Isolation and characterization of an autonomously replicating sequence from Ustilago maydis

Area of Science:

• Molecular Biology
• Genetics
• Mycology

Background:

• Autonomously replicating sequences (ARSs) are crucial for plasmid replication in eukaryotic organisms.
• Understanding ARS function in non-conventional yeasts like Ustilago maydis is essential for advancing genetic manipulation techniques.

Purpose of the Study:

• To isolate and characterize autonomously replicating sequences (ARSs) from the fungus Ustilago maydis.
• To evaluate the efficiency of ARS-containing DNA fragments in enhancing Ustilago maydis transformation.
• To analyze the structural features of a specific U. maydis ARS (UARS1) and its subfragments.

Main Methods:

• Isolation of DNA fragments conferring ARS activity from Ustilago maydis.
• Cloning ARS fragments into an integrative transforming vector to assess transformation efficiency.
• Analysis of plasmid stability and copy number in U. maydis.
• Subcloning and characterization of the UARS1 ARS element.

Main Results:

• Isolated DNA fragments dramatically increased Ustilago maydis transformation efficiency by several thousandfold.
• ARS-containing plasmids were maintained as extrachromosomal elements, replicating autonomously within U. maydis cells.
• The characterized ARS, UARS1, was localized to a 1.7-kilobase fragment and could be reduced to a 383 bp fragment retaining ARS activity.
• The minimal ARS fragment contained direct repeats and sequence motifs similar to those in Saccharomyces cerevisiae ARSs.

Conclusions:

• DNA fragments functioning as ARSs have been successfully isolated from Ustilago maydis.
• These ARSs are effective in enhancing transformation and enabling stable, albeit mitotically unstable, extrachromosomal replication in U. maydis.
• Structural analysis of UARS1 reveals conserved features with ARSs from other yeasts, providing insights into ARS function.