This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.
Aim : A full term pregnancy early in the life drastically decreases the life time risk of the mammary tumor. In animal models, this protection can be mimicked by exogenous estrogen and progesterone treatment. The pregnancy specific mammary factor (PMF) is an un-identified transcription factor in the mammary gland, which binds specifically to the two proximal regions of the -casein gene promoter only in the pregnancy. We hypothesize that the PMF is a progesterone downstream signaling factor, which may be involved in the protective effect on mammary tumorigensis by progesterone.
Our aim i n this study is to identify, clone and characterize the PMF from the mouse mammary gland. Material and methods: Identification and cloning of the PMF was carried out using BD matchmaker one-hybrid library construction kit. The binding sequences of the PMF were cloned into pHIS-2 vector and the cDNA library was generated from the late pregnant mouse mammary gland. The PMF-pHIS-2, cDNA library and yeast reporter expression vector pGADT-7 Rec2 (sma-1 linearized) were co-transformed into the activated yeast Y-187 strain. Co-transformed cells were grown on nutritionally selective media. The surviving colonies on the nutritional selective media with 50 mM of 3-AT were picked. The plasmids were then isolated from these colonies and sequenced. The sequences were identified by BLAST searching the GenBank. Results: We have carried out the yeast one hybrid twice. The resulting cDNAs including: mitochondrial proteins (accession # NC_0050891, NC_039170.7 and NT_039170.7); adrenergic receptor 1 (#NT-096135.5); p-53 related protein (# NW_001030649.1); and hypothetical protein on chromosome 11 (NT_16577.3). Many of these cDNAs were isolated from both experiments. Currently we are purifying the PMF protein and will analyze it by mass spectrophotometry. We will compare our data from the mass spectrophotometery with that of yeast one-hybrid.
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