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An abstract of the article "Nanophytoplankton Diversity Across The Oligohaline Lake Pontchartrain Basin Estuary: A Preliminary Investigation Utilizing Psba Sequences," by J.L. Wee and colleagues is presented.

Regulation of gene expression in plastids may involve molecular components conserved from cyanobacteria-like ancestors. Among prokaryotes, genes are commonly regulated at the transcriptional level by 'two-component' or 'His-Asp' signal transducers, consisting of a 'sensor kinase', which autophosphorylates at a conserved histidine residue, and a cognate response regulator, which is phosphorylated by the sensor kinase at a conserved aspartate residue. A putative His-Asp response regulator gene (trg1: transcriptional regulatory gene 1) has been identified in the estuarine raphidophytic alga Heterosigma akashiwo. The chloroplast-encoded trg1 is 693 bp in length, contains no introns, and yields a conceptual translation product of 231 amino acids, with a predicted mass of 27 kDa. Homology searches suggest that Heterosigma trgl has an omnpR-like identity within the DNA-binding His-Asp family of response regulators. trg1 contains both the phosphorylation and DNA-binding domains which are present in prokaryote response regulators. Quantitative competitive RT-PCR showed that Heterosigma trg1 is expressed at low levels (5 microg per g total RNA). In contrast, psbA (a photosystem II component) transcript is abundant (60 mg per g total RNA). Cell cycle analysis showed that psbA abundance oscillates in response to light but trg1 mRNA levels are invariant. We hypothesize that a His-Asp phosphorelay mechanism may affect chloroplast genome transcription in a manner similar to bacterial signal transduction pathways in which 'sensor kinase' and cognate 'response regulator' proteins interact.

A gene encoding a thioredoxin protein was identified in the chloroplast genome of the rhodophyte Porphyra yezoensis. The P. yezoensis trxA gene contains 324 bp and is transcribed into a 0.7 kb messenger RNA. Analysis of the transcription start site demonstrates that canonical chloroplast -10 and -35 sequences are not present. The deduced amino acid sequence of the thioredoxin gene from the red algae has the greatest similarity to type m thioredoxins, providing strong support for the hypothesis that type m thioredoxins in photosynthetic eukaryotes originated from an engulfed bacterial endosymbiont. Hybridization analysis of nuclear and chloroplast DNAs from several members of the phyla Chromophyta and Rhodophyta using P. yezoensis DNA as a probe demonstrated strong hybridization to the chloroplast and nuclear genomes of Griffithsia pacifica and a weak cross-hybridization to the chromophyte P. foliaceum. The G. pacifica chloroplast gene has a 66% identity with the P. yezoensis DNA, contains conserved active site amino acid residues, but lacks a methionine start codon.

Using CsCl-Hoechst dye or CsCl-ethidium bromide gradients, satellite and nuclear DNAs were separated and characterized in three marine dinoflagellates: Glenodinium sp., and two toxic dinoflagellates, Protogonyaulax tamarensis and Protogonyaulax catenella. In all three dinoflagellates, the lowest density fraction, satellite DNA(1), hybridized to chloroplast genes derived from terrestrial plants and/or other algae. Dinoflagellate chloroplast DNAs exhibited molecular sizes of 114 to 125 kilobase pairs, which is consistent with plastid sizes determined for other chromophytic algae (120-150 kilobase pairs). Mitochondrial DNA was not resolved from nuclear DNA in this system. Two additional satellite DNAs, satellite DNA(2) and satellite DNA(3), recovered from P. tamarensis and P. catenella were similar to one another, both within and between species, when characterized by restriction enzyme analysis. These satellites were 85 to 95 kilobase pairs in size, and exhibited restriction fragments that hybridized to yeast nuclear ribosomal RNA genes. Restriction enzyme analyses and DNA hybridization studies of cpDNA document that the two Protogonyaulax isolates are not evolutionarily identical.

