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Identification of diversified genes that contain immunoglobulin-like variable regions in a protochordate


The evolutionary origin of adaptive immune receptors is not understood below the phylogenetic level of the jawed vertebrates. We describe here a strategy for the selective cloning of cDNAs encoding secreted or transmembrane proteins that uses a bacterial plasmid (Amptrap) with a defective β-lactamase gene. This method requires knowledge of only a single target motif that corresponds to as few as three amino acids; it was validated with major histocompatibility complex genes from a cartilaginous fish. Using this approach, we identified families of genes encoding secreted proteins with two diversified immunoglobulin-like variable (V) domains and a chitin-binding domain in amphioxus, a protochordate. Thus, multigenic families encoding diversified V regions exist in a species lacking an adaptive immune response.

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We thank B. Pryor for editorial assistance, R. Litman for sequence analysis, C. Andrews, J. Wahle and T. Willis for technical assistance and C. Amemiya for comments about the manuscript. Supported by NIH grant AI23338 (to G. W. L) and the H. Lee Moffitt Cancer Center (J. P. C.).

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The authors declare no competing financial interests.

Correspondence to Gary W. Litman.

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Figure 1: Amptrap selection strategy.
Figure 2: Validation of Amptrap cloning based on MHC class II and β2M genes from Raja eglanteria.
Figure 3: Characterization of five families of predicted IgSF proteins from amphioxus.
Figure 4: Diversity of VCBP genes.
Figure 5: Selective expression of VCBP1 in adult amphioxus intestine.