Transcriptional Analysis of Endogenous and Foreign Genes in Chloroplast Transformants of Chlamydomonas

Abstract

Transcription from modified chloroplast genes has been studied in vitro, but only with the recently developed ability to stably introduce foreign DNA into Chlamydomonas reinhardtii chloroplast chromosomes in situ has it become possible to do so in vivo. Cloned chloroplast DNA sequences, into which had been inserted chimeric genes composed of the GUS coding sequence reporter under transcriptional control of chloroplast promoters for the C. reinhardtii atpA, atpB, and rbcL genes, were introduced into the cells on microprojectiles. These constructs become integrated into chloroplast chromosomes by homologous recombination. RNA gel blot analyses demonstrated that a single major 8-glucuronidase (GUS)-hybridizing transcript accumulates in each chloroplast transformant. We have found that: (1) Transcription of the chimeric gene begins at the same site as in the corresponding endogenous chloroplast gene; (2) the rates of transcription in vivo of the atpA:GUS and atpB:GUS genes relative to one another and to other genes are the same as those for the endogenous atpA and atpB genes, respectively, indicating that these promoters are fully functional despite being fused to a foreign gene and being at an alien location on the chloroplast chromosome; (3) in contrast to the atpA and atpB promoters, the rbcL promoter directs transcription of the rbcL:GUS gene at only 1% of the expected rate, suggesting that other features are required for optimal activity of this promoter; and (4) 22 base pairs upstream of the 5' end of the atpB:GUS transcript in the atpB promoter element is sufficient to confer wild-type levels of promoter activity.

Evidence for Nuclear Control of the Expression of the atpA and atpB Chloroplast Genes in Chlamydomonas

“…In each set of protection experiments in which we used low amounts of total RNAs from the two strains (<2.5 u,g), we observed that protection of the rbcL probe was considerably higher than that of the afpA probe. This indicated that rbcL transcripts were particularly abundant in the two strains, an observation that is consistent with the much higher transcription rate for rbcL than for afpA or afpB genes (Blowers et al, 1990).…”

“…Our pulse labeling studies indicated that the a subunit was synthesized in large excess over the p subunit. This cannot be accounted for by differences in transcript availability as both the rates of transcription and steadystate levels of the transcripts are similar for the two genes (Blowers et al, 1990;Leu et al, 1990). Therefore, this difference in the rates of protein synthesis rather points to a regulation at the translational level.…”

An mRNA 3′ Processing Site Targets Downstream Sequences for Rapid Degradation in Chlamydomonas Chloroplasts

“…These strains contained a chimeric petD promoter/5Ј UTR-uidA (␤-glucuronidase) coding region-rbcL 3Ј UTR reporter gene, which was located downstream of and in opposite orientation to the endogenous atpB gene. This insertion site has been used for numerous uidA gene fusions in the past (10,11,19) and does not affect the expression of atpB, which is used as the selectable marker for transformation of the nonphotosynthetic recipient strain CC373 (see "Experimental Procedures").…”

“…2 were based on pDG2 (19). This plasmid contains the petD promoter and 5Ј UTR translationally fused to the uidA coding region and rbcL 3Ј UTR, and the chimeric gene is inserted into the large inverted repeat of the chloroplast genome downstream of the atpB gene (11). We previously engineered a BglII site into the petD 5Ј UTR at position ϩ25 relative to the mature RNA 5Ј end, which lengthens the transcript by 6 nt and is present in pDG2 (19).…”

“…The atpB gene contains a somewhat unusual promoter, with essential elements included in the 5Ј UTR (10). Following transcription, two 5Ј ends can be found in the mature mRNA (11), a situation also found for many other chloroplast transcripts and presumed or proven to result from a primary transcript undergoing partial endonucleolytic cleavage. The region spanning from 10 -89 nt downstream of the atpB stop codon contains an IR sequence predicted to form an AU-rich stem-loop structure, and the mature 3Ј end is found 3-4 nt downstream of the IR (12,13).…”