Studies demonstrating differences in chloroplast structure and biochemistry have been used to formulate hypotheses concerning the origin of algal plastids. Genetic and biochemical experiments indicate that significant variation occurs in ribulose-1,5-bisphosphate carboxylase (Rubisco) when supertaxa of eukaryotic algae are compared. These differences include variations in the organelle location of the genes and their arrangement, mechanism of Rubisco synthesis, polypeptide immunological reactivity and sequence, as well as efficacy of substrate (ribulose bisphosphate and CO2) binding and inhibitor (6-phosphogluconate) action. The structure-function relationships observed among chromophytic, rhodophytic, chlorophytic and prokaryotic Rubisco demonstrate that: (a) similarities among chromophytic and rhodophytic Rubisco exist in substrate/inhibitor binding and polypeptide sequence, (b) characteristic differences in enzyme kinetics and subunit polypeptide structure occur among chlorophytes, prokaryotes and chromophytes/rhodophytes, and (c) there is structural variability among chlorophytic plant small subunit polypeptides, in contrast to the conservation of this polypeptide in chromophytes and rhodophytes. Taxa-specific differences among algal Rubisco enzymes most likely reflect the evolutionary history of the plastid, the functional requirements of each polypeptide, and the consequences of encoding the large and small subunit genes in the same or different organelles.

Eukaryotes contain a chimeric assembly of genomes, each localized in a specialized subcellular compartment. The successful survival of an organism requires that these sequestered genomes be viewed as dependent variables in a coevolutionary complex. This discussion focuses on chloroplast evolution. A selected review of information available on chloroplast diversity is presented, followed by an analysis of the genetic modifications which may have occurred in the conversion of a free-living ancestral photosynthetic prokaryote into an organelle that has an obligately dependent and highly efficient interplay with the nuclear genome., (Copyright © 1986. Published by Elsevier Ltd.)

Paralytic shellfish toxin concentrations were measured and individual toxin profiles were monitored in axenic batch cultures of Protogonyaulax tamarensis and Protogonyaulax catenella. High pressure liquid chromatographic methods were used that allowed the separation of all 12 known paralytic shellfish poisons, including toxins C1, C2, and C3, from a single sample. In isolates of both Protogonyaulax species, total toxin levels were relatively low after inoculation, increased rapidly in early to mid-exponential growth to a value 100 to 300% of that at the initial time point, then decreased by 86 to 95% as the culture aged. Although the concentrations of individual toxins per cell followed the same general pattern as that seen for total moles of toxin per cell, variability in toxin profile with culture age was observed. In P. tamarensis, the mole percent of neosaxitoxin increased substantially from 8 to 44% as total toxin levels per cell decreased. A concomitant decrease in the mole percent of saxitoxin with culture age was noted. Although not as precipitous, changes in the mole percent of specific toxins from P. catenella were also observed. The mole percent of gonyautoxins I and IV increased, while that of gonyautoxins II and III decreased. These data suggest that the toxin profile in isolates of Protogonyaulax can change, sometimes significantly, with changing environmental variables.

This study represents the first report on chloroplast protein synthesis during the synchronous cell growth of a chromophytic (chlorophyll a,c) plant. When the unicellular alga Olisthodiscus luteus is maintained on a 12-hour light:12-hour dark cycle, cell and chloroplast number double every 24 hours. A temporal separation between these two events occurs. Measurements of chloroplast and total cellular protein values suggest that polypeptide synthesis occurs mainly in the light portion of the cell cycle, and pulse chase studies demonstrate that chloroplast proteins made in the light are not degraded in the dark. Data support the following conclusions: (a) a similar complement of chloroplast DNA coded proteins is made at all phases of the light portion of the cell cycle, and (b) chloroplast protein synthesis is a light rather than a cell cycle mediated response.

