Chlamydomonas Reinhardi Research Articles

Marker rescue from bleomycin-treated Chlamydomonas reinhardi.

A new method has been developed for gene transfer in eukaryotic cells. Chlamydomonas reinhardi, a unicellular eukaryotic alga, was treated with a lethal dose of bleomycin, an agent that induces chromosome breakage. Bleomycin-treated cells were mated with untreated cells, and the mixture was plated onto selective agar medium. The progeny that arose contained the genetic markers from the untreated parent plus a subset of the genetic markers from the bleomycin-treated parent. Those markers derived from the untreated parent were stable, whereas those recovered from the bleomycin-treated parent were often unstable. Markers closely linked in the bleomycin-treated parent were usually rescued or lost together, whereas distantly linked or unlinked markers were rescued or lost independently. These results suggest that bleomycin treatment of C. reinhardi leads to the formation of chromosome fragments, and fusion of bleomycin-treated cells to untreated cells results in the rescue of some of these fragments. This procedure provides a new means of gene transfer that may be useful for genetic mapping, genetic engineering and for the study of genetic organization.

Biological effects of surfactants: Part 6—effects of anionic, non-ionic and amphoteric surfactants on a green alga ( Chlamydomonas)

The well known algistatic effect of cationic surfactants was not exhibited by homologous series of amphoteric, anionic and non-ionic types assayed with Chlamydomonas reinhardi. Sulphobetaine amphoterics, 3-(octyl-octadecyldimethylammonio)-1-propanesulphonates, caused growth inhibition when the lipophile contained twelve or more carbon atoms, but only at micellar concentrations. Critical micelle concentration (CMC) of sulphobetaines decreases logarithmically with increasing alkyl chain length and derivatives having eight and ten carbons in the lipophile were below the CMC at the highest concentration tested. Despite their pronounced protein-denaturing properties, sodium higher alkylsulphates did not materially inhibit the growth of Chlamydomonas. This may be due to the fact that the Kraft points of these anionics having fourteen or more carbon atoms were above the culture temperatures, thereby reducing their solution properties. Although less inhibiting than the sulphobetains, the non-ionic alcohol ethoxylates decreased growth with increasing hydrophilicity. The most lipophilic non-ionic showed growth-promoting effects.

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.

Molecular weight and subunit stoichiometry of the chloroplast coupling factor 1 from Chlamydomonas reinhardi.

The molecular weight of the Chlamydomonas reinhardi coupling factor 1 (CF1) is 4.2 X 10(5) as determined by gel exclusion chromatography and sedimentation equilibrium. In addition, a measured sedimentation coefficient of 12.9 S results in a calculated molecular weight of 3.9 X 10(5). These molecular weight estimates are too high to support an alpha 2 beta 2-type subunit stoichiometry and are suggestive of an alpha 3 beta 3-type enzyme. The subunit stoichiometry of the C. reinhardi CF1 was determined from the distribution of label into the subunits of uniformly labeled CF1. An alpha: beta: gamma: epsilon ration of 2.9:2.9:1:1 was obtained.

The effect of food concentration on swimming patterns, feeding behavior, ingestion, assimilation, and respiration by Daphnia1

Swimming patterns, feeding behaviors, and ingestion, filtering, assimilation, and respiration rates were measured for Daphnia magna in food concentrations from 0 to 106 Chlamydomonas reinhardi cells·cm−3, equivalent to 0–20 mg C·liter −1. A Holling type 2 functional response curve or an Ivlev model without a threshold appear to be biologically meaningful descriptions of Daphnia feeding response to different food concentrations. Daphnia has no feeding threshold or reduced filtering activity at low concentrations such as are predicted by optimal foraging models. Above the incipient limiting concentration, constant ingestion rates and an over‐collection of food result in increased rejection rates. These behaviors may cause high respiratory rates and reduced net assimilation rates at the highest food concentration. Daphnia does not orient to or alter swimming patterns in response to algal patches.

