SHEEP ELASTIN GENES - ISOLATION AND PRELIMINARY CHARACTERIZATION OF A 9.9-KILOBASE GENOMIC CLONE

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labeled elastin mRNA (mRNAE). A recombinant containing a 9.9-kb (kilobase) insert was selected from several positive clones by secondary and tertiary screening for further characterization. Positive identification of this elastin clone, designated SE1, was made with radiolabeled mRNAE by hybridization-selected translation and Southern blotting of restriction-enzyme fragments of SE1 DNA. Hybridization of either mRNAE or elastin complementary DNA to restriction fragments of SE1 showed that most of these fragments of SE1 contained elastin-coding sequences. Orientation of the insert was established by preferential hybridization of a short complementary elastin DNA to restriction fragments adjacent to the right arm of Charon 4A. Reciprocal hybridizations of nick-translated SE1 and sheep genomic DNA on Southern blots showed that 2 restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclese-EcoRI digest of total sheep genomic DNA. A subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. EM of SE1-mRNAE hybrids indicated the presence of at least 7 large R-loops. Measurements of these structures indicated that SE1 is likely to contain less than 2 kb of coding sequence and more than 8 kb of intervening sequence, with an average exon size of 120 base-pairs. The elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.