The decapeptide caerulein represents one of the main constituents of the skin secretion of Xenopus laevis. Total mRNA was isolated from skin, transcribed into cDNA and inserted via GC-tailing into the plasmid pUC8. Among the transformants, 300 clones were selected at random and screened with a cDNA primed with the synthetic deoxynucleotide d(AGTCCATCCA), which is complementary to the mRNA region coding for the fragment Trp-Met-Asp-Phe of caerulein. Of nine strongly hybridizing clones, three were sequenced and these were found to contain inserts with very similar nucleotide sequences. The cloned cDNAs code for parts of two different caerulein precursors. These contain one or two copies of caerulein and five additional amino acids located between pairs of arginine residues. The extra glycine at the carboxy terminnus is considered to serve as the signal for amidation, while the tetrapeptide Phe-Ala-Asp-Gly, linked to the amino end of caerulein in these precursors, must be cleaved by an unusual processing mechanism.
For studies on the expression of recombinant DNA, linker fragments have been prepared which serve to introduce specific points of cleavage on the DNA as well as the protein level. Such linkers were designed for cyanogen bromide as well as for collagenase cleavage of fused proteins. The preparation was done according to the triester synthesis scheme using improved and simpler routes to functionalized dinucleotide building blocks. Field desorption mass spectrometry was used as a routine tool for the identification and control of the purity of these units.Synthese von Desoxyoligonucleotid-Fragmenten zur Anwendung in der Genverknupfung unter Einsatz verbesserter priiparativer und analylischer Methoden ',*) Zur Untersuchung der Expression rekombinanter DNS haben wir Oligonucleotide als Genverknupfungsfragmente dargestellt, die spezifische Spaltstellen sowohl in die DNA wie in das zugehorige Protein einfuhren. Ihr Aufbau ist so, daB die Spaltung eines Fusionsproteins entweder durch Bromcyan oder durch Kollagenase erfolgen kann. Die Synthese wurde nach dem Triesterschema durchgefuhrt, wobei schnellere und einfachere Wege zu funktionalisierten Dinucleotid-Baueinheiten entwickelt wurden. Die Felddesorptions-Massenspektrometrie diente als Routinemethode fur den Strukturbeweis und die Reinheitskontrolle dieser Baueinheiten.
Ab"p(CP)Tp(CP)Gib"p(CP)AbZp(CP)Tp(CP)Cb~(CP)Abzp(CP)Tp(CP)G~~ (U), the dodecamer DMTrdCbzp(CP)AbZp(CP)Tp(CP)GibUp(CP)Abzp(CP)AbZp(CP)Tp(CP)Tp(CP)Cbzp(CP)AbZ-p(CP)Tp(CP)GtF (16) and the two nonamers DMTrdGibUp(CP)Gibup(CP)Cb%(CP)Cblp(CP)-Cb"p(CP)Tp(CP)ib"p(CP)CbZp(CP)Az (24) and DMTrdGib"p(CP)Gib"p(CP)Gibup(CP)CbZp(CP)-Cbzp(CP)Tp(CP)Gib"p(CP)Cb'p(CP)A~ (25) were prepared by combining the blocks and using the reaction conditions listed in Tables 2 and 3. The products were worked up as described above Liebigs Ann. Chem. 1984 56'
Zur Untersuchung der Expression rekombinanter DNS werden die Oligonucleotide (IV)‐(VII) als Genverknüpfungsfragmente dargestellt, die spezifische Spaltstellen sowohl in die DNS wie in das zugehörige Protein einführen.
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