Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants

Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 l agarose droplets containing a basal medium plus 25 M NAA and 4 M BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6–8×10⁵ ml^–1 is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Bloom's syndrome and EM9 cells in BrdU-containing medium exhibit similarly elevated frequencies of sister chromatid exchange but dissimilar amounts of cellular proliferation and chromosome disruption

Bloom's syndrome (BS) and EM9 cells both display elevated frequencies of sister chromatid exchange (SCE) following growth for two rounds of DNA replication in bromodeoxyuridine (BrdU)-containing medium. To learn whether hyperresponsiveness to BrdU itself might play a role in causing the SCE elevation, the effects of BrdU on two other parameters, cellular proliferation and chromosome disruption, were examined, comparing the responses of BS and normal lymphoblastoid cells and of EM9 and CHO cells. BS and normal cells responded similarly with respect to growth for 4 days in BrdU-containing medium (0, 1, 3, and 5 g/ml). Chromosome aberrrations were increased only slightly in the BS and normal cells after 2 days in BrdU. CHO cells responded to growth in BrdU-containing medium like BS and normal cells; however, little growth of EM9 was detected at any of the BrdU concentrations employed. CHO and EM9 cells also exhibited strikingly different amounts of chromosome damage following growth in BrdU. After 2 days in 1, 3, and 5 g/ml BrdU 21%, 46%, and 50%, respectively, of the CHO cells had chromosome aberrations in contrast to 92%, 96%, and 98% of the EM9 cells. Most of the aberrations in the BrdU-treated CHO cells consisted of what appeared to be polycentric and ring chromosomes or chromosomes exhibiting telomere association. Acentric fragments were absent from most cells with polycentric and ring chromosomes, indicating either that the abnormal chromosomes were formed during an earlier cell cycle or that the abnormal chromosomes represent a form of association in which the telomeres are apposed so tightly that the juncture between chromosomes cannot be identified microscopically. EM9 cells treated with BrdU exhibited many chromatid and isochromatid gaps and breaks as well as numerous quadriradial, triradial, and complex interchange configurations. In addition, the types of aberrations present in CHO cells also were increased greatly in number. The different responses of BS and EM9 cells to growth in BrdU suggest that the molecular defects in the two cell types are different.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures.

Sodium selenite (Na2SeO3) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 10(-6) M Na2SeO3 resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na2SeO3 concentrations (7.90 X 10(-6) and 1.19 X 10(-5) M) resulted in a three-fold increase in the SCE frequency above background level (6--7 SCEs/cell). Exposure of lymphocytes to 1 X 10(-4) M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 +/- 0.75 while a similar exposure to 2.7 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25--30% and 11--17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.     

Labeling of the Plasma Membrane of Pea Cells by a Surface-localized Glucan Synthetase

When radioactive UDP-glucose is supplied to 1-millimeter-thick slices of pea (Pisum sativum) stem tissue, radioactive glucose becomes incorporated into membrane-bound polysaccharides. Evidence is given that this incorporation does not result from breakdown of UDP-glucose and utilization of the resultant free glucose, and that the incorporation most likely takes place at the cell surface, leading to a specific labeling of the plasma membrane. The properties of the plasma membrane that are indicated by this method of recognition, including the association of K⁺-stimulated ATPase activity with the plasma membrane, resemble properties inferred using other approaches. The membrane-associated polysaccharide product formed from UDP-glucose is largely 1,3-linked glucan, presumably callose, and does not behave as a precursor of cell wall polymers. No substantial amount of cellulose is formed from UDP-glucose in this procedure, even though these cells incorporate free glucose rapidly into cellulose. This synthetase system that uses external UDP-glucose may serve for formation of wound callose.     

Formation of substrate and transition-state analogue complexes in crystals of phosphoglucomutase after removing the crystallization salt.

Crystals of phosphoglucomutase, grown in 2.1 M ammonium sulfate, "desalted", and suspended in a 30% polyoxyethylene-8000/1 M glycine solution as described in the accompanying paper [Ray, W. J., Jr., Puvathingal, J. M., Bolin, J. T., Minor, W., Liu, Y., & Muchmore, S. W. (1991) Biochemistry 30 (preceding paper in this issue)], were treated with glucose phosphates to form an equilibrium mixture of the catalytically active substrate/product complexes. However, this treatment extensively fractured the crystals, even when very dilute solutions of glucose phosphates were used. But formation of the desired complexes was achieved, without fracturing, by introducing the glucose phosphates at high salt concentration, where they do not bind significantly to the enzyme, and maintaining their presence during subsequent sulfate-removal steps, in order to obtain essentially uniform binding throughout the crystal at all times. Although this procedure produced unfractured crystals of the catalytically active complexes, an adjustment in water activity was required to prevent the crystals from slowly liquefying in the presence of the added glucose phosphates. After this adjustment, the quality of diffraction-grade crystals subjected to this treatment was not significantly altered. An even larger adjustment in water activity was required to stabilize crystals that had been largely converted into a mixture of vanadate-based transition-state analogue complexes [cf. Ray, W. J., Jr., & Puvathingal, J. M. (1990) Biochemistry 29, 2790-2801] by means of an analogous procedure. The rationale for, and the implications of, this adjustment of water activity are discussed. The phenomenon of lattice-based binding cooperativity also is discussed together with a possible role for such cooperativity in the fracturing of protein crystals during formation of ligand complexes and possible ways to circumvent such fracturing based on the annealing of crystals at fractional saturation. An assay for quantifying the extent of formation of the vanadate-based transition-state analogue complexes in crystals of phosphoglucomutase is described. A solution to problems associated with producing and maintaining a steady-state in treated crystals is discussed within the context of maximizing the fraction of the crystalline enzyme present as a complex with one such inhibitor, glucose alpha-1-phosphate-6-vanadate. One of these problems, achieving a substantial reduction in sulfate concentration, could not be successfully addressed by employing the desalting procedure used to produce the substrate/product complexes, because of reduced diffusional rates in the final solution.(ABSTRACT TRUNCATED AT 400 WORDS)