Cloning and expression of a novel cDNA encoding shell coat protein, cp4, from the brushtail possum (Trichosurus vulpecula)

The marsupial conceptus is enclosed by several egg coats of evolutionary significance and unknown composition, of which the shell coat in mammals occurs only in marsupials and monotremes. Intact coats are vital to marsupial embryonic development. Towards a better understanding of the marsupial coat proteins, a cDNA sequence (cp4) encoding a shell coat protein was cloned from the brushtail possum. A cDNA library of a zygote stage uterus was screened using a deduced oligonucleotide sequence based on a partial amino acid sequence of the coat protein. This study has confirmed a single copy cp4 gene encoding a unique protein of 306 amino acids, although its Nterminus shares high sequence identity with the C-terminal half of the enzyme alpha-enolase. Using Northern blots, the expression of cp4 in adult tissues showed that cp4 transcript is restricted predominantly to the uterus with two stages of expression occurring in the gravid uterus at early cleavage and bilaminar stages, suggesting an important developmental role for CP4. Using RT-PCR, cp4-specific expression as represented by the T-end 400 bp was present in heart, liver, oviduct, and uterus. Uterine expression reflected the increase found with Northern blot except that expression was low at unilaminar and bilaminar stages. (C) 2003 Wiley-Liss, Inc.