Arie Admon

Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB

Enhancement of RNA polymerase II transcription by the viral transactivator VP16 requires the TFIID complex, which consists of the TATA-binding protein (TBP) and TBP-associated factors (TAFs). Here we report the molecular cloning, expression, and biochemical characterization of Drosophila TAFII40 (dTAFII40), a subunit of TFIID. In vitro protein-protein interaction assays revealed direct binding between dTAFII40 and a 39 amino acid VP16 activation domain. In addition, affinity chromatography indicated a direct binding of the basal factor TFIIB to immobilized dTAFII40. Since VP16 also binds TFIIB, our results suggest a ternary interaction among an activator, a coactivator, and a basal transcription factor. Antibodies directed against dTAFII40 inhibited activation by GAL4-VP16 without affecting basal transcription. These results, taken together with previous studies of Sp1 and dTAFII110, establish that different activators interact with distinct TAFs in the TFIID complex and that TAFs can contact both activators and basal factors.

Fasciclin IV: Sequence, expression, and function during growth cone guidance in the grasshopper embryo

Monoclonal antibody 6F8 was used to characterize and clone fasciclin IV, a new axonal glycoprotein in the grasshopper, and to study its function during growth cone guidance. Fasciclin IV is dynamically expressed on a subset of axon pathways in the developing CNS and on circumferential bands of epithelial cells in developing limb buds. One of these bands corresponds to the location where the growth cones of the Ti1 pioneer neurons make a characteristic turn while extending toward the CNS. Embryos cultured in the 6F8 antibody or Fab exhibit aberrant formation of this axon pathway. cDNA sequence analysis suggests that fasciclin IV has a signal sequence; long extracellular, transmembrane, and short cytoplasmic domains; and shows no homology with any protein in the available data bases. Thus, fasciclin IV appears to be a novel integral membrane protein that functions in growth cone guidance.

OPTIMIZATION OF SOLID PHASE PEPTIDE SEQUENCING

Publisher Summary This chapter describes optimized conditions for use of solid phase support on the 477A sequencer and discusses the benefits of this combination. Solid phase sequencing enables prior chemical and enzymatic modifications of the peptides while attached to the solid support. Sequencing covalently-bound peptides result in longer readable sequences because of the improved signal to noise and higher initial and repetitive yields. These improvements are most significant when attempting to sequence peptides at the 1 to 10 picomole range or when complete sequence of the peptide is necessary. The identification of post translational modification sites and post attachment chemical treatment of the linked peptides are other useful features of covalent sequencing. Shortening cycle time, reducing reagent consumption, and eliminating the need to precycle polybrene-coated filters has resulted in significant savings of time and expense. However, solid phase sequencing cannot be universally applied as many peptides do not have the available carboxyl groups to be covalently coupled to the solid support.