The copper-sensitive/temperature-sensitive phenotype of the Escherichia coli cutE mutant has been complemented by cloning wild-type genomic DNA into the plasmid vector pACYC184 and selecting transformants on medium containing 4 mM copper sulfate and chloramphenicol. One of these complementing clones, designated pCUT1, contained a 5.6-kb BamHI fragment. This recombinant plasmid transformed cutE, allowing wild-type growth of transformants on medium containing copper sulfate. Complementation of copper sensitivity was assessed by comparing both cell survival at increased copper levels and the results of 64Cu accumulation assays. An EcoRI subclone, 2.3 kb in size, was also shown to complement cutE when cloned in both medium- and high-copy-number vectors and was completely sequenced. This clone was mapped on the E. coli physical map at 705.70 to 707.80 kb. A series of subclones was constructed from pCUT1 and used to show that the large open reading frame of the translated sequence was essential for complementation. This open reading frame has a potential upstream promoter region, ribosome-binding site, and transcriptional terminator and encodes a putative protein of 512 amino acids that contains a region showing some homology to a putative copper-binding site.

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The chemical synthesis, on a 10–30 μmol scale, of two series of puromycin analogues is described: the first is the type 3′-N-aminoacyl-puromycin aminonucleoside (e.g. 3′-N-glycyl-PANS or PANS-Gly) in which the O-methyl-L-tyrosyl residue of puromycin is replaced by various aminoacyl residues, and the second is the type NpPANS-Gly in which the 5′-hydroxyl of PANS-Gly is substituted with phosphate, 3′-AMP, 3′-CMP, 3′-GMP, or 3′-UMP. The 3′-N-aminoacyl-PANS derivatives were synthesized either by coupling N-protected amino acids via their N-hydroxysuccinimide esters to PANS in 70% aqueous pyridine or by direct coupling in ethanol or methanol of N-protected amino acids to PANS using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) as condensing agent After removal of the protecting groups, reaction mixtures were purified by preparative thin-layer chromatography on silicic acid, paper chromatography, and paper electrophoresis to give the 3′-N-aminoacyl-PANS derivatives in yields of 40–80%.The 5′-O-nucleotidyl derivatives of PANS-Gly were synthesized by three methods: (i) by the coupling of fully acetylated 3′-mononucleotides to 3′-N-(Boc-glycyl)-PANS, (ii) by the coupling of N4-acetyl-2′,5′-di-O-tetrahydropyranylcytidine or N2,O2′,O5′-tritetrahydropyranylguanosine to 5′-O-phosphoryl-(Boc-glycyl)-PANS, and (iii) by the coupling of N4-acetyl-2′,5′-di-O-tetrahydropyranyl-3′-CMP or N2,O2′,O5′-tritetrahydropyranyl-3′-GMP to 3′-N-Boc-PANS or 3′-N-trifluoroacetyl-PANS followed by aminoacylation of the CpPANS or GpPANS produced with Boc-glycine and EEDQ. Coupling reactions were carried out using either N,N′-dicyclohexylcarbodiimide or 2,4,6-triisopropylbenzenesulfonyl chloride as condensing agents in anhydrous pyridine. After removal of protecting groups, reaction mixtures were purified by paper chromatography and paper electrophoresis to give the NpPANS-Gly derivatives in 3–30% yield.

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