Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.)

Protoplasts were isolated from anther-derived suspension cultures of commercial wheat (Triticum aestivum L. cv. Chris). The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium in the dark. Numerous microcalli were produced under these conditions, some of which differentiated into globular embryos. Upon transfer to a solid medium and exposure to 16h/8h light/dark cycle, the protocalli proliferated and many of the somatic embryos matured. Complete plantlets were obtained and maintained in sterile culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - MES 2-[N-morpholino] ethanesulfonic acid     

The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat.

The TIS11 primary response gene is rapidly and transiently induced by both 12-O-tetradecanoylphorbol-13-acetate and growth factors. The predicted TIS11 protein contains a 6-amino-acid repeat, YKTELC. We cloned two additional cDNAs, TIS11b and TIS11d, that contain the YKTELC sequence. TIS11, TIS11b, and TIS11d proteins share a 67-amino-acid region of sequence similarity that includes the YKTELC repeat and two cysteine-histidine containing repeats. TIS11 gene family members are not coordinately expressed: (i) unlike TIS11, the TIS11b and TIS11d mRNAs are detectable in quiescent Swiss 3T3 cells and are not dramatically induced by 12-O-tetradecanoylphorbol-13-acetate; (ii) cycloheximide superinduction does not occur for TIS11b and TIS11d; and (iii) unlike TIS11, TIS11b expression is extinguished in PC12 pheochromocytoma cells.     

The developmental regulation of single-cell motility in Dictyostelium discoideum

The velocity of single amebae in the absence of a chemotactic signal has been analyzed during growth, development, rapid recapitulation, and dedifferentiation in the cellular slime mold Dictyostelium discoideum. It is demonstrated that (1) the velocity of axenically grown cells in half that of bacterially grown cells, (2) the velocity of bacterially grown cells decreased to roughly the same low level as axenically grown cells approximately 5 hr after the removal of exogeneous bacteria, (3) the velocity remains low for a 7-hr period preceding the onset of aggregation in both axenically and bacterially grown cells, (4) the velocity increases transiently at the onset of aggregation for both axenically and bacterially grown cells, (5) the velocity decreases to a very low level after the formation of loose aggregates and remains at that level at least through the early culminate I stage, (6) the velocity is not stimulated in 13-hr developing cells (finger stage) by inducing rapid recapitulation, (7) the velocity decreases after the erasure event in cultures of 7-hr developing cells (ripple stage) stimulated to undergo dedifferentiation, but the inhibition of the erasure event by the addition of 10(-4) M cAMP does not block this decrease. These results demonstrate that the basal level of single-cell motility in growing cultures is significantly influenced by the nutrient composition of the supporting medium, and that the transient increase in single-cell motility at the onset of aggregation is under the rigid control of the initial developmental program. Both rapid recapitulation and the program of dedifferentiation appear to have no influence on the basal level of single-cell motility.     

Dictyostelium amebae alter motility differently in response to increasing versus decreasing temporal gradients of cAMP

Using a perfusion chamber, we examined the behavior of individual amebae in increasing and decreasing temporal gradients of cAMP. We demonstrated that amebae respond to increasing temporal gradients of cAMP with stimulated motility and to corresponding decreasing temporal gradients with depressed motility. Depressed motility observed in decreasing temporal gradients corresponded to the inhibited levels observed when cAMP was applied at constant concentrations. These results were consistent with a simple model for the motile behavior of amebae in an early aggregation territory in which nondissipating waves of cAMP originate at the aggregation center and travel outward periodically. We conclude that chemotactically responsive amebae can assess whether a temporal gradient of chemoattractant is increasing or decreasing in the absence of a spatial gradient, and can adjust their motility accordingly.     

An analysis of developmental timing in Dictyostelium discoideum

A new method has been developed to assess the minimum complexity and relationships of those pathways (developmental timers) which time the consecutive stages of a developing system (Soll, 1983). This method has been applied to the morphogenetic program of Dictyostelium discoideum and has resulted in (1) a minimum estimate of the number of components comprising the timers for the first seven stages of morphogenesis, (2) a characterization of the temperature sensitivities of these components including demonstration of a reversible timer component, (3) detailed temporal definition of a number of transition points between rate-limiting components including a major branch point for the onset of several independent timer components coincident with the onset of aggregation, and (4) a temporal model for the relationships between the timers of the seven consecutive morphogenetic stages, including several examples of parallel timers.     

