Otitis media is the most common cause of hearing impairment in children and is primarily characterized by inflammation of the middle ear mucosa. Yet nothing is known of the underlying genetic pathways predisposing to otitis media in the human population. Increasingly, large-scale mouse mutagenesis programs have undertaken systematic and genomewide efforts to recover large numbers of novel mutations affecting a diverse array of phenotypic areas involved with genetic disease including deafness. As part of the UK mutagenesis program, we have identified a novel deaf mouse mutant, Jeff ( Jf ). Jeff maps to the distal region of mouse chromosome 17 and presents with fluid and pus in the middle ear cavity. Jeff mutants are 21% smaller than wild-type littermates, have a mild craniofacial abnormality, and have elevated hearing thresholds. Middle ear epithelia of Jeff mice show evidence of a chronic proliferative otitis media. The Jeff mutant should prove valuable in elucidating the underlying genetic pathways predisposing to otitis media.
The functional significance of residues in the RecA protein P-loop motif was assessed by analyzing 100 unique mutants with single amino acid substitutions in this region. Comparison of the effects on the LexA coprotease and recombination activities shows that Pro67 is unique among these residues because only at this position did we find substitutions that caused differential effects on these functions. One mutant, Pro67-->Trp, displays high constitutive coprotease activity and a moderate inhibitory effect on recombination functions. Glu and Asp substitutions result in low level constitutive coprotease activity but dramatically reduce recombination activity. The purified Pro67-->Trp protein shows a completely relaxed specificity for NTP cofactors in LexA cleavage assays and can use shorter length oligonucleotides as cofactors for cleavage of lambda cI repressor than can wild type RecA. Interestingly, both the mutant protein and wild type RecA can use very short oligonucleotides, e.g. (dA)6 and (dT)6, as cofactors for LexA cleavage. We have also found two mutations at position 67, which are completely defective for LexA coprotease activity in vivo but still maintain recombinational DNA repair (Pro67-->Lys) and homologous recombination (Pro67-->Lys and Pro67-->Arg) activities. These findings show that the recombination activities of RecA are mutationally separable from the coprotease function and that Pro67 is located in a functionally important position in the RecA structure.
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