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C57BL/6 female mice (Jackson ImmunoResearch Labs) were kept in sterile microisolators. All the mice, except for those used for DNA break assays (Fig. 3), were young adults (8 to 10 weeks old). Mice were immunized intraperitoneally with 100 μg of (4-hydroxy-3-nitrophenyl)acetyl-chicken γ-globulin (NP-CGG) or CGG precipitated in alum (6). Spleens of immunized mice were taken at times indicated after immunization and dissociated into single-cell suspensions.
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22
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1842279236
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note
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lo GC cells = 29.3 ± 4.0%.
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23
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1842273356
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note
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+ GC B cells.
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24
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1842344406
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note
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Total RNA was recovered from limited numbers of sorted cells using TRIzol reagent (Gibco). After ethanol precipitation, all RNA of each sample was used for first-strand cDNA synthesis in a total volume of 20 μl, using a SuperScript kit (Gibco). We used 2 μl of the cDNA solution for PCR amplification in 50 μl volume reaction with Taq polymerase (Gibco). The primers used for RAG1, RAG2, and Hprt have been described previously (5). The primers for λ5 were 5′-ACTGGTCAGATATCAGTGTC-3′ for reverse transcription; 5′-TTGGGTCTAGTGGATGGTGTC-C-3′ and 5′ CTGACCTAGGATTGTGAGCTGGGT-3′ for PCR amplification.
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κS have been published (14-16).
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1842346340
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note
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Single-letter abbreviations for the amino acid residues are as follows: G, Gly; L, Leu; S, Ser; W, Trp; and Y, Tyr.
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39
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1842399705
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note
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32P-labeled oligonucleotide corresponding to amino acid positions 70 through 74 of the V186.2 gene segment. DNA from positive clones was subjected to sequencing on an Applied Biosystems 373A automated DNA sequencer.
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40
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1842341527
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in preparation
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H 165.1 gene segments. Two hybrid sequences were present: the 215-bp insert described earlier and an in-frame exchange of 30 bp from the C1H4 exon into a productive V186.2 rearrangement.
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We thank J. Cerny and A. Perelson for their review of this manuscript. Supported in part by USPHS grants HL48722 and AI40227 (M.S.S.) and AI24335, AG10207, and AG13789 (G. K). S.R.D. is a recipient of an Arthritis Foundation Postdoctoral Fellowship, M.S.S acknowledges the support of a Leukemia Society Scholarship and G.K. received funding from the Jeanne M. and Joseph P. Sullivan Foundation and the Santa Fe Institute.
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