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Biomedical subjects

H J Bohnert

Publications and source records attributed to H J Bohnert.

At least 91 records · Page 5Linked to original sources
PubMed1989

Expression during Salt Stress and Nucleotide Sequence of cDNA for Ferredoxin-NADP Reductase from Mesembryanthemum crystallinum.

In the facultative halophyte Mesembryanthemum crystallinum (common ice plant) the enzyme ferredoxin-NADP(+)-reductase (FNR) is coded for by a small family of 2 to 3 genes. We have determined the expression characteristics as the plants adapt to high salt and the nucleotide sequence of a full-length cDNA coding for the precursor of this chloroplast-located enzyme. On a developmental scale amounts of FNR transcripts and protein are highest in young emerging leaves. The FNR cDNA is a member of a class of genes whose expression is only slightly affected by salt stress. Even less pronounced than mRNA fluctuations, the amount of FNR protein is unaffected by salt stress. The longest FNR cDNA found was 1,419 nucleotides in size. It consisted of 74 nucleotides 5'-leader sequence, 1,095 nucleotides of protein coding sequence encoding 365 amino acids, and 247 nucleotides 3-region excluding a short poly(A(+)) tail. As expected for a nucleus-coded chloroplast protein an amino terminal transit peptide (52 amino acids in length) was found. The mature FNR protein is predicted to contain 313 amino acids corresponding to a protein of M(r) 35,713. The deduced amino acid sequence of the mature FNR protein is 93.2 and 85.9% identical to those of spinach and pea. The transit peptide of pea and spinach have 55.8 and 69.2% identity with that from ice plant.

Journal Article↗
PubMed1989

Salt stress leads to differential expression of two isogenes of phosphoenolpyruvate carboxylase during Crassulacean acid metabolism induction in the common ice plant.

The common ice plant is a facultative halophyte in which Crassulacean acid metabolism, a metabolic adaptation to arid environments, can be induced by irrigating plants with high levels of NaCl or by drought. This stress-induced metabolic transition is accompanied by up to a 50-fold increase in the activity of phosphoenolpyruvate carboxylase (PEPCase). To analyze the molecular basis of this plant response to water stress, we have isolated and characterized two members of the PEPCase gene family from the common ice plant. The PEPCase isogenes, designated Ppc1 and Ppc2, have conserved intron-exon organizations, are 76.4% identical at the nucleotide sequence level within exons, and encode predicted polypeptides with 83% amino acid identity. Steady-state levels of mRNAs from the two genes differ dramatically when plants are salt-stressed. Transcripts of Ppc1 increase about 30-fold in leaves within 5 days of salt stress. In contrast, steady-state levels of Ppc2 transcripts decrease slightly in leaf tissue over the same stress period. Steady-state levels of transcripts of both genes decrease in roots over 5 days of salt stress. We have used in vitro transcription assays with nuclei isolated from leaves to demonstrate that the increased expression of Ppc1 caused by water stress occurs in part at the transcriptional level.

Adaptation, Physiological↗
PubMed1988

Complete processing of a small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea requires the amino acid sequence Ile-Thr-Ser.

Chloroplast import and processing of two precursor proteins with mutations in the carboxyl-terminal region of the transit peptide were examined in vitro. Deletion mutations were introduced into the 57-amino acid transit peptide of a chloroplast protein, the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, from pea. A mutant, PSd48/57, in which nine carboxyl-terminal amino acids of the transit peptide had been deleted, was imported and processed to a series of 13- to 18-kDa polypeptides including the 14-kDa mature small subunit. In contrast, processing of a mutant, PSd45/57, in which an additional three amino acids had been removed, resulted in a series of polypeptides which did not include the mature small subunit. Whereas PSd48/57 was imported as efficiently as the wild-type precursor, import of PSd45/57 was only 25% as efficient as that of the authentic precursor. The mutant precursor proteins PSd48/57 and PSd45/57 are distinguished by a three-amino acid sequence, Ile-Thr-Ser, located in the carboxyl-terminal region of the transit peptide. We show that all or part of this sequence is required for correct processing.

Amino Acid Sequence↗
PubMed1988

Physical map and protein gene map of cyanelle DNA from the second known isolate of Cyanophora paradoxa (Kies-strain).

