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Using transposon Tn5 insertions to sequence bacteriophage T4 gene 11
1Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213-3890.
DNA (Mary Ann Liebert, Inc.)
|May 1, 1989
Summary
A novel DNA deletion method using Tn5 transposition enables rapid DNA sequencing and gene mapping. This technique was successfully applied to sequence bacteriophage T4 gene 11 and analyze its protein product.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Efficient DNA sequencing and genetic analysis are crucial for understanding gene function.
- Existing methods for generating DNA deletions can be time-consuming and complex.
- Bacteriophage T4 gene 11 encodes the baseplate protein gp11, essential for phage structure and function.
Purpose of the Study:
- To develop a simple, rapid method for creating overlapping DNA deletions for sequencing.
- To apply this method to sequence bacteriophage T4 gene 11.
- To genetically map and sequence mutations within gene 11.
Main Methods:
- Developed a method utilizing Tn5 transposition for generating DNA deletions in cloned fragments.
- Employed a positive selection strategy with a high-copy-number filamid vector.
- Used Tn5 as a portable restriction site and sequencing primer binding site for deletion generation and DNA sequencing.
Main Results:
- Successfully sequenced bacteriophage T4 gene 11 (657 bp), predicting a 219-amino acid protein (gp11).
- Identified translational coupling between phage T4 genes 10, 11, and 12.
- Mapped five amber alleles of gene 11, revealing mutations at codons 206 or 207.
Conclusions:
- The developed Tn5-based deletion method is efficient for DNA sequencing and genetic analysis.
- The sequence data and mutation analysis provide insights into the structure and function of gp11.
- The carboxyl terminus of gp11 is critical for its interaction with baseplate protein P10.