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In 2008, curriculum developers and English instructors at Miyazaki Municipal University (MMU) designed and implemented a new reading curriculum for all freshmen students. Building on the previous curriculum, this new approach attempted to provide a standardized framework of reading instruction that included both an intensive and extensive approach to improving English reading skills. Intensive Reading (IR) focuses on teaching students techniques and strategies to deal with text well beyond their current level. Extensive Reading (ER), on the other hand, requires learners to read vast amounts of material that has been simplified to the learner's appropriate reading level; thereby, improving motivation to read in a foreign language. By combining both an intensive and extensive approch, the MMU curriculum hoped to synthesize the best of both approaches. The purpose of this paper to describe and analyze the 2008 to 2013 MMU reading curriculum.

Communication Strategies (CS) are the methods which speakers use to compensate for difficulties in communicating and studies in CS have tended to focus on students learning a second language. However, bilingual children, especially children who are both bilingual and bicultural, also face many unique difficulties communicating within two different language communities. Having been raised with two languages, these children, however, almost always have one language, known as their primary language, in which they are more proficient. Their other language is known as their subordinate language and is weaker and less fluent in most cases. In order to compensate for this, bilinguals, especially bilingual children, learn unique strategies to meet their communicative needs in both languages. The study below presents a case study on CS use in a seven-year-old, bilingual, bicultural child. First, the author will give an outline of CS research, especially research associated with CS use in bilingual subjects. Then, language samples obtained from story-telling sessions will be analyzed in order to describe the subject's use of CS.

Although participation of PEROXIN11 (PEX11), FISSION1 (FISl), and DYNAMIN-RELATED PROTEIN (DRP) has been well established during induced peroxisome proliferation in response to external stimuli, their roles in cell cycle–associated constitutive replication/duplication have not been fully explored. Herein, bimolecular fluorescence complementation experiments with Arabidopsis thaliana suspension cells revealed homooligomerization of all five PEX11 isoforms (PEX11a-e) and heterooligomerizations of all five PEX11 isoforms with FIS1b, but not FIS1a nor DRP3A. Intracellular protein targeting experiments demonstrated that FIS1b, but not FIS1a nor DRP3A, targeted to peroxisomes only when coexpressed with PEX11d or PEX11e. Simultaneous silencing of PEX11c-e or individual silencing of DRP3A, but not FIS1a nor FIS1b, resulted in ∼40% reductions in peroxisome number. During G2 in synchronized cell cultures, peroxisomes sequentially enlarged, elongated, and then doubled in number, which correlated with peaks in PEX11, FIS1, and DRP3A expression. Overall, these data support a model for the replication of preexisting peroxisomes wherein PEX11c, PEX11d, and PEX11e act cooperatively during G2 to promote peroxisome elongation and recruitment of FIS1b to the peroxisome membrane, where DRP3A stimulates fission of elongated peroxisomes into daughter peroxisomes, which are then distributed between daughter cells.

Although the Japanese are arguably the most literate people on the planet, any teacher of Japanese freshmen university students can tell you that most are not what could be called "readers." Most freshmen come into the university with few or none of the requisite skills needed to handle complex reading material in either their mother tongue or a foreign language. The purpose of this paper, therefore, is to introduce a process of reading that gives students a solid method for coping with reading material that is beyond their level. This process is known as Survey, Question, Read, Recite, Review, or SQ3R. The author will introduce the basic tenets of SQ3R and demonstrate how he has incorporated these into the curriculum of his freshmen reading course. The author will also discuss the results of pre/post-course exams and surveys to show how students have reacted to SQ3R.

