GCRI gene function is required for high-level glycolytic gene expression in Saccharomyces cerevisiae.Recently, we suggested that the CTTCC sequence motif found in front of many genes encoding glycolytic enzymes lay at the core of the GCR1-binding site. Here we mapped the DNA-binding domain of GCR1 to the carboxy-terminal 154 amino acids of the polypeptide. DNase I protection studies showed that a hybrid MBP-GCR1 fusion protein protected a region of the upstream activating sequence of TPI (UASTpI), which harbored the CTTCC sequence motif, and suggested that the fusion protein might also interact with a region of the UAS that contained the related sequence CATCC. A series of in vivo G methylation protection experiments of the native TPI promoter were carried out with wild-type and gcrl deletion mutant strains. The G doublets that correspond to the C doublets in each site were protected in the wild-type strain but not in the gcrl mutant strain. These data demonstrate that the UAS of TPI contains two GCRl-binding sites which are occupied in vivo. Furthermore, adjacent RAPl/GRFI/TUF-and REB1/GRF2/QBP/Y-binding sites in UASTp, were occupied in the backgrounds of both strains. In addition, DNA band-shift assays were used to show that the MBP-GCR1 fusion protein was able to form nucleoprotein complexes with oligonucleotides that contained CTTCC sequence elements found in front of other glycolytic genes, namely, PGK, ENO], PYK, and ADHi, all of which are dependent on GCRI gene function for full expression. However, we were unable to detect specific interactions with CTTCC sequence
The ability to isolate fetal nucleated red blood cells (NRBCs) from the maternal circulation makes possible prenatal genetic analysis without the need for diagnostic procedures that are invasive for the fetus. Such isolation requires antibodies specific to fetal NRBCs. To generate a panel of antibodies to antigens present on fetal NRBCs, a new type of nonimmune phage antibody library was generated in which multiple copies of antibody fragments are displayed on each phage. Antibody fragments specific for fetal NRBCs were isolated by extensive predepletion of the phage library on adult RBCs and white blood cells (WBCs) followed by positive selection and amplification on fetal liver erythroid cells. After two rounds of selection, 44% of the antibodies analyzed bound fetal NRBCs, with two-thirds of these showing no binding of WBCs. DNA fingerprint analysis revealed the presence of at least 16 unique antibodies. Antibody specificity was confirmed by flow cytometry, immunohistochemistry, and immunofluorescence of total fetal liver and adult RBCs and WBCs. Antibody profiling suggested the generation of antibodies to previously unknown fetal RBC antigens. We conclude that multivalent display of antibodies on phage leads to efficient selection of panels of specific antibodies to cell surface antigens. The antibodies generated to fetal RBC antigens may have clinical utility for isolating fetal NRBCs from maternal circulation for noninvasive prenatal genetic diagnosis. Some of the antibodies may also have possible therapeutic utility for erythroleukemia.antibody phage display ͉ monoclonal antibody ͉ single chain Fv ͉ fetal erythroid antibodies I t has long been known that fetal red blood cells (RBCs) routinely leak into the maternal circulation during normal pregnancy (1, 2). More recently, it has been established that a very small number of fetal nucleated RBCs (NRBCs) are also routinely present in the maternal circulation (3, 4). These cells are considered the ideal target for noninvasive DNA prenatal diagnosis, but presently they cannot be readily isolated from the maternal circulation in high enough numbers and purity for routine clinical use. Because the isolation methods for purifying fetal NRBCs from maternal circulation rely on antibody-based separation and detection techniques, progress in this area has been hampered by the relative lack of antibodies to unique fetal erythroid antigenic determinants (5). Well characterized antigens expressed on fetal erythroid cells but not adult RBCs, such as CD71 and CD36, are also expressed on a number of adult white blood cells (WBCs) resulting in contamination by many WBCs in purification techniques relying on these antibodies.Fetal erythroid lineage antigens classically have been identified by massive screening of mAbs produced by conventional murine hybridoma technology using mice immunized with human fetal NRBCs. The majority of antibodies generated by this method are nonspecific and react with irrelevant epitopes present on all human cells. Conventional murine hybridom...
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