Mobile self-splicing group I introns from the psbA gene of Chlamydomonas reinhardtii: highly efficient homing of an exogenous intron containing its own promoter.

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 in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16S rrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. The Cr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/microgram of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp -88 and -194 (relative to the ORF) that stimulated homing and contained a possible bacterial (-10, -35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.

Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes.

Manganese (Mn(2+)) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn(2+)-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn(2+) concentration. Interestingly, at site III, Mg(2+) alone promoted weak, but the same specific cleavage as Mn(2+). When added with Mn(2+), Mg(2+) had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn(2+) cleavage at site III also exhibited lower values of K (Mn(2+) requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn(2+)-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.

A kinetically efficient form of the Chlamydomonas self-splicing ribosomal RNA precursor.

The 23S rRNA gene of Chlamydomonas reinhardtii contains a group IA3 intron, Cr.LSU, whose splicing is essential for cell growth. To better understand Cr.LSU splicing, kinetic analyses were undertaken with 23S.3, a preRNA previously shown to self-splice. Self-splicing of 23S.3 showed biphasic kinetics, with only approximately 33% reacting efficiently. Removal of a region of the 5' exon that could potentially interfere with the intron core (i.e., P3) increased the size (53%) of the active fraction. Replacement of the large P6a-extension by a 20-nt stem-loop further increased the active fraction to approximately 80%. The k(cat) and K(G)(M) for self-splicing (first step) by these latter RNAs were approximately 1 min(-1) and approximately 20 microM, respectively. These results indicate that Cr.LSU is a highly efficient ribozyme whose folding in vitro is impeded by exonic and/or intronic sequences. The implications for in vivo splicing of Cr.LSU are discussed.

Requirement for cytoplasmic protein synthesis during circadian peaks of transcription of chloroplast-encoded genes in Chlamydomonas.

Cycloheximide, an inhibitor of cytoplasmic translation, induced a rapid reduction of 70-80% in levels of mRNA for the chloroplast elongation factor Tu (tufA) in asynchronously growing Chlamydomonas. This effect was shown to be mainly transcriptional, and not restricted to tufA, as transcription of other chloroplast-encoded genes were cycloheximide-sensitive, although not all equally (psbA showed no more than 40% inhibition). Confirmatory evidence that the inhibition of chloroplast transcription was mainly due to blocking cytoplasmic translation was obtained with the cycloheximide-resistant mutant act1, and by using another translation inhibitor, anisomycin. In synchronously growing Chlamydomonas, chloroplast transcription is regulated by the circadian clock, with the daily peak occurring during the early light period. When cycloheximide was added during this period, transcription was inhibited, but not when it was added during the trough period (late light to early dark). Moreover, in synchronized cells switched to continuous light, the drug blocked the scheduled increase in tufA mRNA, but did not remove the pre-existing mRNA. These experiments define two functionally different types of chloroplast transcription in Chlamydomonas, basal (cycloheximide-insensitive) and clock-induced (cycloheximide-sensitive), and indicate that the relative contribution of each type to the overall transcription of a given gene are not identical for all genes. The results also provide evidence for nuclear regulation of chloroplast transcription, thereby obviating the need for an organellar clock, at least for these rhythms.

Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas.

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 atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera.

The catalytic group-I introns of the psbA gene of chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications.

The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1-Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3' end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11-38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas.

Processing of a composite large subunit rRNA. Studies with chlamydomonas mutants deficient in maturation of the 23s-like rrna.

(Cr.LSU). Little is known of the cis and trans requirements or of the processing pathway for this essential RNA. Previous work showed that the ribosome-deficient ac20 mutant overaccumulates an unspliced large subunit (LSU) RNA, suggesting that it might be a splicing mutant. To elucidate the molecular basis of the ac20 phenotype, a detailed analysis of the rrn transcripts in ac20 and wild-type cells was performed. The results indicate that processing of the ITSs, particularly ITS-1, is inefficient in ac20 and that ITS processing occurs after splicing. Deletion of the Cr.LSU intron from ac20 also did not alleviate the mutant phenotype. Thus, the primary defect in ac20 is not splicing but most likely is associated with ITS processing. A splicing deficiency was studied by transforming wild-type cells with rrnL genes containing point mutations in the intron core. Heteroplasmic transformants were obtained in most cases, except for P4 helix mutants; these strains grew slowly, were light sensitive, and had an RNA profile indicative of inefficient splicing. Transcript analysis in the P4 mutants also indicated that ITS processing can occur on an unspliced precursor, although with reduced efficiency. These latter results indicate that although there is not an absolutely required order for LSU processing, there does seem to be a preferred order that results in efficient processing in vivo.

