Karen L. Kindle

Expression of a gene for a light-harvesting chlorophyll a/b-binding protein in Chlamydomonas reinhardtii: effect of light and acetate

In Chlamydomonas reinhardtii, the chlorophyll a/b-binding proteins of photosystem II are encoded in the nucleus by a small family of genes. We have studied the expression of one gene, which we call cabII-1, in a green-in-the-dark strain, which can synthesize chlorophyll in the dark or light, and in a yellow-in-the-dark mutant strain, which is able to make chlorophyll only in the light. In light/dark synchronized cultures of both strains, cabII-1 mRNA abundance increases during the first 6 h of a 12-h light phase, remains high for several hours, then declines. A variety of illumination conditions have been used to analyze the cabII-1 mRNA increase: continuous or intermittent red, blue, or white light, with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. Our results suggest that light induces increased cabII-1 transcript abundance in two ways: 1) by virtue of its role in the light reactions of photosynthesis and 2) by a blue lightstimulated mechanism which is independent of photosynthesis.We have also examined the role of acetate in regulating cabII-1 mRNA levels in the dark. In both green- and yellow-in-the-dark strains, 15 mM Na-acetate, added to synchronized cells in the dark, induces an increase in cabII-1 mRNA abundance with a temporal accumulation pattern very similar to that induced by continuous white light. We suggest that by providing an energy source, acetate stimulates cellular growth, cell cycle progression, and increased cabII-1 mRNA abundance. Interestingly, in cells exposed to light, acetate inhibits the light-induced increase in cabII-1 mRNA abundance by a mechanism which is not yet understood.

Small cis-Acting Sequences That Specify Secondary Structures in a Chloroplast mRNA Are Essential for RNA Stability and Translation

ABSTRACT Nucleus-encoded proteins interact with cis-acting elements in chloroplast transcripts to promote RNA stability and translation. We have analyzed the structure and function of three such elements within the Chlamydomonas petD 5′ untranslated region; petD encodes subunit IV of the cytochromeb 6/f complex. These elements were delineated by linker-scanning mutagenesis, and RNA secondary structures were investigated by mapping nuclease-sensitive sites in vitro and by in vivo dimethyl sulfate RNA modification. Element I spans a maximum of 8 nucleotides (nt) at the 5′ end of the mRNA; it is essential for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nucleus-encoded factor to block 5′→3′ exoribonucleolytic degradation. Elements II and III, located in the center and near the 3′ end of the 5′ untranslated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element III spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressors of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.

Expression of chimeric genes by the light-regulated cabII-1 promoter in Chlamydomonas reinhardtii: a cabII-1/nit1 gene functions as a dominant selectable marker in a nit1- nit2- strain.

In Chlamydomonas reinhardtii, expression of the cabII-1 gene increases dramatically in response to light (cabII-1 encodes one of the light-harvesting chlorophyll a/b-binding proteins of photosystem II). We have used a region upstream of the cabII-1 gene in translational fusions to the bacterial uidA gene (encodes beta-glucuronidase) and transcriptional fusions to the Chlamydomonas nitrate reductase gene (nit1). Chlamydomonas transformants carrying intact copies of the chimeric uidA gene do not express beta-glucuronidase at the level of enzyme activity or mRNA accumulation. Methylation in the cabII-1 promoter region of the introduced gene is extensive in these strains, suggesting that newly introduced foreign genes may be recognized and silenced by a cellular mechanism that is correlated with increased methylation. Transformants that express the chimeric cabII-1/nit1 gene have been recovered. In contrast to the endogenous nit1 gene, the chimeric cabII-1/nit1 gene is expressed in ammonium-containing medium. Moreover, nit1 mRNA accumulation is dramatically stimulated by light, with a time course that is indistinguishable from that of the endogenous cabII-1 gene. The cabII-1/nit1 gene has been used to select transformants in a nit1- nit2- Chlamydomonas strain (CC400G) and should be useful for transformation of the large number of mutants in the Ebersold-Levine lineage, which carry the same mutations.

