C Woltering | PubMed Health

PubMed1998

A phage-based system to select multiple protein-protein interactions simultaneously from combinatorial libraries.

Selectively infective phage (SIP) can be used to identify protein-protein interactions. SIP was modified to facilitate the simultaneous selection of interacting protein pairs from large combinatorial libraries. An interference-resistant phage was constructed which non-covalently, but stably links the genetic information of an interacting pair, encoded separately on phage and phagemid vectors, by co-packaging into heteropolyphages. In a model system, the interaction between a SIP-selected peptide and the intracellular domain of the p75 neurotrophin receptor was detected in the presence of a 10(4)-fold excess of a non-interacting control pair (jun leucine zipper and p75 intracellular domain) via SIP hetero-polyphage transductants. To minimize the redundancy of transductants and to minimize possible ligand exchange generated in a solution-based SIP screening, a filter-based in situ infectivity screening was developed. The combination of the above techniques may provide a powerful system for rapid screening of very large sequence spaces.

Adaptor Proteins, Vesicular Transport↗

PubMed1997

A conserved tissue-specific structure at a human T-cell receptor beta-chain core promoter.

The T-cell receptor (TCR) beta-chain promoters have been characterized as nonstructured basal promoters that carry a single conserved ubiquitous cyclic AMP-responsive element. Our investigation of the human TCR beta gene uncovers a surprisingly complex and tissue-specific structure at the TCR Vbeta 8.1 promoter. The core of the promoter (positions -42 to +11) is recognized by the lymphoid cell-specific transcription factors Ets-1, LEF1, and AML1 as well as by CREB/ATF-1, as is demonstrated in gel shift and footprinting experiments. With the exception of LEF1, these factors activate transcription in T cells. Binding sites at the core region show little conservation with consensus sites. Nonetheless, CREB, Ets-1, and AML1 bind and activate cooperatively and very efficiently through the nonconsensus binding sites at the core promoter region. Moderate ubiquitous activation is further induced by CREB/ATF and Sp1 factors through proximal upstream elements. The tissue-specific core promoter structure is apparently conserved in other T-cell-specifically expressed genes such as the CD4 gene. Our observations suggest that both the enhancer and the promoter have a complex tissue-specific structure whose functional interplay potentiates T-cell-specific transcription.

Base Sequence↗