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Light induces retinal synthesis via photoactivation of a small amount of Chlamydomonas rhodopsin pigment (Foster et al., Proc. Natl. Acad. Sci. USA 85, 6379-6383). A reducing agent [dithiothreitol (DDT) or mercaptoacetic acid (MAA)] also induces retinal synthesis in the dark via a rhodopsin with a chromophore. If the opsin is saturated with retinal and is bleached with light in the presence of a thiol trapping agent, the bleaching becomes irreversible. We conclude that the reducing agent as well as light break a disulfide bond resulting in activation of the rhodopsin and induction of carotenogenesis. Both the chemical and light induction is inhibited by GDPbetaS and pertussis toxin. Breaking the bridge between the 3rd and 5th helix may lead to increased proton accessibility of Asp134 leading to the rolling of the 3rd helix and MetaIIb formation., (Copyright 2000 Academic Press.)

Cultures of Chlamydomonas were exposed to a range of relatively mild stresses for a period of 24 h. These stresses comprised high and low temperatures, osmotic stress, low pH, starvation and toxic stress. They were then allowed to recuperate for around ten vegetative generations under near-optimal conditions in unmodified minimal medium. Fitness was then assayed as the rate of division of isolated cells on agar. We found that there was a strong tendency for stressed cultures to have lower mean fitness and greater standardized variance in fitness than the negative controls which had been cultured throughout in unmodified minimal medium. The same tendency was shown, as expected, by positive controls which received mutagenic doses of ultraviolet irradiation. We concluded that the most reasonable interpretation of these observations is that mild stress increases the genomic rate of mutation. This appears to be the first time that this phenomenon has been noticed in eukaryotes. The response might be adaptive because lineages in which higher mutation rates are elicited by stress can be favourably selected through the production of a few mutants which are fortuitously well adapted to the stressful environment. Other interpretations are not excluded, however. Regardless of the mechanism involved, the elevation of mutation rates under stress will affect the rate of evolutionary response to environmental change and also the maintenance of sexuality.

The length of eukaryotic cilia and flagella depends on the cell cycle-regulated assembly and disassembly of at least 9 doublet and 2 central microtubules, their associated proteins, and the surrounding membrane. In light-synchronized Chlamydomonas cells, flagella assembled to 10-14 microm in length near the beginning of the light period and they disassembled prior to cell division, during the dark period. Flagella on light-synchronized pf18 Chlamydomonas mutants grew to 10-12 microm near the beginning of the light period but shortened by 50% or more by the end of the light period. Flagellar length was cell-cycle regulated: when flagella were amputated at various times during the light period, new flagella regenerated to the lengths of control cells at that time of the light cycle. The later in the cycle pf18 cells were deflagellated, the shorter were the regenerated flagella. Flagellar shortening was not affected, in either pf18 or wild-type (wt) cells, by inhibitors of protein synthesis or of microtubule assembly, so flagellar length cannot depend on protein turnover. Shortening in pf18 was attenuated by Li+, which stimulated flagellar growth in wt cells, by red light, by protein kinase inhibitors, and by the Ca2+ channel blockers La3+ and Cd2+. Shortening was increased by cAMP, Na+, K+, and EGTA. Ca2+-CAM blockers did not affect pf18 shortening but they increased shortening in wt and fa1 cells. We propose that flagellar length is regulated by a signal transduction pathway that is sensitive to Ca2+ levels and red light.

The eyespot apparatus of the unicellular alga Chlamydomonas exhibits a clear directivity, i.e., it perceives light from different directions with different sensitivity. Using a newly constructed confocal microscope we have studied how absorption and reflection of eyespot and cell body shape this directivity. In agreement with previous results the eyespot was found to be highly reflectant, owing to its interference reflector design, but only for yellow light. Light of 490 nm, the maximum of absorption of the photoreceptor, was hardly reflected at all, even when the reflector was "tuned" to lower wavelengths by tilting it relative to the incoming light. The absorption of the carotenoids in the interference reflector also contributed little to the shielding properties of the cell, leaving the major contribution to the cell body. Thus most of the attenuation of light reaching the eyespot from the rear is due to chlorophyll and other pigments within the cell. In its peak around 490 nm the "contrast-ratio" reached a value of 8-10.