Purification, biochemical characterization and protein-DNA interactions of the I-CreI endonuclease produced in Escherichia coli.

I- CreI is a member of the LAGLI-DADG family of homing nucleases; however, unlike most members of this family it contains only a single copy of this signature motif. I- CreI was over-expressed in Escherichia coli, and a simple purification protocol developed that gave reasonably pure protein in high yield. Size-exclusion chromatography and chemical cross-linking indicated that the protein is a dimer in solution. DNA cleavage by I- CreI was absolutely dependent on Mg2+(or Mn2+), and was inhibited by monovalent cations. I- CreI displayed a surprisingly high temperature optimum (>50 degrees C), with full activity occurring even at 70 degrees C. Interestingly, SDS was needed for efficient release of the cleavage products from the protein, indicating formation of very stable DNA-protein complexes. In contrast to these robust characteristics, purified I- CreI was unstable; however, it could be stabilized by the addition of either target or non-target DNA. Mobility shift assays revealed that I- CreI binds to DNA in the absence of Mg2+. Hydroxyl radical footprinting showed that I- CreI strongly protected the backbone of a continuous stretch of at least 12 nt on each strand that were shifted, relative to each other, by 2 bp in the 3'direction. Methylation protection and interference analyses were also performed, and together with the hydroxyl radical footprinting, indicate that I- CreI binds in both the major and minor grooves of its target DNA.

Evidence for light/redox-regulated splicing of psbA pre-RNAs in Chlamydomonas chloroplasts.

Efficient splicing in vivo of most self-splicing group I introns is believed to require proteins, raising the possibility that splicing could be regulated; however, examples of such regulation have been lacking. The Chlamydomonas reinhardtii chloroplast psbA gene contains four large group I introns that self-splice efficiently in vitro, but only under nonphysiological conditions. The psbA gene encodes the D1 protein of photosystem II, which is synthesized at very high rates in the light in order to replace photodamaged protein. We show that psbA pre-mRNAs, containing one or more introns, accumulate in wild-type cells in the dark, apparently due to rate-limited splicing. Analysis of the pre-RNAs indicates that splicing of the four introns does not follow a strict order. Exposure of cells to light induced rapid (15-20 min) decreases in precursor levels of approximately 3-5-fold (depending on the intron), which were accompanied by transient increases in free intron levels. Because light also stimulated psbA transcription approximately 2-fold over the same period, the data suggests that light increases the splicing efficiency of psbA introns approximately 6-10-fold. Similar estimates of the extent of light stimulation were obtained by analyzing precursor decay rates in the presence of actinomycin D. The effect of light is specific for psbA introns, because levels of unspliced 23S pre-RNA did not decrease. The light-induced increase in psbA pre-RNA processing was abolished by inhibitors of photosynthetic electron transport, but not by the ATP synthesis inhibitor, carbonylcyanide m-chlorophenylhydrazone, which actually promoted pre-RNA processing in the dark. Finally, nonphotosynthetic mutants, including the tscA-lacking photosystem I mutant, H13, did not show evidence of light-stimulated RNA processing. However, the light response was restored in photosynthetic transformants of H13 that had been complemented with the tscA gene. These data suggest strongly that light coordinately stimulates splicing of all four psbA introns. Moreover, they demonstrate that this response to light is mediated by photosynthetic electron transport. The implications of these results for the regulation of psbA gene expression are discussed.

Double strand break-induced recombination in Chlamydomonas reinhardtii chloroplasts.