The recent crystal structure of photosystem I (PSI) from Thermosynechococcus elongatus shows two nearly symmetric branches of electron transfer cofactors including the primary electron donor, P(700), and a sequence of electron acceptors, A, A(0) and A(1), bound to the PsaA and PsaB heterodimer. The central magnesium atoms of each of the putative primary electron acceptor chlorophylls, A(0), are unusually coordinated by the sulfur atom of methionine 688 of PsaA and 668 of PsaB, respectively. We [Ramesh et al. (2004a) Biochemistry 43:1369-1375] have shown that the replacement of either methionine with histidine in the PSI of the unicellular green alga Chlamydomonas reinhardtii resulted in accumulation of A(0)(-) (in 300-ps time scale), suggesting that both the PsaA and PsaB branches are active. This is in contrast to cyanobacterial PSI where studies with methionine-to-leucine mutants show that electron transfer occurs predominantly along the PsaA branch. In this contribution we report that the change of methionine to either leucine or serine leads to a similar accumulation of A(0)(-) on both the PsaA and the PsaB branch of PSI from C. reinhardtii, as we reported earlier for histidine mutants. More importantly, we further demonstrate that for all the mutants under study, accumulation of A(0)(-) is transient, and that reoxidation of A(0)(-) occurs within 1-2 ns, two orders of magnitude slower than in wild type PSI, most likely via slow electron transfer to A(1). This illustrates an indispensable role of methionine as an axial ligand to the primary acceptor A(0) in optimizing the rate of charge stabilization in PSI. A simple energetic model for this reaction is proposed. Our findings support the model of equivalent electron transfer along both cofactor branches in Photosystem I.

Diatoms (Chrysophyta) are photosynthetic microorganisms that are abundant in the natural environment and often associated with specific habitat and water quality conditions. Their significance as bioindicators and as exploitable sources of fine chemicals makes them desirable candidates for the study of stress responses. The protein expression of a thermo-intolerant ( Phaeodactylum tricornutum ) and thermo-tolerant ( Chaetoceros muelleri ) diatom following exposure to elevated temperature was investigated using one- and two-dimensional gel electrophoresis and Western blot analysis. It was determined using SDS PAGE with 35 S-methionine labeled proteins and Western blot analysis using pea HSP70 antisera that higher temperatures and longer duration treatment were required to cause a noticeable stress response in C. muelleri compared to P. tricornutum. This may be explained by C. muelleri possessing higher amounts of constitutively expressed heat shock proteins, which allows these cells to rapidly adjust to temperature increases. Two-dimensional gel electrophoresis revealed that putative small heat shock proteins (smHSPs) may appear to play a role during heat stress in both diatoms, which is similar to the response in plants. SDS PAGE data are also presented characterizing the recovery of P. tricornutum after heat shock. These results suggest that there is a lag period between heat shock and stress protein synthesis in these thermo-intolerant cells. This supports the hypothesis that cells without higher amounts of constitutively expressed stress proteins have a greater sensitivity to increased temperature. Work is underway to identify particular stress proteins responsible for conveying thermo-tolerance and to determine if overexpression of these genes in thermo-intolerant diatoms affects their temperature sensitivity.

Photosystem I contains two potential electron transfer pathways between P(700) and F(X). These branches are made up of the electron transfer chain components A, A(0), and A(1). The primary electron acceptor A(0) is a chlorophyll a monomer that could be one or both of the two chlorophyll molecules, eC-A(3)/eC-B(3), identified in the 2.5 A resolution structure. The eC-A(3)/eC-B(3) chlorophylls are both coordinated by the sulfur atom of a methionine. This coordination is highly unusual, as interactions between the acid Mg(2+) and the soft base sulfur are weak. The eC-A(3)/eC-B(3) chlorophylls also are located close to one of the connecting chlorophylls that may link the antenna and the electron transfer chain chlorophylls. Due to their location in the structure, the eC-A(3)/eC-B(3) chlorophylls may play a role in both excitation energy transfer and electron transfer. To test the role of the eC-A(3)/eC-B(3) chlorophylls in electron transfer, Met-684 of PsaA and Met-664 of PsaB have been changed to His, Ser, and Leu. Replacement of either M(A684) or M(B664) results in a significant alteration in growth phenotype. The His and Leu mutants are very light sensitive in the presence of oxygen. Growth is impaired to a greater extent in the B-side mutants. However, all of the mutants are able to grow anaerobically at comparable rates. The His and Ser mutants all accumulate PSI at a level similar to that of wild type, whereas the Leu mutants have reduced amounts of PSI. Ultrafast transient absorbance measurements show that the (A(0)(-) - A(0)) difference signal accumulates in the MH(A684) and MH(B664) mutants under neutral conditions, demonstrating that electron transfer between A(0)(-) and A(1) is blocked or significantly slowed. The results show that both the A-branch and the B-branch of the ETC are active in PSI from Chlamydomonas reinhardtii.

