Functional cybrid plants possessing a Nicotiana genome and an Atropa plastome

Summary Mesophyll protoplasts of plastome chlorophyll-deficient, streptomycin-resistant Nicotiana tabacum were fused with those of wild type Atropa belladonna using the polyethylene-glycol/high pH/high Ca⁺⁺/dimethylsulfoxide method. Protoplasts were cultured in nutrient media suitable for regeneration of tobacco but not Atropa cells. In two experiments, a total of 41 cell lines have been selected as green colonies. Cytogenetic (chromosomal number and morphology) and biochemical (isozyme analyses of esterase, amylase and peroxidase) studies were used to evaluate the nuclear genetic constitution of regenerated plants. To study plastid genetic constitution, restriction endonuclease analysis of chloroplast DNA was performed. Three groups of regenerants have been identified: (a) nuclear hybrids (4 cell lines); (b) Atropa plants, most probably arising from rare surviving parental protoplasts (4 lines) and (c) Nicotiana/Atropa cybrids possessing a tobacco genome and an Atropa plastome (33 lines). Most of cybrids obtained were diploid, morphologically normal plants phenotypically similar to tobacco. Some plants flowered and yielded viable seeds. Part of cybrid regenerants were variegated, variegation being transmitted to sexual progeny. Electron microscopic analysis of the mesophyll cells of variegated leaves revealed the presence of heteroplastidic cells. Analysis of thylakoid membrane polypeptides shows that in the cybrids the content of at least one of the major polypeptides, presumably a chlorophyll a/b binding protein is drastically reduced.     

Variation in mitochondrial translation products in fertile and cytoplasmic male-sterile sugar beets

Summary Intact and functional mitochondria were isolated from sugar beet plants (Beta vulgaris L.) containing normal fertile (F) or cytoplasmic male-sterile (S₁–S₄) cytoplasms. Incorporation of ³⁵S-methionine by mitochondria isolated from both roots and leaves showed approximately 20 major and ten minor translation products. Comparison of the polypeptide synthesis patterns produced by leaf mitochondria from fertile plants of three different species within the genus Beta revealed several taxonomically related differences. Contrary to this, the patterns of polypeptides synthesized by mitochondria from roots and leaves of sugar beet plants containing the F and S₁–S₄ cytoplasms were very similar; in the S₁ and S₂ cytoplasms no qualitative, and only a few quantitative, differences from the F cytoplasm were observed. Thus, in these cases, cytoplasmic male sterility in sugar beet is not correlated with the constitutive expression of variant polypeptides. In the S₃ cytoplasm, however, an additional 6 kDa polypeptide was synthesized and in the S₄ cytoplasm an additional 10 kDa polypeptide was observed when compared with the F cytoplasm. The expression of cytoplasmic male sterility in sugar beet may be associated with these variant polypeptides. The mitochondrial polypeptides synthesized were identical in plants with different nuclear backgrounds but with identical S₁ cytoplasms. Mitochondria from plants with variants of the S₄ cytoplasm in the same nuclear genotype also showed identical patterns of polypeptide synthesis, including the synthesis of the 10 kDa S₄-specific polypeptide. Pulse-chase experiments did not affect the synthesis of this polypeptide.     

The localization of the nuclear protein pool in Xenopus oocytes

It has been demonstrated previously that nuclear proteins in Xenopus oocytes are synthesized in the cytoplasm and maintained in a cellular pool. The present study was performed to determine if any portion of this pool is associated specifically with the nuclear envelope. This was accomplished by first micro-injecting oocytes with [³H]leucine; at various times after injection, nuclear envelope and nucleoplasmic fractions were run on SDS-polyacrylamide gels. In this way labeled polypeptides available in the envelope fraction could be compared to polypeptides which were subsequently incorporated into the nucleoplasm. No evidence was obtained that the nuclear protein pool is associated with the envelope.     

