Overcoming Poor Transgene Expression in the Wild-Type Chloroplast: Creation of Highly Mosquitocidal Strains of .

High-level expression of transgenes in the chloroplast of wild-type Chlamydomonas reinhardtii (C. reinhardtii) remains challenging for many genes (e.g.

Expression of a Synthetic Gene for the Major Cytotoxin (Cyt1Aa) of subsp. in the Chloroplast of Wild-Type .

() strains that are toxic to mosquito larvae because they express chloroplast transgenes that are based on the mosquitocidal proteins of subsp. (Bti) could be very useful in mosquito control. has several advantages for this approach, including genetic controls not generally available with industrial algae.

Toward mosquito control with a green alga: Expression of Cry toxins of subsp. (Bti) in the chloroplast of .

We are developing strains that can be used for safe and sustainable control of mosquitoes, because they produce proteins from subsp. (Bti) in the chloroplast. has a number of advantages for this approach, including genetic controls that are not generally available with industrial algae.

PCR analysis of chloroplast double-strand break (DSB) repair products induced by I-CreII in Chlamydomonas and Arabidopsis.

Homing endonuclease I-CreII has been used to study the consequences and repair of a double-strand break (DSB) in the chloroplast genome of Chlamydomonas and Arabidopsis. Since I-CreII is from a mobile psbA intron of Chlamydomonas, it cleaves the psbA gene of an intronless-psbA strain of Chlamydomonas. And it cleaves specifically in the psbA gene of Arabidopsis, which is naturally intronless.

Reverse transcription of spliced psbA mRNA in Chlamydomonas spp. and its possible role in evolutionary intron loss.

Reverse transcription of mRNA is thought to be an important first step in a model that explains certain evolutionary changes within genes, such as the loss of introns or RNA editing sites. In this model, reverse transcription of mRNA produces cDNA molecules that replace part of the parental gene by homologous recombination. In vivo evidence of reverse transcription of physiologically relevant mRNAs is generally lacking, however, except in genetically engineered cells.

Circadian regulation of chloroplast transcription in Chlamydomonas is accompanied by little or no fluctuation in RPOD levels or core RNAP activity.

In Chlamydomonas growing under 24 h light-dark cycles, chloroplast transcription is under circadian clock control, and peaks early in the morning. The peak (but not trough) requires ongoing cytoplasmic translation, as it is sensitive to cycloheximide (CH). The chloroplast transcriptional apparatus in Chlamydomonas is simpler than in land plants, with only one type of RNA polymerase (RNAP, bacterial) and apparently only one sigma factor (RPOD).

Mapping of the css (chloroplast splicing suppressor) gene(s) to a recombinationally suppressed region of chromosome III in Chlamydomonas reinhardtii.

In previous work, three suppressors of defective group I introns (7151, 71N1, 7120) were isolated from a mutant of Chlamydomonas reinhardtii that had a splicing-deficient chloroplast large subunit (LSU) rRNA intron. Genetic analysis indicated that the 7151 and 71N1 suppressor mutations each involved single nuclear loci, and that the 7151 mutation was dominant. Here we present genetic evidence that the 7120 suppressor also involves a single nuclear locus and that the mutation is dominant in vegetative diploids.

A novel rhodanese is required to maintain chloroplast translation in Chlamydomonas.

Rhodanese-domain proteins (RDPs) are widespread in plants and other organisms, but their biological roles are mostly unknown. Here we report on a novel RDP from Chlamydomonas that has a single rhodanese domain, and a predicted chloroplast transit peptide. The protein was produced in Escherichia coli with a His-tag, but lacking most of the N-terminal transit peptide, and after purification was found to have rhodanese activity in vitro.

Microhomology-mediated and nonhomologous repair of a double-strand break in the chloroplast genome of Arabidopsis.

Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide.

Biochemical and mutagenic analysis of I-CreII reveals distinct but important roles for both the H-N-H and GIY-YIG motifs.

Homing endonucleases typically contain one of four conserved catalytic motifs, and other elements that confer tight DNA binding. I-CreII, which catalyzes homing of the Cr.psbA4 intron, is unusual in containing two potential catalytic motifs, H-N-H and GIY-YIG.

Chlamydomonas chloroplasts can use short dispersed repeats and multiple pathways to repair a double-strand break in the genome.

Certain group I introns insert into intronless DNA via an endonuclease that creates a double-strand break (DSB). There are two models for intron homing in phage: synthesis-dependent strand annealing (SDSA) and double-strand break repair (DSBR). The Cr.