A restriction map of the cyanelle DNA from a different isolate of Cyanophora paradoxa (Kies-strain) was established. The positions of 18 protein genes and the rRNA genes have been located and compared to the positions of these genes from the first isolate of C. paradoxa (Pringsheim-strain). The gene arrangement is absolutely conserved in both cyanelle DNAs. The differences in size (ca. 9 kb) and the unrelatedness in the restriction patterns could be explained by numerous small insertions into intergenic regions of the cyanelle chromosomes.

Cyanobacteria↗
PubMed1988

PEPCase Transcript Levels in Mesembryanthemum crystallinum Decline Rapidly upon Relief from Salt Stress.

Mesembryanthemum crystallinum plants respond to water stress by changing their pathway of carbon assimilation from C(3) to Crassulacean acid metabolism (CAM). Stressed plants are characterized by elevated levels of phosphoenolpyruvate carboxylase (PEPCase) mRNA, protein, and enzyme activity. We wanted to determine whether CAM is a reversible response to environmental conditions or a developmentally programmed adaptation that is irreversibly expressed once induced. Plants were osmotically stressed by irrigation with 500 millimolar NaCl for 12 days to elicit CAM. Salt was then thoroughly flushed from the soil and PEPCase protein and transcript levels were monitored. PEPCase mRNA levels dropped by 77% within 2.5 hours after salt removal. PEPCase activity and polypeptide levels declined more slowly, with a half-life of 2 to 3 days. These results show that PEPCase expression in M. crystallinum is a reversible response to stress that is regulated at the level of transcription or stability of the PEPCase mRNA.

Journal Article↗
PubMed1987

Regions in the transit peptide of SSU essential for transport into chloroplasts.

Deletion mutations, 3-19 amino acids in size, were introduced into the transit peptide (57 amino acids) of a small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase from pea. Transport of the authentic small subunit precursor (pSSU) and of the mutant pSSUs by isolated chloroplasts of pea was examined. We show that the transit peptide contains two different, separated functional regions. A deletion mutation in the central region of the transit peptide, a region purported to be important for function, barely affected transport. Changes in the amino-terminal region of the transit peptide appeared normal. A deletion mutation at the carboxy-terminus of the transit peptide interfered with both transport and processing. From the aberrant processing we suggest that pSSU is matured in more than one step, and that the maturation signal is located within the carboxy-terminal 16 amino acids. The methionine residue at the evolutionarily conserved cleavage site (cysteine-methionine) between the transit peptide and the mature protein is not essential processing.

Chloroplasts↗
PubMed1987

Induction of Crassulacean Acid Metabolism in Mesembryanthemum crystallinum by High Salinity: Mass Increase and de Novo Synthesis of PEP-Carboxylase.

Intact plants of the halophilic species Mesembryanthemum crystallinum were induced to exhibit Crassulacean acid metabolism by irrigation with nutrient solution containing 500 millimolar NaCl. During the induction period, the extractable activity of phosphoenolpyruvate carboxylase (PEPcase) increased approximately 40-fold. This increase was linearly correlated with a mass increase of PEPcase protein as measured by single radial immunodiffusion. De novo synthesis of PEPcase protein was shown by immunoprecipitation of the newly synthesized, radioactively labeled protein in leaf discs from salt-treated plants. Nontreated plants were characterized by a low level of the enzyme and low rates of PEPcase synthesis. Synthesis of this enzyme in leaf discs was correlated with the concentration of NaCl in the nutrient solution during growth.

Journal Article↗
PubMed1987

Salt Stress Increases the Level of Translatable mRNA for Phosphoenolpyruvate Carboxylase in Mesembryanthemum crystallinum.

