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We present a workflow for the design and production of biological networks from high-level program specifications. The workflow is based on a sequence of intermediate models that incrementally translate high-level specifications into DNA samples that implement them. We identify algorithms for translating between adjacent models and implement them as a set of software tools, organized into a four-stage toolchain: Specification, Compilation, Part Assignment, and Assembly. The specification stage begins with a Boolean logic computation specified in the Proto programming language. The compilation stage uses a library of network motifs and cellular platforms, also specified in Proto, to transform the program into an optimized Abstract Genetic Regulatory Network (AGRN) that implements the programmed behavior. The part assignment stage assigns DNA parts to the AGRN, drawing the parts from a database for the target cellular platform, to create a DNA sequence implementing the AGRN. Finally, the assembly stage computes an optimized assembly plan to create the DNA sequence from available part samples, yielding a protocol for producing a sample of engineered plasmids with robotics assistance. Our workflow is the first to automate the production of biological networks from a high-level program specification. Furthermore, the workflow's modular design allows the same program to be realized on different cellular platforms simply by swapping workflow configurations. We validated our workflow by specifying a small-molecule sensor-reporter program and verifying the resulting plasmids in both HEK 293 mammalian cells and in E. coli bacterial cells.

The oligomeric state in solution of four plant annexins, namely Anx23(Ca38), Anx24(Ca32), Anx(Gh1), and Anx(Gh2), was characterized by sedimentation equilibrium analysis and gel filtration. All proteins were expressed and purified as amino-terminal His(n) fusions. Sequencing of the Anx(Gh1) construct revealed distinct differences with the published sequence. Sedimentation equilibrium analysis of Anx23(Ca38), Anx24(Ca32), and Anx(Gh1) suggests monomer-trimer equilibria for each protein with association constants in the range of 0.9 x 10(10)-1.7 x 10(11) M(-2). All four proteins were subjected to analytical gel filtration under different buffer conditions. Observations from this experiment series agree quantitatively with the ultracentrifugation results, and strongly suggest calcium independence of the annexin oligomerization behavior; moreover, binding of calcium ions to the proteins seems to require disassembly of the oligomers. Anx(Gh2) showed a different elution profile than the other plant annexins; while having only a very small trimer content, this annexin seems to exist in a monomer-dimer equilibrium in solution.

A cDNA, cakn, orthologous to the Arabidopsis KN gene, which is involved in vesicle fusion during cell plate formation, was isolated from bell pepper fruit. cakn seems to be monogenic and its expression mainly transcriptionally regulated. During fruit development, transcript and protein levels increase significantly in the early stages in which numerous cell divisions occur, but in the stages corresponding to fruit growth by cell enlargement, whereas the messengers are undetectable, proteins are still faintly present, suggesting a different stability rate for the two types of macromolecules.

This work provides the first three-dimensional structure of a member of the plant annexin family and correlates these findings with biochemical properties of this protein. Annexin 24(Ca32) from Capsicum annuum was purified as a native protein from bell pepper and was also prepared by recombinant techniques. To overcome the problem of precipitation of the recombinant wild-type protein in crystallization trials, two mutants were designed. Whereas an N-terminal truncation mutant turned out to be an unstable protein, the N-terminal His-tagged annexin 24(Ca32) was crystallized, and the three-dimensional structure was determined by x-ray diffraction at 2. 8 A resolution. The structure refined to an R-factor of 0.216 adopts the typical annexin fold; the detailed structure, however, is different from non-plant annexins, especially in domains I and III and in the membrane binding loops on the convex side. Within the unit cell there are two molecules per asymmetric unit, which differ in conformation of the IAB-loop. Both conformers show Trp-35 on the surface. The loop-out conformation is stabilized by tight interactions of this tryptophan with residue side chains of a symmetry-related molecule and enforced by a bound sulfate. Characterization of this plant annexin using biophysical methods revealed calcium-dependent binding to phospholipid vesicles with preference for phosphatidylcholine over phosphatidylserine and magnesium-dependent phosphodiesterase activity in vitro as shown with adenosine triphosphate as the substrate. A comparative unfolding study of recombinant annexin 24(Ca32) wild type and of the His-tag fusion protein indicates higher stability of the latter. The effect of this N-terminal modification is also visible from CD spectra. Both proteins were subjected to a FURA-2-based calcium influx assay, which gave high influx rates for the wild-type but greatly reduced influx rates for the fusion protein. We therefore conclude that the N-terminal domain is indeed a major regulatory element modulating different annexin properties by allosteric mechanisms.

