The Cia5 gene controls formation of the carbon concentrating mechanism in Chlamydomonas reinhardtii.

Wild-type Chlamydomonas reinhardtii cells shifted from high concentrations (5%) of CO2 to low, ambient levels (0.03%) rapidly increase transcription of mRNAs from several CO2-responsive genes. Simultaneously, they develop a functional carbon concentrating mechanism that allows the cells to greatly increase internal levels of CO2 and HCO3-. The cia5 mutant is defective in all of these phenotypes. A newly isolated gene, designated Cia5, restores transformed cia5 cells to the phenotype of wild-type cells. The 6,481-bp gene produces a 5.1-kb mRNA that is present constitutively in light in high and low CO2 both in wild-type cells and the cia5 mutant. It encodes a protein that has features of a putative transcription factor and that, likewise, is present constitutively in low and high CO2 conditions. Complementation of cia5 can be achieved with a truncated Cia5 gene that is missing the coding information for 54 C-terminal amino acids. Unlike wild-type cells or cia5 mutants transformed with an intact Cia5 gene, cia5 mutants complemented with the truncated gene exhibit constitutive synthesis of mRNAs from CO2-responsive genes in light under both high and low CO2 conditions. These discoveries suggest that posttranslational changes to the C-terminal domain control the ability of CIA5 to act as an inducer and directly or indirectly control transcription of CO2-responsive genes. Thus, CIA5 appears to be a master regulator of the carbon concentrating mechanism and is intimately involved in the signal transduction mechanism that senses and allows immediate responses to fluctuations in environmental CO2 and HCO3- concentrations.

Psr1, a nuclear localized protein that regulates phosphorus metabolism in Chlamydomonas.

Understanding the ways in which phosphorus metabolism is regulated in photosynthetic eukaryotes is critical for optimizing crop productivity and managing aquatic ecosystems in which phosphorus can be a major source of pollution. Here we describe a gene encoding a regulator of phosphorus metabolism, designated Psr1 (phosphorus starvation response), from a photosynthetic eukaryote. The Psr1 protein is critical for acclimation of the unicellular green alga Chlamydomonas reinhardtii to phosphorus starvation. The N-terminal half of Psr1 contains a region similar to myb DNA-binding domains and the C-terminal half possesses glutamine-rich sequences characteristic of transcriptional activators. The level of Psr1 increases at least 10-fold upon phosphate starvation, and immunocytochemical studies demonstrate that this protein is nuclear-localized under both nutrient-replete and phosphorus-starvation conditions. Finally, Psr1 and angiosperm proteins have domains that are similar, suggesting a possible role for Psr1 homologs in the control of phosphorus metabolism in vascular plants. With the identification of regulators such as Psr1 it may become possible to engineer photosynthetic organisms for more efficient utilization of phosphorus and to establish better practices for the management of agricultural lands and natural ecosystems.

Nucleus-encoded, plastid-targeted acetolactate synthase genes in two closely related chlorophytes, Chlamydomonas reihardtii and Volvox carteri: phylogenetic origins and recent insertion of introns.

Acetolactate synthase (ALS) catalyzes the first committed step in the synthesis of branched-chain amino acids. In green plants and fungi, ALS is encoded by a nuclear gene whose product is targeted to plastids (in plants) or to mitochondria (in fungi). In red algae, the gene is plastid-encoded. We have determined the complete sequence of nucleus-encoded ALS genes from the green algae Chlamydomonas reinhardtii and Volvox carteri. Phylogenetic analyses of the ALS gene family indicate that the ALS genes of green algae and plants are closely related, sharing a recent common ancestor. Furthermore, although these genes are clearly of eubacterial origin, a relationship to the ALS genes of red algae and cyanobacteria (endosymbiotic precursors of plastids) is only weakly indicated. The algal ALS genes are distinguished from their homologs in higher plants by the fact that they are interrupted by numerous spliceosomal introns; plant ALS genes completely lack introns. The restricted phylogenetic distribution of these introns suggests that they were inserted recently, after the divergence of these green algae from plants. Two introns in the Volvox ALS gene, not found in the Chlamydomonas gene, are positioned precisely at sites which resemble "proto-splice" sequences in the Chlamydomonas gene.