There is now little doubt of the existence of radioprotective mechanisms, or stress responses, that are upregulated in response to exposure to small doses of ionizing radiation and other DNA-damaging agents. Phenomenologically, there are two ways in which these induced mechanisms operate. First, a small conditioning dose (generally below 30 cGy) may protect against a subsequent, separate, exposure to radiation that may be substantially larger than the initial dose. This has been termed the adaptive response. Second, the response to single doses may itself be dose-dependent so that small acute radiation exposures, or exposures at very low dose rates, are more effective per unit dose than larger exposures above the threshold where the induced radioprotection is triggered. This combination has been termed low-dose hypersensitivity (HRS) and induced radioresistance (IRR) as the dose increases. Both the adaptive response and HRS/IRR have been well documented in studies with yeast, bacteria, protozoa, algae, higher plant cells, insect cells, mammalian and human cells in vitro, and in studies on animal models in vivo. There is indirect evidence that the HRS/IRR phenomenon in response to single doses is a manifestation of the same underlying mechanism that determines the adaptive response in the two-dose case and that it can be triggered by high and low LET radiations as well as a variety of other stress-inducing agents such as hydrogen peroxide and chemotherapeutic agents although exact homology remains to be tested. Little is currently known about the precise nature of this underlying mechanism, but there is evidence that it operates by increasing the amount and rate of DNA repair, rather than by indirect mechanisms such as modulation of cell-cycle progression or apoptosis. Changed expression of some genes, only in response to low and not high doses, may occur within a few hours of irradiation and this would be rapid enough to explain the phenomenon of induced radioresistance although its specific molecular components have yet to be identified.

In the green alga Chlamydomonas chlamyrhodopsin fulfills its role as a light sensor by absorbing light and activating photoreceptor channels within the eyespot area. At intense light stimuli, the photoreceptor (P) current triggers a fast and a slow flagellar current that finally leads to backward swimming (stop response). Here we report about probing the photoreceptor current directly at the eyespot. This allows the detection of the whole P current with a size of above 50 pA. The P current appears with a delay of less than 50 microseconds, suggesting that rhodopsin and the P channel are closely coupled or form one ion channel complex. The Ca2+ dependence of the P current has been demonstrated with the established suction technique in a capacitive mode. The P current shows the maximum amplitude at only 300 nM Ca2+, and it gradually declines at higher Ca2+. In addition to Ca2+, the photoreceptor and the fast flagellar current can be carried by Sr2+ and Ba2+. Mg2+ is conducted less efficiently and at high concentrations blocks the photoreceptor channel. A motion analysis of the cells shows that only Ca2+ and Sr2+ can induce physiological stop responses, whereas the large Ba2+ currents cause abnormal long-lasting cell spiraling.

Chlamydomonas exhibits a sequence of a photoreceptor current and two flagellar currents upon stimulation with bright green flashes. The currents are thought to be a prerequisite for the well-known photophobic responses. In the preceding paper, we analyzed the kinetics of these currents and their dependence on extracellular divalent ions. Here, we show that the photoreceptor current can be carried by monovalent ions (K+ > NH4+ > Na+), provided that the driving force is high enough. The small residual photoreceptor current observed in the absence of Ca2+ is able to evoke flagellar currents at low extracellular pH. This demonstrates that signal transduction from the rhodopsin to the flagella is not inevitably dependent on extracellular Ca2+. Double-flash experiments exclude a contribution of intra-rhodopsin charge movements to the photoreceptor current signal. Evidence will be provided for the existence of nonlocalized K+ outward currents, which counterbalance the localized Ca2+ influx and repolarize the cell after a light flash. A model is presented that explains the different pathways for direction changes and phobic responses.