The mechanisms of chloroplast recombination are largely unknown. Using the chloroplast-encoded homing endonuclease I-CreI from Chlamydomonas reinhardtii, an experimental system is described that allows the study of double strand break (DSB)-induced recombination in chloroplasts. The I-CreI endonuclease is encoded by the chloroplast ribosomal group I intron of C.reinhardtii and cleaves specifically intronless copies of the large ribosomal RNA (23S) gene. To study DSB-induced recombination in chloroplast DNA, the genes encoding the I-CreI endonuclease were deleted and a target site for I-CreI, embedded in a cDNA of the 23S gene, was integrated at an ectopic location. Endonuclease function was transiently provided by mating the strains containing the recombination substrate to a wild-type strain. The outcome of DSB repair was analyzed in haploid progeny of these crosses. Interestingly, resolution of DSB repair strictly depended upon the relative orientation of the ectopic ribosomal cDNA and the adjacent copy of the 23S gene. Gene conversion was observed when the 23S cDNA and the neighbouring copy of the 23S gene were in opposite orientation, leading to mobilization of the intron to the 23S cDNA. In contrast, arrangement of the 23S cDNA in direct repeat orientation relative to the proximal 23S gene resulted in a deletion between the 23S cDNA and the 23S gene. These results demonstrate that C.reinhardtii chloroplasts have an efficient system for DSB repair and that homologous recombination is strongly stimulated by DSBs in chloroplast DNA.

Transcription of tufA and other chloroplast-encoded genes is controlled by a circadian clock in Chlamydomonas.

Levels of mRNA for the chloroplast-encoded elongation factor Tu (tufA) showed a dramatic daily oscillation in the green alga Chlamydomonas reinhardtii, peaking once each day in the early light period. The oscillation of tufA mRNA levels continued in cells shifted to continuous light or continuous dark for at least 2-3 days. Run-off transcription analyses showed that the rate of tufA transcription also peaked early in the light period and, moreover, that this transcriptional oscillation continued in cells shifted to continuous conditions. The half-life of tufA mRNA was estimated at different times and found to vary considerably during a light-dark cycle but not in cells shifted to continuous light. Light-dark patterns of transcription of several other chloroplast-encoded genes were examined and also found to persist in cells shifted to continuous light or dark. These results indicate that a circadian clock controls the transcription of tufA and other chloroplast-encoded genes.

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1.

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identified in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [32P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.

The atpF group-II intron-containing gene from spinach chloroplasts is not spliced in transgenic Chlamydomonas chloroplasts.

In order to determine whether the group-II trans-splicing machinery of the chloroplast of Chlamydomonas reinhardtii can splice a heterologous group-II cis intron, the atpF gene of spinach was transferred into the chloroplast genome of C. reinhardtii using the atpX expression vector. The atpF gene contains a group-II intron which, like other higher plant chloroplast introns, does not self-splice in vitro. The chimeric transgene was expressed at high levels, based on the accumulation of the precursor; however, spliced products could not be detected by Northern blotting, or by RT-PCR coupled with Southern-blot hybridization of the amplified products with an exon-junction probe. These results indicate that the spinach atpF intron is not spliced in transgenic C. reinhardtii chloroplasts. Thus, splicing of chloroplast introns mediated by cellular factors may be species-specific; alternately, the group-II splicing machinery of C. reinhardtii is specific for trans spliced introns.

Control of lhc gene transcription by the circadian clock in Chlamydomonas reinhardtii.

Transcription of nuclear lhc genes has been shown to be under circadian clock control in angiosperms. but many aspects of this regulation have not been elucidated. Unicellular organisms, such as the green alga Chlamydomonas reinhardtii, offer significant advantages for the study of cellular clocks. Therefore, we have asked whether lhc gene expression is regulated by a circadian clock in C. reinhardtii. The mRNA for a photosystem I chlorophyll a/b apoprotein showed a strong diurnal rhythm in cells growing under 12 h/12 h light/dark (LD) cycles; the mRNA accumulated and then declined during the light period reaching very low levels at mid-dark. A similar diurnal pattern was documented for rbcS mRNA. In LD-grown cells shifted to continuous light, the ca. 24 h rhythm of lhca1 mRNA continued for at least 2 cycles. In LD-grown cells shifted to continuous darkness the rhythm of lhca1, but not rbcS2, mRNA also continued, although at lower absolute levels than in LD-grown cells. Also, in the cells shifted to continuous dark, the lhca1 mRNA rhythm persisted in the absence of significant cell division. Pulse-labelling with 32PO4 and sensitivity to actinomycin D demonstrated that control of lhca1 (and rbcS) is mainly transcriptional. However, it was also shown that the half-life of lhca1 mRNA (and rbcS2) is short (1-2 h) and may also vary somewhat during a cycle. We conclude that a cellular, circadian clock regulates lhca1 transcription in C. reinhardtii.