Role of silicon in diatom metabolism : X. Polypeptide labelling patterns during the cell cycle, silicate starvation and recovery in Cylindrotheca fusiformis

The soluble polypeptides from Cylindrotheca fusiformis were labelled with [³⁵S]O₄²⁻ and resolved by two-dimensional gel electrophoresis. More than 600 polypeptides were detected upon a 26-day exposure to X-ray film. Analysis of the labelling pattern during the cell cycle show that labelling of at least 208 polypeptides changes; the majority, however, remain unchanged. Most of the changes occur in the beginning of the cell cycle and typically involve increases; those occurring in the second half of the cycle typically involve decreases. Light or its absence affects apparent protein turnover and the labelling rates of several polypeptides. Polypeptide labelling during the cell cycle was used as a reference to analyse the effect of silicate deprivation on diatom metabolism. In the absence of silicate, protein turnover increases: however, the addition of silicate counteracts but does not fully reverse this change. Silicate starvation affects the program of synthesis for several polypeptides, but in general the program of polypeptide labelling continues up to the S phase of the cell cycle. Addition of silicate to silicate-starved cells causes the appearance of four hitherto undetected polypeptides.     

Salt-induced conformational change of random copolymers (Lys50Tyr50)n and (Lys50Phe50)n studied by 1H- and 13C-nuclear magnetic resonances. Aggregation behavior of helical segments

¹H- and ¹³C-nmr studies of conformational transitions of random amino acid copolymers containing aromatic residues (Lys⁵⁰Tyr⁵⁰)_n and (Lys⁵⁰Phe⁵⁰)_n in the presence of neutral salts were performed to serve as models of the aggregation behavior of polypeptides of biological significance. The ¹H and ¹³C signal intensities of Tyr and Phe residues decreased preferentially with increasing concentration of neutral salts such as NaCl and NaClO₄. This behavior contrasts with that of (Lys)_n in the presence of similar neutral salts, where the displacement of the ¹³C signal is clearly seen on transition from the random-coil to the helical conformation. On the basis of the previous conformational studies, the loss of the peak areas is ascribed to the presence of immobilized helical segments by hydrophobic interaction between aromatic side chains. The remaining resonances are due to the residual random-coil regions, since the values of nuclear Overhauser enhancements and chemical shifts are unchanged in the presence and absence of the neutral salts.     

The identification of polypeptides synthesised during the acquisition of teichoic acid synthetic activity in Bacillus licheniformis

An attempt has been made to identify proteins synthesised during induction of teichoic acid synthesis in Bacillus licheniformis ATCC 9945. The proteins are recognised as those produced on the change from teichuronic acid to teichoic acid synthesis that occurs after the transfer of the bacteria from phosphate-limited to phosphate-rich conditions. B. licheniformis was grown in phosphate-limiting conditions in the presence of threonine to stimulate threonine uptake. The bacteria were then transferred to phosphate-rich conditions and were pulsed-labelled with [¹⁴C]threonine during the change to teichoic acid synthesis. All of the proteins were extracted from the cells with sodium dodecyl sulphate and were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Radioactive polypeptides were identified by fluorography of the polyacrylamide gels. The radioactive polypeptides that were formed on change from teichuronic acid to teichoic acid synthesis were compared with the polypeptides present in a membrane sub-fraction that had high teichoic acid-synthesising activity. The labelling of nine polypeptides with [¹⁴C]threonine was dependent on new RNA synthesis. Of these nine polypeptides, five were also present in the membrane sub-fraction with the highest teichoic acid-synthesising activity.     

A comparative study on the two classes of heterogeneous nuclear ribonucleoprotein particles separated in metrizamide density gradient,by electrophoresis of proteins and chase experiments. Evidence for two distinct subfractions of HnRNP in mammalian nuclei

Heterogeneous nuclear ribonucleoprotein particles (HnRNP) were separated in metrizamide density gradients, into two fractions migrating to 1.31 g ml^-1 and 1.18 g ml^-1, respectively. Proteins associated with each of these fractions were analysed by SDS-acrylamide gel electrophoresis. It is shown that the whole proteins extracted from these two metrizamide fractions exhibit clearly different electrophoretic patterns: 1.31 HnRNP particles contain as major polypeptide chains molecules with molecular weights ranging from 40 000 to 65 000, while major polypeptides of 1.18 HnRNP are banding in the 30 000–40 000 molecular weight region of the gels. Both fractions contain numerous other associated polypeptide chains whose molecular weights are above 65 000. A possible kinetic relationship between these two HnRNP classes was investigatedin vivo by performing chase experiments. No clear evidence for a precursor-product relationship was found. Implications arising from these structural and kinetic observations, and problems relating to nuclear maturation of pre-messenger RNA, are discussed.     

