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The length at which the N terminus of nascent proteins becomes available to antibodies during their synthesis on ribosomes was determined. Three different proteins, bovine rhodanese, bacterial chloramphenicol acetyltransferase and MS2 coat protein, were synthesized with coumarin at their N terminus in a cell-free system derived from Escherichia coli. A(More)
Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.psbA4 intron homes efficiently from a plasmid into the chloroplast psbA gene in Chlamydomonas, but little(More)
Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 and Cr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector(More)
Homing endonuclease I-CreII has been used to study the consequences and repair of a double-strand break (DSB) in the chloroplast genome of Chlamydomonas and Arabidopsis. Since I-CreII is from a mobile psbA intron of Chlamydomonas, it cleaves the psbA gene of an intronless-psbA strain of Chlamydomonas. And it cleaves specifically in the psbA gene of(More)
The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5' untranslated regions of the(More)
Fluorescently labeled rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system. A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide. Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in(More)
Synthesis of rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction. The ribosome-bound protein is enzymatically inactive but can be(More)
The use of synthetic tRNA for in vitro protein engineering was tested in a coupled transcription/translation system prepared from Escherichia coli. DNA sequences similar to the natural tRNA(Ala/UGC) gene from E. coli but with different anticodons were synthesized in vitro, cloned into a DNA plasmid, and then transcribed in vitro with T7 RNA polymerase. The(More)
The fate of the amino termini of nascent polyalanine, polyserine, and polylysine was monitored by fluorescence techniques as each was translated on Escherichia coli ribosomes. A coumarin probe was placed at the alpha-amino group of a synthetic elongator alanyl-tRNA or a synthetic initiator alanyl-tRNA or at the epsilon-amino group of natural lysyl-tRNA, and(More)