Promoters active in mammalian cells (pSV40) and in plants (pNOS) permit expression in yeast of the Tn5 APH(3') II gene

Abstract Four plasmids were constructed by associating Escherichia coli and yeast selection markers and replication origins to a structural gene coding for aminoglycoside phosphotransferase (APH(3')) controlled by different flanking sequences. We used the two bacterial genes of Tn5 (APH(3')II) and Tn903 (APH(3')I) as such and the chimeric pSVneo (APH(3')II) and pNOSneo (APH(3')II) constructs, functional in mammalian and plant cells, respectively. Yeast clones resistant to G418 were obtained with all plasmids except with that bearing the bacterial APH(3')II gene. The three plasmids harbouring the functional APH genes, however, conferred different levels of G418 resistance to yeast.     

Interspecific hybrids between Brassica napus L. and B. oleracea L. developed by embryo culture

Summary Interspecific hybrids between Brassica napus and B. oleracea are difficult to produce, and previous attempts to transfer economic characters from one species to the other have largely been unsuccessful. In these studies, oilseed rape cv. Tower (2n38) (B. napus) was crossed with broccoli and kale (2n18) (B. oleracea), and hybrid plants were developed from embryos in culture by either organogenesis or somatic embryogenesis. In rape × broccoli, F₁ plants were regenerated from hybrid embryos and the plants produced viable selfed seeds. F₅ plants (2n38) homozygous for white flower colour were selected for high oil content (47%) and Line 15; a selection from these plants produced fertile hybrids with rape, broccoli and kale without embryo culture. In reciprocal crosses between oilseed rape cv. Tower and an aphid resistant diploid kale, 28 and 56 chromosome F₁ hybrid plants were regenerated from somatic embryos. The 56 chromosome plants were self-fertile and it was concluded from F₂ segregation ratios that a single dominant gene controls resistance to cabbage aphid in kale. The 28 chromosome F₁'s were self-sterile, but these and the 56 chromosome F₁'s could be backcrossed to rape and kale. A cross between the F₁ (2n56) and a forage rape resulted in the selection of a cabbage aphid (Brevicoryne brassicae L.) resistant line (Line 3). Both Line 15 and Line 3 can serve as bridges for gene interchange between B. campestris, B. napus and B. oleracea, which has not been possible hitherto. Hybridisations between rape and tetraploid kale produced F₁ plants with 37 chromosomes. One F₂ plant possessed coronal scales and the inheritance was shown to be controlled by a single recessive gene unlinked to petal colour.This paper is dedicated to Mr. T. P. Palmer, a colleague and close friend who retired from the DSIR as Assistant Director of the Crop Research Division in September 1984     

Reaction centers from three herbicide-resistant mutants of Rhodobacter sphaeroides 2.4.1: sequence analysis and preliminary characterization

Many herbicides that inhibit photosynthesis in plants also inhibit photosynthesis in bacteria. We have isolated three mutants of the photosynthetic bacterium Rhodobacter sphaeroides that were selected for increased resistance to the herbicide terbutryne. All three mutants also showed increased resistance to the known electron transfer inhibitor o-phenanthroline. The primary structures of the mutants were determined by recombinant DNA techniques. All mutations were located on the gene coding for the L-subunit resulting in these changes Ile²²⁹ Met, Ser²²³ Pro and Tyr²²² Gly. The mutations of Ser²²³ is analogous to the mutation of Ser²⁶⁴ in the D1 subunit of photosystem II in green plants, strengthening the functional analogy between D1 and the bacterial L-subunit. The changed amino acids of the mutant strains form part of the binding pocket for the secondary quinone, Q _b . This is consistent with the idea that the herbicides are competitive inhibitors for the Q _b binding site. The reaction centers of the mutants were characterized with respect to electron transfer rates, inhibition constants of terbutryne and o-phenanthroline, and binding constants of the quinone UQ₀ and the inhibitors. By correlating these results with the three-dimensional structure obtained from x-ray analysis by Allen et al. (1987a, 1987b), the likely positions of o-phenanthroline and terbutryne were deduced. These correspond to the positions deduced by Michel et al. (1986a) for Rhodopseudomonas viridis.Abbreviations ATP adenosine 5-triphosphate - Bchl bacteriochlorophyll - Bphe bacteriopheophytin - bp basepair - cyt c²⁺ reduced form of cytochrome c - DEAE diethylami-noethyl - EDTA ethylenediamine tetraacetic acid - Fe²⁺ non-heme iron atom - LDAO lauryl dimethylamine oxide - Pipes piperazine-N,N-bis-2-ethane-sulfonic acid - PSII photosystem II - RC reaction center - SDS sodium dodecylsulfate - Tris tris(hydroxy-methyl)aminomethane - UQ₀ 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50     