Annexins interact in a calcium-dependent manner with membrane phospholipids. Although their exact function is not known, annexins have been proposed to be involved in a variety of cellular processes. To determine whether plant annexins are implicated in cell division, we have isolated cDNAs encoding annexin from TBY2 cells. Based on sequence analysis, these cDNAs fall into two families, differing mainly by deletions or insertions in their 5'- and 3'-untranslated regions. The two annexins Ntp32.1 and Ntp32.2 encoded by these cDNAs are homologous to p32 from bell pepper (Cap32.1): we propose that these Solanaceae annexins constitute a distinct type which we call Sp32 annexins. There are two genes (Ntan.1 and Ntan.2) derived from the separate progenitor species of Nicotiana tabacum and analysis of Southern blots is consistent with the presence of these two genes. We show that Sp32 transcript amounts are developmentally modulated in tobacco plants: RNA levels are highest in growing and dividing tissues. Sp32 annexin gene expression is also regulated in TBY2 cultured cells: transcripts and proteins are detected only in exponentially growing cells. In synchronized TBY2 cells, Sp32 annexin transcripts are expressed at the G2/M transition, in the M phase and at the G1/S transition. These results are the first evidence that the expression of plant annexins is modulated during the cell cycle. The Sp32 annexin proteins accumulate during the cell cycle and peak at the end of mitosis. Immunolocalization shows that the majority of Sp32 annexins is present in intercellular junctions, forming a ring structure under the plasma membrane. Since annexins are known to bind secretory vesicles during exocytosis, their localization at cell junctions suggests that annexins could be involved in cell wall maturation.

In bell pepper, a gene encoding a major plastid-lipid associated protein is expressed as both partially and totally spliced transcripts (respectively PAP2 and PAP1). Although PAP is present as a single-copy gene in the bell pepper genome, Southern blots using PAP2 as a probe revealed multiple homologous copies. Analyses of the intronic sequence of PAP2 showed the existence of a 206bp short interspersed repetitive element (SINE) belonging to the Ts family of retrotransposons (Yoshioka et al., 1993). Comparison with PAP sequences in other Solanaceae species suggested that the structure of the gene is highly conserved: the two introns are inserted at the same position. However, the Ts insertion found in bell pepper is absent in tobacco and tomato. Studies using RT-PCR showed that in these latter species only totally spliced transcripts of PAP are present. On the other hand, RNA analyses of tobacco plants transformed with the bell pepper PAP revealed the presence of both totally and incompletely spliced transcripts. Altogether our results support the hypothesis that the Ts insertion into the first intron of PAP results in a splicing defect of the corresponding pre-mRNA. Based on the presence of peculiar, previously unidentified Ts elements, a possible horizontal transmission of Ts elements from animals to plants is discussed.

We have isolated a cDNA (PAP) corresponding to a single nuclear gene that encodes an approximately 30-kD major protein of bell pepper (Capsicum annuum L.) fruit chromoplasts. RNA and protein analyses revealed that, although at a low level, this gene is also expressed in every organ of the plant, the amount of the corresponding transcript and protein dramatically increasing in the latter stages of fruit development. Western-blot and immunocytochemical analyses of purified chloroplasts from leaves and fruits and of chromoplasts from red fruits showed that the encoded protein is the major component of plastoglobules and fibrils and is localized on the outer surface of these lipid structures. Analyses of PAP in plants belonging to different taxa revealed that it is expressed and highly conserved in both monocotyledonous and dicotyledonous plants. The presence of the protein in plastids not differentiating into chromoplasts indicates that PAP is expressed irrespective of the ontogeny of various plastid lines. In light of our results and since the encoded protein, identical to that previously named ChrB or fibrillin, is present in plastoglobules from several species and accumulates in the fibrils of bell pepper chromoplast, we propose to designate it as a plastid-lipid-associated protein.

