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Biomedical subjects

H J Bohnert

Publications and source records attributed to H J Bohnert.

At least 73 records · Page 4Linked to original sources
PubMed1991

Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli.

Procedures were developed for 95 and 80% purification to homogeneity of the large subunit (L) and small subunit (S) of ribulose 1,5-bisphosphate carboxylase/oxygenase (L8S8) from Synechococcus PCC 6301, each expressed separately in Escherichia coli. Purified L had a low specific activity in the absence of S (0.075 mumol CO2 fixed/mg holoenzyme/min). Following elution on a Pharmacia Superose 6 or 12 gel filtration column, 50% of the purified L appeared as the octamer, L8. The rest was in equilibrium with lower polymeric species and/or was retained on the column. Large and small subunits assembled rapidly into the L8S8 holoenzyme that had high specific activities, 6.2 and 3.1 mumol CO2 fixed/mg holoenzyme/min for the homologous Synechococcus L8S8 and the hybrid Synechococcus L-pea S L8S8, respectively. The CO2 dependence for carbamylation of L8 was compared to that of L8S8 as a function of pH and CO2 concentration. The pH dependence indicated an apparent pKa for L8 of 8.28 and for L8S8 of 8.15, suggesting that S may influence the pKa of the lysine involved in carbamylation. The Kact for CO2 at pH 8.4 were similar for L8 (13.5 microM) and L8S8 (15.5 microM). L8 bound 2-[14C]carboxy-D-arabinitol 1,5-bisphosphate (CABP) tightly so that most of the bound [14C]CABP survived gel filtration. A major amount of the L8-[14C]CABP complex appeared as larger polymeric aggregates when eluted in the presence of E. coli protein.

Chromatography, Ion Exchange↗
PubMed1991

Modulation of the tight binding of carboxyarabinitol 1,5-bisphosphate to the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase.

The large subunit (L) of ribulose 1,5-bisphosphate carboxylase/oxygenase (rubisco) from Synechococcus PCC 6301 was expressed in Escherichia coli, purified as the octamer L8, and analyzed for its ability to tightly bind the transition state analog, 2-carboxyarabinitol 1,5-bisphosphate (CABP). [14C]CABP remained tightly bound to L8 after challenging with [12C]CABP and gel filtration, indicating that L8 alone without the small subunit (S) could tightly bind CABP. Binding of CABP to L8 induced a shift in the gel filtration profile due to apparent aggregation of L8. Aggregation did not occur with the L8S8-CABP complex nor with L8-CABP in the presence of 150 mM MgCl2. If ionic strength was increased with either KCl or MgCl2 during or after the binding of [14C]CABP to L8, [14C]CABP in the complex exchanged with [12C]CABP and was lost from the protein. Ionic strength strongly affected the rate constant (k4) for [14C]CABP dissociation from the L8-[14C]CABP complex, but had little effect on k4 for the L8S8-CABP complex. The differences in CABP binding characteristics between the L8-CABP and L8S8-CABP complexes demonstrate that S is intimately involved in maintaining the stability of the tight binding of CABP to the active site. These are the same interactions stabilizing the intermediate, 3-keto-2-carboxyarabinitol 1,5-bisphosphate, to native rubisco during CO2 fixation.

Binding Sites↗
PubMed1990

Increased expression of a gene coding for NAD:glyceraldehyde-3-phosphate dehydrogenase during the transition from C3 photosynthesis to crassulacean acid metabolism in Mesembryanthemum crystallinum.

We utilized differential plaque hybridization to identify three cDNA clones for transcripts which increase in abundance during the salinity-induced transition from C3 photosynthesis to crassulacean acid metabolism (CAM) in Mesembryanthemum crystallinum. Although there are differences in the abundance of these transcripts in unstressed tissue, steady-state levels of all three increased within 30 h following irrigation with 0.5 M NaCl. One cDNA encodes the cytosolic form of glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating) (NAD-GAPDH], an enzyme involved in the production of phosphoenolpyruvate for CO2 fixation at night and the conversion of pyruvate to storage carbohydrate during the day. Coding region and 3'-noncoding sequence probes were used to examine the expression of NAD-GAPDH transcripts in leaf and root tissue. We show that the gene encoding the NAD-GAPDH cDNA is expressed in both leaf and root tissue during C3 photosynthesis and CAM. NAD-GAPDH transcript levels increase rapidly in leaf (but not in root) tissue during the transition to CAM. Our data indicate that the predominant NAD-GAPDH transcript expressed during C3 photosynthesis and CAM is encoded by a single gene in M crystallinum. These results imply that the transition to CAM in some cases involves an upward readjustment in the level of a gene product expressed during C3 photosynthesis, rather than the expression of a CAM-specific isoform with unique regulatory or kinetic properties.