In vitro expression of partial resistance toPhytophthora palmivora by shoot cultures of papaya

Shoot apices ofCarica papaya were multiplied in vitro on solidified nutrient media supplemented with -naphthyl-acetic acid and 6-benzylaminopurine. The micropropagated shoots were inoculated in vitro, through a stem wound, with a sporangial suspension (1.2×10⁴ sporangia ml^-1) ofPhytophthora palmivora. The symptoms exhibited by the shoots in vitro were similar to those described previously for infection of the whole plant in the field. The time taken for the host tissue to become brown and to wilt and the time to sporulation of the pathogen were all recorded for each shoot of four varieties of papaya challenged with each of ten isolates ofP. palmivora. Significant differences were observed between host-pathogen combinations for these variables and host-specificity was detected amongst the isolates ofP. palmivora. The time taken for the shoot to wilt was positively correlated with the time to sporulation of the isolated but both these variables were negatively correlated with the time to browning of the shoot. In vitro selection for disease resistance will be useful during breeding programmes involving papaya genotypes which are maintained through clonal propagation.     

Isolated cytoskeletons of human blood platelets: dark-field imaging of coiled and uncoiled microtubules

Detergent extraction of human blood platelets pre-treated with Taxol to stabilize microtubules allows isolation of marginal band (MB) cytoskeletons. We studied MB cytoskeleton structure using dark-field light microscopy and negative stain electron microscopy (EM). Dark-field illumination clearly demonstrated the "hoop" shape of MB cytoskeletons in unfixed suspensions where the microtubule coils had a mean diameter of 2.87 microns (+/- 0.18 micron, SD). Microtubules were uncoiled by brief exposure to trypsin (2 ng/micrograms protein) or by NaCl (154-600 mM) but not by DNase I, which removed approximately 40% of total actin, but had no effect on dark-field images of microtubule coils. As microtubules uncoiled, a single fiber emerged from the hoop and gradually lengthened as the brightness of the hoop diminished; these fibers correspond to the single microtubules seen by EM. Polypeptides of coiled and uncoiled MB cytoskeletons were analyzed by SDS-PAGE. When microtubules became uncoiled, no changes in the major components (alpha- and beta-tubulin, IEF-51K, or actin) were found. However, a number (greater than 10) of minor polypeptides, each less than 5% of total cytoskeletal protein and with an Mr ranging from 80,000- greater than 260,000, were decreased in "uncoiled" MB cytoskeletons. These results implicate one or more of these minor polypeptides in maintenance of hoop integrity. Dark-field light microscopy thus provides an approach toward investigating the mechanism(s) involved in maintaining the microtubule coil of the platelet marginal band.     

Selection of an atrazine-resistant tobacco cell line having a mutant psbA gene

Summary A mutant cell line that shows high resistance to the photosynthesis-inhibiting herbicide atrazine was selected from cultured photomixotrophic Nicotiana tabacum cv. Samsun NN cells by repeated exposure to toxic levels of the herbicide. This resistance was confirmed by measurements of Hill reaction activity in isolated thylakoid membranes. Nucleotide sequencing revealed that the resistant cell line had a point mutation in its chloroplast psbA gene. The 264th codon, AGT (serine) was changed to ACT (threonine) in this mutant. This new type of mutation also conferred moderate cross-resistance to diuron and subsequently was stable in the absence of continued selection pressure.     

