Plasmid reorganization during integrative transformation in Hansenula polymorpha

Area of Science:

• Molecular Biology
• Yeast Genetics
• Biotechnology

Background:

• Integrative transformation in Hansenula polymorpha using plasmids with the LEU2 gene often resulted in instability and plasmid loss on non-selective media.
• Transformants exhibited reorganized plasmids capable of replication within H. polymorpha.

Purpose of the Study:

• To investigate the molecular basis of plasmid instability and reorganization during H. polymorpha transformation.
• To characterize the DNA segments involved in plasmid reorganization and their functional role.

Main Methods:

• Isolation and characterization of reorganized plasmids from unstable H. polymorpha transformants.
• Southern hybridization analysis using DNA probes derived from additional DNA segments.
• Sequence analysis of captured chromosomal fragments.

Main Results:

• Reorganized plasmids contained additional H. polymorpha DNA segments homologous to multiple chromosomal regions.
• These segments functioned as H. polymorpha autonomous replicating sequences (HARS), conferring high transformation efficiency and autonomous replication.
• Sequence analysis revealed AT-rich blocks within the captured fragments, characteristic of HARS.

Conclusions:

• Plasmid instability in H. polymorpha transformants is caused by the capture of chromosomal DNA fragments containing HARS.
• These captured HARS enable autonomous plasmid replication, leading to plasmid maintenance.
• Similar reorganization events were observed with other selectable markers and yeast species.