ABSTRACT. N-ras mutations were examined in DNA samples extracted from the spleen of CBA/Ca mice that developed myeloid leukemia (ML) following exposure to radiations of different qualities. A total of 17 ML cases, i.e. 5 cases of neutron- induced and 12 cases of photon- (3 gamma-ray and 9 x-ray) induced ML were included in the study along with 12 DNA samples from the bone marrow cells of control mice. Polymerase chain reaction-single strand conformational polymorphisms (PCR-SSCP) and the direct sequencing of PCR products were used to analyze three regions of the N-ras gene: (i) a 128 base-pair (bp) long portion of exon I (codons 2-37); (ii) a 103 bp long portion of exon II (codons 48-82); and (iii) a 107 bp long portion of exon III (codons 118-150). PCR-SSCP mobility shifts indicated mutations within only exon II of the N-ras gene. Such mutations were more prevalent in samples from mice exposed to fast neutrons. The exact type and location of these mutations were then determined by direct DNA sequencing. Silent point mutations, i.e. base transitions at the third base of codons 57 (GA*C*->GA*T*), 62 (CA*A*->CA*C*), or 70 (CA*G* ->CA*A*) were present only in mice that developed ML after exposure to fast neutrons. A base transversion at the third base of codon 61 (CA*A* ->CA*C*) was also observed in some ML cases. DNA sequencing demonstrated that ML samples contained normal as well as mutated DNA sequences. The higher frequency of N-ras mutations in neutron- induced ML suggested that fast neutrons are more effective in inducing genomic instability at the N-ras region of the genome. More importantly, N- ras mutations are not the initiating event in radiation leukemogenesis. This conclusion was supported by the finding that N-ras mutations were detected only in mice with an overt leukemic phenotype but not in mice with minimal tissue infiltration of leukemic cells, suggesting that the disease may be present prior to the presence of N-ras mutations. Alternatively, N-ras may be present in these mice but a large number of normal spleen cells in these mice interferes with the detection of mutation in a small population of leukemic cells.

Keywords: N-ras, mutation, radiation, leukemogenesis, murine.

Reprint requests to: K. Noy Rithidech, Brookhaven National Laboratory, Medical Department, P.O. Box 5000, Upton, NY 11973-5000, phone: (516) 344-2672, fax: (516) 344-5311, email: rithidech@bnlarm.bnl.gov.

ABSTRACT. CD59 (membrane inhibitor of reactive lysis, protectin) is a membrane protein whose functions include the inhibition of the insertion of the ninth component of complement into the target membrane. It belongs to a superfamily of proteins including Ly-6, elapid snake venom toxins, and urokinase receptor (UPAR); the members of the superfamily have a similar structure that includes four (in mammals five) disulfide bridges that maintain a three-dimensional conformation consisting of a central core, three finger-like "loops" extending from it and a small loop near the carboxyl end. We have used site directed mutagenesis to explore three aspects of the structure of CD59: 1) the role of the disulfide bridges in expression and function of the molecule; 2) the location of epitopes reacting with monoclonal antibodies to the molecule; and 3) the parts of the molecule that are critical to its function in inhibiting complement lysis. Mutant molecules in which the disulfides maintaining the finger-like loops (Cys3-Cys26, Cys19-Cys39, and Cys45-Cys63) were removed were not expressed on the cell surface. The mutation of the disulfide (Cys6-Cys13) resulted in no change in expression or function. The mutation of Cys64-Cys69 maintaining the small loop resulted in an expressed molecule with increased functional activity. The major epitope for 6 of 7 monoclonal antibodies was centered on Arg53 as the mutation 53Arg->Ser resulted in a loss of interaction with these antibodies, as did the deletion of four nearby residues (Leu54 - Asn57). The alteration 55Arg->Ser resulted in loss of reactivity for some but not other antibodies. The reactivity with one monoclonal antibody, H19, was abrogated by the mutations 61Tyr->Gly and 61Tyr->Ala. Functional activity of the molecule was not adversely altered by mutations in the first and second loops; however, the 61Tyr->Gly mutation was non- functional. The mutation of 61Tyr->His diminished function but changes 61Tyr->Ala and 61Tyr->Phe had no effect on function. We conclude that the functional site of CD59 is located in this region of the molecule.

Keywords: CD59, mutation, function, structure.

Reprint requests to: Wendell F. Rosse, M.D., Box 3934, Duke University Medical Center, Durham NC, 27710, phone: (919) 684-3724, fax: (919) 681-8477, e-mail: rosse001@mc.duke.edu.