A high-resolution 13C-nmr study of collagenlike polypeptides and collagen fibrils in solid state studied by the cross-polarization–magic angle-spinning method. Manifestation of conformation-dependent 13C chemical shifts and application to conformational characterization

We have recorded high-resolution ¹³C-nmr spectra of collagen fibrils in the solid state by the cross-polarization–magic-angle-spinning(CP–MAS)method and analyzed the spectra with reference to those of collagenlike polypeptides. We used two kinds of model polypeptides to obtain reference ¹³C chemical shifts of major amino acid residues of collagen (Gly, Pro, Ala, and Hyp): the 3₁-helical polypeptides [(Gly)_nII, (Pro)_nII, (Hyp)_n, and (Ala? Gly? Gly)_nII], and the triple-helical polypeptides [(Pro? Gly? Pro)_n and (Pro? Ala? Gly)_n]. Examination of the ¹³C chemical shifts of these polypeptides, together with our previous data, showed that the ¹³C chemical shifts of individual amino acid residues are the same, within experimental error (±0.5 ppm), among different polypeptides with different primary sequences, if the conformations are the same. We found that the ¹³C chemical shifts of Ala residues of the 3₁-helical (Ala? Gly? Gly)_n and triple-helical (Pro? Ala? Gly)_n are significantly displaced, compared with those of the α-helix, β-sheet, and silk I form, and can be utilized as excellent probes to examine conformational features of collagen-like polypeptides. Further, the ¹³C chemical shifts of Gly and Pro residues in the triple-helical polypeptides are substantially displaced from those found in (Gly)_nII and (Pro)_nII of the 3₁-helix, reflecting further conformational change from the 3₁-helix to the supercoiled triple helix. In particular, the ¹³C chemical shifts of Gly C ? O carbons of the triple-helical polypeptides are substantially displaced upfield (4.1–5.1 ppm), with respect to those of the 3₁-helical polypeptides. These displacements are interpreted by that Gly C ? O of the former is not involved in NH …? O ? C hydrogen bonds, while this carbon of the latter is linked by these kinds of hydrogen bonds. On the basis of these ¹³C chemical shifts, as reference data for the collagenlike structure, we were able to assign the ¹³C-nmr peaks of Gly, Ala, Pro, and Hyp residues of collagen fibrils, which are in good agreement with the values expected from the model polypeptides mentioned above. We also discuss a plausible conformational change of collagen fibrils during denaturation.     

Effects of Prolonged UV-B Enhanced Fluorescent Radiation on Some Marine Microalgae

The eukaryotic unicellular microalgae Chlorella salina, Dicrateria inornata, and Isochrysis galbana were grown under control (fluorescent 20 W m^–2) and UV-B enhanced (UV-B^E, 0.5 W m^–2) fluorescent radiation. The growth rate showed marginal increase under UV-B^E. Decrease in protein content was observed in Dicrateria cells but in Chlorella an initial increase up to 4 d and in Isochrysis an increase at days 4 and 5 was noted. The chlorophyll a content showed marked increase in Chlorella and Isochrysis but in Dicrateria a decline was found. UV-B^E reduced the photosynthetic activity in all three species, but the reduction was larger in Chlorella and Dicrateria. Fluorescence excitation spectra for F₆₈₂ in Chlorella cells grown for 5 d under UV-B^E showed reduction in all peaks. In contrast to this, in Dicrateria and Isochrysis cells, the 530 and 590 nm excitation peaks increased with an appreciable decrease in the 466 nm peak. SDS-PAGE analysis revealed significant decrease in the contents of 47, 33, and 23 kDa polypeptides in Chlorella cells. In Dicrateria cells, significant loss in the content of 55, 38, and 18 kDa polypeptides was observed. The content of low molecular mass polypeptides (15 kDa) remained unaffected. Isochrysis cells were more stable in preserving the content of thylakoid polypeptides.     

A precursor of the reserve-protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons

Developing cotyledons of Phaseolus vulgaris L. were labeled for 30 min with [³H] amino acids, homogenized, and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [³H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm^-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl₂ indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER, and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [¹⁴C] amino acids and [³H] glucosamine or with [¹⁴C] amino acids and [³H] mannose, the [³H]/[¹⁴C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility on SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.     