Amino Acid Sequence↗
PubMed1990

Direct screening of a small genome: estimation of the magnitude of plant gene expression changes during adaptation to high salt.

Mesembryanthemum crystallinum (common ice plant), a facultative halophyte with a genome size of 393,000 kb, was used to estimate the magnitude of changes in gene expression in response to environmental stress by excess salt. Such treatment induces a water-conserving pathway of carbon assimilation (CAM) which is, at least in part, transcriptionally controlled. From a genomic library, 200 phage containing approximately 3200 kb (0.8% of the genome) were randomly selected. The inserts in these clones could be divided into four classes ranging from highly repetitive DNA (class I clones) to single-copy DNA (class IV clones). The inserts of the 166 clones of classes II to IV were digested with various restriction enzymes and the fragments were analyzed by hybridization with radioactively labelled mRNA isolated from stressed and unstressed leaves. We found that a total of approximately 140 DNA fragments hybridized with the RNA probe. Among those, several differentially regulated transcripts were observed. Stress-dependent fluctuation of mRNA abundance was verified by Northern analyses: one mRNA, not detectable in unstressed leaves, appeared in stressed leaves, while steady-state levels of three transcripts decreased during stress. All regulated signals are derived from low abundance mRNAs, which may be missed during screening of cDNA libraries. We conclude from these results that, for the entire genome, on the order of more than one hundred genes are differentially regulated in response to salt stress.

Blotting, Southern↗
PubMed1990

The cyanelle S10 spc ribosomal protein gene operon from Cyanophora paradoxa.

In Cyanophora paradoxa photosynthetic organelles termed cyanelles perform the functions of chloroplasts in higher plants, while the structural and biochemical characteristics of the cyanelle are essentially cyanobacterial. Our interest in studying the evolutionary relationship between cyanelles and chloroplasts led us to focus on cyanelle-encoded genes of the translational apparatus, specifically genes equivalent to those of the bacterial S10 and spc operons. The structure of a large ribosomal protein gene cluster from cyanelle DNA was characterized and compared with that from plastids and bacteria. Sequences of the following cyanelle genes encompassing 4.8 kb are reported here: 5'-rpl22-rps3-rpl16-rps17-rpl14-rpl5-rps8-rpl6-rpl18- rps5-3'. Cyanelles contain five more ribosomal protein genes than do higher plant chloroplasts and four more genes than Euglena gracilis plastids in the S10/spc region of this gene cluster. The gene encoding rpl36 is absent, in contrast to the case in other plastid DNAs. These genes, including the previously characterized genes rpl3, rpl2 and rps19, are transcribed as a primary transcript of approximately 7500 nucleotides. The occurrence of transcripts smaller than this presumptive primary transcript suggests that it is processed into defined segments. Transcription terminates 3' of rps5 where a 40 bp hairpin with one mismatch (-42.2 kcal) may be folded. Immediately downstream of rps5 an open reading frame, ORF492, is contained on a separate transcript. A comparison of gene content, operon structure and deduced amino acid sequence of the genes in the S10 and spc operons from different organisms supports the notion that cyanelles are intermediary between known plastids and cyanobacteria.

Amino Acid Sequence↗
PubMed1990

Developmental control of crassulacean Acid metabolism inducibility by salt stress in the common ice plant.