A chloroplast group I intron undergoes the first step of reverse splicing into host cytoplasmic 5.8 S rRNA. Implications for intron-mediated RNA recombination, intron transposition and 5.8 S rRNA structure.

The chloroplast 23 S rRNA gene of Chlamydomonas reinhardtii contains a self-splicing group I intron, Cr.LSU. Incubation of total RNA from C. reinhardtii with [alpha-32P]GTP produces a GTP-labeled RNA that is derived from Cr.LSU, but is approximately 95 nucleotides longer (and referred to as IA). We show that IA is derived from an intermolecular reaction of Cr.LSU with cytoplasmic 5.8 S rRNA; the 3'-terminal G of the intron becomes ligated to A64. This reaction, which was reproduced using synthetic RNAs, is identical to the first step of reverse splicing. The second step of reverse splicing did not occur with any efficiency, even though both P1 and P10-like helices can form between the reactive region of 5.8 S rRNA and the IGS of Cr.LSU. Cloning of IA provided a chimeric Cr.LSU precursor with the normal 3' exon substituted by 95 nucleotides of 5.8 S; this precursor spliced poorly despite having a potential P10 helix (between the 3'-exon and the IGS). The inefficient reverse splicing of Cr.LSU into 5.8 S RNA and poor forward splicing of the chimera may be explained by competition between helices P1 (between the 5'-exon and IGS) and P10; competition between P1 and P10 is apparently not a factor in forward splicing of the wild-type LSU precursor. The sequence of the 5.8 S gene of C. reinhardtii was determined, and the published RNA sequence revised. A secondary structure model of C. reinhardtii 5.8 S rRNA was proposed incorporating the requirement for base-pairing to the IGS of Cr.LSU. The resulting structure resembles that recently proposed for yeast 5.8 S rRNA, and has considerable advantages over previous models of C. reinhardtii 5.8 S rRNA. These data describe a novel example of a naturally occurring ribozyme reacting with a naturally occurring RNA of the same cell; implications for ribozyme-mediated RNA recombination and intron transposition via reverse splicing are discussed.

Intracellular translocation of a 28 kDa GTP-binding protein during osmotic shock-induced cell volume regulation in Dunaliella salina.

The primary aim of this study was to determine if small GTP-binding proteins play a role in the conspicuous and much-examined volume control process in Dunaliella salina. We confirmed the previous identification by Rodriguez et al. (Rodriguez Rosales, M.P., Herrin, D.L. and Thompson, G.A., Jr. (1992) Plant Physiol. 98, 446-451) of small GTP-binding proteins in the green alga Dunaliella salina and revealed the presence of at least five such proteins, having molecular masses of approx. 21, 28, 28.5, 29 and 30 kDa. These proteins were concentrated largely in the endoplasmic reticulum (ER) and in an intermediate density organelle fraction (GA) containing mainly Golgi vesicles, mitochondria and flagella. The chloroplast fraction and plasma membrane contained the 28-kDa GTP-binding protein exclusively, while the cytosol contained both the 28-kDa component and small amounts of a 21-kDa GTP-binding protein. Immunodetection analysis showed that the D. salina 28-kDa protein cross-reacted strongly with a polyclonal antibody raised against a Volvox carteri yptV1 type GTP-binding protein. This antibody was utilized for quantitative GTP-binding protein measurements as described below. Certain anti-GTP-binding protein antibodies derived from non-plant sources, namely, monoclonal antibodies raised against yeast and mouse ypt1 GTP-binding proteins, cross-reacted not only with the D. salina 28-kDa protein but also the 29-kDa component. The 30-kDa GTP-binding protein of D. salina did not bind the antibodies mentioned above but did cross-react with an anti-yeast ypt1 polyclonal antibody. None of the D. salina GTP-binding proteins reacted positively with polyclonal antibodies raised against SEC4, rab1 or rab6 proteins. When D. salina cells were subjected to hypoosmotic swelling by abruptly reducing the NaCl concentration of their medium from 1.7 M to 0.85 M, the increase in cell surface area was accompanied by a substantial translocation of the 28-kDa GTP-binding protein from the ER and GA fractions to the plasma membrane, chloroplast and cytosolic fractions, as determined by quantitative [32P]GTP binding and [125I]antibody binding on nitrocellulose blots. This translocation increased the content of the 28-kDa component in the plasma membrane, chloroplast and cytosol by 3-4-fold. No net movement of the 30-kDa GTP-binding protein from either the ER or GA fractions was observed following hypoosmotic shock.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of a cDNA encoding the 20-kDa photosystem I light-harvesting polypeptide of Chlamydomonas rinhardtii.

