Architectural specificity in chromatin structure at the TATA box in vivo: Nucleosome displacement upon β-phaseolin gene activation

Extensive studies of the β-phaseolin (phas) gene in transgenic tobacco have shown that it is highly active during seed embryogenesis but is completely silent in leaf and other vegetative tissues. In vivo footprinting revealed that the lack of even basal transcriptional activity in vegetative tissues is associated with the presence of a nucleosome that is rotationally positioned with base pair precision over three phased TATA boxes present in the phas promoter. Positioning is sequence-dependent because an identical rotational setting is obtained upon nucleosome reconstitution in vitro. A comparison of DNase I and dimethyl sulfate footprints in vivo and in vitro strongly suggests that this repressive chromatin architecture is remodeled concomitant with gene activation in the developing seed. This leads to the disruption of histone-mediated DNA wrapping and the assembly of the TATA boxes into a transcriptionally competent nucleoprotein complex.

Role of intrinsic synaptic circuitry in collicular sensorimotor integration

The superficial gray layer of the superior colliculus contains a map that represents the visual field, whereas the underlying intermediate gray layer contains a vector map of the saccades that shift the direction of gaze. These two maps are aligned so that a particular region of the visual field is represented directly above the neurons that orient the highest acuity area of the retina toward that region. Although it has been proposed that the transmission of information from the visuosensory to the motor map plays an important role in the generation of visually guided saccades, experiments have failed to demonstrate any functional linkage between the two layers. We examined synaptic transmission between these layers in vitro by stimulating the superficial layer while using whole-cell patch-clamp methods to measure the responses of intermediate layer neurons. Stimulation of superficial layer neurons evoked excitatory postsynaptic currents in premotor cells. This synaptic input was columnar in organization, indicating that the connections between the layers link corresponding regions of the visuosensory and motor maps. Excitatory postsynaptic currents were large enough to evoke action potentials and often occurred in clusters similar in duration to the bursts of action potentials that premotor cells use to command saccades. Our results indicate the presence of functional connections between the superficial and intermediate layers and show that such connections could play a significant role in the generation of visually guided saccades.

Rapid mass spectrometric peptide sequencing and mass matching for characterization of human melanoma proteins isolated by two-dimensional PAGE.

We report a general mass spectrometric approach for the rapid identification and characterization of proteins isolated by preparative two-dimensional polyacrylamide gel electrophoresis. This method possesses the inherent power to detect and structurally characterize covalent modifications. Absolute sensitivities of matrix-assisted laser desorption ionization and high-energy collision-induced dissociation tandem mass spectrometry are exploited to determine the mass and sequence of subpicomole sample quantities of tryptic peptides. These data permit mass matching and sequence homology searching of computerized peptide mass and protein sequence data bases for known proteins and design of oligonucleotide probes for cloning unknown proteins. We have identified 11 proteins in lysates of human A375 melanoma cells, including: alpha-enolase, cytokeratin, stathmin, protein disulfide isomerase, tropomyosin, Cu/Zn superoxide dismutase, nucleoside diphosphate kinase A, galaptin, and triosephosphate isomerase. We have characterized several posttranslational modifications and chemical modifications that may result from electrophoresis or subsequent sample processing steps. Detection of comigrating and covalently modified proteins illustrates the necessity of peptide sequencing and the advantages of tandem mass spectrometry to reliably and unambiguously establish the identity of each protein. This technology paves the way for studies of cell-type dependent gene expression and studies of large suites of cellular proteins with unprecedented speed and rigor to provide information complementary to the ongoing Human Genome Project.

Both N-terminal myosin-binding and C-terminal actin-binding sites on smooth muscle caldesmon are required for caldesmon-mediated inhibition of actin filament velocity

It has been suggested that the tethering caused by binding of the N-terminal region of smooth muscle caldesmon (CaD) to myosin and its C-terminal region to actin contributes to the inhibition of actin-filament movement over myosin heads in an in vitro motility assay. However, direct evidence for this assumption has been lacking. In this study, analysis of baculovirus-generated N-terminal and C-terminal deletion mutants of chicken-gizzard CaD revealed that the major myosin-binding site on the CaD molecule resides in a 30-amino acid stretch between residues 24 and 53, based on the very low level of binding of CaDΔ24–53 lacking the residues 24–53 to myosin compared with the level of binding of CaDΔ54–85 missing the adjacent residues 54–85 or of the full-length CaD. As expected, deletion of the region between residues 24 and 53 or between residues 54 and 85 had no effect on either actin-binding or inhibition of actomyosin ATPase activity. Deletion of residues 24–53 nearly abolished the ability of CaD to inhibit actin filament velocity in the in vitro motility experiments, whereas CaDΔ54–85 strongly inhibited actin filament velocity in a manner similar to that of full-length CaD. Moreover, CaD1–597, which lacks the major actin-binding site(s), did not inhibit actin-filament velocity despite the presence of the major myosin-binding site. These data provide direct evidence for the inhibition of actin filament velocity in the in vitro motility assay caused by the tethering of myosin to actin through binding of both the CaD N-terminal region to myosin and the C-terminal region to actin.

Patterned library analysis: A method for the quantitative assessment of hypotheses concerning the determinants of protein structure

Site-directed mutagenesis and combinatorial libraries are powerful tools for providing information about the relationship between protein sequence and structure. Here we report two extensions that expand the utility of combinatorial mutagenesis for the quantitative assessment of hypotheses about the determinants of protein structure. First, we show that resin-splitting technology, which allows the construction of arbitrarily complex libraries of degenerate oligonucleotides, can be used to construct more complex protein libraries for hypothesis testing than can be constructed from oligonucleotides limited to degenerate codons. Second, using eglin c as a model protein, we show that regression analysis of activity scores from library data can be used to assess the relative contributions to the specific activity of the amino acids that were varied in the library. The regression parameters derived from the analysis of a 455-member sample from a library wherein four solvent-exposed sites in an α-helix can contain any of nine different amino acids are highly correlated (P < 0.0001, R2 = 0.97) to the relative helix propensities for those amino acids, as estimated by a variety of biophysical and computational techniques.

Mice with a targeted mutation in the thyroid hormone β receptor gene exhibit impaired growth and resistance to thyroid hormone

Patients with mutations in the thyroid hormone receptor β (TRβ) gene manifest resistance to thyroid hormone (RTH), resulting in a constellation of variable phenotypic abnormalities. To understand the molecular basis underlying the action of mutant TRβ in vivo, we generated mice with a targeted mutation in the TRβ gene (TRβPV; PV, mutant thyroid hormone receptor kindred PV) by using homologous recombination and the Cre/loxP system. Mice expressing a single PVallele showed the typical abnormalities of thyroid function found in heterozygous humans with RTH. Homozygous PV mice exhibit severe dysfunction of the pituitary–thyroid axis, impaired weight gains, and abnormal bone development. This phenotype is distinct from that seen in mice with a null mutation in the TRβ gene. Importantly, we identified abnormal expression patterns of several genes in tissues of TRβPV mice, demonstrating the interference of the mutant TR with the gene regulatory functions of the wild-type TR in vivo. These results show that the actions of mutant and wild-type TRβ in vivo are distinct. This model allows further study of the molecular action of mutant TR in vivo, which could lead to better treatment for RTH patients.