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The rates of emergence of H-2b- and H-2d- variants from an H-2b+/ H-2d+ heterozygous mouse leukemia cell line were determined by fluctuation analyses. The results show that the wild type cells throw off the H-2b- variants at about 10(-5) per cell per generation and the H-2d- variants at 10(-6) per cell per generation. The reason for this tenfold difference in the rates for the two symmetrical variants is not clear. The fluctuation analyses also indicate that the variants exist in the wild type population prior to exposure to the selective agents.

Using immunoselection with an H-2Kk-specific monoclonal antibody following mutagenesis on an (H-2k/H-2d) F1 cell line we have obtained variants that do not react with the selecting monoclonal antibody but continue to react with other monoclonal antibodies directed against the same gene product. The mutants fall into two classes based on their serological profile. This phenotype is suggestive of a structural mutation in the selected gene. If the genetic change involved is a point mutation (as opposed to a deletion), one should be able to obtain revertants. Using the fluorescence-activated cell sorter, we have been able to obtain from one of the monoclonal-antibody-nonreactive mutants cells that do bind the selecting antibody. In order to prove that the presumptive revertant is not a contaminant wild-type cell that inadvertently got mixed into the resistance mutant, we first introduced an outside marker, resistance to the purine analogue 2-amino-6-mercaptopurine (6-thioguanine), into the monoclonal-antibody-resistant mutant. The revertants obtained using the cell sorter continue to express the nonselective phenotype of resistance to 6-thioguanine, showing that they are not wild-type cells. In addition, their serological characteristics are different from those of either the wild-type cells or the hybridoma-resistant mutants from which they were derived. Based on the serological analyses, it would seem that we have isolated at least three variant forms of the H-2Kk gene product.

Variants that no longer express an entire H-2 haplotype were readily isolated, by immunoselection with antisera directed against the haplotype, from an H-2b/H-2d heterozygous Friend leukemia cell line carrying a Robertsonian translocation of the chromosomes bearing the H-2 genetic region. These variants can be denoted as being of the phenotype H-2b- H-2d+ or H-2b+ H-2d-. Some of the H-2b- H-2d+ variants: (1) lack the restriction enzyme fragments characteristic of the missing H-2b haplotype, as assessed by Southern blot analysis; (2) express more cell surface H-2d antigens than wild-type cells, as assessed by flow microfluorimetry; and (3) appear to have become homozygous for the more active H-2d-linked allele at the Glyoxalase I locus. These variants thus seem to have lost genetic material corresponding to the H-2b haplotype and may have gained genetic material corresponding to the H-2d haplotype. These results are consistent with the possibility that these variants were generated by mitotic recombination.