We have isolated a 454-bp cDNA that encodes a novel fruit specific defensin from bell pepper (Capsicum annuum). The encoded 75-amino-acid polypeptide contains an N-terminal domain characteristic of a signal peptide and a 48-amino-acid mature domain named J1. The mature protein, from which the N-terminal amino acid sequence was determined, contains eight cysteines that from four intramolecular disulfide bridges, suggesting a monomeric form for J1. In healthy fruits J1 is undetectable at the green stage but high levels accumulate during ripening. In wound areas of the green fruit the accumulation of J1 dramatically increased, suggesting a role for J1 in the plant's defense response. Moreover, we have demonstrated that J1 possesses an antifungal activity. We have isolated and characterized the corresponding two homologous genes (j1-1 and j1-2) that exist in the bell pepper genome. Both genes are interrupted by the insertion, at the same position, of one intron of 853 bp for j1-1 and 4900 bp for j1-2. Northern blot and reverse transcriptase-polymerase chain reaction and restriction fragment length polymorphism analyses revealed that j1-1 transcripts are present only in fruits, only in trace amounts in mature green fruits, and that they accumulate to high levels in fully ripe fruits, whereas no j1-2 transcripts were detected in the samples monitored.

Alien are highly repeated plant transposable elements characterized by their small size (approx. 400 bp), high A + T content, target site specificity, potential to form stable secondary structures and possession of a conserved 28-bp terminal inverted repeat (TIR). Besides the TIR, they contain subterminal inverted repeat motifs (SIRM), as well as the 5'-CATGCAT domain which has been reported to be a cis-acting regulatory element of gene expression in some plant species. Although they were first identified in the intron of the bell pepper (Capsicum annuum) Sn-2 gene and in the promoter region of the potato starch phosphorylase-encoding gene, Alien arranged in tandem are present in the promoter of patatin class-II genes. PCR on the bell pepper genomic DNA using the Alien TIR consensus sequence as primer yielded DNA fragments of nearly 400 bp. These fragments have characteristics of transposable elements and contain numerous motifs reminiscent of Alien elements. Importantly, PCR on genomic DNA extracts from various monocotyledonous and dicotyledonous plants using the TIR consensus sequence as primer and subsequent hybridization with different Alien probes revealed that these elements are ubiquitously present and highly repeated in the genomes of higher plants.

Several lines of evidence indicate that annexins, as calcium-dependent phospholipid-binding proteins, are involved in a variety of plant cellular processes. We were interested in determining if annexins are implicated in the highly regulated fruit development of bell pepper. By differential screening of several cDNA libraries, we isolated a full-length cDNA of 1180 bp encoding an annexin. Northern blot analyses show a differential expression pattern of the transcripts during the early stages of development and during ripening. Immunoblots using antiserum raised against p33/p35 from maize reveal that cross-reactive polypeptides of about 30 kDa are present at each stage of fruit development in bell pepper. We partially purified the annexins from seedlings and green fruits. At least one annexin of 32 kDa is present in these plant tissues.

In Bidens pilosa (cv. radiata), a non-injurious stimulus induces a local and transient change in membrane potential, and an injurious stimulus induces a transmitted electrical signal described as the combination of an action potential and a slow wave. We have studied calmodulin gene expression after these stimuli. When the stimulus is non-injurious, calmodulin mRNA accumulation is only increased in the stimulated region. In contrast, when the stimulus is injurious, mRNA accumulation takes place in both wounded and distant, unwounded tissue. We propose that the slow wave plays a role in the long-distance transmission of a wound-induced information in plants.

In this study we have constructed a number of plants (cybrids), in which the nuclear genome of Nicotiana plumbaginifolia is combined with the plastome of Atropa belladonna, or the nuclear genome of N. tabacum with plastomes of Lycium barbarum, Scopolia carniolica, Physochlaine officinalis or Nolana paradoxa. Our biochemical and immunological analyses prove that in these cybrids the biogenesis of the chlorophyll a/b binding proteins (CAB) of the light harvesting complex II (LHCII) is altered. Besides normal sized CAB polypeptides of 27, 25.5 and 25 kDa, which become less abundant, the cybrids analyzed have additional polypeptides of 26, 24.5 and 24 kDa. Direct protein micro-sequencing showed that at least two truncated 26 kDa CAB polypeptides in plant cells containing a nucleus of N. plumbaginifolia and plastids of A. belladonna are encoded by the type 1 Lhcb genes. These polypeptides are 11-12 amino acids shorter at the N-terminus than the expected size. Based on the available data we conclude that the biogenesis of the LHCII in vivo may depend on plastome-encoded factor(s). These results suggest that plastome-encoded factors that cause specific protein degradation and/or abnormal processing might determine compartmental genetic incompatibility in plants.