Feeding selectivities and relative ingestion rates of Daphnia and Bosmina1

The feeding selectivities and feeding rates of Daphnia rosea and Bosmina longirostris were measured in mixtures of 4C‐labeled algae (Chlamydomonas reinhardi) and 3H‐labeled bacteria (Aerobacter aerogenes). Daphnia showed no preference for one over the other. Selectivity coefficients (algal clearance : bacterial clearance rate) for Bosmina ranged from 2.8 to 13.7 depending on both previous feeding history and the relative abundance of the two foods. At high concentrations of Chlamydomonas alone the ingestion rates of the two cladocerans per unit body weight were not significantly different, while at low food concentrations (500–10,000 cells ml−1) the relative ingestion rate of Bosmina was 4.8 to 1.6× higher than that of Daphnia. Differences in feeding behavior are attributed to interspecific differences in morphology and feeding mode. The ability of Bosmina to feed efficiently at low food concentrations and to feed selectively may invalidate inferences about zooplankton competition from theoretical models of optimal body size alone.

Membrane biogenesis in Chlamydomonas reinhardi 137 +: Cell-cycle variations in the synthesis of phospholipids of non-photosynthetic membranes

To monitor the biogenesis of non-photosynthetic membranes during Chlamydomonas reinhardi 137 + vegetative development, the syntheses of phosphatidylethanolamine (PE) and phosphatidylcholine (PC), the alga's two major extra-thylakoid phospholipids, have been examined through the synchronous cycle. Synthesis of both phospholipids is largely confined to the photoperiod (mid-to-late G1), as is the accretion of cellular polar glycerolipid, with negligible lipogenesis in the dark (S, M, and early-to-mid G1). Coincidence between the cyclic variations of non-thylakoid and of thylakoid polar glycerolipid production during the Chlamydomonas cell cycle indicates that the synthesis of membrane molecules serves to both modulate and coordinate the biogenesis of the various cellular membranes in these actively-cycling cells.

Initiation of cell wall lysis in gametes of Chlamydomonas reinhardi by isolated flagella of the complementary mating type

Mating in the unicellular green alga Chlamydomonas reinhardi involves initial agglutination of the flagella of plus and minus gametes followed by cell wall loss in preparation for cell fusion. It has been reported that agglutination and wall loss can also be caused by isolated flagella. Contradictory results have been reported concerning the efficacy of isolated flagella in these reactions, possibly due to differences in isolation procedures. We have found that gametic flagella prepared in 7% sucrose show both specific adhesion and initiation of lytic factor release when mixed with intact gametes. Evidence for multiple flagellar receptors involved in the initial stages of mating have been presented in previous studies. Here we report that gametic flagella isolated during regeneration regain the ability to agglutinate intact gametes of the opposite mating type when less than 3 μm in length. The initiation of cell wall loss is delayed until the average flagellar length is greater than 4 μm. This result supports the view that the flagellar membrane is heterogeneous along its longitudinal axis.

Activation for cell fusion in Chlamydomonas: analysis of wild-type gametes and nonfusing mutants.

Gametes of Chlamydomonas reinhardi become activated for cell fusion as the consequence of sexual adhesion between membranes of mating-type plus and minus flagella. By using tannic acid plus en bloc uranyl acetate staining, and by fixing at very early stages in the mating reaction, we have demonstrated the following. (a) Activation of the minus mating structure entails major modifications in the structure of the organelle, causing it to double in size and to concentrate surface coat material, termed fringe, into a central zone. (b) The unactivated plus mating structure is endowed with fringe that moves with the tip of the actin-filled fertilization tubule during activation. Pre-fusion images suggest the occurrence of a specific recognition event between the plus and minus fringes. (c) Gametes carrying the imp-1 mutation fail to form a fringe and are unable to fuse. The imp-1 mutation is linked to the mating-type plus (mt+) locus, suggesting that the gene specifying the synthesis or insertion of fringe is encoded in this sector of the genome. (d) Gametes carrying the imp-11 mutation fail to form both a normal fringe and a normal submembranous density beneath the fringe, and are also unable to fuse. The imp-11 mutation converted a wild-type minus cell into a pseudo-plus strain; a model to explain this conversion proposes that the normal imp-11 gene product represses plus-specific genes concerned with Chlamydomonas gametogenesis.

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.

