Restriction endonuclease analysis of plastid DNA from tomato, potato and some of their somatic hybrids

Summary The buoyant density and endonuclease restriction patterns of potato (Solanum tuberosum L.) and tomato (Lycopersicon esculentum) ptDNA were examined and compared with those of their somatic hybrids. The plastids from these plants, both of which belong to the family of Solanaceae, contain a single DNA species whose density of 1.697 gcm^-3 and size of approximately 156 kbp are similar to those of ptDNA from other higher plants. The Sal I restriction patterns were indistinguishable; however, those obtained with Kpn I, Pst I, and Eco RI disclosed that each species possesses a unique ptDNA. These observations suggest a relatively recent divergence of both species. Of the twelve hybrid lines screened, eight contained exclusively potato ptDNa and four contained only tomato ptDNA at a 0.1–3% level of detection. Rearrangements of modifications of DNA were not detected. The plastid identities of three hybrid lines that had previously been analyzed by isoelectric focusing of RuBPcase subunits (Melchers et al. 1978) agreed with those determined by restriction endonuclease analysis.Abbreviations used in the text ptDNA plastid DNA, chloroplast DNA - cDNA copy DNA - RuBPcase ribulose bisphosphate carboxylase/oxygenase - LSU large subunit of RuBPcase - kbp kilobase pairs - SDS sodium dodecyl sulfate - SSC standard saline citrate - IEF isoelectric focusing     

Interaction of Cytokinins and Abscisic Acid During Regulation of Stomatal Opening in Bean Leaves

Effects of benzyladenine (BA) and abscisic acid (ABA) applied separately or simultaneously on parameters of gas exchange of Phaseolus vulgaris L. leaves were studied. In the first two experimental sets) 100 M ABA and 10 M BA were applied to plants sufficiently supplied with water. Spraying of leaves with ABA decreased stomatal conductance (g _s) and in consequence transpiration rate (E) and net photosynthetic rate (P _N) already 1 h after application, but 24 h after application the effect almost disappeared. 10 M BA slightly decreased gas exchange parameters, but in simultaneous application with ABA reversed the effect of ABA. Immersion of roots into the same solutions markedly decreased gas exchange parameters and 24 h after ABA application the stomata were completely closed. The effect of ABA was ameliorated by simultaneous BA application, particularly after 1-h treatment. In the third experimental set, plants were pre-treated by immersing roots into water, 1 M BA, or 100 M ABA for 24 h and then the halves of split root system were dipped into different combinations of 1 M BA, 100 M ABA, and water. In plants pre-treated with ABA all gas exchange parameters were small and they did not differ in plants treated with H₂O+H₂O, H₂O+BA, or BA+BA. In plants pre-treated with BA or H₂O, markedly lower values of P _N were found when both halves of roots were immersed in ABA. Further, the effects of pre-treatment of plants with water, 1 M BA, 100 M ABA, or ABA+BA on the development of water stress induced by cessation of watering and on the recovery after rehydration were followed. ABA markedly decreased gas exchange parameters at the beginning of the experiment, but in its later phase the effect was compensated by delay in development of water stress. BA also delayed development of water stress and increased P _N in water-stressed leaves. BA reversed the effect of ABA at mild water stress. Positive effects of BA and ABA pre-treatments were observed also after rehydration.     

Geographic structuring of chloroplast DNA genotypes inTiarella trifoliata (Saxifragaceae)

Tiarella trifoliata comprises varietieslaciniata, trifoliata, andunifoliata, and is distributed from southeastern Alaska to northern California. We analyzed restriction site variation of chloroplast DNA (cpDNA) using 23 endonucleases in 76 populations representing the entire geographic range of the species and the three recognized varieties. We also employed comparative restriction site mapping of PCR-amplified chloroplast DNA fragments using 16 restriction endonucleases. This species exhibits low cpDNA restriction site variation. No differentiation is evident among varieties of this species based on cpDNA data; some plants of each variety were characterized by each of the two major cpDNA types detected. The two major cpDNA clades, which differ by only a single restriction site mutation, are geographically structured. A northern clade comprises populations from Alaska to central Oregon; most populations analyzed from southern Oregon and California form a southern clade. Populations that possess the typical northern cpDNA type also occur disjunctly to the south at high elevations in the Siskiyou—Klamath Mountain area of southern Oregon and northern California. Conversely, the southern cpDNA type is found disjunctly to the north in the Olympic Peninsula of Washington. Both geographic areas characterized by disjunct cytoplasms are considered glacial refugia.Tiarella trifoliata joins two other species,Tolmiea menziesii andTellima grandiflora, in having well-demarcated northern and southern cpDNA lineages. All three species have similar life-history traits and geographic distributions. We suggest that glaciation may have played a major role in the formation of the cpDNA discontinuities present in these three taxa. The pronounced relationship between cpDNA variation and geographic distribution suggests the potential applicability of intraspecific phylogeography to plants via the analysis of intraspecific cpDNA variation. These three examples also join a rapidly growing data base which indicates that cytoplasms are often geographically structured within species and species complexes.     

