CONSERVATION OF ANTIFREEZE PROTEIN-ENCODING GENES IN TANDEM REPEATS

The antifreeze protein (AFP) multigene family in winter flounder is made up of genes in tandem repeats and others that are linked, but irregularly spaced. The close spacing of the tandemly repeated genes has provided an opportunity to determine how well nearest-neighbour AFP genes from the tandem repeats resemble each other, the genes elsewhere in the tandem repeats, and the genes outside the tandem repeats. Four pairs of tandemly repeated genes were sequenced. Two pairs were identical and probably represent independent clones of the same chromosomal region. The six unique genes coded for one or other of the two major AFPs, HPLC-6 and HPLC-8. Their comparison over a region of 900 bp, from just after the CAAT box to just before the polyadenylation signal, showed a maximum of 26 single-bp changes and no major insertions or deletions. Nearest-neighbour genes had almost as many changes as genes elsewhere in the tandem repeats. However, these six genes were much more homogeneous than five AFP genes from outside the tandem repeats, none of which encode a major AFP component, and all but one had large insertions, deletions, or rearrangements. Despite the similarity of the genes in tandem repeats, three variants due to short insertions or deletions in the 3'-flanking DNA were interspersed, and genes coding for HPLC-6 and HPLC-8 were nearest neighbours. It is suggested that enhanced opportunities for recombination between repeats have helped keep these genes more uniform than those outside the tandem repeats. Potential 'hot-spots' for recombination are the oligo(dG) tract in the intron, which shows length variation from gene to gene, and the 33-bp repeats in the 3'-exon.