The rhodopsin nature of the photoreceptor for the behavioural light responses in Chlamydomonas has originally been revealed by action spectroscopy. Meanwhile most physiological experiments and the identification of all-trans-retinal in cell extracts favour that this chlamyrhodopsin contains an all-trans-type retinal chromophore with strong similarity to the light sensors SR I and SRII from Halobacteria. Reconstitution of retinal-deficient cells with [3H]retinal identified a single retinal protein with a MW of 30,000. Chlamyrhodopsin triggers a photoreceptor current in the eyespot region resulting in direction changes or phototaxis. Furthermore, when the light stimulus oversteps a critical level, two flagellar currents appear, which are the basis for photophobic responses. The physiological, electrophysiological and biochemical experiments suggest that all behavioural responses are triggered by a single rhodopsin-type receptor.

While measuring action spectra for phase-shifting the circadian clock of Chlamydomonas, we observed that light pulses started near the phase response curve (PRC) "breakpoint" caused a reduction of the amplitude of the phototactic rhythm and two unexpected effects: (1) nonmonotonic fluence response curves (FRCs), and (2) shortening of the period of the subsequent free-running rhythm. The reduction of the rhythm's amplitude is dependent upon both the fluence and wavelength of the light pulse. The results are consistent with the amplitude being dependent upon the perceived "strength" of the stimulus, and with the nonmonotonic FRCs and reduced amplitude reflecting a light-induced change of the pacemaker's state variables to a region of the phase plane close to the "singularity." The period change that is evoked by single stimuli exhibits novel characteristics: large changes in period and a phase specificity that correlates with "singular" behavior. These period changes also appear to be a function of the stimulus strength, but indirectly; the magnitude of the period change is most strongly correlated with the magnitude of the light-induced phase shift. These results are interpreted in the context of limit cycle models of circadian clocks, and are used to suggest new tactics for measuring action spectra of light-induced clock resetting.

In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)+ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40,000 clones in each library revealed 44 clones which hybridize preferentially with a [32P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [32P] cDNA probe synthesized from poly(A)+ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.

A real-time automated method was developed for simultaneous measurements of phototactic orientation (phototaxis) and step-up photophobic response of flagellated microorganisms. Addition of all-trans retinal restored both photoresponses in a carotenoid-deficient mutant strain of Chlamydomonas reinhardtii in a dose-dependent manner. The phototactic orientation was biphasic with respect to both the light intensity and the concentration of retinal. All-trans retinal was more effective than 11-cis retinal to regenerate both photobehavioral responses. Analogs having locked 11-cis configurations and a phenyl ring in the side chain also induced photoresponses, although at concentrations more than two orders of magnitude higher than all-trans retinal. According to the present assay method, the responses were hardly detectable in cells incubated with retinal analogs in which the 13-ene was locked in either its trans or cis configuration. The results strongly suggest that the isomerization of the 13-14 double bond is important for photobehavioral signal transduction and that a single retinal-dependent photoreceptor controls both phototactic and photophobic responses.

Seven naturally occurring furoquinoline alkaloids were investigated for their photobiological activity using arg-1 cells of Chlamydomonas reinhardtii. UV-A-mediated toxicity of the compounds was calculated from the colony-forming ability of the treated cells. The UV-A-mediated mutagenicity was measured by counting the number of Arg+ revertants induced by the treatment. Dictamnine was found to be the strongest mutagen as well as the most toxic compound of the group. The mutagenic activities were measured as mutation frequencies at equal substance concentration and ranked in the following order: An increase in the number of substituents on the lateral aromatic nucleus greatly decreased the photomutagenicity. Except for evolitrine, a similar ranking order was found as reported for the dark mutagenicity of these compounds in Salmonella typhimurium strain TA98. Based on the result that furoquinolines are able to intercalate into DNA, we assume that the different mutagenic potencies may reflect differences in the geometry of the intercalation complex, which is important for the subsequent photochemical reaction.

Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt+ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt- parent, arise at a low frequency. Mutations at the mt(+)-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt- parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically with the mat-3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt+ parent.