Fatty acid composition and degree of fatty acid saturation during temperature stress in thermo-intolerant ( Phaeodactylum tricornutum ) and thermo-tolerant ( Chaetoceros muelleri ) marine diatoms were investigated. A greater number of fatty acids were observed in C. muelleri than in P. tricornutum regardless of treatment. The major fatty acids detected were 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1(n-9)c, 18:2(n-6) and 20:5(n-3) with additional fatty acids 18:1(n-9)t and 20:4(n-6) detected in C. muelleri . Short duration (2 h) temperature increase above optimal growth temperature had a greater effect on fatty acid composition in C. muelleri than in P. tricornutum and the degree of fatty acid saturation was affected more by temperature in C. muelleri than in P. tricornutum during both short and long duration (24 h) treatments. Total protein assay results suggest that P. tricornutum , but not C. muelleri , was undergoing stress under our growing conditions although lipids in both diatoms were affected by increased temperature. Immunodetection of proteins with anti-rubisco indicates that the rubisco large subunit was undergoing greater turnover in C. muelleri than in P. tricornutum. However, the integrity of rubisco as a suitable indicator of lipid status needs further study. This work supports the hypothesis that a particular temperature, and not treatment duration, has the greater effect on changes in fatty acid composition. Furthermore, changes in fatty acid composition and degree of fatty acid saturation occurred more quickly in the diatoms in response to increased temperature than previously observed in nutrient starvation studies. Since diatom lipids represent an important resource for growth and reproduction of marine animals, the rapid alteration of their lipid composition under temperatures normally encountered in marine environments warrants further study.

Time-of-flight mass spectrometry has the potential to replace gel electrophoresis for rapid and accurate analysis of DNA sequencing mixtures. However, impurities in the Sanger sequencing reaction solutions can complicate and degrade the mass spectrometric performance. Therefore, a fast purification procedure is necessary for mass spectrometric analysis. Two affinity methods were tested for the capture of the target fragments: a probe strand, complementary to the primer used to initiate synthesis of the Sanger fragments, is immobilized to a solid support either before or after hybridization so that the impurities are readily separated by filtering and washing the support material. The approaches were tested using mock sequencing mixtures assembled from synthetic DNA strands, to which representative impurities could be added. The results showed that the latter method has better overall yield. The recovered fragments were analyzed by matrix-assisted laser desorption/ionization.

The PsaA and PsaB polypeptides form the reaction center core heterodimer of photosystem I (PSI). Both PsaA and PsaB are predicted to have 11 hydrophobic domains, although it is unclear how both polypeptides fold within the thylakoid membrane. If all 11 hydrophobic regions form membrane-spanning domains, the N- and C-terminus must be located on opposite sides of the membrane. The C-terminus of PsaB is very conserved in a wide range of organisms and may be important for PSI assembly or function. Using chloroplast transformation in Chlamydomonas reinhardtii we have generated a series of C-terminal extension and deletion mutants of the PsaB polypeptide. Analysis of these mutants and spontaneous revertants indicates that the C-terminus may be extended by at least 14 amino acids without impairing PSI assembly. Deletion of amino acids 732-736 also has no impact on PSI, whereas deletion of amino acids 727-736 results in no accumulation of the complex. The site of truncation in the 727-736 deletion coincides with the end of the hydrophobic domain XI supporting a location of the C-terminus of PsaB on the lumenal side of PSI.

Photosystem I is a member of the iron-sulfur center or type I reaction centers. The primary electron donor in photosystem I is a chlorophyll a dimer termed P700. The biophysical properties of P700 are well understood, but the protein environment that gives it such unique properties is unknown. We have characterized site-directed mutants of the photosystem I reaction center protein PsaB and identified an amino acid, His-656, that interacts closely with one of the P700 chlorophylls. Mutation of His-656 to Asn or Ser increases the oxidation midpoint potential of P700/P700+. by 40 mV. The P700/P700+. optical difference spectra show the appearance of a new bleaching band at 667 nm. Electron nuclear double resonance spectroscopy indicates a significant increase in the hyperfine coupling corresponding to methyl protons at position 12 of the spin carrying chlorophyll a of P700+. The implication of these results to current structural models of the photosystem I reaction center is discussed.

