Pantropic retroviral vectors integrate and express in cells of the malaria mosquito, Anopheles gambiae.

The lack of efficient mechanisms for stable genetic transformation of medically important insects, such as anopheline mosquitoes, is the single most important impediment to progress in identifying novel control strategies. Currently available techniques for foreign gene expression in insect cells in culture lack the benefit of stable inheritance conferred by integration. To overcome this problem, a new class of pantropic retroviral vectors has been developed in which the amphotropic envelope is completely replaced by the G glycoprotein of vesicular stomatitis virus. The broadened host cell range of these particles allowed successful entry, integration, and expression of heterologous genes in cultured cells of Anopheles gambiae, the principle mosquito vector responsible for the transmission of over 100 million cases of malaria each year. Mosquito cells in culture infected with a pantropic vector expressing hygromycin phosphotransferase from the Drosophila hsp70 promoter were resistant to the antibiotic hygromycin B. Integrated provirus was detected in infected mosquito cell clones grown in selective media. Thus, pantropic retroviral vectors hold promise as a transformation system for mosquitoes in vivo.

Rapid chemical kinetic techniques for investigations of neurotransmitter receptors expressed in Xenopus oocytes

Xenopus laevis oocytes have been used extensively during the past decade to express and study neurotransmitter receptors of various origins and subunit composition and also to express and study receptors altered by site-specific mutations. Interpretations of the effects of structural differences on receptor mechanisms were, however, hampered by a lack of rapid chemical reaction techniques suitable for use with oocytes. Here we describe flow and photolysis techniques, with 2-ms and 100-μs time resolution, respectively, for studying neurotransmitter receptors in giant (≈20-μm diameter) patches of oocyte membranes, using muscle and neuronal acetylcholine receptors as examples. With these techniques, we find that the muscle receptor in BC3H1 cells and the same receptor expressed in oocytes have comparable kinetic properties. This finding is in contrast to previous studies and raises questions regarding the interpretations of the many studies of receptors expressed in oocytes in which an insufficient time resolution was available. The results obtained indicate that the rapid reaction techniques described here, in conjunction with the oocyte expression system, will be useful in answering many outstanding questions regarding the structure and function of diverse neurotransmitter receptors.

Mode matches and their locations in the hydrophobic free energy sequences of peptide ligands and their receptor eigenfunctions

Patterns in sequences of amino acid hydrophobic free energies predict secondary structures in proteins. In protein folding, matches in hydrophobic free energy statistical wavelengths appear to contribute to selective aggregation of secondary structures in “hydrophobic zippers.” In a similar setting, the use of Fourier analysis to characterize the dominant statistical wavelengths of peptide ligands’ and receptor proteins’ hydrophobic modes to predict such matches has been limited by the aliasing and end effects of short peptide lengths, as well as the broad-band, mode multiplicity of many of their frequency (power) spectra. In addition, the sequence locations of the matching modes are lost in this transformation. We make new use of three techniques to address these difficulties: (i) eigenfunction construction from the linear decomposition of the lagged covariance matrices of the ligands and receptors as hydrophobic free energy sequences; (ii) maximum entropy, complex poles power spectra, which select the dominant modes of the hydrophobic free energy sequences or their eigenfunctions; and (iii) discrete, best bases, trigonometric wavelet transformations, which confirm the dominant spectral frequencies of the eigenfunctions and locate them as (absolute valued) moduli in the peptide or receptor sequence. The leading eigenfunction of the covariance matrix of a transmembrane receptor sequence locates the same transmembrane segments seen in n-block-averaged hydropathy plots while leaving the remaining hydrophobic modes unsmoothed and available for further analyses as secondary eigenfunctions. In these receptor eigenfunctions, we find a set of statistical wavelength matches between peptide ligands and their G-protein and tyrosine kinase coupled receptors, ranging across examples from 13.10 amino acids in acid fibroblast growth factor to 2.18 residues in corticotropin releasing factor. We find that the wavelet-located receptor modes in the extracellular loops are compatible with studies of receptor chimeric exchanges and point mutations. A nonbinding corticotropin-releasing factor receptor mutant is shown to have lost the signatory mode common to the normal receptor and its ligand. Hydrophobic free energy eigenfunctions and their transformations offer new quantitative physical homologies in database searches for peptide-receptor matches.