Photoinhibition was induced in a cyanobacterium strain, Synechocystis 6714, and a green alga, Chlamydomonas reinhardtii, by exposing them to light intensities from 1000 to 4000 microE/(m2.s) at various temperatures. The photoinhibition process was followed by measurements of chlorophyll fluorescence and oxygen evolution. During exposure to high light, fluorescent active reaction centers II became low fluorescent inactive centers. This process involved several reversible and irreversible steps. The pathway from the active state to the inactive low fluorescent state was different in Synechocystis and Chlamydomonas. In the latter there was a reversible intermediary step characterized by an increase of F0. This state was stable at 5 degrees C and slowly reversible at room temperature. The high F0 fluorescence level corresponded to a state of photosystem II centers that were inactive for oxygen evolution. An F0 decrease occurred in the dark in the absence of protein synthesis and was correlated to a restoration of oxygen evolution. Further experiments suggested that the existence of the intermediate fluorescent state is due to modified closed centers in which the reduced primary acceptor is less accessible to reoxidation. In cyanobacteria this reversible state was not detected. In both organisms, the decrease of Fmax reflected an irreversible damage of photosystem II centers. These centers need replacement of proteins in order to be active again. The quenching of Fmax and the irreversible inhibition of oxygen evolution were slowed down in both organisms by decreasing the temperature of the photoinhibitory treatment from 34 to 5 degrees C. We conclude that low temperature protected the reaction center II from irreversible photodamage.

Photoautotrophic growth of Chlamydomonas reinhardtii was shown to be independent of the presence of atmospheric oxygen. Under constant light and photoautotrophic conditions, C. reinhardtii grew equally well in either air or 367 PPM CO2-in-He. During 12-h light-dark cycles, the cells in air grew substantially faster than those grown in CO2-in-He, indicating a significant role for O2 in dark metabolism. Although cells grown under CO2-in-He were not supplied any exogenous O2, photosynthetic water splitting resulted in the liberation of O2. The effect of photoevolved O2 on the growth of C. reinhardtii was examined (1) by measuring the amount of O2 consumed by photosynthesizing algae, (2) by growing the algae heterotrophically on acetate in the dark and supplied with O2 generated by photoautotrophically grown cells, and (3) by determining the minimum level of O2 needed to stimulate CO2 evolution from cells suspended in minimal medium supplemented with acetate. The results from these investigations indicated that exogenous O2 was not required for photoautotrophic growth by C. reinhardtii and that this alga grew in an anoxic environment if supplied with CO2 and light.

Expression of the nuclear coded heat shock protein HSP22 in Chlamydomonas reinhardtii y-1 cells is light regulated at the level of transcript accumulation. In dark grown cells, containing a non-differentiated plastid, light has an additional regulatory effect on the accumulation of HSP22. When such cells are exposed to heat stress in the light, poly(A)+ RNA hybridizing with the HS22 probe is synthesized at levels comparable with those found in cells pre-illuminated for 3 h (greening) prior to the heat shock. However, this RNA is poorly translated in vitro and HSP22 does not accumulate in vivo. HS22 mRNA efficiently translated in vitro is induced in dark grown cells only when chloroplast differentiation has been initiated by exposure to the light for 3 h. In these cells HSP22 accumulates during heat shock. Inhibition of plastid translation activity during light-dependent chloroplast development prevents accumulation of HSP22 in vivo. However the HS22 mRNA formed in this case can be efficiently translated in vitro. Light requirement for the accumulation of HSP22 during heat stress is exhibited also by wild type C. reinhardtii cells which possess a differentiated chloroplast irrespective of the light conditions during cell growth. However dark grown wild type cells do not require pre-illumination for developing the ability to accumulate HSP22 during heat stress in the light.

Spectral and photochemical parameters were described of the mutant strain of Chlamydomonas reinhardii 516--3a, in which the ratio chlorophyll a/P700 is about 20. Experiments were carried out with this strain for investigating earlier observed for Chlorella effect of burning out of deeps in absorption spectra at 4.2K resulting from irradiation with monochromatic light of the wavelength 698 nm. Arguments are presented in favour of the assumption that phototransformation of P700 is responsible for the formation of deeps. It has been found from the width of the deeps that the lifetime of the excited states of the centres responsible for the formation of deeps is between 16 ps and 65 ps.