Chloroplast DNA (ctDNA) from the marine chromophytic alga, Olisthodiscus luteus, has been isolated using a whole cell lysis method followed by CsCl-Hoechst 33258 dye gradient centrifugation. This DNA, which has a buoyant density of 1.691 grams per cubic centimeter was identified as plastidic in origin by enrichment experiments. Inclusion of the nuclease inhibitor aurintricarboxylic acid in all lysis buffers was mandatory for isolation of high molecular weight DNA. Long linear molecules (40 to 48 micrometers) with considerable internal organization comprised the majority of the ctDNA isolated, whereas supertwisted ctDNA and open circular molecules averaging 46 micrometers were occasionally present. Also observed in this study were folded ctDNA molecules with electron dense centers ("rosettes") and plastid DNA molecules which have a tightly wound "key-ring" center. The ctDNA of Olisthodiscus has a contour length that is median to the size range reported for chlorophytic plants.A minor component of the total cellular DNA, which originates from a DNase insensitive cellular structure, has a buoyant density of 1.694 grams per cubic centimeter. This DNA consists predominantly of linear molecules, but open circles 11.5 micrometers in length and rare 22-micrometer dimers were also present.This study represents the first analysis of the extranuclear DNA of a chromophytic alga.

Changes in the physiological state of the multiplastidic alga Olisthodiscus luteus result in a shift in chloroplast complement from 33 to 21 plastids. The effect of this induced change in organelle complement on nuclear and chloroplast DNA levels has been analyzed. Data suggest that the absolute amount of chloroplast and nuclear DNA found within a cell remains constant but that the amount of chloroplast DNA per plastid is inversely proportional to the number of chloroplasts to which that DNA must be distributed.

Isolation of high molecular weight ribosomal RNA from the wall-less alga Olisthodiscus luteus and the angiospermous plant Sauromatum guttatum is described. It has been found that a buffer which contains magnesium must be used to successfully isolate Olisthodiscus rRNA whereas the isolation of intact Sauromatum rRNA requires a buffer system containing a high amount of the chelator EDTA. Sauromatum but not Olisthodiscus extracts were contaminated with ribonuclease unless the inhibitor diethylpyrocarbonate was used during the ribonucleic acid extraction procedure. Nuclease levels were monitored by coincubating [3H]-labeled Escherichia coli ribosomal RNA with the experimental RNA samples. The effects of detergents on the isolation and quantitation of RNA are presented, and methods to avoid loss of highly thermolabile plant ribosomal RNA species are discussed.

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5' non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.

Information on the ctDNA protein coding profile of the Chlorophyta, Rhodophyta, and Chromophyta might provide clues to the evolutionary mechanism(s) by which plants diverged into these three phylogenetic groups. The purpose of this study was to examine the ctDNA protein coding profile of the chromophytic plant Olisthodiscus luteus. Whole cells were labeled in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Control experiments demonstrate that the chloroplast proteins labeled in vivo by this technique form a distinct subset of the total proteins synthesized by the cell. Approximately 50 plastid proteins (35 soluble, 15 membrane) were detected after two-dimensional gel electrophoresis and fluorography. Three ctDNA-coded proteins, the large subunit of ribulosebisphosphate carboxylase, the apoprotein of the P700-chlorophyll a-protein complex, and the "photogene" were identified. These proteins are also coded by chlorophytic ctDNA. Unexpectedly, the ctDNA of Olisthodiscus was shown to code for the small subunit of ribulosebisphosphate carboxylase. The gene for this enzyme subunit is nuclear coded in all chlorophytic plants that have been analyzed.

The kinetic complexity of chloroplast DNA isolated from the chromophytic alga Olisthodiscus luteus has been determined. Using optical reassociation techniques, it was shown that the plastid DNA of this alga reacted as a single component with a second order rate constant of 4.1 molar(-1) and second(-1) (Cot((1/2)) 0.24 molar second) under conditions equivalent to 180 millimolar Na(+) and 60 degrees C. Given the 92 x 10(5) dalton complexity calculated for this chloroplast genome, an Olisthodiscus cell contains 650 plastome copies. Although this complement remains constant throughout the growth cycle of the organism, the ploidy level of an individual chloroplast shows significant plasticity and is dependent upon the number of chloroplasts present per cell. Experiments with the DNA fluorochrome Hoechst dye 33258 (bisbenzimide) demonstrate that plastids isolated from all phases of cell growth each possess a ring-shaped nucleoid containing detectable DNA. Olisthodiscus chloroplast DNA showed no sequence mismatch when thermal denaturation profiles of reassociated chloroplast DNA were examined, thus all plastome copies are essentially identical. Finally, reassociation studies demonstrated that no foldback (short inverted repeat) sequences were present in the plastid genome although significant hairpin loop structures were observed in control nuclear DNA samples.

