Form I Rubiscos from non-green algae are expressed abundantly but not assembled in tobacco chloroplasts.

Non-green algae have Rubiscos that are phylogenetically distinct from their counterparts in green algae and higher plants. Some non-green-algal Rubiscos are more specific for CO2, relative to O2, than higher-plant Rubiscos, sometimes coupled with lower Michaelis constants for CO2. If these Rubiscos could be substituted for the higher-plant enzyme, and if they functioned successfully in the higher-plant chloroplast and were regulated appropriately, they would improve the CO2 use and quantum efficiency of higher-plant photosynthesis. To assess the feasibility of expressing non-green algal Rubiscos in higher-plant chloroplasts, we inserted the rbcLS operons from the rhodophyte Galdieria sulphuraria and the diatom Phaeodactylum tricornutum into the inverted repeats of the plastid genome of tobacco, leaving the tobacco rbcL gene unaltered. Homoplasmic transformants were selected. The transgenes directed the synthesis of abundant amounts of transcripts and both subunits of the foreign Rubiscos. In some circumstances, leaves of the transformants with the P. tricornutum Rubisco contained as much foreign Rubisco protein as endogenous tobacco Rubisco (>30% of the soluble leaf protein). However, the subunits of the foreign Rubiscos were not properly folded and/or assembled. All the foreign large subunits and most of the foreign small subunits were recovered in the insoluble fractions of leaf extracts. Edman sequencing yielded the expected N-terminal sequences for the foreign small subunits but the N-termini of the foreign large subunits were blocked. Accumulation of large amounts of denatured foreign Rubisco in the leaves, particularly of the P. tricornutum transformants, caused a reduction in the amount of tobacco Rubisco present, with concomitant reductions in leaf CO2 assimilation and plant growth.

Directed mutation of the Rubisco large subunit of tobacco influences photorespiration and growth.

The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment. In 0.3% (v/v) CO2, the mutant and wild-type plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO2-fixing rate and its ability to distinguish between CO2 and O2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O2 concentrations. Consistent with the reduced CO2 fixation rate at saturating CO2, the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.

Ribulose-1,5-bisphosphate carboxylase/oxygenase activase deficiency delays senescence of ribulose-1,5-bisphosphate carboxylase/oxygenase but progressively impairs its catalysis during tobacco leaf development.

Transgenic tobacco (Nicotiana tabacum L. cv W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) activase grew more slowly than wild-type plants in a CO2-enriched atmosphere, but eventually attained the same height and number of leaves. Compared with the wild type, the anti-activase plants had reduced CO2 assimilation rates, normal contents of chlorophyll and soluble leaf protein, and much higher Rubisco contents, particularly in older leaves. Activase deficiency greatly delayed the usual developmental decline in Rubisco content seen in wild-type leaves. This effect was much less obvious in another transgenic tobacco with an antisense gene directed against chloroplast-located glyceraldehyde-3-phosphate dehydrogenase, which also had reduced photosynthetic rates and delayed development. Although Rubisco carbamylation was reduced in the anti-activase plants, the reduction was not sufficient to explain the reduced photosynthetic rate of older anti-activase leaves. Instead, up to a 10-fold reduction in the catalytic turnover rate of carbamylated Rubisco in vivo appeared to be the main cause. Slower catalytic turnover by carbamylated Rubisco was particularly obvious in high-CO2-grown leaves but was also detectable in air-grown leaves. Rubisco activity measured immediately after rapid extraction of anti-activase leaves was not much less than that predicted from its degree of carbamylation, ruling out slow release of an inhibitor from carbamylated sites as a major cause of the phenomenon. Nor could substrate scarcity or product inhibition account for the impairment. We conclude that activase must have a role in vivo, direct or indirect, in promoting the activity of carbamylated Rubisco in addition to its role in promoting carbamylation.

Effects of Ambient CO2 Concentration on Growth and Nitrogen Use in Tobacco (Nicotiana tabacum) Plants Transformed with an Antisense Gene to the Small Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase.