Following exposure to UV light the synthesis of six polypeptides (112, 100, 89, 76, 71 and 65 kDa) was found to be enhanced or induced in the alga Chlamydomonas reinhardtii. Treatment with 4-nitroquinoline-N-oxide (NQO) resulted in the enhanced/induced synthesis of six polypeptides with molecular masses similar to those enhanced following exposure to UV light. Heat shock resulted in the enhanced synthesis of five polypeptides (89, 76, 71, 60 and 22 kDa), three of which (89, 76 and 71 kDa) had apparently identical mobilities to polypeptides whose synthesis was enhanced following UV treatment.

The effect of caffeine on repair was studied in the green alga Chlamydomonas reinhardtii. Treatment of UV-irradiated wild-type (UVS+) cells with a sublethal level of caffeine caused a significant increase in survival compared to untreated UV-irradiated cells. Caffeine did not affect survival in the repair-deficient strain UVSE1, which is deficient in repair of UV-induced damage carried out by enzymes associated with recombination during meiosis. A significant increase in survival in the presence of caffeine was observed in the repair-deficient strain UVSE4 in which recombination during meiosis is not affected. Treatment of zygotes homozygous for UVS+, UVSE1, or UVSE4 with sublethal levels of caffeine caused marked increases in recombination frequency in UVS+ and UVSE4 zygotes and no increase in recombination in UVSE1 zygotes. These results indicate that caffeine increases recombination in normal strains. Increased opportunity for recombination caused by caffeine would not result in increased recombination frequency in the UVSE1 strain, assuming limited-recombination enzyme activity in this strain. The observed increase in survival following UV-irradiation in the presence of caffeine in strains having normal recombination would therefore be associated with a caffeine-induced increase in opportunities for recombination repair.

We report here on the UV-induced vanadate-dependent cleavage of the alpha and beta heavy chains of the outer arm dynein from Chlamydomonas flagella. Both polypeptides are cleaved at a single site (termed the V1 site) by UV irradiation in the presence of Mg2+, ATP, and vanadate. The alpha chain yields fragments of Mr 290,000 and 190,000. Fragments of Mr 255,000 and 185,000 are obtained from the beta chain. Ultraviolet irradiation of the alpha and beta chains in the presence of vanadate and Mn2+ (but no nucleotide) induces cleavage of both molecules at sites (termed the V2 sites) distinct from the V1 sites. The single V2 site within the beta chain is located 75,000 daltons from the site of V1 cleavage within the Mr 255,000 V1 fragment. The alpha chain contains three distinct sites of V2 cleavage; all are located within the Mr 290,000 V1 fragment, 60,000, 90,000, and 100,000 daltons from the site of V1 cleavage. From these studies, we estimate the masses of the alpha and beta heavy chains to be 480,000 and 440,000 daltons, respectively.

Two new UV-sensitive mutants of Chlamydomonas, UVS10 and UVS11, were isolated. Both behave as single nuclear mutations. UVS10 was mapped to linkage group I. UVS11 is a separate, unlinked mutation but has not yet been located to a specific linkage group. Both mutants are proficient in the excision of pyrimidine dimers from nuclear DNA. The survival of UV-irradiated UVS11 is increased when plated in the presence of 1.5 mM caffeine, similar to wild-type. Caffeine has no effect on the survival of UV-irradiated UVS10. UV-irradiated UVS11 frequently divides at least once before dying, in contrast to UVS10 or wild-type. UVS11 also exhibits a much increased frequency of mutation to streptomycin resistance after UV irradiation.