Ice plant (Mesembryanthemum crystallinum) is a facultative halophyte that responds to water stress in the form of drought or high salinity by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM), a physiological adaptation that increases water conservation. Although CAM is clearly environmentally controlled, and reversible upon removal of water stress, the competence to switch is developmentally determined. We have demonstrated this by measuring three parameters in the expression of a gene encoding a stress-specific isoform of a key enzyme of CAM, phosphoenolpyruvate carboxylase (PEPCase, Ppc1): (a) protein accumulation; (b) steady-state amounts of mRNA; and (3) transcriptional activity in isolated nuclei. Young plants (3 weeks of age) show little induction of PEPCase protein, mRNA, or transcription when stressed. In contrast, salt stress elicits a strong induction at all three levels of expression at 6 weeks of age. By 9 weeks of age, plants have already accumulated PEPCase protein and mRNA without being stressed. More importantly, transcriptional activation of Ppc1 by salt stress in 9-week-old plants is no longer observed despite an increase of both Ppc1 mRNA and protein. From these results we suggest that a developmental program exists that regulates PEPCase transcription and mRNA stability. This program appears to be synchronized with the climatic conditions in the plant's native environment.

Journal Article↗
PubMed1989

Deletion of the carboxyl-terminal portion of the transit peptide affects processing but not import or assembly of the small subunit of ribulose-1,5-bisphosphate carboxylase.

Import of the small subunit of ribulose-1,5-biphosphate carboxylase/oxygenase into the chloroplast has been proposed to involve two proteolytic cleavages which convert the 20-kDa precursor (pSSU) into the mature 14-kDa subunit (SSU) via an 18-kDa intermediate. A deletion mutant (PSd48/57) of pSSU which lacks 10 amino acids in a conserved region in the carboxyl-terminal portion of the transit peptide is converted into a series of 16-18-kDa polypeptides in addition to the mature 14-kDa SSU when imported into isolated pea chloroplasts. We examined import and processing of this mutant pSSU to determine whether the 16-18-kDa SSUs undergo further maturation in the chloroplast stroma to yield 14-kDa SSU. The ratio of incorrectly processed to 14-kDa SSU is stable up to 60 min following import. This indicates that processing of PSd48/57 involves a single proteolytic cleavage which occurs during or immediately following transit across the chloroplast envelope. The carboxyl-terminal portion of the transit peptide confers either sequence specificity for the processing protease or provides a three-dimensional structure necessary for consistent cleavage at the mature amino terminus of SSU. Incorrectly processed SSUs were incorporated into the holoenzyme demonstrating that removal of the entire transit sequence is not necessary for assembly of the holoenzyme.

Amino Acid Sequence↗
PubMed1989

Evolutionary relationship of psbA genes from cyanobacteria, cyanelles and plastids.

The psbA gene is part of the reaction center of photosystem II in cyanobacteria and the plastids of higher plants. Its primary sequence is highly conserved among all species investigated so far and its sequence shows homologies with the L and M subunits of the reaction center of photosynthetic bacteria. We have analyzed the psbA homolog from a eukaryotic alga, Cyanophora paradoxa, where the gene is encoded on cyanelle DNA. These cyanelles are surrounded by a murein sacculus and resemble cyanobacteria in many other characteristics, although they are genuine organelles that functionally replace plastids. Analysis of the gene revealed a psbA protein identical in length (360 codons) with the cyanobacterial counterpart. The overall sequence identity is, however, more pronounced between cyanelle psbA and the shorter (353 amino acids) psbA product found in higher plants. These data strongly support the postulated bridge position of cyanelles between chloroplasts and free-living cyanobacteria.

Amino Acid Sequence↗
PubMed1989

Expression of the CAM-form of phospho(enol)pyruvate carboxylase and nucleotide sequence of a full length cDNA from Mesembryanthemum crystallinum.

We have determined the complete nucleotide sequence of a full length cDNA encoding the Crassulacean acid metabolism (CAM) isogene of phospho(enol)pyruvate carboxylase (PEPCase). The cDNA clone, 3348 bp in length, was obtained from mRNA isolated from Mesembryanthemum crystallinum (common ice plant) which had undergone salt stress and subsequent induction of CAM. The long open reading frame encodes PEPCase (EC 4.1.1.31) with a predicted molecular mass of 110533 daltons. The deduced amino acid sequence of the ice plant PEPCase is most similar to that from maize having an amino acid identity of 74.9%. Sequence identity in corresponding regions of the PEPCase proteins from Escherichia coli and the cyanobacterium Anacystis nidulans are 41.4% and 33.5%, respectively. A compilation of the four amino acid sequences permitted the identification of phylogenetically conserved regions within the proteins which may play a role in the function of this important enzyme in plant metabolism. Gene specific probes from 3' coding and noncoding regions of the cDNA clone used to probe genomic Southern blots established that this PEPCase gene is present in one copy in the nuclear genome of M. crystallinum. Transcripts arising from this gene increase dramatically when M. crystallinum is irrigated with 0.5 M NaCl, a stress which induces this plant to switch the primary fixation of CO2 from C3 (Calvin cycle) to CAM mode. The salt-induced mRNA encodes a PEPCase isoform which is undetectable in plants in the C3 mode as demonstrated by Northern hybridization.

