavior during chimeric development. Aside from a small number of cell-cell recognition genes, however, little is known about the genetic basis of self/non-self recognition in this species. Based on the key hypothesis that there should be differential expression of genes if D. discoideum cells were interacting with non-clone mates, we performed transcriptomic profiling study in this species during clonal vs. chimeric development. The transcriptomic profiles of D. discoideum cells in clones vs. different chimeras were compared at five different developmental stages using a customized microarray. Effects of chimerism on global transcriptional patterns associated with social interactions were observed.ResultsWe find 1,759 genes significantly different between chimera and clone, 1,144 genes associated significant strain differences, and 6,586 genes developmentally regulated over time. Principal component analysis showed a small amount of the transcriptional variance to chimerism-related factors (Chimerism: 0.18%, Chimerism × Timepoint: 0.03%). There are 162 genes specifically regulated under chimeric development, with continuous small differences between chimera vs. clone over development. Almost 60% of chimera-associated differential genes were differentially expressed at the 4 h aggregate stage, which corresponds to the initial transition of D. discoideum from solitary life to a multicellular phase.ConclusionsA relatively small proportion of over-all variation in gene expression is explained by differences between chimeric and clonal development. The relatively small modifications in gene expression associated with chimerism is compatible with the high level of cooperation observed among different strains of D. discoideum; cells of distinct genetic backgrounds will co-aggregate indiscriminately and co-develop into fruiting bodies. Chimeric development may involve re-programming of the transcriptome through small modifications of the developmental genetic network, which may also indicate that response to social interaction involves many genes with individually small transcriptional effect.

Dataset Information

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum.

Publications

Self-cleaving peptides for expression of multiple genes in Dictyostelium discoideum.

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