Intracellular carbonic anhydrase is essential to photosynthesis in Chlamydomonas reinhardtii at atmospheric levels of CO2. Demonstration via genomic complementation of the high-CO2-requiring mutant ca-1.

Genomic complementation of the high-CO2-requiring mutant ca-1-12-1C of Chlamydomonas reinhardtii was achieved by transformation with DNA pools from an indexed cosmid library of wild-type genomic DNA. Transformation of mutant cells with cosmid DNA from two microtiter plates in the library produced colonies that grew phototrophically at atmospheric CO2 levels. Transformations with cosmid DNA from each of the rows and files of the two plates pinpointed one well in each plate with a cosmid bearing the targeted gene. Sequencing of cosmid subclones revealed a gene encoding a recently identified C. reinhardtii chloroplast carbonic anhydrase (CAH3). Transformations with chimeric constructs combining different portions of the wild-type and mutant genes indicated the presence of a mutation in the 5'-half of the gene. Comparison of mutant and wild-type gene sequences in this region revealed a G-to-A substitution in the mutant gene, which produced a nonsense codon. The data presented demonstrate that the carbonic anhydrase produced from the CAH3 gene is essential to the inorganic carbon-concentrating mechanism in C. reinhardtii and that genomic complementation can be a facile and efficient means for isolating genes associated with defects affecting photosynthesis and other physiological processes in this eukaryotic green alga.

A Three-Component Enzyme System Catalyzes the O Demethylation of the Herbicide Dicamba in Pseudomonas maltophilia DI-6.

An enzyme activity which converts dicamba (2-methoxy-3,6-dichlorobenzoic acid) to 3,6-dichlorosalicylic acid in vitro has been detected in cell lysates of Pseudomonas maltophilia DI-6. Phenyl-Sepharose column chromatography of a partially purified lysate resulted in the separation of this enzyme into three separate protein components tentatively identified as an oxygenase, a ferredoxin, and a reductase. The activity of dicamba O-demethylase was dependent on oxygen and required NADH and Mg(sup2+).

Automated sampling and RNA isolation at room temperature for measurements of circadian rhythms in Chlamydomonas reinhardtii.

The development of techniques allowing the unattended collection of RNA from cell samples at room temperature makes practical accurate and facile monitoring of circadian rhythms in Chlamydomonas reinhardtii. The utility of these methods was demonstrated by collecting RNA samples for three days from cells maintained in continuous darkness. Every hour, cells were automatically collected and lysed with buffer containing SDS and proteinase K. Samples were maintained at room temperature with little or no evidence of degradation of RNA. Strong, non-damping circadian rhythms of cab mRNA abundance were measured. Free-running rhythms of about 24 h were measured from cultures maintained at 16, 20, 25 and 30 degrees C, thus demonstrating temperature compensation of circadian period. Simultaneous collections from cultures previously entrained to 12 h light/12 h dark cycles of opposite phase displayed circadian rhythms of cab mRNA abundance that were in phase with their previous entraining light cycles. Thus, this result suggests that the measured circadian rhythms of cab mRNA abundance was not an artifact of the collection procedure.

Analysis of dicamba degradation by Pseudomonas maltophilia using high-performance capillary electrophoresis.

A method based on high-performance capillary electrophoresis (HPCE) was developed for the simultaneous analysis of dicamba and its metabolites in media containing the bacterium Pseudomonas maltophilia. Dicamba, 3,6-dichlorosalicylic acid (DCSA), and related products were extracted from media samples using ether under acidic conditions and injected onto an HPCE system containing a pH 10.0 running buffer. Baseline resolution between these compounds was obtained at 30 kV with a total run time of 6 min on a 50-microns i.d. x 50-cm capillary. The linear range for dicamba and DCSA detected at 274 nm extended from 0 to 100 mg/liter and the dynamic range for both compounds extended to 2000 mg/liter. The within-run precision was +/- 5% or less throughout the entire concentration range studied. The limits of detection for dicamba and DCSA in the media samples were 6 and 2 mg/liter. This corresponded to detection limits of 0.3 and 0.1 ng, respectively, in the injected extracts. With this method it was possible to conduct preliminary studies examining the kinetics of dicamba metabolism in P. maltophilia.