Distinct roles for the 5' and 3' untranslated regions in the degradation and accumulation of chloroplast tufA mRNA: identification of an early intermediate in the in vivo degradation pathway.

Elongation factor Tu in Chlamydomonas reinhardtii is a chloroplast-encoded gene (tufA) whose 1.7-kb mRNA has a relatively short half-life. In the presence of chloramphenicol (CAP), which freezes translating chloroplast ribosomes, a 1.

Unusual metal specificity and structure of the group I ribozyme from Chlamydomonas reinhardtii 23S rRNA.

Group I intron ribozymes require cations for folding and catalysis, and the current literature indicates that a number of cations can promote folding, but only Mg2+ and Mn2+ support both processes. However, some group I introns are active only with Mg2+, e.g.

Mg2+ mimicry in the promotion of group I ribozyme activities by aminoglycoside antibiotics.

Aminoglycoside antibiotics inhibit several types of ribozymes, including group I introns, by displacing critical Mg2+ ions. However, they stimulate activity of the small hairpin ribozyme. We show here that aminoglycosides promote self-splicing of the Cr.

Assessing the relative importance of light and the circadian clock in controlling chloroplast translation in Chlamydomonas reinhardtii.

Previous work has shown that transcription of a number of chloroplast-encoded genes, including those for photosynthesis, are under circadian clock control in Chlamydomonas reinhardtii. However, some of these genes encode long-lived mRNAs that are also subject to translational control. Rates of synthesis of the major chloroplast translation products vary dramatically (10-20-fold) during light-dark (LD) cycles, peaking in the light period.

Structure, circadian regulation and bioinformatic analysis of the unique sigma factor gene in Chlamydomonas reinhardtii.

In higher plants, the transcription of plastid genes is mediated by at least two types of RNA polymerase (RNAP); a plastid-encoded bacterial RNAP in which promoter specificity is conferred by nuclear-encoded sigma factors, and a nuclear-encoded phage-like RNAP. Green algae, however, appear to possess only the bacterial enzyme. Since transcription of much, if not most, of the chloroplast genome in Chlamydomonas reinhardtii is regulated by the circadian clock and the nucleus, we sought to identify sigma factor genes that might be responsible for this regulation.

Chloroplast RNA processing and stability.

Primary chloroplast transcripts are processed in a number of ways, including intron splicing, internal cleavage of polycistronic RNAs, and endonucleolytic or exonucleolytic cleavages at the transcript termini. All chloroplast RNAs are also subject to degradation, although a curious feature of many chloroplast mRNAs is their relative longevity. Some of these processes, e.

Expression, purification, and biochemical characterization of the intron-encoded endonuclease, I-CreII.

The ORF of the Cr.psbA4 intron of Chlamydomonas reinhardtii mediates efficient intron homing, and contains an H-N-H and possibly a GIY-YIG motif. The ORF was over-expressed in Escherichia coli without non-native amino acids, but was mostly insoluble.

A horizontally acquired group II intron in the chloroplast psbA gene of a psychrophilic Chlamydomonas: in vitro self-splicing and genetic evidence for maturase activity.

The majority of known group II introns are from chloroplast genomes, yet the first self-splicing group II intron from a chloroplast gene was reported only recently, from the psbA gene of the euglenoid, Euglena myxocylindracea. Herein, we describe a large (2.6-kb) group II intron from the psbA gene (psbA1) of a psychrophilic Chlamydomonas sp.

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth.

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6-10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear.

A cosmid vector containing a dominant selectable marker for cloning Chlamydomonas genes by complementation.

Cosmid vectors containing a dominate selectable marker (ble) for complementation cloning of genes in Chlamydomonas reinhardtii were created. The usefulness of these vectors, which differ in the orientation of the ble cassette, was demonstrated by transforming C. reinhardtii to phleomycin resistance, by constructing a large library (approximately 5 x 10(5) recombinants) in one of them using DNA from a C.

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii.

Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.

FUGOID: functional genomics of organellar introns database.

FUGOID is a web-based, taxonomically broad organelle intron database that collects and integrates various functional and structural data on organellar (mitochondrial and chloroplast) introns. The main information provided by FUGOID includes intron sequence, subclass, resident ORF, self-splicing capability, host gene, protein factor(s) involved in splicing, mobility, insertion site, twintron, seminal references and taxonomic position of host organism. It is implemented in a relational database management system, allowing sophisticated, user-friendly searching, data entry and revision.