CHARACTERIZATION OF A MARSUPIAL SPERM PROTAMINE GENE AND ITS TRANSCRIPTS FROM THE NORTH-AMERICAN OPOSSUM (DIDELPHIS-MARSUPIALIS)

A synthetic oligonucleotide primer, designed from marsupial protamine protein-sequence data [Balhorn, R., Corzett, M., Matrimas, J. A., Cumm ins, J. & Faden, B. (1989) Analysis of protamines isolated from two ma rsupials, the ring-tailed wallaby and gray short-tailed opossum, J. Ce ll. Biol. 107] was used to amplify, via the polymerase chain reaction, protamine sequences from a North American opossum (Didelphis marsupia lis) cDNA. Using the amplified sequences as probes, several protamine cDNA clones were isolated. The protein sequence, predicted from the cD NA sequences, consisted of 57 amino acids, contained a large number of arginine residues and exhibited the sequence ARYR at its amino termin us, which is conserved in avian and most eutherian mammal protamines. Like the true protamines of trout and chicken, the opossum protamine l acked cysteine residues, distinguishing it from placental mammalian pr otamine 1 (P1 or stable) protamines. Examination of the protamine gene , isolated by polymerase-chain-reaction amplification of genomic DNA, revealed the presence of an intron dividing the protamine-coding regio n, a common characteristic of all mammalian P1 genes. In addition, ext ensive sequence identity in the 5' and 3' flanking regions between mou se and opossum sequences classify the marsupial protamine as being clo sely related to placental mammal P1. Protamine transcripts, in both bi rds and mammals, are present in two size classes, differing by the len gth of their poly(A) tails (either short or long). Examination of opos sum protamine transcripts by Northern hybridization revealed four dist inct mRNA species in the total RNA fraction, two of which were enriche d in the poly(A)-rich fraction. Northern-blot analysis, using an intro n-specific probe, revealed the presence of intron sequences in two of the four protamine transcripts. If expressed, the corresponding protei n from intron-containing transcripts would differ from spliced transcr ipts by length (49 versus 57 amino acids) and would contain a cysteine residue.