Cloning of chicken embryo tRNA genes using single stranded nucleosomal DNA highly enriched for tRNA complementary sequences.
DNA from chicken embryo nucleosome tetramers (about 760 base pairs in size) was enriched for tRNA genes by RPC-5 chromatography. The enriched DNA was hybridized with chicken embryo total tRNA and the hybridized DNA isolated utilizing a) avidinbiotin interaction, b) diazobenzyloxymethyl paper, and c) high temperature RPC-5 chromatography. The obtained single stranded DNA highly enriched for tRNA complementary sequences was hybridized with total DNA from nucleosome monomers (140--190 base pairs in size) and the excess of non hybridized monomer nucleosome DNA removed by Sepharose 4B chromatography. The hybrid molecules obtained were made fully double stranded by incubation with E. coli DNA polymerase I, DNA ligase, and exonuclease III. DNA was inserted into plasmid pBR322 by G-C joining procedure and the recombinant DNA used to transform the E. coli strain chi 1776. More than 70% of the transformants obtained hybridize to chicken embryo total tRNA.
D000818:Animals; D002642:Chick Embryo; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D011088:DNA Ligases / Q000378:metabolism; D004256:DNA Polymerase I / Q000378:metabolism; D004274:DNA, Recombinant / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D005092:Exonucleases / Q000378:metabolism; D005796:Genes; D009693:Nucleic Acid Hybridization; D012343:RNA, Transfer / Q000235:genetics*; D014170:Transformation, Genetic
D004274:DNA, Recombinant; D004277:DNA, Single-Stranded; D012343:RNA, Transfer; D004256:DNA Polymerase I; D005092:Exonucleases; D011088:DNA Ligases