Formation of putrescine and cinnamoyl putrescines in tobacco cell cultures

A p-fluorophenylalanine- (PFP) resistant cell line of Nicotiana tabacum and wild type cells accumulating high and low levels of cinnamoyl putrescines, respectively, were used to study the formation of putrescine in the biosynthesis of cinnamoyl putrescines. Labelled arginine and ornithine were equally well incorporated into the main conjugates caffeoyl and feruloyl putrescine. Trapping experiments indicated that both amino acids were decarboxylated for putrescine biosynthesis. Nearly all alcohol-extractable radioactivity from the labelled amino acids was found as cinnamoyl putrescines in the PFP-resistant cell line, whereas wild type cells retained significant radioactivity in the amino acids. The enzyme activities of arginine and ornithine decarboxylases in the resistant cell line were increased 3- to 6-fold.     

Isolation and properties of recombinant DNA from a plasmid and filamentous phage

In the course of studying extrachromosomal DNA with composite replicons, a hybrid has been constructed by the in vitro recombination of the filamentous phage M13mp2 DNA (RF) and plasmid pUR222 (ApR). Both parental DNAs contain a fragment of lac-operon (ca. 800 bp), which includes the distal end of lacI gene, lacPO segments, and the lacZ gene proximal region coding for 145 N-terminal amino acid residues of beta-galactosidase and thus providing for alpha-complementation, the effect being cancelled with a polynucleotide insertion at the unique EcoRI site in the lacZ gene segment. E. coli BMH71-18 cells were transformed with the ligated mixture of EcoRI restricts of both DNAs. A phage-like nucleoprotein was isolated from colourless plaques (on the Xgal- and IPTG-supplemented medium); its deproteinization yielded a DNA which contains the ApR-determinant and, according to PAGE, structurally specific staining, restriction analysis, sequencing by the Sanger procedure, and electron microscopy data, is a linear double-stranded molecule comprising the phage and plasmid genomes in an equimolar ratio. Since the hybrid DNA does not display the alpha-complementation effect, both bacterial inserts are in the opposite orientation. Transformation of both phage (F+) and plasmid (F-) hosts with the hybrid DNA led to cultures which, after precipitation of the nucleoprotein from the extracellular medium and deproteinization, afforded the same composite DNA.(ABSTRACT TRUNCATED AT 250 WORDS)