Amino Acid Sequence↗
PubMed1989

Effect of mutations on the binding and translocation functions of a chloroplast transit peptide.

We studied transport and binding to intact chloroplasts of 10 mutants in three regions of the transit peptide of a precursor to the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase [3-phospho-D-glycerate carboxy-lyase (transphosphorylating), E.C.4.1.1.39]. Transport was assayed in a reconstituted system using isolated pea chloroplasts and radioactively labeled precursor. Binding to the chloroplast envelope was assayed in a similar manner using chloroplasts pretreated with nigericin. Most mutants showed a dramatically decreased capacity of binding, although some of them transported relatively well. The accumulation of the mutant proteins inside the chloroplast as a function of time was examined. Although the authentic small subunit precursor was imported rapidly, uptake of most mutant precursors was considerably slower and continued until the last time point examined. In terms of assigning functions to individual regions, we found that at least the middle region and parts of the amino and the carboxyl termini of the transit peptide are more important for receptor binding than for translocation. A two-step processing mechanism has been postulated for the maturation of the small subunit precursor. This model predicts the occurrence of processing intermediates. When precursors carrying carboxyl-terminal deletions were presented to the chloroplast, no defined intermediates could be detected. Instead, a number of proteins, probably resulting from aberrant processing, accumulated simultaneously inside the chloroplasts.

Amino Acid Sequence↗
PubMed1989

Identification of an assembly domain in the small subunit of ribulose-1,5-bisphosphate carboxylase.

The mature small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in higher plants contains a highly conserved sequence of 16 amino acids that is absent in the SSUs of cyanobacteria. To determine whether this region of the SSU of higher plants has a specific function, portions of the SSU genes (rbcS) of pea (Pisum sativum) and the cyanobacterium Anacystis nidulans were fused to create chimeric genes that either lacked or contained the coding sequence for the 16 conserved amino acids. Precursor proteins synthesized in vitro from the chimeric genes were incubated with isolated pea chloroplasts to assay import and assembly into the holoenzyme. Fusion proteins lacking the 16-amino acid sequence were imported and processed but failed to assemble with endogenous large subunit. Addition of the region from a pea rbcS containing the 16 amino acids to the rbcS of Anacystis enabled the imported SSU fusion protein to assemble with pea large subunit. This 16-amino acid sequence is encoded by a separate exon in certain rbcS genes of higher plants. We propose that the conserved 16-amino acid sequence constitutes a domain acquired to facilitate assembly of the eukaryotic holoenzyme.

Amino Acid Sequence↗
PubMed1989

Time Course of mRNA Induction Elicited by Salt Stress in the Common Ice Plant (Mesembryanthemum crystallinum).

In the facultative halophyte Mesembryanthemum crystallinum (common ice plant), irrigation with solutions containing NaCl induces an alternate mode of carbon dioxide fixation, Crassulacean acid metabolism (CAM). The salt stress protocol which we have established facilitates the study of CAM induction and the correlation of changes in metabolism and gene expression. We have studied the time course of mRNA induction for phosphoenolpyruvate carboxylase (PEPCase) (gene: ppc) and several other enzymes of carbon metabolism during stress. While CAM is not fully established for at least 10 days after the start of stress, mRNA amounts for PEPCase and for other CAM enzymes, such as Pyruvate orthophosphate dikinase, increase between day 2 and 3 after stress induction. Increases continue for at least 5 days. Concomitant with the increase of CAM transcripts, fluctuations in the mRNA amounts for genes rbcS and cab were observed. Transcript levels for these proteins decreased several-fold during a 3 to 4 day period.

Journal Article↗