The use of binary transcriptional systems offers many advantages for experimentally manipulating gene activity, as exemplified by the success of the Gal4/UAS system in Drosophila . To expand the number of applications, a second independent transactivator (TA) is desirable. Here, we present the optimization of an additional system based on LexA and show how it can be applied. We developed a series of LexA TAs, selectively suppressible via Gal80, that exhibit high transcriptional activity and low detrimental effects when expressed in vivo. In combination with Gal4, an appropriately selected LexA TA permits to program cells with a distinct balance and independent outputs of the two TAs. We demonstrate how the two systems can be combined for manipulating communicating cell populations, converting transient tissue-specific expression patterns into heritable, constitutive activities, and defining cell territories by intersecting TA expression domains. Finally, we describe a versatile enhancer trap system that allows swapping TA and generating mosaics composed of Gal4 and LexA TA-expressing cells. The optimized LexA system facilitates precise analyses of complex biological phenomena and signaling pathways in Drosophila .
The analysis of large molecules by matrix-assisted laser desorption time-of-flight mass spectrometry (MALD/TOF) is established as a powerful technique for the determination of molecular mass.',' The analysis of biopolymers such as prot e i n~,~-~ oligonucleotide^,^-^ and polysaccharideslO*'l by MALD/TOF provides molecular mass information faster and with more accuracy than previously available techniques such as polyacrylamide gel electrophoresis." Another class of large molecules for which this technique should provide important information is synthetic organic polymers. MALD/TOF has several advantages over conventional polymer molecular mass techniques such as gel permeation chromatography and vis-~0 m e t r y . I~ It provides absolute molecular masses as opposed to relative values, the entire distribution is determined instead of an average value for the molecular mass and molecular composition information is obtained since the molecular masses of single oligomers are measured. Previous results on polygly~ols~*'~ have been encouraging. Here we show results demonstrating the power of MALD/TOF for the determination of molecular mass distribution, polymer composition, and end group for selected water-soluble polymers.Measurements were performed using a Bruker REFLEXTM MALD/TOF mass spectrometer. This time-of-flight instrument is fitted with a reflectron and dual microchannel plate detector for high resolution analysis and with a postacceleration detector in the linear mode for lower resolution molecular mass distribution measurements. Samples were prepared in a sinapinic acid matrix at a mole ratio of to with a total loading of polymer around 20 to 100 pico-moles (this is based on the manufacturer's value for the average molecular mass). Ions were formed by laser desorption at 337 nm (N2 laser, 3 ns pulse width, lo7 to lo* 0.2 mm2 spot) and accelerated with 10-33 kV. The negative ions were detected in all cases, and ions less than m/z 1000 were removed with a pulsed deflector. The poly(acry1ic acid) sample (weight-average molecular mass," M, = 3000 Da) is supplied from Polysciences, Inc. and poly(styrene sulfonate) (peak-average molecular mass,I5 M, = 200000 Da) is from Polymer Laboratories. The polymers were supplied as the neutralized sodium salts of the acids. Since salts have been shown to have a deleterious effect on MALD ionization,6 the polymers were prepared in the acid form by ion-exchange with a column of Amberlite IRN-77 (Rohm and Haas Co.) ion-exchange resin followed by freeze-drying. The MALD/TOF mass spectrum of poly(styrene sulfonic acid) is shown in Fig.
Some properties of matrix-assisted laser desorption have been studied using single-ion-counting methods and a time-to-digital converter. The methods allow examination of the process for irradiances near the reported threshold for observation with a transient recorder. All measurements were made using bovine insulin as a test compound. We present direct evidence that an irradiance threshold near 10(6) W cm-2 exists for ion production, and that the process is a collective effect, either involving a large number of molecular ions (approximately 10(4) in a successful event or none at all. Above the threshold, the yield is found to scale with a high power (4th to 6th) of the irradiance. Measurements of initial velocity distributions indicate an axial velocity spread corresponding to approximately 50 eV and a radial velocity spread corresponding to approximately 2.4 eV. Thus the ejection or extraction mechanism appears to be strongly asymmetric.
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