V(D)J recombination of chromosomally integrated, wild-type deletional and inversional substrates occur at similar frequencies with no preference for orientation.
Efficient and correct recombination of V(D)J substrates results in the generation of antibodies. The RSS substrates are oriented in two directions with respect to each other: deletional and inversional. Deletional recombination results in the formation of the coding joint and excision of the intervening sequences. Inversional recombination retains all the genomic sequences and forms both a coding joint and a signal joint. A bias for deletional recombination has been characterized with specific loci in vivo and recapitulated in experiments using extrachromosomal substrates. We constructed retroviral substrates of RSS in the deletional and inversional orientation. We introduced the substrates into wild-type and scid pre-B cells and measured the frequency of functional recombination in addition to open/shut recombination. We also mutated the RSSs to determine whether mutated sequences influenced orientation bias. We show that pre-B cells recombine the wild-type substrates at a 1.6 ratio of deletion:inversion. Nonamer mutated substrates recombined with a deletional bias whereas heptamer mutated substrates recombined with an inversional bias. A spacer length mutation and drastic mutations in the RSS abolish all recombination. These results suggest that there is no orientation bias with wild-type RSSs but that orientation bias occurs when RSSs are mutated.
Shuh
Maureen
M
Department of Biological Sciences, Loyola University New Orleans, New Orleans, LA 70118, USA. mshuh@loyno.edu
Hixson
Douglas C
DC
D000818:Animals; D001483:Base Sequence; D015321:Gene Rearrangement; D007136:Immunoglobulins / Q000235:genetics* / Q000276:immunology; D051379:Mice; D016513:Mice, SCID; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction
D007136:Immunoglobulins