Growth of the R1 progeny of a tobacco plant (Nicotiana tabacum) transformed with an antisense gene to the small subunit of ribulose-1,5-carboxylase/oxygenase (Rubisco) was analyzed under 330 and 930 [mu]bar of CO2, at an irradiance of 1000 [mu]mol quanta m-2 s-1. Rubisco activity was reduced to 30 to 50% and 13 to 18% of that in the wild type when one and two copies of the antisense gene, respectively, were present in the genome, whereas null plants and wild-type plants had similar phenotypes. At 330 [mu]bar of CO2 all antisense plants were smaller than the wild type. There was no indication that Rubisco is present in excess in the wild type with respect to growth under high light. Raising ambient CO2 pressure to 930 [mu]bar caused plants with one copy of the DNA transferred from plasmid to plant genome to achieve the same size as the wild type at 330 [mu]bar, but plants with two copies remained smaller. Differences in final size were due mostly to early differences in relative rate of leaf area expansion (m2 m-2 d-1) or of biomass accumulation (g g-1 d-1): within less than 2 weeks after germination relative growth rates reached a steady-state value similar for all plants. Plants with greater carboxylation rates were characterized by a higher ratio of leaf carbon to leaf area, and at later stages, they were characterized also by a relatively greater allocation of structural and nonstructural carbon to roots versus leaves. However, these changes per se did not appear to be causing the long-term insensitivity of relative growth rates to variations in carboxylation rate. Nor was this insensitivity due to feedback inhibition of photosynthesis in leaves grown at high partial pressure of CO2 in the air (pa) or with high Rubisco activity, even when the amount of starch approached 40% of leaf dry weight. We propose that other intrinsic rate-limiting processes that are independent of carbohydrate supply were involved. Under plentiful nitrogen supply, reduction in the amount of nitrogen invested in Rubisco was more than compensated for by an increase in leaf nitrate. Nitrogen content of organic matter, excluding Rubisco, was unaffected by the antisense gene. In contrast, it was systematically lower at elevated pa than at normal pa. Combined with the positive effects of pa on growth, this resulted in the single-dose antisense plants growing as fast at 930 [mu]bar of CO2 as the wild-type plants at 330 [mu]bar of CO2 but at a lower organic nitrogen cost.

Reduction of ribulose biphosphate carboxylase activase levels in tobacco (Nicotiana tabacum) by antisense RNA reduces ribulose biphosphate carboxylase carbamylation and impairs photosynthesis.

The in vivo activity of ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco) is modulated in response to light intensity by carbamylation of the active site and by the binding of sugar phosphate inhibitors such as 2'-carboxyarabinitol-1-phosphate (CA 1P). These changes are influenced by the regulatory protein Rubisco activase, which facilitates the release of sugar phosphates from Rubisco's catalytic site. Activase levels in Nicotiana tabacum were reduced by transformation with an antisense gene directed against the mRNA for Rubisco activase. Activase-deficient plants were photosynthetically impaired, and their Rubisco carbamylation levels declined upon illumination. Such plants needed high CO2 concentrations to sustain reasonable growth rates, but the level of carbamylation was not increased by high CO2. The antisense plants had, on average, approximately twice as much Rubisco as the control plants. The maximum catalytic turnover rate (k cat) of Rubisco decreases in darkened tobacco leaves because of the binding of CA 1P. The dark-to-light increase in k cat that accompanies CA 1P release occurred to similar extents in antisense and control plants, indicating that normal levels of activase were not essential for CA 1P release from Rubisco in the antisense plants. However, CA 1P was released in the antisense plants at less than one-quarter of the rate that it was released in the control plants, indicating a role for activase in accelerating the release of CA 1P.

Effects of mutations at residue 309 of the large subunit of ribulosebisphosphate carboxylase from Synechococcus PCC 6301.

Previous studies [G. S. Hudson et al. (1989) J. Biol. Chem. 265, 808-814] showed that the faster turnover rates and lower affinities for CO2 of ribulosebisphosphate carboxylase/oxygenases from C4 plants, compared to C3 and C3/C4 plants, were specified by the chloroplast-encoded large subunits. In pairs of closely related C3 and C4 species from three genera, these kinetic changes were accompanied by only three to six amino acid residue substitutions, depending on the genus. None of these substitutions occurred near the active site and only one, 309Met (C3) to Ile (C4), was common to all three genera. Unlike the plant carboxylases, the highly homologous enzyme from the cyanobacterium Synechococcus PCC 6301 folds and assembles properly when its rbcL and rbcS genes are coexpressed in Escherichia coli. Furthermore, the cyanobacterial enzyme has Ile at position 309 of the large subunit, a high turnover number, and a poor affinity for CO2. 309Ile was replaced with Met and several other residues by site-directed mutagenesis of the cyanobacterial rbcL. Met and Leu were tolerated at this position with no alteration in the kinetic or structural properties of the assembled holoenzyme. However, substitution with Val, Gly, Trp, or Arg prevented the assembly of the subunits. The indifference to Met or Ile at this position, as well as the tolerance for Leu which is not observed with any natural ribulosebisphosphate carboxylase, leads to the conclusion that either the 309Met/Ile substitution has no effect on the kinetic properties of the plant enzyme, despite the correlation apparent in previous studies, or the cyanobacterial enzyme is sufficiently different from the plant enzyme in other respects that the influence of residue 309 is masked.

Reduction of ribulose-1,5-bisphosphate carboxylase/oxygenase content by antisense RNA reduces photosynthesis in transgenic tobacco plants.