The Light Harvesting System of the Diatom Cyclotella cryptica. Isolation and Characterization of the Main Light Harvesting Complex and Evidence for the Existence of Minor Pigment Proteins*

The main chlorophyll a/c light harvesting complex of the diatom Cyclotella cryptica was isolated by sucrose density gradient centrifugation. It consisted of two polypeptides of Mrs 18000 and 22000. Both polypeptides and fragments thereof, obtained by formic acid treatment, were blocked at their N-ter-mini. An antiserum raised against the two subunits selectively immunolabeled the thylakoid within the chloroplasts. The subunits were nuclear encoded and could be immunoprecipitated from poly (A)⁺ RNA as precursor proteins in the Mr range of 20000 to 24000. The existence of minor chlorophyll protein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions. Low light grown cells contained more fucoxanthin and less β-carotene relative to chlorophyll a than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthin increased five-fold relative to chlorophyll a under high light conditions. Western-immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorophyll a/c antenna complexes demonstrated that, beside the dominating chlorophyll a/c light harvesting complex, minor antenna complexes might exist, which, in part, seem to react to the light climate applied.     

COMPARATIVE MEASUREMENTS OF NUCLEAR DNA IN A HETEROTHALLIC AND A SELF-FERTILE ISOLATE OF THE MYXOMYCETE,DIDYMIUM IRIDIS

Comparative measurements were made of the nuclear Feulgen-DNA content of a heterothallic and a self-fertile isolate of the myxomycete Didymium iridis. Plasmodial nuclei of both isolates contain the diploid amount of DNA. The replicated diploid (4C) values for the heterothallic and the self-fertile isolates are 5.66 and 5.95, respectively. Myxamoebae, however, are quite dissimilar in their nuclear DNA content. Those of the heterothallic isolates, Honduran 1–2 (A¹) and Panamanian 2–4 (A⁷), have mean values of 3.81 and 3.69, whereas myxamoebae of the self-fertile Philippine-1 isolate were found to have a mean value of 6.07. Myxamoebae of the Ph-1 isolate are, therefore, at the same ploidy level as the Ph-1 Plasmodium. Mean DNA values for Ph-1 sporangial nuclei were in category 4C. Measurement of the DNA content of mitotic metaphases in sporangia at T = 6 hr confirmed that the mean DNA content of both Ph-1 myxamoebae and plasmodial nuclei is equivalent to 4C. It is concluded that nuclear phase alternance is lacking in the Ph-1 isolate and that the Plasmodium of this isolate develops by apogamy.     

Nuclear effect on mitochondrial protein expression of the CMS Owen cytoplasm in sugar beet

In sugar beet, cytoplasmic male sterility (CMS) is conferred by the Owen mitochondrion (Svulg). In order to find polypeptides specific to this cytoplasm and putatively involved in CMS, we assessed the protein expressions of Svulg and a non-sterilizing mitochondrion (Nvulg) by in organello protein synthesis of mitochondria isolated from leaves. Given the hydrophobicity of mitochondrial translation products, we compared the in organello synthesis polypeptides of both cytoplasms with an acid-base two-dimensional electrophoresis adapted to hydrophobic protein separation. To evaluate the possible effect of nuclear background, we assessed the mitochondrial protein expression in three different nuclear backgrounds by using three near-isogenic-line pairs. While three to four variant polypeptides were revealed for each nuclear context, each variant polypeptide was specific to a nuclear-cytoplasmic context. Although this study did not enable us to unambiguously find any variant polypeptide related to CMS, we did observe an effect of the nucleus on mitochondrial gene expression. Received: 25 April 2000 / Accepted: 17 October 2000     

Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro

A soluble Ad2 DNA synthesizing complex was prepared from Ad2-infected KB cell nuclei and purified by exclusion chromatography on a BioGel A-50m column. The purified complex was able to synthesize DNA from all regions of the virus genome, as indicated by $\text{Eco}$RI restriction endonuclease analysis of $\text{in vitro}$ labeled DNA. Experiments were performed to identify Ad2-induced early polypeptides present in the complex. Ad2-infected and mock-infected cells were labeled with [³⁵S]methionine 7–10 h postinfection, then incubated for 8 h to allow the ³⁵S-labeled early polypeptides to become associated with the complex. The polypeptides in the purified complex and each of the cell fractions were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. The major components of the purified complex were the 73K DNA binding phosphoprotein and 11K, two adenovirus 2-induced early polypeptides. The 11K has a preferred nuclear location. Small quantities of other Ad2-induced early proteins, 21K, 15K, and possibly 8.3K were also associated with the complex.     

