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Published on: May 25, 2018
A comparative hybridization analysis of yeast DNA with Paramecium parafusin- and different
1Department of Cell Biology, Nencki Institute of Experimental Biology, Warsaw, Poland. wyroba@nencki.gov.pl
Abstract:
Molecular probes designed for the parafusin (PFUS), the Paramecium exocytic-sensitive phosphoglycoprotein, gave distinct hybridization patterns in Saccharomyces cerevisiae genomic DNA when compared with different phosphoglucomutase specific probes. These include two probes identical to segments of yeast phosphoglucomutase (PGM) genes 1 and 2. Neither of the PGM probes revealed the 7.4 and 5.9 kb fragments in Bgl II-cut yeast DNA digest detected with the 1.6 kb cloned PFUS cDNA and oligonucleotide constructed to the PFUS region (insertion 3--I-3) not found in other species. PCR amplification with PFUS-specific primers generated yeast DNA-species of the predicted molecular size which hybridized to the I-3 probe. A search of the yeast genome database produced an unassigned nucleotide sequence that showed 55% identity to parafusin gene and 37% identity to PGM2 (the major isoform of yeast phosphoglucomutase) within the amplified region.
Insights
Molecular probes targeting parafusin (PFUS) revealed unique DNA fragments in yeast, distinct from phosphoglucomutase (PGM) genes. This suggests a novel gene in yeast with partial similarity to parafusin and PGM2.
Area of Science:
- Molecular Biology
- Genomics
- Biochemistry
Background:
- Paramecium parafusin (PFUS) is an exocytic-sensitive phosphoglycoprotein.
- Phosphoglucomutase (PGM) enzymes are crucial in carbohydrate metabolism.
Purpose of the Study:
- To investigate the presence and characteristics of parafusin-like sequences in Saccharomyces cerevisiae.
- To differentiate PFUS-related sequences from known yeast PGM genes.
Main Methods:
- Hybridization of molecular probes (cloned PFUS cDNA, oligonucleotides, PGM gene segments) to yeast genomic DNA.
- Restriction digestion (Bgl II) and Southern blot analysis.
- Polymerase Chain Reaction (PCR) amplification using PFUS-specific primers.
- Yeast genome database search and sequence homology analysis.
Main Results:
- PFUS-specific probes identified unique 7.4 and 5.9 kb DNA fragments in Bgl II-digested yeast DNA, not detected by PGM probes.
- PCR amplification yielded yeast DNA fragments that hybridized to a PFUS-specific probe.
- A novel unassigned yeast genomic sequence showed 55% identity to parafusin and 37% identity to PGM2.
Conclusions:
- Saccharomyces cerevisiae possesses a unique gene with homology to parafusin and PGM2.
- This finding expands the understanding of phosphoglycoprotein diversity in eukaryotes.