Isolation, purification, and characterization of coupling factor 1 from Chlamydomonas reinhardi

Chloroplast thylakoid particles were prepared from wild-type Chlamydomonas reinhardi by gentle sonication. These particles catalyzed phenazine methosulfate dependent photophosphorylation with rates ranging from 300 to 700 mumol of adenosine 5'-triphosphate (ATP) formed (mg of chlorophyll)-1h-1. Photophosphorylation was not sensitive to tentoxin but was sensitive to an anticoupling factor 1 (CF1) antiserum preparation made against spinach CF1. The C. reinhardi chloroplast CF1 was isolated from thylakoid particles by either chloroform or ethylenediaminetraacetic acid extraction. The former enzyme appeared to be missing the gamma subunit and did not reconstitute with partially resolved thylakoid particles. The latter enzyme reconstituted with partially resolved particles and had a specific activity at 37 degrees C of 2-5 umol of ATP hydrolyzed (mg of protein)-1 min-1. The enzyme utilized both MnATP and MgATP. CaATP was a poor substrate, and SrATP was not hydrolyzed. The enzyme was not activated by heat or proteolysis but was stimulated approximately 2-fold by 50 mM dithiothreitol. Alcohols reversibly stimulated the ATPase activity of the enzyme 5-25-fold. Ethanol, 20%, dramatically lowered the temperature optimum from approximately 75 to approximately 45 degrees C and slightly lowered the pH optimum from 8.5 to 8.2. Ethanol had no effect on the activation energy of the ATPase reaction (17 +/- 1.7 kcal/mol). The kinetics of the ATPase reaction catalyzed by the C. reinhardi enzyme are complex. Both free divalent cations and divalent cation ATP inhibited the activity of the enzyme. The apparent Km for MgTAP (55 uM free Mg2+) was approximately 0.2 mM.

Differential activity of DNA methyltransferase in the life cycle of Chlamydomonas reinhardi.

Two molecular weight forms of DNA (cytosine-5-)-methyltransferase [S-adenosyl-L-methionine:DNA (cytosine-5-)- methyltransferase, EC 2.1.1.37], both active in assays in vitro, were isolated from the green alga Chlamydomonas reinhardi at various stages of the life cycle. The enzyme with Mr 60,000 was found in vegetative cells and gametes of both male (mt-) and female (mt+) mating types. The enzyme with Mr 200,000 was specific to gametic cells and zygotes, which are the only stages at which methylation of chloroplast DNA occurs in vivo. Chloroplast DNA from gametes was shown to be methylated on both strands at most if not all methylation sites and the Mr 200,000 enzyme was shown to methylate both unmethylated and hemimethylated sites, the latter at an elevated rate. Micrococcus luteus DNA showed the same nearest-neighbor frequencies of methylation after methylation by each molecular weight component. The data suggest strongly that the Mr 200,000 enzyme is the active multimeric form of the Mr 60,000 enzyme and that it acts as both initiation and maintenance methylase. It is proposed that methylation of chloroplast DNA in female gametes and zygotes is regulated by assembly of the multimeric Mr 200,000 active enzyme, which in turm determines the maternal inheritance of chloroplast DNA.

A pleiotropic mutant of Chlamydomonas reinhardi showing cell wall abnormalities and altered phosphatase activities

A mutant (PD 12 ts of the green alga Chlamydomonas reinhardi has been isolated in which both neutral and alkaline phosphatase activities are altered when grown at 35°C. Defects in cell wall morphology have been detected in this mutant. How the two phenotypic effects of this pleiotropic mutation are related is discussed.

The sterols and fatty acids from purified flagella ofChlamydomonas reinhardi

AbstractPurified flagella of the eukaryotic algaChlamydomonas reinhardi have a sterol composition (55% ergosterol [24β‐methylcholesta‐5,7,22‐trans‐trien‐3β‐ol] and 45% 7‐dehydroporiferasterol [24β‐ethylcholesta‐5,7,22‐trans‐trien‐3β‐ol]) identical of that of the whole algal cell. Fatty acids isolated fromC. reinhardi flagella were identified as 16∶0, 18∶0, 18∶1, 18∶2 and 18∶3. Whole cell fatty acids included 14∶0, 16∶2 and 16∶3 in addition to those found in the flagella. Triunsaturates comprised 22.9% of the flagellar fatty acids and 76.4% of those from the whole cell.

Flagellar adhesion and deadhesion in Chlamydomonas gametes: effects of tunicamycin and observations on flagellar tip morphology.

