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Structure of Physarum actin gene locus ardA: a nonpalindromic sequence causes inviability of phage lambda and
Abstract:
Previously we reported that approx. 80% of the genome from the plasmodial slime mold Physarum polycephalum, including all the actin genes, can be cloned only in recBC- sbcB- Escherichia coli hosts [Nader et al., Proc. Natl. Acad. Sci. USA 82 (1985) 2698-2702]. We have now sequenced the actin gene locus ardA. The nucleotide sequence of its coding region is flanked by the typical putative regulatory sequences for transcription initiation and polyadenylation. The coding region is interrupted by five introns, all located at novel positions with regard to those of previously analysed actin genes. Within the ardA gene we have located a 360-bp fragment which comprises exon V and parts of its flanking introns. This region suppresses plaque formation of recombinant lambda phages and causes recA-independent deletions in phages and plasmids. In contrast to our previous hypothesis, this sequence is not a DNA palindrome, but consists of five (dA) X (dT)- and (dG) X (dC)-homopolymers. Both termination of replication and partial unwinding of duplex DNA under torsional stress were detected within the unstable 360-bp region in vitro.
Insights
Physarum polycephalum actin gene ardA sequencing revealed novel intron positions. A 360-bp unstable region within the gene, composed of homopolymers, causes DNA deletions and replication termination in vitro.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Previous work showed Physarum polycephalum genome cloning requires specific E. coli hosts.
- Actin genes are crucial for cellular structure and function.
Purpose of the Study:
- To sequence the Physarum polycephalum actin gene locus ardA.
- To characterize a novel unstable DNA region within the ardA gene.
Main Methods:
- DNA sequencing of the ardA gene.
- In vitro assays to study DNA replication and stability.
Main Results:
- The ardA gene coding region contains five introns at novel positions.
- A 360-bp fragment within ardA, comprising homopolymers, suppresses phage plaque formation.
- This fragment induces recA-independent deletions in phages and plasmids.
- In vitro studies detected replication termination and DNA unwinding within the unstable region.
Conclusions:
- The ardA gene structure in Physarum polycephalum differs significantly from other known actin genes.
- The identified unstable homopolymer region has implications for DNA replication and genome stability.