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Direct transfer of coliphage lambda DNA from Escherichia coli to cellular slime mold Dictyostelium discoideum
Abstract:
Dictyostelium discoideum myxamoebae were cultured with Escherichia coli cells infected with lambda phage in the presence of chloramphenicol. After eliminating the uningested bacteria by repeated centrifugation in a Percoll gradient, we examined the myxamoeba cytoplasm (not the food vacuole) for the presence of phage DNA. A significant amount of DNA extracted from the myxamoebae was hybridizable with purified phage lambda DNA, and capable of forming phage particles when packaged in vitro with phage lambda proteins. The EcoRI restriction maps of the phages recovered from the plaques were identical to that of the infecting phage. These results strongly suggest that phage DNA molecules were taken up by the cellular slime mold cells and that at least some fraction existed in intact form.
Insights
Dictyostelium discoideum cells took up intact lambda phage DNA from bacteria. This DNA was functional, forming new phage particles, indicating successful cellular uptake and persistence.
Area of Science:
- Molecular Biology
- Cell Biology
- Microbiology
Background:
- Dictyostelium discoideum is a model organism for studying cellular processes.
- Bacteriophages are viruses that infect bacteria and have potential applications in biotechnology.
- Understanding DNA uptake mechanisms in eukaryotic cells is crucial for gene delivery and synthetic biology.
Purpose of the Study:
- To investigate the uptake and integrity of lambda phage DNA within Dictyostelium discoideum myxamoebae.
- To determine if internalized phage DNA can remain intact and functional within the cellular slime mold.
Main Methods:
- Culturing Dictyostelium discoideum with Escherichia coli infected with lambda phage.
- Separating myxamoebae from uningested bacteria using Percoll gradient centrifugation.
- Extracting DNA from myxamoebae and assessing its hybridizability with lambda phage DNA.
- Packaging extracted DNA in vitro to assess phage particle formation.
- Analyzing recovered phages using EcoRI restriction mapping.
Main Results:
- A significant amount of DNA from myxamoebae hybridized with lambda phage DNA.
- DNA extracted from myxamoebae was capable of forming functional phage particles when packaged with phage proteins.
- EcoRI restriction maps of recovered phages were identical to the infecting phage, confirming DNA integrity.
Conclusions:
- Dictyostelium discoideum myxamoebae are capable of taking up intact lambda phage DNA.
- Internalized phage DNA can persist in a functional form within the cellular slime mold cytoplasm.
- This study demonstrates a potential mechanism for DNA delivery into eukaryotic cells using bacteriophages.