The chloroplast DNA of the chromophytic alga Olisthodiscus luteus has been physically mapped with four restriction enzymes. An inverted repeat of 22 kilobase pairs is present in this 150-kilobase-pair plastid genome. The inverted repeat contains the genes for the large and small subunit polypeptides of ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) and also codes for the 32,000-dalton Q(B) protein. These observations demonstrate that significant differences exist in chloroplast genome structure and organization among major plant taxa.

Ribulose-1,5-bisphosphate carboxylase (RuBPCase) was purified from the marine chromophyte Olisthodiscus luteus. This study represents the first extensive analysis of RuBPCase from a chromophytic plant species as well as from an organism where both subunits of the enzyme are encoded on the chloroplast genome. The size of the purified holoenzyme (17.9 Svedberg units, 588 kilodaltons) was determined by sedimentation analysis and the size of the subunits (55 kilodaltons, 15 kilodaltons) ascertained by analytical sodium dodecyl sulfate gel electrophoresis. This data predicts either an 8:9 or 8:8 ratio of the large to small subunits in the holoenzyme. Amino acid analyses demonstrate that the O. luteus RuBPCase large subunit is highly conserved and the small subunit much less so when compared with the chlorophytic plant peptides. The catalytic optima of pH and Mg(2+) have been determined as well as the response of enzyme catalysis to temperature. The requirements of NaHCO(3) and Mg(2+) for enzyme activation have also been analyzed. The Michaelis constants for the substrates of the carboxylation reaction (CO(2) and ribulose bisphosphate) were shown to be 45 and 48 micromolar, respectively. Competitive inhibition by oxygen of RuBPCase-catalyzed CO(2) fixation was also demonstrated. These data demonstrate that a high degree of RuBPCase conservation occurs among widely divergent photoautotrophs regardless of small subunit coding site.

There are almost no data describing chloroplast genome organization in chromophytic (chlorophyll a/c) plants. In this study chloroplast ribosomal operon placement and gene organization has been determined for the golden-brown alga Olisthodiscus luteus. Ribosomal RNA genes are located on the chloroplast DNA inverted repeat structure. Nucleotide sequence analysis, demonstrated that in contrast to the larger spacer regions in land plants, the 16S-23S rDNA spacer of O. luteus is only 265 bp in length. This spacer contains tRNA(Ile) and tRNA(Ala) genes which lack introns and are separated by only 3 bp. The sequences of the tRNA genes and 16S and 23S rDNA termini flanking the spacer were examined to determine homology between O. luteus, chlorophytic plant chloroplast DNA, and prokaryotes.

Two extranuclear DNA species have been isolated from the marine alga Olisthodiscus luteus. Rapid lysis of cells followed by the immediate addition of CsCl to the lysate was critical to the preservation of these satellite DNA species. Restriction endonuclease analysis demonstrates a molecular weight of 99 x 10(6) for chloroplast DNA and 23 x 10(6) for a second satellite species. The origin of the second satellite is not known. However, this smaller satellite DNA which originates from a nonnuclear, DNAse insensitive cellular component, displays no sequence homology with ctDNA by hybridization experiments. Constancy of restriction endonuclease fragment patterns of chloroplast and second satellite species during all phases of the growth cycle, whether cultures were maintained synchronously or asynchronously, was demonstrated.

The classification of plants has traditionally been dependent upon the comparative analysis of morphological and biochemical data. In this paper the use of molecular probe analysis of chloroplast DNA (ctDNA) is used to expand the data base used in taxonomic studies. Chloroplast DNA size, homogeneity, the global arrangement of ctDNA structure, gene content, gene cluster array and gene sequence determination are discussed as useful criteria in the analysis of phylogenetic relationships.

