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Expression and characterization of six Sry loci from the SHR Y chromosome
Author(s) -
Underwood Adam,
Ely Daniel,
Turner Monte,
Milsted Amy
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a476-b
Subject(s) - testis determining factor , microbiology and biotechnology , recombinant dna , antiserum , biology , hmg box , gene , dna , transfection , y chromosome , genetics , antibody , dna binding protein , transcription factor
Recently we described six Sry loci on a single Y chromosome of SHR. To determine if these loci have the potential to express functional proteins we cloned the coding region of each into eukaryotic (pcDNA3.1) and prokaryotic (pIVEX2.3) expression constructs. Western blot analysis with a commercially available antiserum was used to confirm the presence of recombinant eukaryotic Sry (reSry), expressed in transiently transfected CHO cells, and recombinant prokaryotic Sry (rpSry), expressed in BL21(De3) cells. Six reSry and six rpSry proteins were detected. Only the rpSry proteins were detected using a peptide antibody against amino acids 117–130 of rat Sry; this antibody did not detect reSry proteins. One explanation for why the antibody did not react with reSry is the presence of a potential PKC phosphorylation site at amino acid 124. Results from these studies demonstrate that we can express and detect recombinant Sry and suggest that reSry is potentially phosphorylated. DNA affinity purification was successfully performed with reSry and rpSry proteins using oligonucleotides containing an Sry response element, indicating that all reSry and rpSry interact with consensus Sry DNA response elements. The relative DNA binding affinity of each protein with and without posttranslational modification for Sry response elements can now be addressed. Supported by NIH RO1‐HL71579‐01A3 and UA FRG 1653.