Site-specific recombination promotes linkage between trimethoprim- and sulfonamide resistance genes. Sequence characterization of dhfrV and sulI and a recombination active locus of Tn21

Summary A new gene for trimethoprim resistance, dhfrV, found in several plasmid isolates with different characteristics, was sequenced and found to correspond to a peptide of 157 amino acids showing 75% similarity with the previously characterized, drug resistant dihydrofolate reductase of type I. The sequenced surroundings of dhfrV in plasmid pLMO20, were found to be almost identical with genetic areas surrounding resistance genes in transposon Tn21 and in R plasmid R388. The trimethoprim resistance genes of pLMO20 and R388 and the spectinomycin resistance gene of Tn21 could be regarded as having been inserted, by recombination, into an evolutionary older structure containg the sulfonamide resistance gene, sulI. The latter gene was sequenced and found to correspond to a peptide of 279 amino acids and with a molecular weight of 30126 daltons. The inserted genes were found to be governed by a promoter situated in the highly conserved structure and also controlling expression of sulI. The insertion points of the different resistance genes were precisely defined, and at the 3 ends of the inserted genes inverted repeats allowing the formation of stem and loop structures were found. Similar structures were found at the 3 ends of the antibiotic resistance genes in Tn7, which could indicate similar recombination mechanisms to be effective in the evolutionary construction of all these different resistance elements.     

Point mutations in the 23 S rRNA genes of four lincomycin resistant Nicotiana plumbaginifolia mutants could provide new selectable markers for chloroplast transformation

Summary Experiments designed to establish stable chloroplast transformation require selectable marker genes encoded by the chloroplast genome. The antibiotic lincomycin is a specific inhibitor of chloroplast ribosomal activity and is known to bind to the large ribosomal subunit. We have investigated a defined region of the chloroplast 23 S rRNA genes from four lincomycin resistant Nicotiana plumbaginifolia mutants and from wild-type N. plumbaginifolia. The mutants LR415, LR421 and LR446 have A to G transitions at positions equivalent to the nucleotides 2058 and 2059 in the Escherichia coli 23 S rRNA. The mutant, LR400, possesses a G to A transition at a position corresponding to nucleotide 2032 of the E. coli 23 S rRNA.     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Monoclonal antibodies to a fern spermatozoid detect novel components of the mitotic and cytokinetic apparatus in higher plant cells

Summary The aim of this study was to search for uncharacterized components of the plant cytoskeleton using monoclonal antibodies raised against spermatozoids of the fernPteridium (Marc et al. 1988). The cellular distribution of crossreacting immunoreactive material during the division cycle in wheat root tip cells was determined by immunofluorescence microscopy and compared to the fluorescence pattern obtained with antitubulin. Five antibodies are of special interest. Pas1D3 and Pas5F4 detect a diffuse cytoplasmic material, which, during mitosis, follows the distribution of microtubules (MTs) at the nuclear surface and in the preprophase band (PPB), spindle and phragmoplast. The immunoreactive material codistributes specifically with MT arrays of the mitotic apparatus and does not associate with interphase cortical MTs. Pas5D8 is relevant to the PPB and spatial control of cytokinesis. It binds in a thin layer at the cytoplasmic surface throughout the cell cycle, except when its coverage is transiently interrupted by an exclusion zone at the PPB site and later at the same site when the phragmoplast fuses with the parental cell wall.Pas2G6 reacts with a component of basal bodies and the flagellar band in thePteridium spermatozoid and recognizes irregularly shaped cytoplasmic vesicles in wheat cells. During interphase these particles form a cortical network.Pas6D7 binds to dictyosomes and dictyosome vesicles. At anaphase the vesicles accumulate at the equator and subsequently condense into the cell plate.Abbreviations MT microtubule - PPB preprophase band