Sequence of the chloroplast DNA region of Chlamydomonas reinhardii containing the gene of the large subunit of ribulose bisphosphate carboxylase and parts of its flanking genes

Specific sequence elements in the 5′ untranslated regions of rbcL and atpB gene mRNAs stabilize transcripts in the chloroplast of Chlamydomonas reinhardtii

“…59 UTR sequences (A), predicted RNA secondary structures (B), and dimethylsulfate modification (B, C) of the nucleotides of rbc L and atpB gene transcripts that were analyzed in this study for the presence of stability elements+ The total lengths of the rbc L and atpB 59 UTRs is 92 and 340 nt, respectively (Dron et al+, 1982;Woessner et al+, 1986)+ A: The 59 sequences fused in this study to the bacterial uidA gene+ Gray boxes in the sequences covering positions ϩ38 to ϩ47 in the rbc L 59 UTR and positions ϩ31 to ϩ42 in the atpB 59 UTR mark the regions found in this work to be important for transcript stability+ The individual single base changes introduced into the 59 UTR sequences are shown in the underlined sequences below the gray boxed elements+ B: Secondary structures of the rbc L and atpB 59 sequences as predicted by the mfold program (Zuker et al+, 1999)+ Boxed bases denote the location of the stability elements defined in this study (compare with A)+ The two atpB 59 end structures shown correspond to the two transcripts probably present in the chloroplast as concluded from the primer extension and DMS methylation data+ The atpB 59 UTR structure shown to the right lacks the 25 terminal nucleotides of the primary transcript shown to the left+ Labels at the sequences indicate susceptibility to methylation with DMS as estimated from the autoradiographs of C+ I: heavy methylation; ᭹: medium methylation; ⅙ : weak methylation+ Note that bases predicted by the mfold program to be unpaired are predominantly modified, and that modification of atpB 59 sequences is consistent with the presence of the two different structures shown (see text for explanation)+ C: Methylation of rbc L and atpB 59 UTR sequences+ Autoradiographs show the primer extension products of control and DMSmethylated 59 sequences of rbc L and atpB transcripts alongside sequencing ladders that serve as molecular weight markers (to the left of the samples loaded from left to right in the order A, T, G, C)+ Methylation blocks movement of the reverse transcriptase along ribonucleic acid strands+ The control lanes are loaded with samples that were not treated with DMS but were otherwise processed like the DMS-modified samples+ Numbers to the right of the autoradiograms refer to the positions of the 59 terminal nucleotides of the cDNAs, taking the 59 terminal nucleotide of the full-length cDNAs as ϩ1+ Note that the methyl- In the 59 UTR of the atpB gene, positions ϩ31 to ϩ42 are important for accumulation of transcripts of atpB 59 end:GUS genes in the chloroplast of Chlamydomonas. Nucleotide changes in this region resulted in decreases in levels of GUS transcripts of 59% for a change in position ϩ33 to almost 100% due to a mutation in position ϩ38 (Fig+ 2)+ However, in general, mutations in the sequence of the atpB 59 UTR did not result in reductions of GUS transcript levels as conspicuous as those resulting from changes in the rbc L 59 UTR+ It is possible that the presence of two species of atpB 59 UTR:GUS transcripts in our samples-primary and processed (Fig+ 1B)-whose structures and/or stabilities could be affected differently by the introduced atpB 59 end mutations, is responsible for the less pronounced effect on atpB 59 UTR:GUS transcript abundance+ Nonetheless, apart from the ϩ35 mutation that even caused transcripts to accumulate to levels that were three times higher than levels of GUS transcripts in control cells (Fig+ 2), the effects are qualitatively comparable to the effects on GUS transcript abundance in chloroplast Abundance of chimeric GUS transcripts in Chlamydomonas chloroplast transformants harboring rbc L 59 end:GUS and atpB 59 end:GUS genes+ Total RNA was isolated from transformants growing in 12-h light/12-h dark cycles at 11 h in the dark (rbc L:GUS transformants) or 1 h in the light (atpB:GUS transformants)+ RNA sampl...…”