Using a fruit-specific cDNA as a probe we isolated and sequenced the two corresponding homologous genes (Sn-1 and Sn-2) of the bell pepper (Capsicum annuum) genome. Both genes have a single intron and numerous unusual long inverted repeat sequences. The introns share 87% homology and Sn-2 contains one 450 bp additional sequence with structural features of a transposable element, which is highly repetitive in the bell pepper genome. Surprisingly, analysis in data banks showed that genes encoding the potato starch phosphorylase (EC 2.4.1.1) and patatin contain a similar element, named Alien, in their 5'-upstream region. Alien elements are characterized by a conserved 28 bp terminal inverted repeat (TIR), small size, high AT content, potential to form stable DNA secondary structures and they have probably been inserted in TA target sites. Interestingly, the TIR of the Alien elements shares high homology with sequences existing in the TIR of extrachromosomal linear pSKL DNA plasmid of Saccharomyces kluyveri. Northern blot analyses detected Sn-1 transcripts principally in the red fruit whereas no Sn-2 transcripts were detected in neither of the samples monitored. Western blot analyses detected a 16.8 kDa Sn protein principally in the ripe red fruit and wounded areas of green unripe fruit. A comparison of the deduced amino acid sequence of Sn-1 with protein sequences in data banks revealed a significant homology with proteins likely involved in the plant's disease resistance response. Analyses at the subcellular level showed that Sn-1 is localized in the membrane of vacuoles.

The ascorbate peroxidase (APX) system was studied in Capsicum annum during fruit ripening. A large increase in organelle APX activity was found during chloroplast-chromoplast transition whereas only a slight difference was detected in total fruit extracts. On native gels, four different isoforms were found in total fruit extracts but the patterns for red and green fruit were quite different. In isolated organelles, six isozymes were found and a comparison of the patterns showed significant differences. A cDNA encoding a cytosolic APX was cloned and sequenced. The corresponding transcript was shown to increase 3-4-fold during fruit ripening.

We have isolated cDNA and genomic clones corresponding to a gene highly and specifically expressed at the late stage of fruit-ripening in bell-pepper. Antibodies raised against the expressed protein allowed determination of the cellular localization of the gene product. The encoded protein is present only in chromoplasts from fully-ripe red fruits, as shown by Western analysis and import experiments. The corresponding mRNA accumulates to high levels during ripening at orange and red stages, but is not detected in yellow varieties impaired in the synthesis of ketocarotenoids. Several lines of evidence indicate that the encoded protein is an oxido-reductase involved in the synthesis of capsanthin and capsorubin.

Screening of a lambda gt11 cDNA expression library of Euglena gracilis with antibodies directed against histones H2 from maize resulted in the isolation of a full-length cDNA for a histone H2A. The open-reading frame of 408 bp corresponded to a protein of 136 amino acid residues (14 kDa). Despite the presence of a poly(A) tail, which is typical of plant histone mRNA but not of animal histone mRNA, the size of the deduced protein and its percentage of homology were closer to animal histone H2As than to plant or lower eukaryotic histone H2A. Sequence alignment revealed that the Euglena H2A protein was characterized by a shorter C-terminus and a N-terminus which extended 10 residues past the animal H2A. In contrast to other organisms studied, the expression of the Euglena H2A gene appeared to be almost constant during an entire life-cycle and presented no cell-stage-specific expression during development. Similar results are obtained for another histone gene, H3, and for beta-tubulin. Regulation of gene expression at a post-transcriptional level seems to be a general feature of Euglena.

We have determined the nucleotide sequence of the plastid psbL gene from bell pepper. This gene has an ACG as a first codon. Isolation of RNA from pepper leaves and ripe fruits and subsequent sequencing of the psbL cDNA revealed that this ACG codon is post-transcriptionally edited into an AUG initiation codon in both leaves and fruits. These data indicate that the RNA editing machinery which exists in chloroplasts is still functional in chromoplasts from ripe fruits.

