Molecular cloning of bovine (Bos taurus) cDNA encoding a 94-kDa glucose-regulated protein and developmental changes in its mRNA and protein content in the mammary gland.
We isolated and sequenced cDNA clones encoding a 94-kDa glucose-regulated protein (GRP94) from a cDNA library constructed using bovine (Bos taurus) mammary gland poly(A)(+) RNA. The coding nucleotide sequence and the deduced amino acid sequence of bovine GRP94 shared 94.2-88.4% and 98.1-96.5% identity with those of other mammalian species, respectively. The primary structure contained a carboxyl-terminal signal sequence for retention in the endoplasmic reticulum, six potential sites for N-linked glycosylation and two potential adenosine 5'-triphosphate binding sites, similar to other mammalian and avian GRP94 homologues. In Northern blot hybridization using a cDNA probe from the bovine GRP94 cDNA sequence, a transcript 3.0 kb in size was detected. We measured the amounts of GRP94 and its mRNA in mammary glands from cows at various developmental stages of hormonally induced lactation. The highest level of GRP94 mRNA, determined by dot blot analysis, was detected in the developing stage. In contrast to the mRNA level, the amount of protein, determined by immunoblot analysis using rabbit antiserum raised against GRP94 purified from bovine brain, was higher in lactating stages than in others. The increased level of GRP94 mRNA during the developing stage and the maintenance of GRP94 protein during lactation suggest that the synthesis of GRP94 is regulated during mammary development and differentiation, and also that the protein is involved in a function related to lactation.
Watanabe
A
A
Hokkaido Research Station, National Institute of Animal Health, 4 Hitsujigaoka, Sapporo 062-0045, Japan. awata@affrc.go.jp
Uchida
I
I
Nakata
K
K
Fujimoto
Y
Y
Oikawa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D015152:Blotting, Northern; D001940:Breast / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007774:Lactation; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D011061:Poly A; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D018076:DNA, Complementary; D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C018551:glucose-regulated proteins; D011061:Poly A; D004247:DNA