The highly conserved amino acid sequence PCDGPGRGGTC in both photosystem I reaction center core proteins PsaA and PsaB has been predicted to contribute the four cysteine ligands for coordination of the 4Fe-4S iron-sulfur cluster FX, and we have proposed a working model for the binding of PsaC to this domain of the reaction center core heterodimer [Rodday et al. (1993) Photosynth. Res. 36, 1-9]. We have investigated structure-function relationships between this domain and the PsaC subunit by site-directed mutagenesis of the conserved prolines P560 and P564, and the charged residues D562 and R566 in the eucaryotic alga Chlamydomonas reinhardtii. The D562N and R566E mutants did not accumulate the PsaA and PsaB reaction center proteins, indicating that these residues are essential for the stable assembly of photosystem I. The P560A, P560L, and P564L mutants accumulated functional reaction centers but showed an impaired interaction between the reaction center core complex and the PsaC subunit. We observed that the reaction centers of the proline mutants dissociated more readily in urea, and reconstitution of the mutant core preparations using PsaC and Fe-S cluster insertion protocols in vitro were incomplete. We suggest that P560 and D562 contribute to the stability of the FX cluster, most likely by providing essential hydrogen bonding to the C561 ligand. The data obtained from the P564 and R566 replacements provide direct evidence that the intercysteinyl region in PsaB is a domain involved in the interaction between PsaC and the reaction center core.

Chloroplast transformation of Chlamydomonas reinhardtii has developed into a powerful tool for studying the structure, function and assembly of thylakoid protein complexes in a eukaryotic organism. In this article we review the progress that is being made in the development of procedures for efficient chloroplast transformation. This focuses on the development of selectable markers and the use of Chlamydomonas mutants, individually lacking thylakoid protein complexes, as recipients. Chloroplast transformation has now been used to engineer all four major thylakoid protein complexes, photosystem II, photosystem I, cytochrome b 6/f and ATP synthase. These results are discussed with an emphasis on new insights into assembly and function of these complexes in chloroplasts as compared with their prokaryotic counterparts.

The chloroplast genome ofChlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplastatpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). TheatpA-aadA cassette has been placed within the chloroplast DNAEcoRI restriction enzyme fragment 14, or within the chloroplastBamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DNA then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstrate that the chloroplastatpA promoter inatpA-aadA routinely recombines with its endogenous counterpart, resulting in heteroplasmic chloroplast DNA populations that may persist for many generations. The heterologous gene does not require a 3′ inverted repeat sequence for its expression. TheatpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady state level ofatpA-aadA mRNA. However, neither genomic position, gene copy number, or mRNA level have a significant effect on cellular resistance to spectinomycin, nor activity of theaadA gene productin vitro. These results suggest that, in the case ofaadA, the limiting step for expression of this gene is at the translational or post-translational level. TheatpA-aadA cassette should prove a useful model for future studies on the maintenance and expression of heterologous genes inC. reinhardtii chloroplasts.

Regulation of mRNA stability is an important control in the differential accumulation of chloroplast mRNAs that occurs in response to developmental and environmental signals. The mechanism by which differential mRNA accumulation is achieved is unknown. We have examined mRNA accumulation in a chloroplast mutant of Chlamydomonas reinhardtii previously shown to contain a single AT base-pair deletion in the psaB gene. In this mutant, steady-state levels of mRNA from psaB accumulate to a level more than twice that found in cells that have had the mutation repaired by chloroplast transformation. In vivo pulse labeling of RNA shows that increased mRNA accumulation is due to a more stable transcript. We show that inhibitors of chloroplast protein synthesis also increase mRNA accumulation from the psaB gene. The results are consistent with a link between polysome association, active synthesis and stability of psaB transcripts.