“…The DNA segment between positions Ϫ70 and ϩ63 relative to the start of rbc L gene transcription containing the rbc L gene promoter and 63 bp of rbc L 59 UTR sequence was amplified by PCR using the M13 universal primer 59-GT AAAACGACGGCCAGT-39 as 59 primer and the 39-mer 59-TTATCGATCCTAAAATAATCTGTCCGGAAATATAATTTA-39 as 39 primer+ The 39 primer, which is complementary to positions ϩ30 to ϩ68 of the rbc L 59 UTR sequence, was synthesized with a ClaI restriction site [ATCGAT] at its 59 end that was used in subcloning the PCR-amplified DNA fragment into pBluescript SKϩ+ A series of variants of the 39 primer containing single nucleotide changes in the positions shown in Figure 1 were used to amplify rbc L 59 DNA fragments with mutated rbc L 59 UTR sequences+ Amplified rbc L 59 fragments were digested with XhoI, which cuts at the 59 end of the amplified rbc L gene region, and ClaI, which cuts at the restriction site introduced by the 39 primer at position ϩ63 of the rbc L UTR sequence, and cloned into XhoI/ClaI-digested pBluescript SKϩ+ The rbc L 59 DNA fragments were released from pBluescript SKϩ by digestion with XhoI and SmaI and cloned into the XhoI/SmaI-digested transformation vector pCrc32+ DNA fragments from the 59 end of the atpB gene containing the sequence between positions Ϫ120 to ϩ62 relative to the start site of atpB gene transcription were amplified by using the 21-mer 59-GTGCAGTGCCCCCTCGAGGTC-39 as 59 primer and the 45-mer 59-GAATTTAAATATAAAAAGTAT TATTCACTAACGCTTATTTTTTAG-39 as 39 primer+ The latter is complementary to positions ϩ24 to ϩ68 of the atpB 59 UTR sequence and contains a DraI restriction site [TTTAAA] at its 59 end that was used in subcloning of the PCR products into pBluescript SKϩ+ Variants of the 39 primer containing the nucleotide changes shown in Figure 1 were used to amplify atpB 59 DNA segments with mutated 59 UTR sequences+ Amplification products were digested with XhoI, which cuts at the 59 end of the amplified atpB 59 fragment, and DraI, which cuts in the atpB 59 UTR at position ϩ62, and cloned into XhoI/EcoRV-digested pBluescript SKϩ+ The fragments were released by digestion with XhoI and SmaI and cloned into XhoI/SmaI-digested transformation vector pCrc32+ The sequences of all cloned fragments were verified by Sanger dideoxynucleotide sequencing+ It was not checked whether the transcripts of the chimeric constructs are translated into the GUS ( b-glucuronidase) protein+ For transcripts of the atpB 59 end:GUS constructs, translation is unlikely because they lack about 280 bp of the full-length atpB 59 UTR+ Translation of transcripts of the rbc L 59 end:GUS constructs is doubtful because they lack about 30 bp of the rbc L 59 UTR including the putative ribosome-binding (Shine-Dalgarno) motif at positions ϩ71 to ϩ74 (Dron et al+, 1982)+…”

The two beta-tubulin genes of Chlamydomonas reinhardtii code for identical proteins.

“…In an attempt to distinguish between the polypeptides encoded by the two transcripts, we prepared specific DNA probes corresponding to the 3' untranslated region of each transcript by cloning genomic DNA fragments into plasmid vectors. The specific probes were used to select, by hybridization, the 13 or ,B2 transcripts for translation in vitro. Each transcript produced a protein in vitro, suggesting that two translatable mRNAs are present in the cell.…”

“…Codon usage in C. reinhardtii was strongly biased: only 37 different codons were used, as compared with 49, 57, and 58 codons used in chicken, human, and yeast tubulin genes, respectively. Table 1 also shows a comparison of codon usage between the C. reinhardtii nucleus-encoded 1-tubulin genes and the chloroplast-encoded large subunit (LS) gene for ribulose bisphosphate carboxylase (13). The LS protein, containing 475 amino acids, is similar in size to 13-tubulin.…”

“…This biased use of codons in the nuclear-encoded genes contrasts with the codon usage in chloroplast-encoded genes in the same organism. For example, of the 38 leucine residues in ribulose bisphosphate carboxylase, none are encoded by a CUG codon (13). A total of 143 codons in the gene (30% or the total) have A residues at the third base position.…”