Chloroplast DNA inheritance in theStellaria longipes complex (Caryophyllaceae)

Inheritance of chloroplast DNA (cpDNA) was examined in F₁ progenies derived from three crosses and three corresponding reciprocal crosses betweenStellaria porsildii andS. longifolia. Chloroplast DNA restriction fragments were analyzed using methods of nonradioactive digoxigenin-11-dUTP labeling and chemiluminescent detection with Lumi-Phos 530. Distinct interspecific restriction fragment polymorphisms were identified and used to demonstrate the mode of cpDNA inheritance. Mode of cpDNA inheritance differed among crosses. Two crosses in whichS. porsildii, SP2920-21, was the maternal parent exhibited three different types of plastids, maternal, paternal and biparental, among the F₁ hybrids, suggesting a biparental cpDNA inheritance and plastid sorting-out inStellaria.     

Mitochondrial DNA from Podospora anserina. II. Properties of mutant DNA and multimeric circular DNA from senescent cultures.

Summary Mitochondrial (Mt) DNA from mitochondrial mutants of race s Podospora anserina and from senescent cultures of races s and A was examined. In mutants, we observed that fewer full length circles (31 ) were present; instead, smaller circles characteristic for each mutant sudied were found. Eco Rl digestion of these mutant MtDNAs indicated that in certain mutants, although specific fragments were absent, the total molecular weight of the fragments was not much different than wild-type.The properties of senescent MtDNA was strikingly different from either wild-type or mutant Mt DNA. First, a multimeric set of circular DNA was observed for both race s and A, with a monomeric repeat size of 0.89 . These circles ranged in size from 0.89 to greater than 20 ; only one molecule out of some 200 molecules was thought to be of full length (31 ). Density gradient analysis showed that there were two density species: a majority were at the same density as wild-type (1.694 g/cm³) and a second at 1.699 g/cm³. Most of the circular molecules from MtDNA isolated by either total DNA extraction or by extraction of DNA from isolated mitochondria were contained in the heavy DNA fraction. Eco R1 enzymatic digestion indicated that the light DNA had several fragments (amounting to about 23×10⁶ daltons) missing, compared with young, wild-type MtDNA. Heavy senescent MtDNA was not cleaved by Eco R1. Analysis with Hae III restriction endonuclease showed also that light senescent MtDNA was missing certain fragments. Heavy MtDNA of average size 20×10⁶ daltons, yielded only one fragment, 2,500 bp long, by digestion with Hae III restriction endonuclease. Digestion of heavy DNA with Alu I enzyme yielded 10 fragments totalling 2,570 bp. By three criteria, electron-microscopy, Eco R1 and Hae digestion, we conclude that the heavy MtDNA isolated from senescent cultures of Podospora anserina consisted of a monomeric tandemly repeating subunit of about 2,600 bp length.These results on the properties of senescent MtDNA are discussed with regard to the published properties of the rho ^- mutation in the yeast, S. cerevisiae.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Heteroplasmy of chloroplast DNA in Medicago

Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.Contribution No 88-547-J from the Kansas Agricultural Experiment Station, Manhattan.     

Analysis of cytoplasmic genomes in somatic hybrids between navel orange (Citrus sinensis Osb.) and ‘Murcott’ tangor

Summary Somatic hybrid plants were produced by protoplast fusion of navel orange and Murcott tangor. Hybridity of the plants was confirmed by the restriction endonuclease analysis of nuclear ribosomal DNA. All of the plants (16 clones) were normal, uniform, and had the amphidiploid chromosome number of 36 (2n=2x=18 for each parent). The cpDNA analysis showed that each of the 16 somatic hybrids contained either one parental chloroplast genome or the other. In all cases, the mitochondrial genomes of the regenerated somatic hybrids were of the navel orange type.Contribution No. E-132 of the Fruit Tree Research Station     

