Sheep elastin genes. Isolation and preliminary characterization of a 9.9-kilobase genomic clone
| Autor: | M Harrison, Shigeki Shibahara, Ronald G. Crystal, J M Davidson, P Tolstoshev, C Leach, M P Schafer |
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| Rok vydání: | 1984 |
| Předmět: |
Genotype
Biology Biochemistry law.invention Restriction fragment Nucleic acid thermodynamics law Complementary DNA Animals Genomic library RNA Messenger Cloning Molecular Molecular Biology Southern blot Electrophoresis Agar Gel Genetics Sheep Base Sequence Nucleic Acid Hybridization DNA Cell Biology Molecular biology Elastin Microscopy Electron genomic DNA Genes Protein Biosynthesis DNA Viral biology.protein Recombinant DNA Research Article |
| Zdroj: | Biochemical Journal. 220:643-652 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bj2200643 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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