Modification of human beta-globin locus PAC clones by homologous recombination in Escherichia coli.
We report here modifications of human beta-globin PAC clones by homologous recombination in Escherichia coli DH10B, utilising a plasmid temperature sensitive for replication, the recA gene and a wild-type copy of the rpsL gene which allows for an efficient selection for plasmid loss in this host. High frequencies of recombination are observed even with very small lengths of homology and the method has general utility for introducing insertions, deletions and point mutations. No rearrangements were detected with the exception of one highly repetitive genomic sequence when either the E.COLI: RecA- or the lambdoid phage encoded RecT and RecE-dependent recombination systems were used.
Imam
A M
AM
Erasmus University Rotterdam, CBG and MGC-Department of Cell Biology, PO Box 1738, 3000 DR Rotterdam, The Netherlands. imam@ch1.fgg.eur.nl
Patrinos
G P
GP
de Krom
M
M
Bottardi
S
S
Janssens
R J
RJ
Katsantoni
E
E
Wai
A W
AW
Sherratt
D J
DJ
Grosveld
F G
FG
D017101:Bacteriophage P1 / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005819:Genetic Markers; D005822:Genetic Vectors; D005914:Globins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D010957:Plasmids; D011938:Rec A Recombinases / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D000091243:Ribosomal Protein S9
D029968:Escherichia coli Proteins; D005819:Genetic Markers; D011994:Recombinant Proteins; D000091243:Ribosomal Protein S9; C000717190:RpsI protein, E coli; D005914:Globins; D011938:Rec A Recombinases