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    Isolation of progesterone-dependent complementary deoxyribonucleic acid fragments from rhesus monkey endometrium by sequential subtractive hybridization and polymerase chain reaction amplification

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    Authors
    Ace, Christopher I.
    Balsamo, Melissa
    Le, L. T.
    Okulicz, William C.
    UMass Chan Affiliations
    Department of Obstetrics and Gynecology
    Document Type
    Journal Article
    Publication Date
    1994-03-01
    Keywords
    Animals
    Base Sequence
    DNA, Complementary
    Endometrium
    Female
    Macaca mulatta
    Molecular Sequence Data
    Nucleic Acid Hybridization
    Polymerase Chain Reaction
    Progesterone
    Life Sciences
    Medicine and Health Sciences
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    Link to Full Text
    https://doi.org/10.1210/en.134.3.1305
    Abstract
    The steroid sex hormone progesterone (P) induces the expression of a variety of genes through a signal transduction pathway mediated by the P receptor, a DNA-binding regulator of transcription. To identify genes and gene networks that are P dependent in the rhesus endometrium, we used a powerful technique employing subtractive hybridization coupled with polymerase chain reaction (PCR). Poly(A)+ RNA was isolated from both P-dominant (days 21-23 of artificial menstrual cycles) and estrogen (E)-dominant (days 9-13) endometrium. The two classes of RNA were converted to cDNA, ligated to EcoRI adaptors, and amplified by PCR using an adaptor-complimentary primer. E-dominant cDNA was labeled with biotin, hybridized in excess to P-dominant cDNA (PcDNA), and complexed with streptavidin. Labeled cross-hybrid sequences common to both populations were subtracted by phenol-chloroform extraction. The remaining cDNA fragments were amplified by PCR. After four rounds of hybridization/amplification, the subtracted PcDNA was analyzed for P-dependent sequences by semiquantitive PCR. Initial analysis revealed that housekeeping genes were undetectable in subtracted cDNA, but a previously characterized P-dependent gene was retained. Three of five clones sequenced at random from the subtracted library exhibited P-inducibility/dependency by PCR analysis of E and PcDNA. One of these, an 835-basepair fragment designated H5, may represent a novel P-dependent gene, as no comparable homology could be found with existing sequences in GenBank and Swissprot databases. We estimate that the procedure described here resulted in highly significant enrichment of up-regulated cDNA fragments from P-dominant tissue.
    Source

    Endocrinology. 1994 Mar;134(3):1305-9.

    Permanent Link to this Item
    http://hdl.handle.net/20.500.14038/42163
    PubMed ID
    8119170
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