We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.

SummaryThe acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5Ј untranslated region (UTR). However, when this 5Ј UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5Ј end of the petD 5Ј UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5Ј UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5Ј→3Ј exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5Ј UTR protects the mRNA from 5Ј→3Ј degradation in wild-type cells.

SummaryMolecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5¢ and 3¢ untranslated regions. While it is wellknown that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G) 18 sequence, which impedes both 5¢®3¢ and 3¢®5¢ exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions ±20, +25, +165 or +25/+165 relative to the mature petD 5¢ end, mRNAs accumulate with 5¢ ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5¢ end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5¢®3¢ exoribonucleolytic activity. In the case of the ±20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5¢ end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5¢ ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) ±20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.