The expression of three chloroplastic genes, psbA (the gene for the reaction center D1 protein of PSII), psbC (the gene for the P700 apoprotein of PSI) and psaB (the gene for the intermediate antema of PSII), and a nuclear gene, cab (the gene for the chlorophyll a/bbinding protein), has been investigated during chloroplast development in Euglena gracilis Klebs. The polysomal fraction, mRNA stability and protein turnover were analysed under different conditions of cell greening. The results indicate that the main regulatory step for the nuclear and chloroplastic genes was at the translational level when greening of cells took place on a resting medium. When cell greening took place on a medium with balanced phosphate, carbon and nitrogen sources (nutritional medium), the main regulation occurred posttranscriptionally by mobilization of the transcripts onto the polysomes. These results indicate that in E. gracilis, for a given gene, regulation operates at different levels, and that although light is the principal effector in the regulation of the genes involved in chloroplast development, the effect of metabolites should also be considered. Interestingly, it appears that these various regulation levels are similar for the chloroplastic and nucleocytoplasmic compartments.

Cysteine synthase (O-acetylserine sulfhydrylase) has been purified to homogeneity from bell pepper (Capsicum annuum) fruit chromoplasts. This enzyme consists of two subunits of 35 kDa. Immunocytochemical localization experiments confirmed the plastid location of this enzyme. A full-length cDNA was isolated from an expression library of C. annuum. The deduced peptide sequence revealed high similarity between the C. annuum cysteine synthase and its bacterial counterparts. In vitro transcription and translation of the cDNA and subsequent import experiments demonstrated that the encoded cysteine synthase is located in the plastids. The steady-state level of the cysteine synthase mRNA is almost constant in dark-grown hypocotyls, leaves, and fruits. However, a slight increase in this mRNA level was detected during fruit development (when the 25 S rRNA was taken as an internal standard). Similarly, the cysteine synthase activity in plastids was found to increase during fruit development and reaches the highest levels in the chromoplasts of red fruits. To address the physiological role of this phenomenon, we have shown that cysteine is engaged in the active metabolism of glutathione. Thus, in connection with the previous demonstration of an active tocopherol metabolism, it is concluded that differentiation of chloroplast to chromoplast in C. annuum involves an active synthesis of potential antioxidants or redox modulators.

Cell-specific and light-regulated expression of the beta-glucuronidase (GUS) reporter gene from maize cab-m1 and rbcS-m3 promoter sequences was studied in maize leaf segments by using an in situ transient expression microprojectile bombardment assay. The cab-m1 gene is known to be strongly photoregulated and to be expressed almost exclusively in mesophyll cells (MC) but not in bundle sheath cells (BSC). Expression of GUS from a 1026-base-pair 5' promoter fragment of cab-m1 is very low in dark-grown leaves; GUS expression is increased about 10-fold upon illumination of dark-grown leaves. In illuminated leaves, the ratio of GUS expression in MC vs. BSC is about 10:1. The cab-m1 region between 868 and 1026 base pairs 5' to the translation start confers strong MC-preferred expression on the remainder of the chimeric gene in illuminated leaves, but a region between -39 and -359 from the translation start is required for photoregulated expression. Transcripts of rbcS-m3 are found in BSC but not in MC and are about double in BSC of greening dark-grown seedlings. In contrast to the behavior of the cab-m1-GUS construct, GUS expression driven by 2.1 kilobase pairs of the rbcS-m3 5' region was about twice as high in MC as in BSC of unilluminated dark-grown maize leaves. The number of BSC, but not MC, expressing GUS nearly doubled upon greening of bombarded etiolated leaves. These data suggest that the 5' region of rbcS-m3 used here could be responsible for most of the light-dependent increase in rbcS-m3 transcripts observed in BSC of greening leaves and that transcriptional or posttranscriptional mechanisms are responsible for the lack of rbcS-m3 transcripts in MC.

Screening of a lambda gt11 cDNA expression library with antibodies directed against LHCII allowed the detection of several clones which differ markedly from the previously described cDNAs encoding LHCII apoproteins. A Northern analysis revealed a transcript size of 4.2 kb, whereas clones encoding LHCII hybridize with mRNAs in the range of 7.5 kb. Nucleotide sequencing of 2 clones showed open reading frames of 530 and 331 codons, respectively. Within these reading frames, 5 analogous motifs can be delimited corresponding to coding regions for around 180 amino acids (molecular weight, 18-19 kDa). The 5 segments share between 50% and 80% homology. Comparison with a tomato LHCI sequence indicates conserved regions at the two ends, and a central part highly divergent and containing a large deletion. By hybrid-selected translation, followed by immunoprecipitation with a monoclonal antibody directed against LHCI apoprotein, a protein of around 100 kDa is obtained. In vivo, the same antibody recognizes peptides of around 20 kDa. These results, coupled with our previous observations concerning LHCII, confirm that in Euglena at least, some chloroplast proteins encoded in the nucleus are synthesized by large mRNAs containing the information for several consecutive divergent peptides. Implications for processing and chloroplast import are discussed.