Mesembryanthemum crystallinum responds to salt stress by switching from C(3) photosynthesis to Crassulacean acid metabolism (CAM). During this transition the activity of phosphoenolpyruvate carboxylase (PEPCase) increases in soluble protein extracts from leaf tissue. We monitored CAM induction in plants irrigated with 0.5 molar NaCl for 5 days during the fourth, fifth, and sixth week after germination. Our results indicate that the age of the plant influenced the response to salt stress. There was no increase in PEPCase protein or PEPCase enzyme activity when plants were irrigated with 0.5 molar NaCl during the fourth and fifth week after germination. However, PEPCase activity increased within 2 to 3 days when plants were salt stressed during the sixth week after germination. Immunoblot analysis with anti-PEPCase antibodies showed that PEPCase synthesis was induced in both expanded leaves and in newly developing axillary shoot tissue. The increase in PEPCase protein was paralleled by an increase in PEPCase mRNA as assayed by immunoprecipitation of PEPCase from the in vitro translation products of RNA from salt-stressed plants. These results demonstrate that salinity increased the level of PEPCase in leaf and shoot tissue via a stress-induced increase in the steady-state level of translatable mRNA for this enzyme.

Journal Article↗
PubMed1985

The use of nuclear-encoded sequences to direct the light-regulated synthesis and transport of a foreign protein into plant chloroplasts.

The light-inducible nuclear gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco), produces a precursor protein with an amino-terminal transit peptide which is transported into the plastids and cleaved by a specific proteinase. To test whether the promoter and transit peptide-coding sequences of the small subunit gene can be used to direct the light-inducible synthesis and transport of a foreign protein into chloroplasts, a chimeric gene was constructed consisting of the promoter, first exon and intron as well as part of the second exon of the small subunit Rubisco gene fused to the amino-terminal end of the neomycin phosphotransferase II gene (nptII) of Tn5. Tobacco tissue, as well as whole plants, into which this chimaeric gene was introduced, were resistant to kanamycin. The transcription of the chimaeric gene as well as the NPTII activity of the resulting fusion protein were shown to be light inducible. The fusion protein is processed and located within the chloroplasts of the transformed plants.

Artificial Gene Fusion↗
PubMed1982

Nucleotide sequence of the gene for the M(r) 32,000 thylakoid membrane protein from Spinacia oleracea and Nicotiana debneyi predicts a totally conserved primary translation product of M(r) 38,950.

The gene for the so-called M(r) 32,000 rapidly labeled photosystem II thylakoid membrane protein (here designated psbA) of spinach (Spinacia oleracea) chloroplasts is located on the chloroplast DNA in the large single-copy region immediately adjacent to one of the inverted repeat sequences. In this paper we show that the size of the mRNA for this protein is approximately 1.25 kilobases and that the direction of transcription is towards the inverted repeat unit. The nucleotide sequence of the gene and its flanking regions is presented. The only large open reading frame in the sequence codes for a protein of M(r) 38,950. The nucleotide sequence of psbA from Nicotiana debneyi also has been determined, and comparison of the sequences from the two species shows them to be highly conserved (>95% homology) throughout the entire reading frame. Conservation of the amino acid sequence is absolute, there being no changes in a total of 353 residues. This leads us to conclude that the primary translation product of psbA must be a protein of M(r) 38,950. The protein is characterized by the complete absence of lysine residues and is relatively rich in hydrophobic amino acids, which tend to be clustered. Transcription of spinach psbA starts about 86 base pairs before the first ATG codon. Immediately upstream from this point there is a sequence typical of that found in E. coli promoters. An almost identical sequence occurs in the equivalent region of N. debneyi DNA.

Journal Article↗
PubMed1982

The subcellular localization of DNA components from Cyanophora paradoxa, a flagellate containing endosymbiotic cyanelles.

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsC1 density gradients. The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724 g X cm-3. Several subsfractions in the range from 1.718 g X cm-3 to 1.735 g X cm-3 are contained in this component. This DNA of high complexity was considered to be host nuclear DNA. The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g X cm-3 in one strain and 1.695 g X cm-3 in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 X 10(3) base pairs and comprise about 5% of the total cellular DNA content. Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 X 10(3) base pairs in size, had a density of approximately 1.688 g X cm-3. The second component, about 15 X 10(3) base pairs in size, banded in the density range between 1.710 g X cm-3 and 1.720 g X cm-3. The latter is associated with nuclear DNA. The 48 X 10(3)-base-pair component was located in the cytosol and could be obtained after CsC1/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA. Both these components amounted to approximately 0.5-1% of total DNA. A further DNA component with a complexity of more than 150 X 10(3) base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710-1.720 g X cm-3) and it amounted to 1-2% of the total DNA. Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs.