A complementary DNA for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was cloned from tobacco (Nicotiana tabacum) and fused in the antisense orientation to the cauliflower mosaic virus 35S promoter. This antisense gene was introduced into the tobacco genome, and the resulting transgenic plants were analyzed to assess the effect of the antisense RNA on Rubisco activity and photosynthesis. The mean content of extractable Rubisco activity from the leaves of 10 antisense plants was 18% of the mean level of activity of control plants. The soluble protein content of the leaves of anti-small subunit plants was reduced by the amount equivalent to the reduction in Rubisco. There was little change in phosphoribulokinase activity, electron transport, and chlorophyll content, indicating that the loss of Rubisco did not affect these other components of photosynthesis. However, there was a significant reduction in carbonic anhydrase activity. The rate of CO(2) assimilation measured at 1000 micromoles quanta per square meter per second, 350 microbars CO(2), and 25 degrees C was reduced by 63% (mean value) in the antisense plants and was limited by Rubisco activity over a wide range of intercellular CO(2) partial pressures (p(i)). In control leaves, Rubisco activity only limited the rate of CO(2) assimilation below a p(i) of 400 microbars. Despite the decrease in photosynthesis, there was no reduction in stomatal conductance in the antisense plants, and the stomata still responded to changes in p(i). The unchanged conductance and lower CO(2) assimilation resulted in a higher p(i), which was reflected in greater carbon isotope discrimination in the leaves of the antisense plants. These results suggest that stomatal function is independent of total leaf Rubisco activity.

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]

Two promoters control the aroH gene of Escherichia coli.

The aroH gene from Escherichia coli encodes 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (Trp), one of three isoenzymes which catalyse the first committed step in the biosynthesis of aromatic amino acids and vitamins. S1 mapping and primer extension analysis of in vivo transcripts revealed the presence of two nonoverlapping promoters for aroH. The more distal of these has been described previously and is negatively regulated by the trp repressor. The second promoter is active under conditions of growth in rich medium, and may be involved in ensuring sufficient levels of precursors for the biosynthesis of aromatic vitamins under these growth conditions.

The chloroplast CF0I subunit can replace the b-subunit of the F0F1-ATPase in a mutant strain of Escherichia coli K12.

The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E. coli F0F1-ATPase. However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function. This proposition was investigated directly. A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein. An E. coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene. The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control. Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E. coli b-subunit in the E. coli F0F1-ATPase.

Comparisons of rbcL genes for the large subunit of ribulose-bisphosphate carboxylase from closely related C3 and C4 plant species.

Ribulosebisphosphate carboxylase/oxygenase from C4 plants exhibits higher turnover rates and lower affinities for CO2 than the enzyme from C3 plants or C3-C4 intermediate species. This property is shown to be inherited maternally in reciprocal interspecific crosses between two Flaveria species, and thus must be specified by the chloroplast-encoded large subunits. To investigate the amino acid changes responsible, the chloroplast rbcL genes from three pairs of C3 and C4 species from three genera (Flaveria, Atriplex, and Neurachne) were cloned and sequenced. Comparisons of the predicted amino acid sequences from species of the same genus revealed a limited number of changes within each pair, ranging from three to six, of which only one (309Met (C3) to Ile (C4] was consistently observed. This residue occurs in the loop connecting the carboxyl end of beta strand 5 with the amino end of alpha helix 5 in the alpha/beta barrel of the large subunit, and is close to the active site in a region which makes interdomain and intersubunit contacts. However, it is unlikely that a change of this residue alone is responsible for the alteration of kinetic properties. Nucleotide sequence comparisons of the rbcL genes showed no significant or consistent changes in the promoter and transcribed but nontranslated regions to suggest why rbcL is not expressed in C4 leaf mesophyll cells. It is concluded that mutations in rbcL have led to an alteration of the kinetics but not the expression of ribulose-bisphosphate carboxylase.

Red cell antibody identification by solid phase red cell adherence utilizing dried RBC monolayers.

Recent technological advances in the immobilization and drying of red cell monolayers for use in solid phase red cell adherence (SPRCA) assays have resulted in the development of reagent red cells for antibody screening and identification that are stable at mom temperature. Panels consisting of twelve different RBC samples dried onto individual microplate wells were evaluated with 176 samples whose antibody specificities had previously been determined by conventional hemagglutination techniques. Identification tests performed with dried SPRCA panels proved to be more sensitive and less time consuming than hemagglutination tests. The red cell antigens of dried membranes were shown to be stable and reactive following storage for 120 days at mom temperature.

Spinach chloroplast rpoBC genes encode three subunits of the chloroplast RNA polymerase.

