Cloning of Four DNA Fragments Complementary to Human Thyroglobulin Messenger RNA

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Abstract
Human thyroglobulin mRNA was isolated from Graves'' goiters by size selection of total poly(A)-rich RNA in a sucrose gradient. It sedimented at 33 S, as in other mammalian species, and showed a single component of approximately 8500 bases by gel electrophoresis. c[complementary]DNA was synthesized from the 33-S RNA by using reverse transcriptase in the presence of human placenta ribonuclease inhibitor and in conditions allowing the formation of long transcripts. The latter was made double-stranded using reverse transcriptase and blunt-ended with nuclease S1. After tailing with dCTP and terminal transferase, the double-stranded cDNA was annealed to pBR322 DNA that was cleaved at the endonuclease PstI site and tailed with dGTP. The resulting plasmids were used to transform Escherichia coli C600 cells and 4 cloned recombinants were selected. Each plasmid DNA contained a sequence complementary to human thyroglobulin mRNA by hybridization with a labeled 33-S mRNA, visualization of cDNA .cntdot. mRNA hybrids by EM and filter hybridization selection of mRNA directing the synthesis of immunologically related thyroglobuin peptides in the reticulocyte lysate. The 4 inserted DNA sequences were 1400-1800 base pairs long, 2 of them showing an homologous sequence of 1100 base pairs. The 4 cloned DNA fragments represented 63% of the 8500 bases of human thyroglobulin mRNA.