Ccs1, a Nuclear Gene Required for the Post-translational Assembly of Chloroplast c-Type Cytochromes

Nuclear genes play important regulatory roles in the biogenesis of the photosynthetic apparatus of eukaryotic cells by encoding factors that control steps ranging from chloroplast gene transcription to post-translational processes. However, the identities of these genes and the mechanisms by which they govern these processes are largely unknown. By using glass bead-mediated transformation to generate insertional mutations in the nuclear genome of Chlamydomonas reinhardtii, we have generated four mutants that are defective in the accumulation of the cytochromeb 6 f complex. One of them, strain abf3, also fails to accumulate holocytochromec 6. We have isolated a gene, Ccs1, from a C. reinhardtii genomic library that complements both the cytochrome b 6 f and cytochromec 6 deficiencies in abf3. The predicted protein product displays significant identity with Ycf44 from the brown algaOdontella sinensis, the red alga Porphyra purpurea, and the cyanobacterium Synechocystis strain PCC 6803 (25–33% identity). In addition, we note limited sequence similarity with ResB of Bacillus subtilis and an open reading frame in a homologous operon in Mycobacterium leprae (11–12% identity). On the basis of the pleiotropicc-type cytochrome deficiency in the ccs1mutant, the predicted plastid localization of the protein, and its relationship to candidate cytochrome biosynthesis proteins in Gram-positive bacteria, we conclude that Ccs1 encodes a protein that is required for chloroplast c-type holocytochrome formation.

Transcription ofCABII is regulated by the biological clock inChlamydomonas reinhardtii

The small gene family encoding the chlorophylla/b-binding proteins of photosystem II (CABII orlhcb) is known to exhibit circadian rhythms of mRNA abundance inChlamydomonas reinhardtii. In this study we investigated the role of transcription in the phenomenon. We used as reportersChlamydomonas genes that encode nitrate reductase (NITI) and arylsulfatase (ARS2) transcriptionally fused to sequences upstream of one of theCABII genes (calledCABII-1). We found that both reporters exhibited the same circadian rhythm of mRNA abundance in phase, period, and amplitude as does the endogenousCABII-1 gene. We also evaluated the efficacy of arylsulfatase enzymatic activity as a reporter and found that its half-life is too long to make it a useful reporter of rhythmic transcription during a circadian or diurnal cycle. The amount of mRNA synthesis from theCABII-1 gene was examined byin vivo labeling experiments and a circadian rhythm in transcription rate was demonstrated.In vivo labeling also revealed a circadian rhythm of mRNA synthesis for theCABII gene family as a whole. The results from the transcriptional reporter assays together with thein vivo labeling experiments strongly support the conclusion that the biological clock regulates the transcriptional activity of theCABII-1 gene, and moreover that regulation at the transcriptional level is the predominant mode by which the clock regulates this gene.

The initiation codon determines the efficiency but not the site of translation initiation in Chlamydomonas chloroplasts.

To study translation initiation in Chlamydomonas chloroplasts, we mutated the initiation codon AUG to AUU, ACG, ACC, ACU, and UUC in the chloroplast petA gene, which encodes cytochrome f of the cytochrome b6/f complex. Cytochrome f accumulated to detectable levels in all mutant strains except the one with a UUC codon, but only the mutant with an AUU codon grew well at 24 degrees C under conditions that require photosynthesis. Because no cytochrome f was detectable in the UUC mutant and because each mutant that accumulated cytochrome f did so at a different level, we concluded that any residual translation probably initiates at the mutant codon. As a further demonstration that alternative initiation sites are not used in vivo, we introduced in-frame UAA stop codons immediately downstream or upstream or in place of the initiation codon. Stop codons at or downstream of the initiation codon prevented accumulation of cytochrome f, whereas the one immediately upstream of the initiation codon had no effect on the accumulation of cytochrome f. These results suggest that an AUG codon is not required to specify the site of translation initiation in chloroplasts but that the efficiency of translation initiation depends on the identity of the initiation codon.

Initiation codon mutations in the Chlamydomonas chloroplast petD gene result in temperature-sensitive photosynthetic growth.

