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Deoxyuridylate-hydroxymethylase of bacteriophage SPO1
1Arbeitsgruppe Molekulare Genetik, Lehrstuhl Biologie der Mikroorganismen, Ruhr-Universität Bochum, FRG.
Abstract:
Phage SPO1 of Bacillus subtilis carries hydroxymethyl-deoxyuridylate in place of thymidylate in its DNA. The enzyme, responsible for the conversion of dUMP to HmdUMP, is a dUMP hydroxymethylase, encoded by the SPO1 gene 29. Here we describe the cloning and sequencing of the gene and the overexpression of the gene product. DNA hybridization using the DNA of bacteriophage T4 dCMP-hydroxymethylase gene as a probe, allowed us to identify and map g29 on a 3.9-kb restriction fragment, EcoRI*11. We determined the nucleotide sequence. One of the open reading frames detected, coding for a putative 44.6-kDa protein, showed significant amino acid homologies with all known thymidylate synthases. Gp29 was overexpressed in the pT7 system. Extracts prepared from induced cells show hydroxymethylase activity in a tritium release assay.
Insights
Bacillus subtilis phage SPO1 DNA uses hydroxymethyl-deoxyuridylate (HmdUMP) instead of thymidylate. The SPO1 gene 29 encodes the dUMP hydroxymethylase enzyme responsible for this conversion, which was cloned, sequenced, and overexpressed.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacillus subtilis phage SPO1 DNA incorporates hydroxymethyl-deoxyuridylate (HmdUMP) in place of thymidylate.
- This unique DNA modification is facilitated by a specific enzyme, dUMP hydroxymethylase.
Purpose of the Study:
- To clone and sequence the SPO1 gene 29, which encodes the dUMP hydroxymethylase.
- To overexpress the gene product (Gp29) and confirm its enzymatic activity.
Main Methods:
- DNA hybridization using the T4 bacteriophage dCMP-hydroxymethylase gene as a probe to identify and map gene 29.
- Nucleotide sequencing of the identified gene fragment.
- Overexpression of the Gp29 protein using the pT7 expression system.
- Enzymatic assay (tritium release) to confirm hydroxymethylase activity.
Main Results:
- Gene 29 was successfully identified, mapped to a 3.9-kb EcoRI fragment, and its nucleotide sequence determined.
- An open reading frame predicted a 44.6-kDa protein (Gp29) with homology to thymidylate synthases.
- Induced expression of Gp29 in the pT7 system resulted in detectable hydroxymethylase activity.
Conclusions:
- The SPO1 gene 29 encodes the dUMP hydroxymethylase responsible for HmdUMP incorporation in phage SPO1 DNA.
- The cloned and overexpressed Gp29 enzyme exhibits the predicted hydroxymethylase activity.
- This study elucidates the genetic basis for a unique DNA modification in bacteriophages.