The phototoxicity and the photomutagenicity of the 2 linear furocoumarins 8-methoxypsoralen (8-MOP) and 8-isoamylenoxypsoralen (8-IOP) and of the angular derivative 5'-methylangelicin (5'-MA) were studied in 2 different test systems. In human lymphocytes, sister-chromatid exchange analysis (SCE analysis) was carried out. We found an increase in SCE frequency after treatment with 8-MOP and 5'-MA, but no enhancement was caused by 8-IOP. In the algal system Chlamydomonas reinhardii, 8-MOP and 5'-MA showed a similarly strong influence with regard to phototoxicity; 8-IOP exhibited a weaker effect within the same irradiation time. For photomutagenicity in Chlamydomonas, 8-MOP proved to be the strongest derivative; 8-IOP, however, was capable of inducing about the same numbers of revertants, but only under certain conditions. The angular 5'-MA showed a weak mutagenic activity in contrast to the psoralens. The influence of the structure of the derivatives and of the experimental conditions on the effects obtained are discussed with regard to the therapeutic risk of monofunctional and bifunctional furocoumarins.

A group of 31 mutants of Chlamydomonas reinhardtii with altered phototactic responses was isolated. Growth of all the mutants was normal and, with one exception, they had no gross morphological aberrations. One mutant produced supernumerary flagella. The mutants can be subdivided into five phenotypic classes on the basis of their abnormal responses: slow positive, no reponse, slow negative, fast negative, and variable. Random motility of the mutants was also characterized. About half were indistinguishable from wild type; the others exhibited "jerky" or "twirly" movements instead of relatively straight paths. These mutants can now be used for genetic and biochemical studies of the cellular basis of phototaxis.

Light-dark synchronized Chlamydomonas reinhardtii are most sensitive to irradiation when exposed early in the dark period of a 12 hour light: 12 hour dark regime. At this time the algae are either dividing or preparing for division. Liquid cell cultures of the 137c wild type strain exposed to 9,000 R X-irradiation exhibit 69% mortality. Monolayer cultures on Millipore filters exposed to proton irradiation (1.5 MeV protons from a positive Van de Graff accelerator) exhibit 89% mortality following a dose of 9 kilorad, and 98% mortality following 18 kilorad. Ultrastructural examination of X-irradiated cells over five days following exposure documented chloroplast and nuclear alterations which included non-nuclear condensations, swollen and ruptured nuclear envelopes and disrupted pyrenoid structure. Following both types of irradiation, both live and dead daughter cells were present within the old mother cell wall. Following both types of irradiation, cells were also observed in which there were (1) profiles resembling division conformations, and (2) chromosomes and spindle fibers present at a time not characteristic for such events in synchronized control cultures. No changes distinct to either X-ray or proton irradiation were present, indicating that, after a given level of injury, similar ultrastructural manifestations of cell damage are evident in irradiated cells.

The turnover of photosystem-II proteins during photoinhibition was analyzed in the green alga Chlamydomonas reinhardtii. Changes in the amount of photosystem II core complex polypeptides D1, D2, 44 kDa and 51 kDa, the antennae-CP-29 and light-harvesting-complex-II polypeptides and the water-oxidizing complex polypeptides of 30 kDa, 23 kDa and 16 kDa were monitored by a variety of techniques. Only the D1 and D2 polypeptides were found to turnover during photoinhibition when cells were exposed to ten fold photosynthesis-saturating light (2500 W/m2 for 90 min) at 25 degrees C. While 80% of photosystem-II activity was lost, a reduction of only 20% was observed in the total amount of D1 and D2 proteins. However, inhibition of chloroplast translation by chloramphenicol during photoinhibition resulted in the loss of about 60% of the D1 and 40% of the D2 proteins, as demonstrated by Western blotting and dot blotting of isolated thylakoids, quantitative analysis of immunogold-labeled whole-cell thin sections, and chase of radioactively prelabelled proteins during photoinhibition. We propose that the light-dependent turnover of the D1 protein is a protective mechanism against photoinhibition as far as the removal and replacement of D1 is compatible with the photoinactivation incurred by photosystem II. At light intensities at which the rate of D1 removal becomes limiting, loss of photosystem-II activity exceeds the turnover of D1 and the stability of the D2 protein is impaired as well.

Photoreceptor membranes in the bovine retina may be substituted in reconstructed systems by pigment fractions from Chlamydomonas reinhardtii which account for GTP-dependent activation of cGMP-phosphodiesterase. Fractions from carotinoid-less mutant did not exhibit this capacity. The results obtained reveal significant structural and functional similarity between rhodopsin-like pigment of Ch. reinhardtii and rhodopsin from vertebrates.

