Restriction maps of several recombinant plasmids representing a section of the E. coli K12 chromosome 35,000 bp in size with the genes phoA, proC and phoB were prepared. The orientation of phoA and the exact position of its N-terminal end on this map were determined by identifying a subfragment which carried the phoA promoter and by determining the nucleotide sequence of a 160 bp portion of this subfragment comprising the codons for the N-terminal end of pre-alkaline phosphatase. From this DNA sequence the leader sequence of alkaline phosphatase which consists of 21 amino acids was derived.
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'
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