Cloning and expression of two new prolactin-related proteins, prolactin-related protein-VIII and -IX, in bovine placenta.
BACKGROUND
Prolactin-related proteins (PRPs) are specific proteins of the growth hormone/prolactin (GH/PRL) family in bovine placenta. This study reports the identification and sequencing of a full-length cDNA for two new members of bovine PRPs, bPRP-VIII and -IX, and their localization and quantitative expression in bovine placenta.


METHODS
New bPRP-VIII and -IX were identified from bovine placentome. Localization and quantitative gene expression in the placenta were respectively investigated by in situ hybridization and real-time RT-PCR methods. Recombinant proteins of these genes were produced by a mammalian HEK293 cell expression system.


RESULTS
Full-length bPRP-VIII and -IX cDNA were respectively cloned with 909 and 910 nucleotide open-reading-frames corresponding to proteins of 236 and 238 amino acids. The predicted bPRP-VIII amino acid sequence shared about 40 to 70% homology with other bPRPs, and bPRP-IX had about 50 to 80% homology of others. The two new bPRPs were detected only in the placenta by RT-PCR. mRNA was primarily expressed in the cotyledon and intercotyledonary tissues throughout gestation. An in situ hybridization analysis revealed the presence of bPRP-VIII and -IX mRNA in the trophoblastic binucleate and/or trinucleate cells. bPRP-VIII mRNA was observed in the extra-embryonic membrane on Day 27 of gestation, however, no bPRP-IX mRNA was observed in the extra-embryonic membrane in the same stage of pregnancy by quantitative real-time RT-PCR analysis. Both new bPRP genes were possible to translate a mature protein in a mammalian cell expression system with approximately 28 kDa in bPRP-VIII and 38 kDa in bPRP-IX.


CONCLUSIONS
We identified the new members of bovine prolactin-related protein, bPRP-VIII and -IX. Localization and quantitative expression were confirmed in bovine placenta by in situ hybridization or real-time PCR. Their different temporal and spatial expressions suggest a different role for these genes in bovine placenta during gestation.
Ushizawa
Koichi
K
Developmental Biology Department, National Institute of Agrobiological Sciences, 2 Ikenodai, Tsukuba, Ibaraki 305-8602, Japan. ushizawa@affrc.go.jp
Takahashi
Toru
T
Hosoe
Misa
M
Kaneyama
Kanako
K
Hashizume
Kazuyoshi
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D005260:Female; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D010920:Placenta / Q000166:cytology / Q000378:metabolism*; D010926:Placental Hormones / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011247:Pregnancy; D011388:Prolactin / Q000145:classification; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D018076:DNA, Complementary; D010926:Placental Hormones; D011993:Recombinant Fusion Proteins; D011388:Prolactin