Sequence analysis of a 12,400 base-pair region of the spinach chloroplast genome indicates the presence of three genes encoding subunits of the chloroplast RNA polymerase. These genes are analogous to the rpoBC operon of Escherichia coli, with some significant differences. The first gene, termed rpoB, encodes a 121,000 Mr homologue of the bacterial beta subunit. The second and third genes, termed rpoC1 and rpoC2, encode 78,000 and 154,000 Mr proteins homologous to the N and C-terminal portions, respectively, of the bacterial beta' subunit. RNA mapping analysis indicates that the three genes are cotranscribed, and that a single intron occurs in the rpoC1 gene. No splicing occurs within the rpoC2 gene or between rpoC1 and rpoC2. Furthermore, the data indicate the possibility of an alternative splice acceptor site for the rpoC1 intron that would give rise to a 71,000 Mr gene product. Thus, with the inclusion of the alpha subunit encoded by rpoA at a separate locus, the chloroplast genome is predicted to encode four subunits (respectively called alpha, beta, beta', beta") equivalent to the three subunits of the core enzyme of the E. coli RNA polymerase.

The chloroplast genes encoding subunits of the H(+)-ATP synthase.

Three CF1 and three CF0 subunits of the chloroplast H(+)-ATP synthase are encoded on the chloroplast genome. The chloroplast atp genes are organized as two operons in plants but not in the green alga, Chlamydomonas reinhardtii. The atpBE or ß operon shows a relatively simple organisation and transcription pattern, while the atpIHFA or α operon is transcribed into a large variety of mRNAs. The atp genes are related to those of cyanobacteria and, more distantly, to those of non-photosynthetic bacteria such as E. coli, suggesting a common origin of most F1F0 ATP synthase subunits. Both the chloroplast and cyanobacterial ATP synthases have four F0 subunits, not three as in the E. coli complex. The proton pore of the CF0 is proposed to be formed by the interaction of subunits III and IV.

A gene cluster in the spinach and pea chloroplast genomes encoding one CF1 and three CF0 subunits of the H+-ATP synthase complex and the ribosomal protein S2.

The regions of the spinach and pea chloroplast genomes containing the ATP synthase genes atpA, atpF and atpH have been sequenced. The encoded proteins, CF1 alpha, CF0I and CF0III, are well conserved between spinach and pea, and analogous to the alpha, b and c subunits of the Escherichia coli ATP synthase complex. The atpF gene is split by a single intron, and the exon/intron boundaries have been defined by isolating and sequencing a partial cDNA clone. Two other genes, designated atpI and rps2, located upstream from atpH, have also been sequenced. They encode a 27,000 Mr hydrophobic protein analogous to the F0a subunit of E. coli ATP synthase and a basic protein analogous to the S2 protein of the E. coli 30 S ribosomal subunit. Transcriptional analysis by electron microscopy of RNA-DNA hybrids, Northern blotting and primer extension experiments shows that these genes are transcribed and processed into a complex set of transcripts, with 5' ends mapping upstream from the rps2, atpI and atpH genes.

The short unique region of the B95-8 Epstein-Barr virus genome.

The 12-kbp short unique region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for latent and lytic cycle transcripts. Two latent and three late mRNAs have been detected, the largest of the late transcripts potentially encoding a 143-kDa protein. The region containing oriP, the putative origin of replication of the genome as a plasmid in latently infected B lymphocytes, is shown to contain 21 direct repeats of a 30-bp A+T-rich sequence and a related large inverted repeat.

The BamHI F region of the B95-8 Epstein-Barr virus genome.

The BamHI F region of the B95-8 Epstein-Barr virus (EBV) genome has been sequenced and analysed for transcription signals and open reading frames. S1 mapping and northern blotting with probes from M13 recombinants have been used to search for mRNAs. Four rightward-reading frames encoding basic proteins appear to be expressed by 3'-coterminal early mRNAs. Two leftward-reading frames appear to be expressed by 3'-coterminal early mRNAs.

Two related but differentially expressed potential membrane proteins encoded by the EcoRI Dhet region of Epstein-Barr virus B95-8.

Three mRNAs in the EcoRI Dhet region of Epstein-Barr virus B95-8 were mapped. Their 3' ends are coterminal. A latent gene containing three exons is transcribed from the ED-L1 promoter and was predicted to lead to expression of a 42-kilodalton protein. An unspliced late mRNA is produced by transcription from the ED-L1A promoter within the first intron of the above gene and was predicted to lead to expression of a 28-kilodalton protein corresponding to the C-terminal two-thirds of the 42-kilodalton protein. Both proteins would have hydrophobic N-terminal domains and highly acidic C-terminal domains and were predicted to span the cell membrane. An early promoter (ED-L2) is located in the 3' untranslated region of the above genes and was predicted to lead to expression of a 6.5-kilodalton polypeptide containing a C-terminal hydrophobic region.