SummaryFas is a cell surface protein of the tumor necrosis factor receptor, nerve growth factor receptor, CD40 family, and is involved in the control of lymphocyte apoptosis. A mutation in the Fas gene in MRL/Ipr mice results in massive lymphoproliferation (Ipr) and accelerated autoimmunity.To further study the nature of this defect, Fas mRNA expression was evaluated by reverse transcriptase polymerase chain reaction as well as by Northern blotting. These studies revealed that the wildtype Fas message was produced at ~10-fold lower levels in the Ipr compared with the + + substrain of MRL mice. In addition to the wild-type transcript, lpr mice also synthesized chimeric transcripts
Materials and Methods
Mice. MRL/Ipr (MRL/MpJ-lpr/lpr), MILL/+ + (MRL/MP-+/+), NZB/W F~, and C3H (C3H/HeSnJ) mice were originally obtained from The Jackson Laboratory (Bar Harbor, ME) and subsequently bred at the Hospital for Special Surgery.Northern and Southern Blotting. Single-ceU suspensions were prepared from the thymus, spleen, lymph nodes, and bone marrow of MRL and C3H mice. Total RNA was isolated by the acid guanidinium method (4) and poly(A)+-enriched KNA was obtained by the method of Badley et al. (5) using oligo(dT) cellulose (Collaborative Research, Bedford, MA). RNA was blotted to nylon membranes (Gene Screen; New England Nuclear, Boston, MA) and hybridizations were performed according to the manufacturer's instructions. PCR products were also blotted to nylon membranes and hybridized with DNA or oligonucleotide probes. DNA probes were labeled with [32p]dCTP by the random primer method (6) and oligonucleotides were biotinylated at the 3' termini using terminal transferase (Tdt) (7). After hybridization with oligonucleotides, blots were developed with the chemiluminescent substrate, AMPPD (Tropix, Bedford, MA), and fluorography. All blots were washed under high stringency conditions. PCR and DNA Sequencing. cDNA was synthesized from total KNA with Moloney murine leukemia virus (MoMULV) KT and oligo(dT) as described (8). cDNA was amplified by PCK on a thermal cycler (480; Perkin Elmer Corp., Norwalk, CT) with Taq polymerase (Bethesda Research Laboratories) unless indicated otherwise. The primers used for PCK analysis are shown in Table 1. The conditions used for amplification were: denaturation at 94~ for 1 rain, annealing at 5~ below the melting temperature (Tm) (calculated as described [91) for 1 min, and extension at 72~ for