The aggregation-dependent loss of flagellar adhesiveness in chlamydomonas reinhardi has been correlated with changes in flagellar tip morphology during adhesion and deadhesion. As aggregating mt- and impotent (able to adhere, but not fuse) mt+ gametes begin to disaggregate in the presence of the protein synthesis inhibitor cycloheximide, there is a concomitant change in flagellar tip morphology from the activated bulbous form to the nonactivated tapered shape. the requirement of protein-synthetic activity for the maintenance of flagellar adhesiveness during aggregation may be due in part to turnover of proteins involved in formation or stabilization of activated flagellar tips. Incubation of aggregating gametes with tunicamycin indicates that, like protein synthesis inhibitors, this inhibitor of glycosylation also causes adhering gametes to deadhere. The results suggest that protein glycosylation may be essential for maintenance of adhesiveness during aggregation.

Amide metabolism of Chlamydomonas reinhardi

Chlamydomonas reinhardi can utilise the lower aliphatic amides (C1−C4) as nitrogen sources. Of these only acetamide can serve as a sole carbon source. The acetamide analogue F-acetamide kills cells after conversion to F-acetate and F-citrate. This conversion is controlled by exogenous ammonia and, in part, acetate levels. Only one enzyme and one active site are involved in acetamidase function. Enzymatic analysis indicates an increased substrate range as compared to the growth — supported range, indicating uptake, toxicity or metabolic control restrictions.

Slow fluorescence fluctuations following high light to low light or dark transitions in Chlamydomonas reinhardi

Slow fluorescence transients in Chlamydomonas reinhardi arise after transitions from high light intensities to low light or dark conditions. Characteristics of the newly described transient phenomena include: (a) A slow biphasic decrease in fluorescence yield occurs in the dark, followed by an even slower, hour long, increase in fluorescence. (b) A similar, but faster, fluorescence yield decrease and subsequent increase also occurs during low intensity illumination periods separating high light intervals, or after transitions from high intensity to low intensity light. (c) Short (several seconds) flashes of light given during a dark period have no effect on the dark fluorescence decay, regardless of the flash frequency. Such flash regimes accurately monitor the dark decline of the M2 level by tracing the parallel decay of flash-generated P2 (Kautsky) peaks. However, flashes during a low light illumination period do influence the decay kinetics. Frequent flashes allow decay similar to that occurring in dark, but less frequent flashes inhibit the decrease in fluorescence yield.

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates.

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light-dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.

Rapid Repair of Ionizing Radiation Injury in Chlamydomonas reinhardi

Repair of sublethal damage is an important factor affecting the survival of irradiated cells. An understanding of this phenomenon is fundamental to understanding the action of ionizing radiations. Although multiple components of such repair have been detected in plateau-phase mammalian cells, processes having different repair rates have never been observed previously in proliferating eucaryotic cell systems. This report describes experiments used to study repair of sublethal damage in cells exposed to fast electrons. Split-dose techniques, utilizing two equal doses given at 100 krad/min and separated by intervals as short as 15 sec, followed by clonogenic cell assay, have been used throughout. These experiments demonstrate the existence of at least two independent repair processes in synchronous, exponentially growing cultures of Chlamydomonas reinhardi. Furthermore, each process probably represents repair of a distinctly different kind of damage. The rapid process described in this report is characterized by a mean repair time on the order of 2 to 4 min in contrast to 20 to 30 min for the well-known Elkind-Sutton type repair. Both processes are nearly temperature independent at temperatures above 25/sup 0/C.

Deoxyribonucleic acid methyltransferase from the eukaryote, Chlamydomonas reinhardi.

DNA methyltransferase was purified 310-fold from a green alga, Chlamydomonas reinhardi vegetative cells. The native enzyme of molecular weight 55 000--58 000 catalyzed the transfer of methyl groups from S-adenosylmethionine to the 5 position of cytosine in DNA. Native DNA accepted methyl groups 10-fold more than did denatured DNA. The sequence specificity analysis of methylated deoxycytidine in vitro revealed that the enzyme introduces methyl groups preferentially into sequences containing 5'd(T-mC-R)3'. Kinetic analysis of the reaction indicated that the enzyme obeys a random sequential mechanism. The extent of saturation with methyl groups depends upon the species from which the DNA was obtained. Kinetic analysis of the reaction catalyzed by RNA polymerase II has indicated that DNA methylation decreases the rate of initiation of RNA synthesis, but does not affect the rate of RNA chain elongation.