Gene isolation through genomic complementation using an indexed library of Chlamydomonas reinhardtii DNA.

Hundreds of mutants with defects in a variety of physiologically important functions, such as photosynthesis, respiration, flagellar motility, phototaxis, circadian rhythms and the cell cycle, have been isolated from cultures of Chlamydomonas reinhardtii. In only a few cases have the genes responsible for these mutations been cloned and sequenced. The development of efficient methods for transformation with nuclear genes [7] has allowed the recent demonstration of gene isolation through genomic complementation with a pooled library of C. reinhardtii DNA [9]. To improve the efficiency with which genes complementing a particular mutation can be isolated, we have established an indexed (ordered) cosmid library of 11,280 individual clones contained in the separate wells of 120 microtiter plates. The average insert size is ca. 38 kb. PCR analysis of five sequenced nuclear genes present in the Chlamydomonas library revealed a range from two copies for the alpha 2- and beta 2-tubulin genes to at least seven copies for the argininosuccinate lyase gene. Overall, these five clones were represented an average of > or = 3.4 times in the library. Thus, the probability that any one particular nuclear gene of < 1000 bp will be found in the library is > or = 97%, and the probability that a gene of ca. 10,000 bp will be found in the library is ca. 92%. Rapid screening methods with cosmid DNAs pooled from individual microtiter dishes have been applied successfully. Bacteria containing clones of the argininosuccinate lyase gene have been identified through genomic complementation of a Chlamydomonas mutant bearing an inactive argininosuccinate lyase gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the arylsulfatase gene from the beta 2-tubulin promoter in Chlamydomonas reinhardtii.

Arylsulfatase, produced by Chlamydomonas reinhardtii during sulfur-limited growth, is secreted into the periplasmic space and is readily assayed using a chromogenic substrate. To assess the usefulness of the gene encoding arylsulfatase (ars) as a reporter gene in C. reinhardtii, we have fused the promoter region of the beta 2-tubulin gene (tubB2) to the coding region of an ars genomic clone to form a tubB2/ars chimeric sequence. This construct was introduced into C. reinhardtii, strain CC425 (cw-15, arg-2), via cotransformation with the argininosuccinate lyase gene (which complements the arg-2 lesion) (1). Transformants expressing arylsulfatase (Ars) in sulfur-sufficient medium were isolated and subsequently shown to contain the tubB2/ars gene. RNA analysis determined that tubB2/ars transcripts accumulated in these cells. Abundance of the chimeric transcript increased immediately following deflagellation in a manner similar to that of the endogenous tubB2 transcript. Thus, chimeric genes incorporating ars coding sequences and heterologous promoters can be used to examine regulated gene expression in C. reinhardtii.

A Dinitroaniline-Resistant Mutant of Eleusine indica Exhibits Cross-Resistance and Supersensitivity to Antimicrotubule Herbicides and Drugs.