Synthesis,degradation, and subcellular localization of proteins encoded by the primary response genes TIS7/PC4 and TIS21/PC3

Murine TIS7 and TIS21 cDNAs were cloned from phorbol ester-induced Swiss 3T3 cells. The cognate rat cDNAs. PC4 and PC3, were cloned from nerve growth factor (NGF)-treated PC12 pheochromocytoma cells. The TIS7/PC4 and TIS21/PC3 primary response genes are rapidly and transiently induced in response to serum, phorbol esters, and polypeptide growth factors in quiescent Swiss 3T3 cells and by NGF and other ligands in PC12 cells. In both 3T3 and PC12 cells the appearance of the TIS21/PC3 message precedes that of TIS7/PC4 message following ligand stimulation, suggesting that the TIS21/PC3 protein is likely to be synthesized more rapidly than the TIS7/PC4 protein. Using antisera prepared against recombinant TIS21 and TIS7 proteins, we find that the TIS21/PC3 protein is, indeed, synthesized more rapidly than the TIS7/PC4 protein following stimulation in both 3T3 and PC12 cells. In addition, “pulse-chase” experiments demonstrate that the TIS21/PC3 protein is degraded much more rapidly than the TIS7/PC4 protein. The sequences of the predicted PC3 and PC4 proteins have lead to the speculation that these two proteins may both be secreted from cells following stimulation. The PC4 protein is reported to have some sequence similarity to interferons. The TIS21/PC3 protein contains a presumptive leader sequence. Using our antisera to the recombinant proteins, however, we cannot detect secretion of radiolabelled TIS7/PC4 or TIS21/PC3 protein. Immunohistochemical and subcellular fractionation experiments suggest that the TIS7 protein is a membrane associated, non-nuclear intracellular protein. The TIS21 protein, in contrast, is' a non-nuclear, soluble intracellular protein. © 1994 Wiley-Liss, Inc.     

Structural insights into the functional role of the Hcn sub-domain of the receptor-binding domain of the botulinum neurotoxin mosaic serotype C/D

Botulinum neurotoxin (BoNT), the causative agent of the deadly neuroparalytic disease botulism, is the most poisonous protein known for humans. Produced by different strains of the anaerobic bacterium Clostridium botulinum, BoNT effects cellular intoxication via a multistep mechanism executed by the three modules of the activated protein. Endocytosis, the first step of cellular intoxication, is triggered by the ∼50 kDa, heavy-chain receptor-binding domain (HCR) that is specific for a ganglioside and a protein receptor on neuronal cell surfaces. This dual receptor recognition mechanism between BoNT and the host cell's membrane is well documented and occurs via specific intermolecular interactions with the C-terminal sub-domain, Hcc, of BoNT–HCR. The N-terminal sub-domain of BoNT–HCR, Hcn, comprises ∼50% of BoNT–HCR and adopts a β-sheet jelly roll fold. While suspected in assisting cell surface recognition, no unambiguous function for the Hcn sub-domain in BoNT has been identified. To obtain insights into the potential function of the Hcn sub-domain in BoNT, the first crystal structure of a BoNT with an organic ligand bound to the Hcn sub-domain has been obtained. Here, we describe the crystal structure of BoNT/CD–HCR determined at 1.70 Å resolution with a tetraethylene glycol (PG4) moiety bound in a hydrophobic cleft between β-strands in the β-sheet jelly roll fold of the Hcn sub-domain. The PG4 moiety is completely engulfed in the cleft, making numerous hydrophilic (Y932, S959, W966, and D1042) and hydrophobic (S935, W977, L979, N1013, and I1066) contacts with the protein's side chain and backbone that may mimic in vivo interactions with the phospholipid membranes on neuronal cell surfaces. A sulfate ion was also observed bound to residues T1176, D1177, K1196, and R1243 in the Hcc sub-domain of BoNT/CD–HCR. In the crystal structure of a similar protein, BoNT/D–HCR, a sialic acid molecule was observed bound to the equivalent residues suggesting that residues T1176, D1177, K1196, and R1243 in BoNT/CD may play a role in ganglioside binding.