YY1 is ubiquitously expressed zinc finger DNA binding protein. It can act as a transcriptional repressor or activator and, when binding at the initiator element, as a component of the basal transcription complex. Binding sites for YY1 have been reported in a wide variety of promoters and they exhibit substantial diversity in their sequence. To better understand how YY1 interacts with DNA and to be able to predict the presence of YY1 sites in a more comprehensive fashion, we have selected YY1 binding sites from a random pool of oligonucleotides. The sites display considerable heterogeneity, but contain a conserved 5'-CAT-3' core flanked by variable regions, generating the consensus 5'-(C/g/a)(G/t)(C/t/a)CATN(T/a)(T/g/c)-3', where the upper case letters represent the preferred base. This high degree of flexibility in DNA recognition can be predicted by modeling the interaction of the four YY1 zinc fingers with DNA and a detailed model for this interaction is presented and discussed.

Polyoma gene expression is temporally regulated during productive infection of mouse cells. Early genes are expressed throughout the viral life cycle, but late mRNAs are not detected until after the onset ofDNA replication. At late times, late-strand transcripts represent the great majority of viral-specific RNA in the cell. To learn more about the mechanism by which the early-late switch is regulated, we have carried out a detailed analysis of polyomavirus transcription in mouse NIH 3T6 cells. Nuclei were isolated from cells infected for 6, 12, 18, or 24 hr, and run-on assays were performed. The resulting RNAs were then hybridized to a number of immobilized early-and late-strand-specific probes, which represent the entire polyoma genome. Results indicate that the late promoter is always on, even in the absence of DNA replication. Even though the early-late switch is characterized by a >300-fold difference in the ratio ofsteady-state early-and late-strand RNAs, there is only a 2-fold effect at the level of transcription initiation. Furthermore, the efficiency of termination for late transcripts is very high at early times during infection (>90%) but drops drastically at late times (<40%). In other experiments, we have found an increase in splicing efficiency of late pre-mRNA molecules that parallels the decrease in termination efficiency. These results, taken together with other studies from our laboratory, have led us to propose two possible models for the temporal control of polyomavirus late gene expression.Polyoma is a small, double-stranded, circular DNA virus whose genome contains two transcription units (early and late) and an intergenic control region (Fig.

Background:The rapidly expanding list of pharmacologically important targets has highlighted the need for ways to discover new inhibitors that are independent of functional assays. We have utilized peptides to detect inhibitors of protein function. We hypothesized that most peptide ligands identified by phage display would bind to regions of biological interaction in target proteins and that these peptides could be used as sensitive probes for detecting low molecular weight inhibitors that bind to these sites. Results:We selected a broad range of enzymes as targets for phage display and isolated a series of peptides that bound specifically to each target. Peptide ligands for each target contained similar amino acid sequences and competition analysis indicated that they bound one or two sites per target. Of 17 peptides tested, 13 were found to be specific inhibitors of enzyme function. Finally, we used two peptides specific for Haemophilus influenzae tyrosyl-tRNA synthetase to show that a simple binding assay can be used to detect small-molecule inhibitors with potencies in the micromolar to nanomolar range.Conclusions: Peptidic surrogate ligands identified using phage display are preferentially targeted to a limited number of sites that inhibit enzyme function. These peptides can be utilized in a binding assay as a rapid and sensitive method to detect small-molecule inhibitors of target protein function. The binding assay can be used with a variety of detection systems and is readily adaptable to automation, making this platform ideal for high-throughput screening of compound libraries for drug discovery.

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Myelin basic protein (MBP) is a major structural component of myelin. It is expressed exclusively in myelinating glia (oligodendrocytes in the CNS and Schwann cells in the PNS) and is localized to the cytoplasmic surface of the plasma membrane and myelin membrane produced by these cells. The work described here concerns the mechanism of plasma membrane localization of MBP in myelinating glial cells and whether it involves differentiated functions specific to these cells or general functions of plasma membrane assembly common to all cells. To this end, the subcellular localization of endogenous MBP in mouse oligodendrocytes was compared with that of transiently expressed MBP in monkey fibroblasts (Cos-1 cells) transfected with an MBP expression vector containing cDNA for rat 14K MBP. The steady-state levels of MBP-specific RNA and of MBP polypeptide expressed in the transfected fibroblasts were comparable to the levels expressed in oligodendrocytes in primary culture. MBP localization was analyzed in whole cells by immunofluorescence and in specific intracellular compartments by subcellular fractionation. The results show that MBP expressed in wild-type oligodendrocytes is localized to the plasma membrane. In contrast, MBP expressed in transfected fibroblasts appears dispersed in the cytoplasm and is distributed uniformly among the various subcellular fractions.(ABSTRACT TRUNCATED AT 250 WORDS)