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We have used genomic Southern blots and restriction maps of genomic clones to examine the organization of the antifreeze protein multigene family in the winter flounder. The majority of the approximately equal to 40 antifreeze protein (AFP) genes in this fish are present in 7- to 8-kilobase-pair (kbp) elements of DNA, which are iterated as tandem direct repeats. Each repeat contains a single antifreeze protein gene that is 1 kbp long, and all of these genes have the same transcriptional orientation. Although the repeated elements are highly homologous, they do show some restriction site and restriction length polymorphisms. When flounder genomic DNA is digested with restriction endonucleases that do not cut within the repeats, most of the antifreeze protein genes reside in fragments that are at least 40 kbp long, representing clusters of five or more repeats in tandem. After genomic DNA is digested with Xba I or Xho I, these genes are present in fragments of exceptionally high molecular weight, suggesting that the clusters themselves are grouped together in the genome. The AFP gene locus may have evolved by gene amplification as recently as 10(6) years ago in response to the onset of the Cenozoic ice age.

Antifreeze protein genes are tandemly linked and clustered in the genome of the winter flounder.