Caspase-3 activates endo-exonuclease: Further evidence for a role of the nuclease in apoptosis

Single-strand DNase and poly rAase, activities characteristic of endo-exonuclease, were co-activated in nuclear fractions of HL-60 cells by caspase-3. Activation was accompanied by cleavages of large soluble polypeptides (130–185 kDa) and a 65 kDa inactive chromatin-associated polypeptide related to the endo-exonuclease of Neurospora crassa as detected on immunoblots. The major products seen in vitro were a 77 kDa soluble polypeptide and an active chromatin-associated 34 kDa polypeptide. When HL-60 cells were induced to undergo apoptosis by treating with 50 M etoposide (VP-16) for 4 hours, 77 kDa and 40 kDa polypeptides accumulated in nuclear fractions. Chromatin DNA fragmentation activity was also activated in cytosol and nuclear extract either by pre-treating the cells in vivo with VP-16 or by treating the cytosol in vitro with caspase-3 or dATP and cytochrome c. Endo-exonuclease activated by caspase-3 in cytosol-derived fractions augmented chromatin DNA fragmentation activity in vitro. Endo-exonuclease is proposed to act in vivo in conjunction with the caspase-activated DNase (CAD) to degrade chromatin DNA during apoptosis of HL-60 cells.     

Chloride relations of photosystem II membrane preparations depleted of,and resupplied with,their 17 and 23 kDa extrinsic polypeptides

Photosystem II membranes prepared from thylakoids of Phytolacca americana chloroplasts were depleted of their intrinsic 17 and 23 kDa polypeptides, and the effects of a reconstitution of these polypeptides on the Cl^– requirements of O₂ evolution activity were analyzed. It was found that the activating effectiveness of limiting amounts of added Cl^– was increased several fold by an addition of the 23 kDa polypeptide. When it was supplemented by the 17 kDa species, only a small additional increase occurred, but Cl^– retention in Cl^– free media was enhanced greatly. Addition of the 17 kDa polypeptide alone was without effect because it is known that it cannot bind to its native site unless the 23 kDa polypeptide is in place.Optimal enhancements of the effectiveness of activating added Cl^– were observed when the assays were done in the presence of the reconstituting polypeptides. When the reconstituting treatment with the polypeptides, and the assay of the Cl^– relations, were separated, it was advantageous to have Cl^– present in the reconstituting medium, and not to add Ca²⁺, another cofactor of photosynthetic water oxidation. Those requirements are attributed to the labilizing effects Cl^– free conditions and divalent cations have on the association of especially the 23 kDa polypeptides with the water oxidizing complex, and to a possible aggregation of the membranes under the influence of Ca²⁺ which might have impeded proper polypeptide binding.Abbreviations Chl Chlorophyll a+b - Mes 4-morpholineethanesulfonic acid - PSII photosystem II - SDS and LDS sodium or lithium dodecylsulfate     

PROTOPLAST FUSION AND GENETIC COMPLEMENTATION OF PIGMENT MUTATIONS IN THE RED MICROALGA PORPHYRZDZUM SP.1

Protoplasts from two green pigment mutants of Porphyridium sp. (UTEX 637) containing a low phycoerythrin level were fused by exposure to polyethylene glycol (MW 6000) combined with a short heat shock (45° C, 5 min). Following regeneration on agar plates, red colonies arose in which complementation of the phycoerythrin deficiency had occurred. The complementation frequency was estimated to be 0.2%. Eight progeny showing red pigmentation were isolated and purified by consecutive transfers on agar plates. Characterization of the fusion progeny revealed that their phycobiliprotein and chlorophyll contents per cell were higher than those of their parental mutant strains and, in most strains, similar to that of the wild type. The fusion products proved to be stable over many growth cycles. The DNA content of the wild type and of the parental mutant strains was about 0.05 pg-cell^?1. Fusion progeny strains showed a variable DNA content: a few fusants contained the same amount of DNA as the wild type and the parental strains, while others had about 50% more DNA per cell. The DNA content of one of the progeny strains (CF1c) was double that of the wild type (0.1 pg. cell^?1). Cells of this fusion progeny contained one nucleus per cell, which suggests that nuclear fusion and the formation of a stable diploid followed cell fusion. Analysis of phycobilisome components of CF1c revealed complementation of linker polypeptides associated with phycoerythrin (γ subunits). CF1c contained, like the wild-type strain, four linker polypeptides; all of these were absent in one parental strain and one was absent in the second. To the best of our knowledge, this is the first report of protoplast fusion, formation of somatic hybrids, and the apparent completion of a parasexual cycle in a red microalga.