The action spectra of five eukaryotic organisms and the prokaryote, Escherichia coli, were examined over the wavelength range, 254-320 nm. Both the repair competent and three repair defective strains (E. coli, Caenorhabditis elegans, Saccharomyces) were examined. Tetrahymena pyriformis action spectra were performed with and without the excision repair inhibitor caffeine present. Others have observed that lethality, mutation, and the production of pyrimidine dimers show much the same wavelength dependence as DNA absorption. The results presented here demonstrate several action spectra which deviate from the DNA absorption spectra. Ultraviolet sensitization ratios (repair competent/repair defective) were also examined and were shown to change over the wavelength range. These findings suggest that DNA may not be the only important chromophore leading to cell death in the uv wavelength range studied. Since uv-B is of major importance in solar uv damage, these findings may also yield important implications for solar uv studies.

A study was made of the ultrastructure of plastids of three mutant types of Chlamydomonas reinhardi which are phenotypically revealed either in the light or in the darkness as yellow mutants. Characteristics of pigments for each mutant have been given. Mutant Y-4 unable to synthesize chlorophyll either in the light or in the darkness shows a complete reduction of photosynthesizing membranes. Mutant Y-1 capable of synthesizing chlorophyll develops a normal system of photosynthesizing membranes. The dark synthesis of chlorophyll in this mutant is broken, the mutant accumulates only carotenoids, the membrane system of its plastid being reduced. On the contrary, mutant Y-3 has in the darkness a complete set of pigments and a well developed membrane system. In the light this mutant yellows due to chlorophyll photodestruction that is followed by destruction of the membrane system of chloroplasts.

Non-Mendelian genes in Chamydomonas reinhardtii are inherited in a uniparental (UP) fashion. Most zygotes and their progeny receive UP genes only from the mt(+) or maternal parent. However, a few exceptional zygotes are also found in which the mt(-) or paternal UP genome is transmitted. Most of the exceptional zygotes are biparental in that their progeny segregate UP genes transmitted by both parents. As a result, biparental zygotes have been extensively used to study the rules governing UP inheritance. The frequency of biparental zygotes can be greatly increased if the maternal parent is irradiated with ultraviolet light prior to mating. Based principally on studies with ultraviolet-induced biparental zygotes, Sager has argued that a vegetative cell contains two copies of the UP genome and that the progeny of a biparental zygote receive a copy derived from each parent. Results reported in this paper with spontaneous and ultraviolet-induced biparental zygotes do not support the two copy model, but argue for a mulitple copy model with most of the copies normally being transmitted by the maternal parent. A multiple copy model which accounts for both Sager's results and ours is presented.

UVS1 is a mutant of Chlamydomonas reinhardi defective in the dark repair of pyrimidine dimers in nuclear DNA. All of the pyrimidine dimers in nuclear DNA can be repaired upon exposure to photoreactivating light immediately after irradiation. However, none of the dimers in nuclear DNA are repaired by photoreactivation if the irradiated cells are incubated in the dark for 24 h in growth medium. Pyrimidine dimers in chloroplast DNA that are unrepaired during the 24 h post-irradiation incubation can be repaired by photoreactivation. Treatment with methyl methanesulfonate to give a similar survival as the fluence of ultraviolet light did not lead to the inactivation of nuclear photoreactivating enzyme after 24 h in the dark. Assay for photoreactivating enzyme in cell-free extracts showed that about 80% of the photoreactivating enzyme activity disappears after incubating ultraviolet-irradiated cells in the dark for 24 h.

A modified Ames assay was developed that used an arginine-requiring mutant of Chlamydomonas reinhardii as an indicator organism. By counting the numbers of colonies formed and Arg+ revertants, we studied the influence of a rat-liver S9 mix (S9 fraction plus co-factors) on the toxic and mutagenic activities of 8-MOP plus black light. Both activities were decreased after pre-incubation of 5 micrograms 8-MOP/ml with S9 mix at 37 degrees C. S9 fractions without co-factors were almost completely inactive.