Site-directed mutagenesis has been used to introduce specific amino acid changes into the photosystem I reaction center in the green alga Chlamydomonas reinhardtii. Plasmids containing mutated copies of the chloroplast psaB gene, encoding a polypeptide of the photosystem I reaction center heterodimer, were introduced into the chloroplast genome by particle bombardment. Successful transformants were selected by two procedures. The first involved complementation of a nonphotosynthetic mutant of Chlamydomonas, CC-2341 (ac-u-g-2.3), which has a frameshift mutation in the psaB gene, and selection of photosynthetic transformants on minimal medium. The second procedure utilized a co-transformation procedure with a plasmid containing a rRNA gene that confers spectinomycin resistance. Homologous replacement of the psaB gene was confirmed by screening for a unique restriction enzyme site within the transforming psaB sequences. These procedures have been used to specifically mutate a highly conserved proline-cysteine motif suggested to be important in coordinating the [4Fe-4S] iron-sulfur center Fx. Our results show that the cysteine is essential for assembly of the photosystem I reaction center although the adjacent proline fulfills no identifiable function. The approach described in this paper will be of value to future studies of the structure, function, and assembly of photosystem I.

Photosystem I (PSI) is a multisubunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photosynthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of Photosystem I. C. reinhardtii is also a particularly favorable organism for chloroplast transformation because it contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adenyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This article describes the procedures for chloroplast transformation.

Photosystem I (PSI) is a multisubunit pigment-protein complex that uses light energy to transfer electrons from plastocyanin to ferredoxin. Application of genetic engineering to photosynthetic reaction center proteins has led to a significant advancement in our understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Chlamydomonas reinhardtii provides a system particularly amenable to analyze the structure-function relationship of Photosystem I. C. reinhardtii is also a particularly favorable organism for chloroplast transformation because it contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method. Exogenous cloned cpDNA can be introduced into the chloroplast by using this biolistic gene gun method. DNA-coated tungsten or gold particles are bombarded onto cells. Upon its entry into chloroplasts, the transforming DNA is released from the particles and integrated into the chloroplast genome through homologous recombination. The most versatile chloroplast selectable marker is aminoglycoside adenyl transferase (aadA), which can be expressed in the chloroplast to confer resistance to spectinomycin or streptomycin. This article describes the procedures for chloroplast transformation.

Background Although echocardiography is commonly used to evaluate cardiac function after MI, CMR may provide more accurate functional assessment but has not been adequately compared with echo. The primary study objective was to compare metrics of left ventricular volumes and global and regional function determined by cardiac magnetic resonance (CMR) and echocardiography (echo) in patients (pts) with recent myocardial infarction (MI). Methods To compare CMR with echo, 47 consecutive patients (pts 70% male; mean age = 66 ± 11 years) with MI >6 wks previously and scheduled for imaging evaluation were studied by both echo and CMR within 60 min of each other. Readers were blinded to pt information. Pearson's correlation coefficient, paired t-tests, and chi-square tests were used to compare CMR and echo measures. Further comparisons were made between pts and 30 normal controls for CMR and between pts and published normal ranges for echo. Results Measures of volume and function correlated moderately well between CMR and echo (r = 0.54 to 0.75, all p < 0.001), but large and systematic differences were noted in absolute measurements. Echo underestimated left ventricular (LV) volumes (by 69 ml for end-diastolic, 35 ml for end-systolic volume, both p < 0.001), stroke volume (by 34 ml, p < 0.001), and LV ejection fraction (LVEF) (by 4 percentage point, p = 0.02). CMR was much more sensitive to detection of segmental wall motion abnormalities (p < 0.001). CMR comparisons with normal controls confirmed an increase in LV volumes, a decrease in LVEF, and preservation of stroke volume after MI. Conclusion This intra subject comparison after MI found large, systematic differences between CMR and echo measures of volumes, LVEF, and wall motion abnormality despite moderate inter-modality correlations, with echo underestimating each metric. CMR also provided superior detection and quantification of segmental function after MI. Serial studies of LV function in individual patients should use the same modality.

