Using transposon Tn5 insertions to sequence bacteriophage T4 gene 11.
A simple and rapid method of creating an overlapping set of deletions in cloned DNA in preparation for sequencing has been developed. The method is based on a positive selection for Tn5 transposition into the cloned DNA fragment on a high-copy-number filamid, resolution of potential filamid dimers by filamentous phage infection, and the use of Tn5 both as a "portable" restriction enzyme site for in vitro DNA deletion and a "portable" sequencing primer binding site to initiate DNA sequencing reactions using a custom primer complementary to the outside ends of IS50. This new method has been utilized to sequence bacteriophage T4 gene 11, encoding the T4 baseplate protein gp11. The coding sequence of gene 11 is 657 bp in length, and predicts a primary structure of 219 amino acids that agrees well with the biochemical data previously obtained. DNA sequence around gene 11 suggests that the expression of genes 10, 11, and 12 of phage T4 are translationally coupled. Plasmids carrying deletions generated using this method have been used to map genetically five amber alleles of gene 11. These amber alleles were sequenced to confirm the proposed reading frame. The five amber alleles actually represent two different mutational changes at either codon 206 or 207, changing these adjacent glutamine codons to amber. The position of these amber alleles lends support to earlier studies identifying the carboxyl terminus of gp11 as essential in the interaction of P11 with baseplate protein P10 (Plishker and Berget, 1984).
Barrett
B K
BK
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA 15213-3890.
Berget
P B
PB
D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000187:drug effects / Q000235:genetics*; D005821:Genetic Techniques; D008969:Molecular Sequence Data; D010957:Plasmids; D015183:Restriction Mapping / Q000379:methods; D013604:T-Phages / Q000235:genetics*
D004251:DNA Transposable Elements; D004279:DNA, Viral