Lactoferrin (LF) is synthesized and secreted by the uterine epithelial cells during proestrus and estrus. There is no detectable lactoferrin protein in uterine epithelium during metestrus and diestrus. A high level of lactoferrin was detected by both immunostaining and in situ hybridization in uterine epithelial cells at day 1 through day 3 of pregnancy, but disappeared completely at day 4 of pregnancy. Genomic clones containing LF gene sequences were isolated from the 129/J mouse genomic library. We found that the LF gene is organized similar to the human transferrin gene with 17 exons separated by 16 introns. Primer extension analysis demonstrated a prominent transcription initiation site at 32 bp 5' to the translation start codon. The promoter region contains two SP1 sites (-914 to -908, -535 to -529) and one AP2 site (-515 to -508). A CAAT box and a noncanonical TATA box were located at -72 and -32 respectively. An estrogen responsive element consensus sequence is located at -353 to -327. The putative ERE in mLF gene shows high homology to the Xenopus vitellogenin 2A ERE with a mutation at 3' half of the palindrome (G to A). Gel mobility shift assay, using nuclear protein extract (NPE) prepared from control and DES-treated immature mouse uterus and lung, indicates that specific protein-DNA complexes are formed with the DNA fragment containing the putative ERE. A synthetic oligonucleotide vitellogenin 2A ERE competes with the mLF ERE region for NPE effectively. DNase I footprinting with estrogen-stimulated immature mouse uterine NPE indicates that the putative mLF ERE region are protected. The past year, this laboratory has also isolated another estrogen-stimulated mouse uterine secretory protein, 63 kDa. Polyclonal antibody against 63 kDa protein was raised in rabbit. We are in the process of determining its N-terminal amino acid and partial sequencing. Human lactoferrin cDNAs from both mammary gland and bone marrow were isolated. Full length cDNA from mammary gland library was sequenced. We found one deletion near the end of the coding sequences which caused a frameshift at the 3' end of the protein.
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