Photosystem I, a membrane protein complex found in all oxygenic photosynthetic organisms, uses light energy to transfer electrons from plastocyanin to ferredoxin. Light energy captured by antenna chlorophylls is transferred rapidly to the primary electron donor, P700. The excited primary donor then initiates electron transfer through a series of secondary acceptors A0, A1, FX, FB and A three-dimensional structural model of the Photosystem I complex at 4 A resolution is used to describe the spatial organization of cofactors. The chemical identity of each of the electron transfer cofactors based on current spectroscopic information is discussed in relation to the available structural information. The use of specific mutagenesis to probe the structure and function of individual subunits and to investigate the interaction of cofactors with the protein environment is then described. The combined results from structural, spectroscopic and molecular analysis are generating a unified model of the structure, function and evolution of Photosystem I and related reaction centers.

Dinoflagellates are marine unicellular eukaryotes that exhibit unique features including a very low level of basic proteins bound to the chromatin and the complete absence of histones and nucleosomal structure. A cDNA encoding a protein with a strong homology to the TATA box-binding proteins (TBP) has been isolated from an expressed sequence tag library of the dinoflagellate Crypthecodinium cohnii. The typical TBP repeat signature and the amino acid motives involved in TFIIA and TFIIB interactions were conserved in this new TBP-like protein. However, the four phenylalanines known to interact with the TATA box were substituted with hydrophilic residues (His(77), Arg(94), Tyr(171), Thr(188)) as has been described for TBP-like factors (TLF)/TBP-related proteins (TRP). A phylogenetic analysis showed that cTBP is intermediate between TBP and TLF/TRP protein families, and the structural similarity of cTBP with TLF was confirmed by low affinity binding to a consensus' TATA box in an equivalent manner to that usually observed for TLFs. Six 5'-upstream gene regions of dinoflagellate genes have been analyzed and neither a TATA box nor a consensus-promoting element could be found within these different sequences. Our results showed that cTBP could bind stronger to a TTTT box sequence than to the canonical TATA box, especially at high salt concentration. Same binding results were obtained with a mutated cTBP (mcTBP), in which the four phenylalanines were restored. To our knowledge, this is the first description of a TBP-like protein in a unicellular organism, which also appears as the major form of TBP present in C. cohnii.

Alveolates are a diverse group of protists that includes three major lineages: ciliates, apicomplexa, and dinoflagellates. Among these three, it is thought that the apicomplexa and dinoflagellates are more closely related to one another than to ciliates. However, this conclusion is based almost entirely on results from ribosomal RNA phylogeny because very few morphological characters address this issue and scant molecular data are available from dinoflagellates. To better examine the relationships between the three major alveolate groups, we have sequenced six genes from the non-photosynthetic dinoflagellate, Crypthecodinium cohnii: actin, beta-tubulin, hsp70, BiP, hsp90, and mitochondrial hsp10. Beta-tubulin, hsp70, BiP, and hsp90 were found to be useful for intra-alveolate phylogeny, and trees were inferred from these genes individually and in combination. Trees inferred from individual genes generally supported the apicomplexa-dinoflagellate grouping, as did a combined analysis of all four genes. However, it was also found that the outgroup had a significant effect on the topology within alveolates when using certain methods of phylogenetic reconstruction, and an alternative topology clustering dinoflagellates and ciliates could not be rejected by the combined data. Altogether, these results support the sisterhood of apicomplexa and dinoflagellates, but point out that the relationship is not as strong as is often assumed.

Publisher Summary Application of genetic engineering to reaction center proteins has led to a significant advancement in understanding of primary electron transfer events and the role of the protein environment in modulating these processes. Despite the presence of an active homologous recombination system, transformation of the chloroplast genome has several inherent complications. First, plants and many green algae are unable to grow in the absence of photosynthesis. Second, most eukaryotes contain several to hundreds of chloroplasts with each organelle containing 80–100 copies of chloroplast (cp) DNA. Third, three cell membranes and a cell wall act as a barrier to exogenously added DNA. Several of these limitations are overcome by using the green alga Chlamydomonas reinhardtii , which contains only a single chloroplast and grows heterotrophically when supplemented with acetate. Chlamydomonas has, therefore, served as a model organism for the development of chloroplast transformation procedures and the study of photosynthetic mutants generated using this method.