High frequency shoot regeneration from leaf explants of muskmelon

Efficient in vitro plant regeneration systems are critical for many purposes including plant transformation. Current regeneration systems for melon (Cucumis melo L.) plants generally utilize cotyledon explants; regeneration from melon leaves has received limited attention. We investigated several factors that influence regeneration from melon leaves including: genotype growth conditions and age of the source plant, leaf age, explant orientation, gelling agent, and the addition of silver nitrate and sulfonylurea herbicide to the culture media. Critical factors that influenced regeneration were preculture conditions of the donor plants, leaf size, and the use of silver nitrate and Phytagel in the medium. The best results were obtained with 3–4 cm diam leaves excised from pot grown greenhouse or growth chamber plants cultured on MS medium with 5 M IAA, 5 M BA, 1 M ABA, 30 M silver nitrate and 2.6 g l^-1 Phytagel. Low concentratons of sulfonylurea herbicide (0.25 mg l^-1 DPX-M 6316) also enhanced regeneration. Under optimized conditions 80–100% of the explants regenerated, with 10–100 shoots per explantAbbreviations ABA abscisic acid - BA benzyladenine - IAA indole-3-acetic acid - MS Murashige and Skoog medium - NAA naphthalene acetic acid     

Mapping of genes on the chloroplast DNA of Spirodela oligorhiza

With the use of spinach chloroplast RNAs as probes, we have mapped the rRNA genes and a number of protein genes on the chloroplast DNA (cpDNA) of the duckweed Spirodela oligorhiz. For a more precise mapping of these genes we had to extend the previously determined [14] restriction endonuclease map of the duckweed cpDNA with the cleavage sites for the restriction endonucleases Sma I and Bgl I. The physical map indicates that duckweed cpDNA contains two inverted repeat regions (18 Md) separated by two single copy regions with a size of 19 Md and 67 Md, respectively.By hybridization with spinach chloroplast rRNAs it could be shown that each of the two repeat units contains one set of rRNA genes in the order: 16S rRNA gene — spacer — 23S rRNA gene — 5S rRNA gene.A spinach chloroplast mRNA preparation (14S RNA), which is predominantly translated into a 32 Kilodalton (Kd) protein [9], hybridized strongly to a DNA fragment in the large single copy region, immediately outside one of the inverted repeats. With another mRNA preparation (18S), which mainly directs the in vitro synthesis of a 55 Kd protein [9], hybridization was observed with two DNA regions, located between 211° and 233° and between 137° and 170°, respectively. Finally, with a spinach chloroplast genomic probe for the large subunit of ribulose 1,5-bisphosphate carboxylase [17], hybridization was found with a DNA fragment located between 137° and 158° on the map.     

Progeny analysis of the interspecific somatic hybrids: Nicotiana tabacum (CMS) + Nicotiana sylvestris with respect to nuclear and chloroplast markers

Summary The progeny of a fusion experiment involving N. sylvestris protoplasts and X-irradiated protoplasts of the cytoplasmic male sterile Line 92 (N. tabacum nucleus and alien, male-sterility inducing, cytoplasm) were analyzed. Three groups of somatic hybrid plants resulted: Type A, Type B-1 and Type B-2. These as well as their androgenic progenies and the progenies resulting from their pollination with N. tabacum or N. sylvestris were followed with respect to several nuclear and cytoplasmic traits. Those controlled by the nuclear genome were plant and flower morphologies; those controlled by genetic information in the cytoplasm were tentoxin sensitivity (affecting the coupling factor of chloroplast ATPase), the large subunit of ribulose bisphosphate carboxylase and the restriction endonuclease pattern of plastid DNA. A further cytoplasmic trait investigated (exact site of genetic control not known) was male sterility. The examinations of the somatic-hybrid groups and their respective progenies indicated that: Type A plants have N. sylvestris nuclei and Line 92 plastids; Type B-1 plants also have Line 92 plastids but their genome is composed of N. sylvestris and N. tabacum nuclei; Type B-2 plants with impaired male fertility had N. sylvestris plastids and N. sylvestris nuclei.     