We present the nucleotide sequence and derived amino-acid sequence of three genes located in the small single copy region of the maize plastid chromosome. Two genes have some homology to respiratory NADH dehydrogenase subunits. They have been observed in Marchantia and tobacco and are designated ndhD and ndhE. The maize genes code for polypeptides of 58 and 11.3 kilodaltons respectively. The gene psaC is found upstream of ndhD; it encodes an 8.7-kilodalton iron-sulfur protein of PSI. The genes ndhD and psaC are cotranscribed in maize, and give rise to an RNA of 2.1 kilobases. The pool of RNAs from this transcription unit increases about three-fold within the first 10 hours of illumination of dark-grown maize seedlings. On the other hand, ndhE is transcribed independently.

The major Chlamydomonas reinhardi y-1 chloroplast membrane polypeptides-I + II, IV, V(a + b)-have been isolated by use of preparative sodium dodecyl sulfate-gel electrophoresis.Rabbit antisera prepared against these polypeptides interact with intact membranes as well as membrane fractions containing these specific antigens. Antisera against polypeptides I + II partially inactivate photosystem I. Antisera against polypeptide IV prevent inactivation of the Hill reaction and inhibit photooxidation of diphenylcarbazide with dichlorophenolindophenol as an electron acceptor. The protection of the Hill reaction by IV antiserum, measured with whole membranes, is partially hindered by the presence of V antiserum, which, by itself, causes inhibition of diphenylcarbazide photooxidation.We concluded that parts of the primary structure of these polypeptides are exposed in situ and can be recognized in whole membranes by antisera prepared against sodium dodecyl sulfate-denatured antigens. Polypeptides I and II, and polypeptides IV, Va, and Vb are associated with photosystem I and photosystem II, respectively.

The chlorophyll-protein complexes I and II have been isolated and anlyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis during greening and degreening of Chlamydomonas reinhardi y-1. At all stages of membrane formation, the complexes, when present, have a constant composition. Chlorophyll-protein complex I consists of a major polypeptide(s) of molecular weight 64,000 synthesized in the chloroplast, to which about 29 chlorophyll a molecules are bound. The complex is not detected when other polypeptides of chloroplastic origin, related to both Photosystem I and Photosystem II activities, are not synthesized. However, Photosystem I activity can develop in membranes in which chlorophyll-protein complex I is not detectable. Chlorophyll-protein complex II consists of two polypeptides of cytoplasmic origin, molecular weights 24,000 and 22,000, which bind 12 chlorophylls (a and b). The chlorophyll-protein complex II can be detected in membranes in which the development of photosystem II activity is prevented. Clipping of a Mr = 2000 fragment(s) from the Mr = 22,000 polypeptide following trypsin digestion of membranes, does not affect the complex. The detection of the complexes is possible only in membranes in which the simultaneous synthesis of both the chlorophyll and the corresponding polypeptides occurs. The 28,000 dalton polypeptide, reported to be present in the chlorophyll-protein complex II, comigrates with the complex but apparently is not part of the complex itself. The apparent molecular weight of the chlorophyll-protein complexes I and II are 88,000 and 28,000, respectively. The minimal true value for complex I is 89,000 or 154,000 and for complex II is 56,000.

A cDNA library made from Euglena polyadenylated RNA has been constructed in the expression vector lambda gt11. The library was screened with antibodies directed against the LHCP apoproteins. Several putative LHCP clones were isolated and analyzed. We present the nucleotide and derived amino-acid sequence of an 800 bp long insert containing a poly A tail. On an amino-acid basis, the Euglena LHCP shows a 60% homology with the corresponding pea gene. Northern blot analysis revealed an unusual size of 7.5 kb. This extremely large mRNA is also recognized by a LHCP probe from Chlamydomonas. Hybrid-selected translation produces 2 proteins of 90-110 kd equivalent in size of the polypeptides immunoprecipitated from the translation products of polyadenylated RNAs.