An in silico evaluation of Tn916 as a tool for generalized mutagenesis in Haemophilus influenzae Rd.
The transposon Tn916 was evaluated as a tool for generalized mutagenesis of the genome of Haemophilus influenzae. This was achieved in silico by searching the genome sequence of H. influenzae Rd for the published Tn916 target site consensus sequence 5' TT/ATTTT(N)6AAAAAA/TA. This search identified 16 putative target sites. In subsequent experiments, integration of Tn916 did not occur at any of these sites. Using the nucleotide sequences of these observed integration sites, a new consensus sequence, 5' TTTTT(N)xAAAAA (4 < or = x < or = 7), was derived. This sequence reflects the curve-twist-curve DNA topology which is a feature common to all Tn916 integration sites. A search of the H. influenzae Rd genome using the new consensus sequence identified 167 potential target sites, representing approximately 1% of the total genome. Only 80 of these sites were located within ORFs. The presence of such a limited number of target sites places severe constraints on the use of Tn916 as a tool for generalized mutagenesis of the genome of H. influenzae.
University of Manchester, School of Biological Sciences1.800, Stopford Building, Oxford Road, Manchester M13 9PTUK.
Sarah L
SL
Hosking
Molecular Infectious Diseases Group, University of Oxford, Institute of Molecular MedicineJohn Radcliffe Hospital, Headington, Oxford OX3 9DSUK.
Mary E
ME
Deadman
Molecular Infectious Diseases Group, University of Oxford, Institute of Molecular MedicineJohn Radcliffe Hospital, Headington, Oxford OX3 9DSUK.
E Richard
ER
Moxon
Molecular Biology Data Centre, IMSU, University of OxfordHeadington, OxfordUK.
John F
JF
Peden
Molecular Infectious Diseases Group, University of Oxford, Institute of Molecular MedicineJohn Radcliffe Hospital, Headington, Oxford OX3 9DSUK.
Nigel J
NJ
Saunders
University of Manchester, School of Biological Sciences1.800, Stopford Building, Oxford Road, Manchester M13 9PTUK.
Nicola J
NJ
High
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D017931:DNA Primers / Q000235:genetics; D004251:DNA Transposable Elements / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016680:Genome, Bacterial; D006193:Haemophilus influenzae / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D015183:Restriction Mapping
D017931:DNA Primers; D004251:DNA Transposable Elements; D004269:DNA, Bacterial