The chloroplast petD gene encodes subunit IV of the cytochrome b6/f complex and is required for photosynthetic electron transport. We have created Chlamydomonas strains in which the initiation codon of the petD gene has been changed to AUU or AUC. These mutants can grow photosynthetically at room temperature, but not at 35 degrees C. The accumulation of subunit IV during photosynthetic or heterotrophic growth at room temperature is reduced to 10–20% of the wild‐type level; petD mRNA abundance is reduced to approximately 50% of the wild‐type amount. Pulse labeling experiments indicate that at room temperature, subunit IV translation proceeds at 10–20% of the wild‐type rate. Cells grown heterotrophically at 35 degrees C accumulate < 5% as much subunit IV as wild‐type cells grown under the same conditions, and < 1% as much subunit IV as wild‐type cells grown at room temperature. We conclude that translation initiation in these mutants is inefficient, leading to decreased translation and accumulation of subunit IV. At 35 degrees C, translational inefficiency leads directly or indirectly to insufficient accumulation of subunit IV to support photosynthetic growth.

A NUCLEAR MUTATION THAT AFFECTS THE 3' PROCESSING OF SEVERAL MRNAS IN CHLAMYDOMONAS CHLOROPLASTS

We previously created and analyzed a Chlamydomonas reinhardtii strain, [delta]26, in which an inverted repeat in the 3[prime] untranslated region of the chloroplast atpB gene was deleted. In this strain, atpB transcripts are unstable and heterogeneous in size, and growth is poor under conditions in which photosynthesis is required. Spontaneous suppressor mutations that allow rapid photosynthetic growth have been identified. One strain, [delta]26S, retains the atpB deletion yet accumulates a discrete and stable atpB transcript as a consequence of a recessive nuclear mutation. Unlike previously isolated Chlamydomonas nuclear mutations that affect chloroplast mRNA accumulation, the mutation in [delta]26S affects several chloroplast transcripts. For example, in the atpA gene cluster, the relative abundance of several messages was altered in a manner consistent with inefficient mRNA 3[prime] end processing. Furthermore, [delta]26S cells accumulated novel transcripts with 3[prime] termini in the petD-trnR intergenic region. These transcripts are potential intermediates in 3[prime] end processing. In contrast, no alterations were detected for petD, atpA, or atpB mRNA 5[prime] ends; neither were there gross alterations detected for several other mRNAs, including the wild-type atpB transcript. We suggest that the gene identified by the suppressor mutation encodes a product involved in the processing of monocistronic and polycistronic messages.

Nuclear Transformation: Technology and Applications

Nuclear transformation is now simple and efficient, and it has revolutionized the kinds of questions that can be addressed using Chlamydomonas reinhardtii as a model system. The highest rates of transformation are obtained using C. reinhardtii genes that complement auxotrophic mutations as selectable markers. Cotransformation is efficient, so effects of mutations engineered in vitro can readily be tested by reintroducing the altered genes into an appropriate mutant strain together with a selectable marker. Because nearly all nuclear transformation events result from apparently random integration of the introduced plasmid into chromosomal DNA, it is possible to generate large numbers of insertional mutants. The subsequent isolation of the disrupted genes by virtue of the molecular tag provides a very powerful means for cloning genes with known mutant phenotypes. Alternatively, since nuclear transformation is so efficient, genes with good selectable phenotypes can be isolated by complementing appropriate recessive mutations with pools of DNA from an indexed genomic library. Recently, expression of eubacterial genes that confer antibiotic resistance to C. reinhardtii has been reported; these genes should be useful as dominant selectable markers in any genetic background. However, transformants are recovered with these bacterial genes at only 1 % the rate obtained by transformation with C. reinhardtii genes, and in about half the cases the introduced genes are silenced under nonselective conditions, sometimes a problem with C. reinhardtii genes as well. Although an active homologous recombination system allows efficient recombination between simultaneously introduced plasmids, the rate of gene-targeted homologous integration events is very low. Further understanding of the factors that limit the expression of reintroduced genes and the rate of gene-targeting could lead to substantial improvements in the capability of manipulating the nuclear genome and of generating or phenocopying mutations corresponding to cloned genes.

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.