Double-stranded protamine cDNA: synthesis and characterization

Abstract

Double-stranded protamine complementary DNA (cDNA) was synthesized from a protamine mRNA template via the single-stranded cDNA intermediate using avian myeloblastosis virus reverse transcriptase. Synthesis at 37 and 46 °C resulted in similar overall yields (≥ 18%), although the initial rate of synthesis was higher at 46 °C than at 37 °C. The DNA of the second strand of the double-stranded cDNA product was 84% resistant to prolonged digestion with excess S1 nuclease. The S1 nuclease resistant material ranged in size from 235 to 100 base pairs (bp) with an average length of 185 bp. Analysis of the products released from double-stranded protamine cDNA by depurination indicated that there were a number of cytosine-rich oligopyrimidine tracts in protamine mRNA, namely C4, C4U1, C5U1, C6U1, C6U4, and C7U1. On the basis of the amino acid sequences for rainbow trout protamines, C5U1, C6U1 and C7U1 must be located within the noncoding regions. Double-stranded protamine cDNA was cleaved at least once by the restriction endonucleases HaeIII and HhaI and in several places by HpaII. These restriction endonucleases cleave at sequences which have a high probability of occurring within the coding region of protamine mRNA, again based on the known amino acid sequences of the rainbow trout protamines.