Centrifugation, Density Gradient↗
PubMed1981

The mitochondrial genomes of Ustilago cynodontis and Acanthamoeba castellanii.

Mitochondrial DNA from Ustilago cynodontis has been investigated in several of its properties. Its dG + dC content is equal to 33.5%; its buoyant density (1.698 g/cm3) is higher, by 5 mg/cm3, and its melting temperature (82.5 degrees C) is lower than expected for a bacterial DNA having the same base composition; the first derivative of its melting curve indicates a large compositional heterogeneity, its molarity of elution from hydroxyapatite is high, 0.28 M phosphate, and allows its partial separation from nuclear DNA. Degradation by micrococcal nuclease indicates that about 25% of the DNA is formed by stretches having no more than 15% dG + dC. Finally, the unit size of mitochondrial genome is about 50 X 10(6). In most of its properties, the mitochondrial genome of U. cynodontis presents strong analogies with that of Saccharomyces cerevisiae. A parallel investigation on mitochondrial DNA from Acanthamoeba castellanii which has as genome unit size of only 27 X 10(6), has shown that this shares with the former the dG + dC content (32.9%), the melting temperature (82.5 degrees C), a large compositional heterogeneity and a very similar pattern of micrococcal nuclease degradation; its buoyant density (1.692 g/cm3) and its molarity of elution from hydroxyapatite (0.25 M phosphate) are, however, normal, probably because of a different short-sequence pattern and the fact that its dA + dT-rich stretches are shorter, on the average.

Amoeba↗
PubMed1981

Compositional heterogeneity of the chloroplast DNAs from Euglena gracilis and Spinacia oleracea.

The chloroplast genomes of Euglena gracilis and Spinacia oleracea were investigated in their compositional heterogeneity, by using different experimental approaches. Euglena chloroplast DNA has a dG + dC content of 28%. Preparations averaging 20 x 10(6) in molecular weight exhibit a gross heterogeneity in their elution profiles from hydroxyapatite and in their buoyant densities because the rRNA genes have a high rG + rC content. Finer analysis by melting, buoyant density of restriction fragments and micrococcal nuclease degradation have revealed an extended compositional heterogeneity. From micrococcal nuclease digestion data, approximately 30% of the chloroplast genome is as low as 12% in its dG + dC content, whereas 10% is higher than 60% dG + dC. Since the average dG + dC content of large restriction endonuclease fragments varied to a lesser extent, most of dA + dT-rich sequences must occur in short stretches interspersed with dG + dC-rich stretches. Spinach chloroplast DNA (dG + dC = 36.5%) did not exhibit any gross compositional heterogeneity in its hydroxyapatite elution or in its buoyant density profile. But the higher resolution methods of melting, bouyant densities of restriction fragments and micrococcal nuclease degradation revealed a high degree of heterogeneity which appears to be due to interspersion of short DNA stretches of different base composition. About 30% of genome is as low as 22% in dG + dC, while 10% is higher than 60% in dG + dC.

Base Composition↗
PubMed1980

Spinach chloroplast mRNA for a 32 000 dalton polypeptide: size and localization on the physical map of the chloroplast DNA.

The RNA from chloroplasts of young spinach plants contains mRNAs which are translated in a cell-free protein synthesizing system from lysed rabbit reticulocytes. Using [35S]methionine to label the products of translation, the main peptides have apparent molecular weights of 55,000, 45,000, 40,000, 32,000, 20,000 and 17,000. The sizes of the mRNAs as estimated by sucrose gradient centrifugation under non-denaturing conditions are between 12 and 23 S. A prominent product synthesized with RNA isolated from young spinach plants has an approximate molecular weight of 32,000. The mRNA for this 32,000 dalton protein sediments at 14 S on sucrose gradient. The position of the DNA sequence coding for this mRNA on the restriction site map of spinach chloroplast DNA was determined by hybridization and hybrid-arrested translation. A preliminary transcription map shows that in RNA from spinach chloroplasts which is larger than 10 S different RNA species are present which hybridize to nearly all fragments of the larger of the two single-copy regions of the chloroplast DNA.

Chloroplasts↗