A dinitroaniline-resistant (R) biotype of Eleusine indica (L.) Gaertner. (goosegrass) is demonstrated to be cross-resistant to a structurally non-related herbicide, amiprophosmethyl, and supersensitive to two other classes of compounds which disrupt mitosis. These characteristics of the R biotype were discovered in a comparative test of the effects of 24 different antimitotic compounds on the R biotype and susceptible (S) wild-type Eleusine. The compounds tested could be classified into three groups based upon their effects on mitosis in root tips of the susceptible (S) biotype. Class I compounds induced effects like the well known mitotic disrupter colchicine: absence of cortical and spindle microtubules, mitosis arrested at prometaphase, and the formation of polymorphic nuclei after arrested mitosis. The R biotype was resistant to treatment with some class I inhibitors (all dinitroaniline herbicides and amiprophosmethyl) but not all (e.g. colchicine, podophyllotoxin, vinblastine, and pronamide). Roots of the R biotype, when treated with either dinitroaniline herbicides or amiprophosmethyl, exhibited no or only small increases in the mitotic index nor were the spindle and cortical microtubules affected. Compounds of class II (carbamate herbicides and griseofulvin) cause misorientation of microtubules which results in multinucleated cells. Compounds of class III (caffeine and structually related alkaloids) cause imcomplete cell walls to form at telophase. Each of these last two classes of compounds affected the R biotype more than the S biotype (supersensitivity). The cross-resistance and high levels of resistance of the R biotype of Eleusine to the dinitroaniline herbicides and the structurally distinct herbicide, amiprophosmethyl, indicate that a mechanism of resistance based upon metabolic modification, translocation, or compartmentation of the herbicides is probably not operative.

The relatively large beta-tubulin gene family of Arabidopsis contains a member with an unusual transcribed 5' noncoding sequence.

We have characterized the beta-tubulin gene family of Arabidopsis thaliana. Five distinct genes were cloned and analyzed by restriction enzyme mapping and cross-hybridization studies. Three of the genes appear to be dispersed, whereas two others are linked within 1.5 kb of one another. The two linked genes are closely related and appear to have resulted from a fairly recent duplication. The three dispersed genes do not cross-hybridize to one another or to the two linked genes under highly stringent hybridization conditions, suggesting that they arose from more ancient duplications. From Southern analysis we estimate that there are a total of between six and ten beta-tubulin genes in Arabidopsis. Additional analyses indicate that the gene family is equal in size or larger than those in other plants, but significantly smaller than those in related Brassica species. Sequence determination of one of the Arabidopsis genes revealed a highly unusual transcribed leader sequence. The leader contains two fairly long tracks of adenines. One is located toward the 5' end of the mRNA and the other is just before the initiation codon. A track of uridines is located between the adenine tracks. This leader can form two different secondary structures that may have regulatory significance.

Acetosyringone promotes high efficiency transformation of Arabidopsis thaliana explants by Agrobacterium tumefaciens.

High frequency transformation of Arabidopsis thaliana leaf explants has been obtained using a disarmed Ti plasmid containing the coding region of a neomycin phosphotransferase gene (NPT II) as a selectable marker. The rate of transformation ranged from 55 to 63 percent when acetosyringone (AS), a natural wound response molecule, was added to an Agrobacterium tumefaciens culture prior to incubation with leaf segments. Without acetosyringone, the transformation rate was approximately 2 to 3 percent. Calli resistant to G418 were regenerated into mature flowering plants in the presence of 10 µg/ml G418. Southern analysis and neomycin phosphotransferase assays confirmed the insertion and expression of the NPT II gene in regenerated Arabidopsis plants.

A small-scale five-hour procedure for isolating multiple samples of CsCl-purified DNA: application to isolations from mammalian, insect, higher plant, algal, yeast, and bacterial sources.

A rapid and simple procedure is described for obtaining CsCl-purified DNA from multiple small samples of cells or tissue. The DNA is recovered in a high-molecular-weight form (greater than or equal to 50 kb) that is readily cleaved with restriction enzymes. Sufficient quantities of DNA (10-50 micrograms) are recovered to allow multiple analyses by Southern blotting and most cloning procedures. The isolation procedure involves addition of intact cells or powders of frozen tissues directly to a simple lysis buffer containing detergent (sodium dodecyl sulfate or sodium sarcosinate) and high concentrations of EDTA. Ultra-high-speed centrifugation of CsCl gradients allows the isolation of DNA from 10 different samples in as little as 5 h. Applications are described for mammalian cells (HeLa cells), insect tissues (Drosophila melanogaster adults and pupa, Manduca sexta pupa, and Musca domestica pupa), higher plant tissues (Vicia faba leaves and meristems), algal cells (walled and wall-less Chlamydomonas reinhardi), yeast cells (Saccharomyces cerevisiae), and bacterial cells (Escherichia coli spheroplasts for preparation of both chromosomal and plasmid DNA). The procedure can be scaled up with larger sample sizes and longer centrifugation times to provide bulk quantities of DNA.

