Protamine 1 gene sequence from the primate Saguinus imperator isolated with PCR using consensus oligonucleotides.

Protamines are extremely variable and positively charged proteins which act by compacting the DNA in the sperm nucleus of many species (1). Their precise role and evolutionary pathways are not well understood at present probably because of the variable nature of these proteins and the corresponding sperm-associated structures together with the limited number of species with known protamine nucleotide sequences. In order to facilitate the isolation and sequencing of other protamine genes we have designed consensus oligonucleotides by comparison of the protamine PI sequences from human, mouse and bull and tested them as PCR primers. Using this approach we report the isolation and sequence of the protamine 1 gene from the primate Saguinus imperator. PCR conditions were 5 pmoles of each primer (5'-ACCTGCTCACAGGTTGGCTG-3', 5' -CATTTATTGACAGGTGGCA-3'), MgCl2 at 1.5 mM, dNTPs at 200 /iM each, 1 unit of Taq I DNA polymerase, 200 ng of genomic DNA in a final volume of 50 fi\, an initial denaturation at 95°C for 10 min, 35 cycles of 2 min at 95°C, 2 min at 55°C, 3 min at 72°C and a final extension at 72°C for 10 min. PCR products were cloned into the EcoRV site of Bluescript KS+ (Stratagene) using the double Geneclean procedure (Bio 101) and JM109 as a host. 7 independent colonies were isolated, single stranded template DNA produced using R408 helper phage and sequenced with Sequenase (USB). This primate gene is 81 % identical to that of human and also contains one intron. The predicted amino-acid sequence reveals 12 differences with that of human. Interesting to note is the existence of a C-terminal tyrosine, present in rodents, lagomorphs and birds but not in the more closely related human protamine (2, 3). This approach should facilitate the determination of other protamine gene sequences, their evolution and the essential protamine domains and gene regions.