Time-resolved absorption and fluorescence spectroscopy were used to investigate the energy and electron transfer processes in the detergent-isolated photosystem I core particles from the site-directed mutant of Chlamydomonas reinhardtii with the histidine-656 of PsaB replaced by asparagine [HN(B656) mutation]. The specific mutation near the primary donor molecule results in a 40 mV increase in the P700/P700+ midpoint potential [Webber, A. N., Su Hui, Bingham, S. E., Kass, H., Krabben, L., Kuhn, M., Jordan, R., Schlodder, E.,Lubitz, W. (1996) Biochemistry 35, 12857-12863]. There is no indication that the HN(B656) mutation affects the spectral distribution of the antenna pigments. However, the lifetime of the trapping process measured independently by transient absorption and fluorescence spectroscopy in the mutant PSI core antenna is increased by a factor of approximately 2 (approximately 65 ps compared to approximately 30 ps in the wild-type PSI). This implies that the trapping process in the PSI antenna is limited by the process where the primary donor molecule directly participates. The HN(B656) mutation results in the appearance of a new bleaching band at 670 nm in the spectrum which is due to formation of P700+ upon photooxidation. The difference spectrum of the photoreduction of the possible primary acceptor, A0 in the mutant PSI is very similar to wild type, indicating that it is unaffected by the HN(B656) mutation. Possible mechanisms for slowing of the trapping process and the appearance of a new band in the P700 - P700+ difference spectrum of the HN(B656) PSI are discussed.

The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles. Factors that determine the rate of mRNA decay in chloroplasts are not well understood. Chloroplast mRNAs typically contain an inverted repeat sequence within the 3' untranslated region that can potentially fold into a stem-loop structure. These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established. Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA. To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3' flanking sequences of psaB or extended the open reading frame into the 3' inverted repeat. The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii. Mutants lacking the 3' stem-loop exhibited a 75% reduction in the level of psaB mRNA. The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis. Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability. When the translation reading frame was extended into the 3' inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs. The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo. Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.

The chloroplast psaB gene encodes one of the polypeptides of the photosystem I reaction center heterodimer that coordinates the electron transfer components P700, A0, and A1. Histidine residues in the most highly conserved region of the PsaB protein are predicted to coordinate the P700 reaction center chlorophyll(s) and the initial electron acceptor, A0. Oligonucleotide-mediated site-directed mutagenesis and chloroplast transformation of Chlamydomonas reinhardtii have been used to determine the importance of these conserved histidines in photosystem I reaction center biogenesis and function. It is demonstrated that these histidine residues are essential for stable accumulation of the photosystem I reaction center. Protein pulse-labeling shows that changing the histidine residues impairs a post-translational step in reaction center assembly. Photosystem I complexes from the mutants have been characterized by Electron Nuclear Double Resonance and Electron Spin Echo Envelope Modulation spectroscopy to determine the impact of any mutations on P700+. In all cases we determine that spectroscopic characteristics of P700+ remain unchanged. The implications of these results to current models of the photosystem I reaction center and related bacterial reaction centers are discussed.

In order to deregulate arginine biosynthesis in Synechococcus sp. PCC7942, d-arginine-resistant cell lines were selected following ethyl methanesulfonate mutagenesis of wild-type (WT) cells. Three of these arginine-producing mutant (APM) cell lines, APM1, APM31 and APM40, were putative regulatory mutants based upon secretion of l-arginine into their growth medium. HPLC of lyophilized post-harvest supernatants of APM 31 and 40 resolved two predominant amino acids, arginine and citrulline. In-vitro activity of N-acetylglutamate kinase (NAGK), the proposed regulatory enzyme of the arginine pathway, was about 100-fold less sensitive to l-arginine inhibition in extracts from APM 31 and 40 than the enzyme in WT extracts. The enzyme from APM 1 was 20-fold less sensitive to l-arginine inhibition than WT. The most likely site of mutation in each of the APM cell lines is in the gene for NAGK, rendering the enzymes insensitive to l-arginine feedback control. These strains can be utilized for the phototrophic production of arginine.

