Abstract
Murine V kappa-J kappa recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain VH-D-JH assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both kappa and heavy chain genes. We have examined the origin of an unusual amino acid, Ile, found at the site of V kappa-J kappa recombination in antigalactan antibodies, by sequence analysis of the corresponding rearranged and germline genes. Results indicate that the Ile codon can be generated by use of a single nucleotide 3' of the V kappa segment in combination with the second and third nucleotides of the first codon of J kappa 5 or J kappa 4. However, several antigalactan antibodies express Ile in combination with J kappa 2. An Ile codon cannot be generated by recombination in any reading frame between germline V kappa and J kappa 2 segments. These results suggest that the origin of the Ile codon in lines using J kappa 2 may represent a novel even in murine light chain assembly, possibly similar to the de novo addition of nucleotides observed in heavy chain gene recombination.
Highlights
MethodsBlotting was performed as described by Southern [6]
To determine the nucleotide sequence encoding expressed antigalactan light chains, cDNA clones were prepared from poly (A) + mRNA prepared from XRPC24 (X24) tumor tissues [7]. cDNAs were C-tailed, inserted
Murine V.-J, recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain V .. -DJ assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both K and heavy chain genes
Summary
Blotting was performed as described by Southern [6]. Filters were hybridized at 65 °C in 3X SSC, 10% dextran sulfate followed by three washes in 0.1 x SSC, 0.1 % SDS at 37'C, and a final wash in 0.1 x SSC, 0 .1 % SDS at 65'C. In some experiments hybridization was at 37°C in 50% formamide followed by three washes at 37°C in 0.2x SSC, 0.2% SDS and a final wash in 0.2x SSC, 0.2% SDS at 65°C. CDNA clones were constructed as previously described [7]. The BALB/c myeloma library in Charon 4A was kindly provided by Dr Laurel Eckhardt, Columbia University, New York. Sequencing was performed either by Maxam-Gilbert chemical modification [8] or M 13 subdoning and didexoxy chain termination [9]
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