Repetitive DNA sequences in methotrexate- and methasquin-sensitive and -resistant Chinese hamster cell lines

DNA purified from a Chinese hamster cell line of lung fibroblast origin (DC83F) was analyzed by density gradient centrifugation and by gel electrophoresis after restriction endonuclease digestion in order to fractionate discrete repetitive fractions within the total DNA. No obvious satellite DNAs were resolved using the CsCl or Ag-Cs₂SO₄ density gradient conditions described herein. However, analysis of the digestion products of a battery of restriction endonucleases indicated that three of these enzymes, EcoR ₁, HaeIII, and XhoI, yielded discrete fragments which could be visualized with EtBr staining or identified by scintillation counting of [ ³ H] DNA. DNAs from several highly ( hundredfold increased resistance) antifolate-resistant sublines of DC-3F, characterized by a large homogeneously staining region (HSR) in the chromosome complement, were examined with both techniques and compared to the parental, antifolate-sensitive cell line DNA. The density gradient profiles and electrophoretic patterns of restriction endonuclease digests were identical among all the cell lines examined and were indistinguishable from those of the parental DC-3F DNA.This work was supported in part by grants to the Sloan-Kettering Institute and to J.L.B. and P.W.M. by the National Institutes of Health and the Fairchild New Frontiers Fund. Portions of this study were presented at the 18th Annual Meeting of the American Society for Cell Biology held in San Antonio, Texas, November 4–8, 1978.     

Rapid immunodetection of Escherichia coli

A filtration flow-through design was used to develop the rapid immunodetection of Escherichia coli. Polyclonal anti-E. coli IgG was conjugated to small, 0.8 Blue latex beads. Cells were mixed with conjugated beads in the presence of anti-E. coli monoclonal IgM. The suspension was then filtered through a 5 nitrocellulose membrane. The cell-containing complexes were effectively collected on the filter, forming a blue spot. The method produced reliable detection of E. coli at a concentration of 10⁵ cells ml^–1, which is a current benchmark figure for urinary tract infection (UTI) diagnosis.     

On the use of Avena protoplasts to study chloroplast development

Different methods were tested to isolate protoplasts from etiolated, partially greened, and light-grown leaves of Avena sativa. Preparations with high yields and high photosynthetic capacities (time of illumination 4 h) were obtained when small transverse leaf segments were incubated for 2 h at 30°C in 2% cellulysin (Calbiochem), 0.6 M mannitol, and 0.5% bovine serum albumin (BSA) at pH 5.6, without shaking. As measured by light-dependent O₂ evolution or fixation of labeled bicarbonate, protoplasts exhibited rates of up to 124 mol per mg of chlorophyll per h at 20°C and saturating bicarbonate, which were nearly identical to those found with intact leaves. The assay conditions necessary for this activity were 0.6 M sorbitol, 50 mM N-2-hydroxy-ethylpiperazine-N-2-ethane sulfonic acid (pH 7.6), and 10 mM NaHCO₃. If plastids were isolated from these protoplasts, sorbitol was 0.45 M, including 10 mM ethylenediaminetetraacetate (EDTA). under these conditions, rates of photosynthesis were up to 125 (light-grown) and 71 (6 h illuminated) mol O₂ evolved or ¹⁴CO₂ fixed per mg of chlorophyll per h, compared to 3.5 mol·mg chl^-1·h^-1 obtained with mechanically isolated plastids. With this system, CO₂-dependent O₂ evolution was already detected after 3 h of illumination of etiolated tissue, but could only be observed at pH values between 7.6 and 8.6, in the presence of EDTA. At lower pH (7.3) or at pH 7.6 in the absence of EDTA, light-dependent O₂ evolution up to 24 h of greening was only measurable with 3-phosphoglycerate as the substrate. The possible effects of EDTA in this respect as well as the advantages of using protoplasts or plastids isolated from protoplasts for developmental studies are discussed.Abbreviations BSA bovine serum albumin - EDTA ethylenediamine tetraacetic acid - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane-sulphonic acid - MES 2(N-morpholino) ethane sulphonic acid - PGA 3-phosphoglycerate     

Effect of the antioxidant ionol (BHT) on growth and development of etiolated wheat seedlings: control of apoptosis, cell division, organelle ultrastructure, and plastid differentiation

