No abstract

Mutational studies of regions that make up the oligomeric interface within the RecA protein filament structure have shown that F217 is an important determinant of RecA function and oligomer stability. All substitutions, other than Tyr and Cys, completely inhibit RecA activities and exhibit a substantial decrease in protein filament stability [Skiba, M. C., and Knight, K. L. (1994) J. Biol. Chem. 269, 3823-3828; Logan, K. M., et al. (1997) J. Mol. Biol. 266, 306-316]. Although the RecA crystal structure exhibits no obvious constraints that explain this mutational stringency, the structure does reveal a hydrophobic pocket in the neighboring monomer that may accommodate the F217 side chain. Together with the F217C mutation, we have introduced a series of Cys substitutions within the interacting surface on the neighboring monomer and have tested for disulfide formation under various conditions, e.g., with or without ATP and ssDNA. We show that the location of F217 in the crystal structure is in general agreement with its position in the catalytically active RecA-ATP-DNA complex. Functional studies with the mutant proteins support the idea that ATP-induced movement of the wild-type F217 side chain toward this hydrophobic pocket is important in mediating allosteric changes in the RecA protein structure.

Primary cell cultures from twitcher (galactocerebrosidase deficient) mice were made by enzymatic dispersion and explantation of skin obtained from 3-d-old littermates of a twi+/twi X twi+/twi mating. Galactocerebrosidase activity remained deficient for two twitcher cell lines, TM-1 and TM-2, and both lines demonstrated an initial period of growth decline, followed by accelerated growth. The TM-2 line has been subcultured for more than 3.5 yr, has a modal chromosome number of 63, a doubling time of approximately 16 h, and has remained galactocerebrosidase deficient throughout its life span. These data indicate this to be an established twitcher cell line that can be continuously maintained in culture as a transformed galactocerebrosidase-deficient mouse cell line. This established line was rendered 6-thioguanine resistant so that the cells could be fused with control human fibroblasts and selected for hybrid lines in hypoxanthine-aminopterin-thymidine medium. Also, the established twitcher cells were crossed with neomycin-resistant control human fibroblasts and selected in G418 medium. Several of the hybrid lines from both crosses had higher than deficient levels of galactocerebrosidase activity initially, followed by a decrease to twitcher levels during subculture, whereas other lines retained high levels of activity. These results indicate that twitcher-human somatic cell hybrids will express galactocerebrosidase activity and thus may be useful for determining the human chromosome or chromosomes associated with this expression.

No abstract

The inherited deficiency of galactosylceramide beta-galactosidase (E.C. 3.2.1.46: galactocerebrosidase) activity results in globoid cell leukodystrophy in humans (Krabbe disease) and in mice (twitcher mutant). To determine whether Krabbe patients' cells complement twitcher cells to produce, in hybrid combination, greater than deficient levels of galactocerebrosidase activity, five separate crosses were made between an established twitcher mouse cell line and five cell strains from unrelated Krabbe disease patients. A total of 57 twitcher mouse/Krabbe somatic cell hybrid lines developed from all of these crosses were deficient in galactocerebrosidase activity despite the presence of human chromosomes 14 or 17, which have been previously implicated as bearing the galactocerebrosidase gene. A control cross between twitcher mouse/positive control human fibroblasts resulted in 14 of 21 independent hybrid lines that expressed higher than deficient levels of galactocerebrosidase activity. The lack of complementation between Krabbe disease patient and twitcher mutant mouse cells provides further evidence that the twitcher mouse is an authentic murine model for Krabbe disease and supports the hypothesis that the mutations in both species are within the structural gene for the galactocerebrosidase enzyme.