A cDNA encoding the precursor to a major 20-kDa thylakoid polypeptide of Chlamydomonas reinhardtii (P22), previously localized to the photosystem I light-harvesting complex (LHCI), was characterized. N-terminal sequencing of P22 identified the precursor cleavage site. Genomic Southern blots and polymerase chain reaction analyses show that the gene for P22 (Lhca1*1) is single-copy and contains at least one intron. Northern-blot analyses show that Lhca1*1 mRNA is highly regulated in light-dark synchronized cells. The primary sequence and predicted topology of P22 has features characteristic of light-harvesting chlorophyll a/b-binding proteins from higher plants. Sequence comparisons indicate that P22 has significantly greater identity with the Type-I LHCI protein of tomato, compared to other LHC proteins. This result suggests that the divergence of LHCI proteins into the classes found in higher plants may have occurred early in evolution, prior to the separation of green algae and land plants.

Cleavage and recognition pattern of a double-strand-specific endonuclease (I-creI) encoded by the chloroplast 23S rRNA intron of Chlamydomonas reinhardtii.

Several group-I introns have been shown to specifically invade intron-minus alleles of the genes that contain them. This type of intron mobility is referred to as 'intron homing', and depends on restriction endonucleases (ENases) encoded by the mobile introns. The ENase cleaves the intron-minus allele near the site of intron insertion, thereby initiating gene conversion. The 23S (LSU) rRNA-encoding gene (LSU) of the chloroplast genome of Chlamydomonas reinhardtii contains a self-splicing group-I intron (CrLSU) that has a free-standing open reading frame (ORF) of 163 codons. Translation of CrLSU intron RNA in cell-free systems produces a polypeptide of approx. 18 kDa, the size expected for correct translation of the ORF. The in vitro-synthesized 18-kDa protein cleaves plasmid DNA that contains a portion of LSU where the intron normally resides, but lacking the intron itself. Cleavage by the intron-encoded enzyme (I-CreI) occurs 5 bp and 1 bp 3' to the intron insertion site (in the 3'-exon) in the top (/) and bottom (,) strands, respectively, resulting in 4-nt single-stranded overhangs with 3'-OH termini. We also show that the recognition sequence of I-CreI spans the cleavage site and is 24 bp in length (5'-CAAAACGTC,GTGA/GACAGTTTGGT).

Regulation of chlorophyll apoprotein expression and accumulation. Requirements for carotenoids and chlorophyll.

Chlorophyll apoprotein accumulation and expression were examined in mutants of Chlamydomonas reinhardtii blocked at specific steps of carotenoid or chlorophyll synthesis. In the absence of carotenoids: 1) apoproteins of the core and light-harvesting complexes of photosystem I (CCI and LHCI, respectively) and photosystem II (CCII and LHCII, respectively) do not accumulate; 2) mRNAs for the CCI, CCII, and LHCII apoproteins accumulate to normal levels; and 3) synthesis of the chlorophyll apoproteins is differentially affected, or in some cases, not affected. In the absence of chlorophylls: 1) the apoproteins fail to accumulate; 2) mRNA levels for CCI and CCII apoproteins are relatively unchanged; 3) levels of LHCII apoprotein mRNA, but not rates of LHCII mRNA synthesis, are reduced in a light-dependent chlorophyll-synthesis mutant (ya12); and 4) synthesis of chlorophyll apoproteins is differentially affected or not affected in the case of several chloroplast-encoded apoproteins. These results demonstrate a direct role for carotenoids as well as chlorophylls in the stabilization of certain chlorophyll apoproteins and, for others, possibly in their translation. The data also indicate a role for chlorophyll synthesis in the stability of LHCII mRNA.