Ionol (BHT), a compound having antioxidant activity, at concentrations in the range 1-50 mg/liter (0.45·10^-5-2.27·10^-4 M), inhibits growth of etiolated wheat seedlings, changes the morphology of their organs, prolongs the coleoptile life span, and prevents the appearance of specific features of aging and apoptosis in plants. In particular, BHT prevents the age-dependent decrease in total DNA content, apoptotic internucleosomal fragmentation of nuclear DNA, appearance in the cell vac-uole of specific vesicles with active mitochondria intensively producing mtDNA, and formation of heavy mitochondrial DNA ( = 1.718 g/cm³) in coleoptiles of etiolated wheat seedlings. BHT induces large structural changes in the organization of all cellular organelles (nucleus, mitochondria, plastids, Golgi apparatus, endocytoplasmic reticulum) and the formation of new unusual membrane structures in the cytoplasm. BHT distorts the division of nuclei and cells, and this results in the appearance of multi-bladed polyploid nuclei and multinuclear cells. In roots of etiolated wheat seedlings, BHT induces intensive synthesis of pigments, presumably carotenoids, and the differentiation of plastids with formation of chloro- or chromoplasts. The observed multiple effects of BHT are due to its antioxidative properties (the structural BHT analog 3,5-di-tert-butyltoluene is physiologically inert; it has no effect similar to that of BHT). Therefore, the reactive oxygen species (ROS) controlled by BHT seem to trigger apoptosis and the structural reorganization of the cytoplasm in the apoptotic cell with formation of specific vac-uolar vesicles that contain active mitochondria intensively producing mtDNA. Thus, the inactivation of ROS by BHT may be responsible for the observed changes in the structure of all the mentioned cellular organelles. This corresponds to the idea that ROS control apoptosis and mitosis including formation of cell wall, and they are powerful secondary messengers that regulate dif-ferentiation of plastids and the Golgi apparatus in plants.     

A method for the deletion of restriction sites in bacterial plasmid deoxyribonucleic acid

Summary A general method has been developed for the deletion of restriction endonuclease sites in bacterial plasmid DNA. The procedure involves partial digestion of the covalently closed circular plasmid DNA with an appropriate restriction endonuclease under conditions which allow accumulation of unit-length linear DNA molecules, controlled digestion of the exposed 5 ends with the 5-exonuclease, and in vivo recircularization of the resulting linear DNA in a bacterial host cell. The method has been used for the deletion of one of the two EcoRI sites in the plasmid pML2 (colE1-Km). Two of the resulting plasmids, pCR1 and pCR11, have a single EcoRI cleavage site, but retain genetic determinants specifying resistance to colicin E1 and kanamycin, and thus may be useful as vectors for the cloning and amplification of DNA in bacteria.     

Adventitious shoot regeneration from leaf explants of tissue cultured and greenhouse-grown raspberry

Adventitious shoot regeneration was observed using leaf-petiole explants from shoot-proliferating cultures of Comet red raspberry (Rubus idaeus L.). A maximum regeneration rate of 70% (3.7 shoots/explant) was obtained using 4.5–9.1 M (1–2 mg l^–1) N-phenyl-N-1,2,3-thiadiazol-5-ylurea (thidiazuron or TDZ) with 2.5–4.9 M (0.5–1 mg l^–1) 1H-indole-3-butanoic acid (IBA) or 2.3 M (0.5 mg l^–1) TDZ with 4.9 M (1 mg l^–1) IBA in modified Murashige-Skoog medium. TDZ was more effective than N-(phenylmethyl)-1H-purin-6-amine (BA) at promoting regeneration in combinations tested with IBA (maximum 50% regeneration rate; 1.8 shoots/explant). Variation in the agar concentration or incubation temperature, orientation or scoring of the leaf-petiole explants and use of separate leaf or petiole explants had no effect on shoot regeneration. Incubation in the dark for 1, 2 or 3 weeks prior to growth in the light did not influence the percent regeneration rate but depressed the number of adventitious shoots. Explant source, from micropropagated shoots or greenhouse-grown plants, had an effect on shoot regeneration that was genotype dependent. Only 8 of 22 (36%) raspberry cultivars were capable of regeneration from leaf explants derived from greenhouse-grown plants.     

Chloroplast DNA variability in the genus Helianthus: restriction analysis and S1 nuclease mapping of DNA-DNA heteroduplexes

Chloroplast DNA (cpDNA) from 36 wild species of the genus Helianthus has been analysed with three restriction endonucleases (Bam HI, Hind III and Sst I). Out of the 71 restriction sites described on the reference cpDNA (sunflower cpDNA), three insertions/deletions and seven site modifications were detected during the survey of the other cpDNAs.Since restriction mapping showed only a very limited fraction of the DNA variability, we chose to adapt the S1 nuclease mapping technique to detect fine variations between chloroplast genomes. For this purpose, DNA-DNA heteroduplexes obtained between sunflower and wild-species DNAs were digested by S1 nuclease and the resulting mismatches were detected by classical endonuclease restriction and hybridization methods. The S1 nuclease mapping results were confirmed by sequencing one S1 nuclease-sensitive region detected between cultivated sunflower and two perennial wild-type species.As a result of these analyses, it appeared that the combination of restriction mapping and S1 nuclease mapping might be helpful to differentiate taxonomically close cytoplasms.