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Characterization of the termini of linear plasmids from Nectria haematococca and their use in construction of an
1Department of Plant Pathology, USDA, Agricultural Research Service, University of Wisconsin-Madison 53706.
Abstract:
The mitochondria of isolate FS37 from Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contain two linear plasmids, pFSC1 and pFSC2, of 9.2 and 8.3 kbp, respectively. Evidence for a protein blocking the 5' termini of these plasmids was obtained from exonuclease digestion experiments. A single protein band with an apparent Mr of 80 K was labeled when the DNA-protein complex of either plasmid was reacted with [125I] Bolton-Hunter reagent and then digested with DNase I. DNA sequence analysis of the termini of both plasmids revealed long inverted repeats of 1,211 bp (pFSC1) and 1,027 bp (pFSC2). No sequence similarity was found between the terminal inverted repeats (TIRs) of pFSC1 and pFSC2, nor was any similarity identified between the TIRs of these plasmids and sequences of TIRs from other linear DNAs. A restriction fragment containing the TIR of pFSC1 conferred autonomous replication when incorporated into an integrative transformation vector of Ustilago maydis.
Insights
Mitochondrial linear plasmids in Nectria haematococca were found to have unique 5' end protein caps and terminal inverted repeats (TIRs). These TIRs enable autonomous replication in other organisms, suggesting potential for genetic engineering.
Area of Science:
- Molecular Biology
- Mycology
- Genetics
Background:
- Mitochondria harbor extrachromosomal DNA elements, including linear plasmids, in various organisms.
- Nectria haematococca mating population I (Fusarium solani f. sp. cucurbitae) contains two distinct linear mitochondrial plasmids, pFSC1 and pFSC2.
Purpose of the Study:
- To characterize the structure and properties of the linear mitochondrial plasmids pFSC1 and pFSC2 from Nectria haematococca.
- To investigate the nature of the 5' termini of these plasmids and their potential for replication.
Main Methods:
- Exonuclease digestion experiments to detect 5' end blocking.
- Radioactive labeling of DNA-binding proteins followed by DNase I digestion.
- DNA sequencing of plasmid termini to identify repeat structures.
- Functional analysis of a terminal inverted repeat (TIR) in a heterologous transformation system.
Main Results:
- Evidence of a protein cap at the 5' termini of both pFSC1 and pFSC2.
- Identification of long, unique terminal inverted repeats (TIRs) of 1,211 bp (pFSC1) and 1,027 bp (pFSC2).
- A restriction fragment containing the pFSC1 TIR conferred autonomous replication in Ustilago maydis.
Conclusions:
- The linear mitochondrial plasmids of Nectria haematococca possess unique structural features, including 5' end protein association and distinct TIRs.
- The identified TIRs possess functional replication origins, demonstrating potential for use in genetic manipulation and heterologous expression systems.