Abstract
We have used the polymerase chain reaction (PCR) in an attempt to clone and sequence the exons and hitherto unavailable contiguous flanks of all members of the small VH9 germline gene family from inbred mouse strains and sublines that have had a common ancestry within the last century, and to analyze the molecular evolution of these sequences. Fifteen genuine germline genes were isolated (designated VH9.1 through VH9.15) from strains and sublines of DBA, BALB, 129 and C57BL inbred mice. Of the 15 genuine isolates, nine are novel: seven sequences from DBA strains and sublines (VH9.3 to VH9.9) and two sequences from C57BL strains (VH9.13 and VH9.14). We have identified sequencing errors and PCR recombinant artefacts in previously published sequences. We detected no sequence divergence of individual genes shared by the strains and sublines studied. However, we isolated two genes from DBA strains and sublines, VH9.1 and VH9.3, that differ only by five nucleotides encoding three amino acid changes that are concentrated within a 33 nucleotide (11 codon) region. Of these 11 codons, eight encode a putative antigen binding site. There were no differences in the remaining 733 nucleotides sequenced (including both 5′ and 3′ flanking regions). Potential explanations for the generation of VH9.1 and VH9.3 are discussed.
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Acknowledgements
The contributions of the authors were as follows. E.J.S. conceived the soma-to-germline hypothesis and proposed that VH9 sequences be isolated in order to facilitate a study of the molecular evolution of a small gene family. R.V.B. proposed that the project encompass sequence isolation from sublines of a variety of available inbred mouse strains. E.J.S. and R.V.B. supervised the development and implementation of protocols. P.Z., A.F., K.A.H. and K.L.P. performed the polymerase chain reaction amplification and cloning work. P.Z., A.F., K.A.H. and Mr. C. McCrae of the Biomolecular Resource Facility, The John Curtin School of Medical Research (Australian National University, Canberra, Australia), performed the automated DNA sequencing. R.V.B. and A.F. drafted the manuscript. Experiments using animals were done with the permission of the Animal Ethics Committee at The John Curtin School of Medical Research (Protocol No. J.CB.55.00, Prof. A. Müllbacher). Part of this work was supported by Australian Research Council grant A09700026 to E.J.S. and R.V.B. The experiments described herein were performed in full compliance of the current laws within Australia.
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Accession numbers: Nucleotide sequence data are available in the DDBJ/EMBL/GenBank databases under Accession numbers AY169677–AY169691
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Zylstra, P., Franklin, A., Hassan, K.A. et al. Molecular evolution of VH9 germline genes isolated from DBA, BALB, 129 and C57BL mouse strains and sublines. Immunogenetics 55, 182–188 (2003). https://doi.org/10.1007/s00251-003-0565-x
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DOI: https://doi.org/10.1007/s00251-003-0565-x