It has been suggested that cyanobacteria served as the ancestors for rhodophytic algae whose chloroplasts contain chlorophyll a and phycobilins, and that a rodophyte served as the plastid source for chromophytic plants that contain chlorophylls a and c. Although organellar DNA has been used to assess phylogenetic relatedness among terrestrial plants and green algae whose chloroplasts contain chlorophylls a and b, few data are presently available on the molecular profile of plastid DNA in chromophytes or rhodophytes. In this study the chloroplast genome of the rhodophytic, filamentous alga Griffithsia pacifica has been characterized. DNA was purified from isolated chloroplasts using protease k treatment and sodium dodecyl sulfate lysis followed by density centrifugation in Hoechst-33258 dye-CsCl gradients. Single and double restriction enzyme digests demonstrate that the DNA prepared from purified chloroplasts has a genome size of about 178 kilobase pairs (kb). A restriction map of this chloroplast genome demonstrates that it is circular and, unlike the chloroplast DNA (cpDNA) in most other plants, contains only a single ribosomal DNA operon. DNA was also purified from the mitochondria that co-isolated with chloroplasts. Mitochondrial DNA consists of molecules that range in size from 27 to 350 kb based on restriction endonuclease digestion and electron microscopic analysis.

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B(12) to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B(12), plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.

Nuclear DNA of the marine chromophytic alga Olisthodiscus luteus was analyzed in this study. Reassociation kinetics analysis has shown that 440-nucleotide DNA fragments from the genome of this alga contain 4% foldback, 58% repetitive, and 34% single-copy sequences. Precise analysis using isolated single-copy DNA revealed that Olisthodiscus has a large haploid DNA content of 1.66 x 10(-12) g/cell. For determination of the organization of single-copy and repetitive sequences within this genome, DNA fragments 3000 nucleotides in length were reassociated to C0t= 100 M . s. At this low C0t value 89% of the DNA bound to hydroxylapatite, suggesting that single-copy and repetitive elements are interspersed. The lengths of the duplexed repetitive DNA on these 3000-nucleotide fragments were measured by electron microscopy after digestion with S1 nuclease which removed the unreassociated single-copy DNA regions. Of these repetitive sequences, 68% were shorter than 1200 nucleotide pairs in length and had a modal length of 350 nucleotide pairs. A minor class of longer (to 4000 nucleotide pairs) repetitive sequences was also observed.

Ribulose-1,5-bisphosphate carboxylase (Rubisco) from the algae Olisthodiscus luteus (chromophyte) and Griffithsia pacifica (rhodophyte) are remarkably similar to each other. However, both enzymes differ significantly in the structure and function when compared to Rubisco from green algae and land plants. Analysis of purified Rubisco from O. luteus and G. pacifica indicates that the size of the holoenzyme and stoichiometry of the 55 and 15 kilodalton subunit polypeptides are approximately 550 kilodaltons and eight:eight for both algae. Antigenic determinants are highly conserved between the O. luteus and G. pacifica enzymes and differ from those of the spinach subunit polypeptides. Sequence similarity between the two algal large subunits has been further confirmed by one-dimensional peptide mapping. Substrate ribulose bisphosphate has no effect on the rate of CO(2)/Mg(2+) activation of O. luteus and G. pacifica enzymes which contrasts to the extensive inhibition of spinach Rubisco activation at similar concentrations of this compound. In addition, the Michaelis constant for CO(2) and the inhibition constant for 6-phosphogluconate are similar for the O. luteus and G. pacifica catalyzed carboxylation reaction. Both values are intermediate to those observed for the tight binding spinach enzyme and weak binding prokaryotic (Rhodospirillum rubrum) enzyme. The biochemical similarities documented between O. luteus and G. pacifica may be due to a common evolutionary origin on the chromophytic and rhodophytic chloroplast but could also result from the fact that both subunit polypeptides are chloroplast DNA encoded in these algal taxa.