Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone.

J. M. Davidson, S. Shibahara, M. P. Schafer, M. Harrison, C. Leach, P. Tolstoshev, Ronald Crystal

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

A sheep genomic library containing sheep DNA in the bacteriophage vector Charon 4A was screened for elastin-gene sequences with partially purified, 32P-labelled 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 radiolabelled mRNAE by hydridization-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 two restriction fragments of SE1 contained sequence elements which were repeated at high frequency in a restriction-endonuclease-EcoR1 digest of total sheep genomic DNA. In the accompanying paper [Davidson, Shibahara, Boyd, Mason, Tolstoshev & Crystal (1984) Biochem. J. 220, 653-663], it is shown that a subcloned fragment of this elastin gene quantitatively and specifically hybridized to mRNAE sequences in sheep tissue RNA. Electron microscopy of SE1-mRNAE hybrids indicated the presence of at least seven 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. Thus the elastin gene is distributed over an extended region of the sheep genome and contains numerous intervening and coding sequences.

Original languageEnglish
Pages (from-to)643-652
Number of pages10
JournalThe Biochemical journal
Volume220
Issue number3
DOIs
Publication statusPublished - 1 Jan 1984
Externally publishedYes

    Fingerprint

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Davidson, J. M., Shibahara, S., Schafer, M. P., Harrison, M., Leach, C., Tolstoshev, P., & Crystal, R. (1984). Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone. The Biochemical journal, 220(3), 643-652. https://doi.org/10.1042/bj2200643