Repeated consensus sequence and pseudopromoters in the four coordinately regulated tubulin genes of Chlamydomonas reinhardi.

The 5' coding and promoter regions of the four coordinately regulated tubulin genes of Chlamydomonas reinhardi have been mapped and sequenced. DNA sequencing data shows that the predicted N-terminal amino acid sequences of Chlamydomonas alpha- and beta-tubulins closely match that of tubulins of other eucaryotes. Within the alpha 1- and alpha 2-tubulin gene set and the beta 1- and beta 2-tubulin gene set, both nucleotide sequence and intron placement are highly conserved. Transcription initiation sites have been located by primer extension analysis at 140, 141, 159, and 132 base pairs upstream of the translation initiator codon for the alpha 1-, alpha 2-, beta 1-, and beta 2-tubulin genes, respectively. Among the structures with potential regulatory significance, the most striking is a 16-base-pair consensus sequence [GCTC(G/C)AAGGC(G/T)(G/C)--(C/A)(C/A)G] which is found in multiple copies immediately upstream of the TATA box in each of the four genes. An unexpected discovery is the presence of pseudopromoter regions in two of the transcribed tubulin genes. One pseudopromoter region is located 400 base pairs upstream of the authentic alpha 2-tubulin gene promoter, whereas the other is located within the transcribed 5' noncoding region of the beta 1-tubulin gene.

Coordinate regulation of the four tubulin genes of Chlamydomonas reinhardi.

During cell division and during the induction of tubulin synthesis that accompanies flagellar regeneration in Chlamydomonas reinhardi, four tubulin mRNAs of discrete molecular sizes are produced. During induction two beta tubulin mRNAs (2.47 kb and 2.34 kb) and two alpha tubulin mRNAs (2.26 kb and 2.13 kb) are synthesized in high abundance and in a closely coordinated fashion. Combined data from restriction enzyme mapping (i.e., Southern analysis) of genomic DNA and of Charon 30 recombinant clones bearing inserts of Chlamydomonas tubulin genes provide direct evidence for four distinct tubulin genes in this organism. Dot-blot analysis of the level of hybridization of a 32p nick-translated beta tubulin cDNA to genomic DNA from gametic cells and to a clone containing the beta 1 tubulin gene indicate that each beta 1 tubulin gene is present in one copy per cell. Additional hybridization experiments employing fragments of cDNA clones which selectively anneal to either the 3' or 5' portions of the two alpha tubulin genes or to one or both of the two beta tubulin genes suggest that each tubulin gene is actively transcribed to give rise to one of the four tubulin mRNAs. These observations further suggest that at most four basic types of tubulin subunits are produced by Chlamydomonas and that the heterogeneity of tubulin subunits reported to exist in the flagellar axoneme must arise as a result of post-translational modification.

Tubulin induction in C. reinhardii: requirement for tubulin mRNA synthesis.

Flagellar excision in Chlamydomonas reinhardii triggers a rapid and extensive induction of tubulin synthesis. Cloned plasmids, pFT beta 1 and pFT beta 2, carrying cDNA inserts complementary to beta-tubulin mRNA, have been prepared and used to demonstrate a direct requirement for tubulin mRNA synthesis during tubulin induction. Increased tubulin mRNA synthesis is detected within 5 min after deflagellation. During the 45 min peak period of tubulin synthesis, tubulin mRNA accumulates to levels 15- to 35-fold higher than those found in control (non-deflagellated) cells. In addition, there appears to be a direct correlation between tubulin mRNA concentrations and the levels of tubulin production during the induction and deinduction cycle that accompanies flagellar regeneration. Amiprophosmethyl (APM), a compound we reported earlier as a selective inhibitor of tubulin synthesis in deflagellated cells, is shown to block the accumulation of tubulin mRNA following flagellar excision and to cause the rapid loss of tubulin mRNA from cells treated at the peak of induction.