In vivo analysis of Chlamydomonas chloroplast petD gene expression using stable transformation of beta-glucuronidase translational fusions.
We have used the Escherichia coli beta-glucuronidase (uidA) gene as a reporter gene to localize the promoter and analyze the function of the 5' untranslated region (UTR) of the Chlamydomonas chloroplast petD gene. Using particle bombardment, petD-uidA transcriptional and translational fusion genes were introduced into the chloroplast genome in the large inverted repeat flanking the atpB gene. In transformants carrying a petD-uidA transcriptional fusion, uidA mRNA accumulated but was not translated. However, in a translational fusion that included the entire petD 5' UTR, uidA mRNA accumulated and a high level of beta-glucuronidase activity was detected. When approximately 70% of the petD 5' UTR was deleted from the translational fusion, uidA mRNA accumulation and beta-glucuronidase activity decreased 4- to 6-fold and 8-fold, respectively. Run-on transcription assays demonstrated that all strains transcribe the uidA gene at equivalent rates. Our results show that sequences essential for translation reside in the petD 5' UTR and also that sequences within the 5' UTR directly or indirectly affect mRNA stability. The expression of beta-glucuronidase under the control of chloroplast transcriptional and translational signals will facilitate further studies of chloroplast gene regulatory mechanisms.
Sakamoto
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Boyce Thompson Institute for Plant Research, Cornell University, Ithaca, NY 14853.
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D000818:Animals; D001483:Base Sequence; D016825:Chlamydomonas reinhardtii / Q000235:genetics*; D002736:Chloroplasts; D003573:Cytochrome b Group / Q000096:biosynthesis / Q000235:genetics*; D045346:Cytochrome b6f Complex; D004926:Escherichia coli / Q000201:enzymology; D015870:Gene Expression; D005966:Glucuronidase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006651:Histocytochemistry; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D006180:Proton-Translocating ATPases / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D015183:Restriction Mapping; D014170:Transformation, Genetic
D003573:Cytochrome b Group; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D045346:Cytochrome b6f Complex; D005966:Glucuronidase; D006180:Proton-Translocating ATPases