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Trichoderma reesei sequences that bind to the nuclear matrix enhance transformation frequency
N J Belshaw1, S Hakola, H Nevalainen
1Institute of Food Research, Norwich, UK.
Molecular & General Genetics : MGG
|October 28, 1997
Summary
Three Trichoderma reesei DNA fragments, identified as matrix attachment regions (MARs), enhance integrative transformation frequency in T. reesei. These MARs do not appear to promote plasmid replication but offer a mechanism for improved transformation efficiency.
Area of Science:
- Molecular Biology
- Genetics
- Mycology
Background:
- The Trichoderma reesei genome was investigated for elements that could facilitate plasmid manipulation.
- Nuclear matrix attachment regions (MARs) are crucial for genome organization and DNA replication.
- Understanding MARs in T. reesei can aid in genetic engineering of this industrially important fungus.
Purpose of the Study:
- To isolate and characterize DNA elements from T. reesei that promote autonomous plasmid replication in Saccharomyces cerevisiae.
- To determine if these elements function as matrix attachment regions (MARs) in T. reesei.
- To assess the effect of these MARs on the integrative transformation frequency of T. reesei.
Main Methods:
- Isolation of DNA fragments (trs1, 2, 3) from T. reesei based on their ability to promote plasmid replication in S. cerevisiae.
- In vitro and in vivo binding assays to identify MARs by their specific interaction with the T. reesei nuclear matrix.
- Sequence analysis of identified MARs to determine their A+T content and identify conserved motifs.
- Transformation experiments in T. reesei using plasmids with and without the identified MARs.
Main Results:
- Three DNA fragments (trs1, 2, and 3) were isolated and identified as matrix attachment regions (MARs) in T. reesei.
- These MARs are AT-rich sequences with no significant homology but share motifs common to eukaryotic MARs.
- The presence of trs elements in plasmids significantly enhanced the integrative transformation frequency of T. reesei by up to fivefold.
- No evidence supported the hypothesis that these MARs promote efficient plasmid replication in T. reesei.
Conclusions:
- The isolated T. reesei trs elements function as MARs and are involved in enhancing integrative transformation efficiency.
- A mechanism for MAR-mediated enhancement of transformation frequency in T. reesei is proposed.
- These findings provide valuable tools for improving genetic manipulation of T. reesei.