A whole genome shotgun gene fusion method for isolation of translation initiation sites in Escherichia coli: identification of Haemophilus influenzae translation initiation sites in E. coli.
We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, Maryland, USA.
K
K
McKenney
J
J
Tian
S
S
Nunes-Duby
J
J
Hoskins
P
P
Reddy
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion / Q000379:methods*; D001483:Base Sequence; D018387:Codon, Initiator / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005686:Galactokinase / Q000235:genetics / Q000302:isolation & purification; D005798:Genes, Bacterial / Q000235:genetics*; D016678:Genome; D006193:Haemophilus influenzae / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D014176:Protein Biosynthesis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018387:Codon, Initiator; D005686:Galactokinase