To better understand how diatoms are capable of responding to environmental stress, protein expression during heat treatment of a thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) diatom (Chrysophyta) was investigated. The stress response is a universal and conserved mechanism of cell survival to unfavorable conditions. Typically, a 10 to 15° C temperature elevation above cell growth optimal causes constitutively expressed proteins to decrease and heat shock proteins (HSPs) to increase. HSPs are categorized by molecular weight among five classes with each apparently specialized for a particular function that enhances cell survival. One-dimensional SDS-PAGE of diatoms subjected to heat treatment revealed that P. tricornutum exhibited a typical stress response, but C. muelleri did not exhibit a characteristic response even at a greatly elevated temperature (50° C). This result was confirmed by total soluble protein assays. Chaetoceros muelleri may contain higher basal levels of HSPs than P. tricornutum allowing C. muelleri to better tolerate elevated temperatures. Western blot analysis using pea HSP70 (70 kDa) antisera of heat-treated P. tricornutum and C. muelleri validated the hypothesis that thermo-tolerant cells contain higher levels of constitutively expressed HSPs. Two-dimensional gel electrophoresis of heat-treated cells indicate that the small HSPs (17–30 kDa) played a role in the stress response similar to that found in vascular plants. Ongoing work is focused on the manipulation of the stress response through over-expression of key hsp genes.

A chimeric octopine synthase-neomycin phosphotransferase (ocs-nptII) gene was used to transform Chlamydomonas reinhardtii to kanamycin resistance. Southern hybridization using DNA isolated from one transformant, T6. 1, indicated that the entire ocs-nptII gene and at least part of the plasmid were integrated into nuclear DNA. Neomycin phosphotransferase II activity has been detected in T6.1 cell extracts. Northern hybridizations, employing a radiolabeled ocs-nptII sequence, revealed a T6.1 transcript approximately the same size as a homologous transcript isolated from E. coli carrying the nptII gene. Although T6.1 is an extremely rare examiple of a stable C. reinhardtii transformant, its occurence nevertheless indicates that bacterial genes can be expressed in the nucleus of the alga.

Chlorella vulgaris was grown photosynthetically in batch culture under nitrogen sufficiency or nitrogen limitation. The starch content of the cells was measured as the amount of glucose released by enzymic hydrolysis of partially purified starch. Nitrogen sufficient algae contained approximately 20% of their dry weight as starch, whereas in nitrogen limited cells starch comprised up to 55% of the cellular dry weight. Starch production was pH dependent; optimal production of starch was achieved between pH 7.5 and 8.0. Optimal growth of C. vulgaris occurred at pH 7.0. Carbon yield experiments showed that for every gram of carbon consumed 0.5 g of starch (glucose) could be recovered.

The gene for the large subunit (LS) of ribulose-1,5,-bisphosphate carboxylase of Euglena gracilis Z chloroplast DNA has been mapped by heterologous hybridization with DNA restriction fragments containing internal sequences from the Zea mays and Chlamydomonas reinhardii LS genes. The Euglena LS gene which has the same polarity as the Euglena rRNA genes has been located with respect to Pst I, Pvu I, and HindIII sites within the Eco RI fragment Eco A. The region of Euglena chloroplast DNA complementary to an 887 bp internal fragment from the Chlamydomonas chloroplast LS gene is interrupted by a 0.5-1.1 kbp non-complementary sequence. This is the first chloroplast protein gene located on the Euglena genome, and the first evidence for an intervening sequence within any chloroplast protein gene.

A chloroplast photosystem I reaction center mutation.ac-u-g-2.3. ofChlamydomonas reinhardtii has been complemented with a wild typepsaB gene to restore photosynthetic competence. The mutation was mapped in thepsaB coding sequence by chloroplast transformation using subcloned restriction fragments ofpsaB. The mutation was found to be a single base pair deletion resulting in a reading frame shift and premature termination of the polypeptide. Transformants were verified by insertion of a site-directed mutation which created a new restriction enzyme site. These transformations demontrate the feasibility of insertion of site-directed mutations into thepsaB gene in order to elucidate amino acid residues involved in photosystem I assembly and function.

A new route leading to a simple and rapid procedure employing Methyl Phosphoroditetrazolide (MPDT) as condensing agent for polynucleotide synthesis is described.