Amplification of the yeast nuclear gene MRS3 confers suppression of a mitochondrial RNA splice defect
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T Söllner1, C Schmidt, C Schmelzer
1Institut für Genetik und Mikrobiologie, Universität München, Federal Republic of Germany.
Abstract:
The MRS3 gene cloned in the multicopy plasmid YEp13 suppresses the mitochondrial splice defect exerted by mutation M1301 in the group II intron bI1. In this article we report on the behavior of the MRS3 gene cloned in the integration vector pEMBLYi27 and in the CEN4-ARS vector YCp50. Transformation of mutant M1301 cells with these recombinant vectors produced transformants, the majority of which showed the original splice defect and contained the recombinant vectors in single or low copy; a minority, however, was splicing competent and showed exceptionally high copy numbers of the MRS3 gene. These latter transformants had either the pEMBLYi27/MRS3 sequence repeated at least 20 times in tandem at the chromosomal site of the MRS3 gene or they had the YCp50/MRS3 sequence established as a multicopy plasmid lacking the copy number control usually exerted by the CEN4 sequence in this plasmid.
Insights
High copy numbers of the MRS3 gene are required to suppress mitochondrial splice defects. This study investigated MRS3 gene behavior in different yeast vectors, revealing copy number is critical for function.
Area of Science:
- Molecular Biology
- Yeast Genetics
Background:
- Mitochondrial gene expression relies on precise RNA splicing.
- Group II introns are essential for mitochondrial mRNA maturation.
- The MRS3 gene plays a role in suppressing mitochondrial splice defects.
Purpose of the Study:
- To investigate the behavior of the MRS3 gene when cloned into different yeast vectors.
- To determine the relationship between MRS3 gene copy number and its ability to suppress mitochondrial splice defects.
- To analyze the integration and plasmid establishment of MRS3-containing vectors.
Main Methods:
- Cloning the MRS3 gene into integration (pEMBLYi27) and CEN-ARS (YCp50) vectors.
- Transformation of yeast mutant M1301 cells with recombinant vectors.
- Analysis of transformant phenotypes (splice competence) and vector copy numbers.
- Investigating tandem repeat formation and plasmid copy number control.
Main Results:
- Most transformants with low-copy MRS3 vectors retained the original splice defect.
- A minority of transformants became splicing competent, correlating with high MRS3 gene copy numbers.
- High copy numbers were achieved through tandem chromosomal integration or multicopy plasmid formation bypassing normal controls.
Conclusions:
- Suppression of the M1301 mitochondrial splice defect by the MRS3 gene is dependent on high gene copy number.
- Vector choice and integration mechanism significantly influence MRS3 gene expression and function.
- The MRS3 gene's ability to correct splicing defects is strongly correlated with its cellular abundance.