Development and germination of pecan somatic embryos derived from liquid culture

Aggregates of globular and pre-globular stage somatic embryos from suspension cultures of pecan (Carya illinoensis Koch) were cultured on solidified media for embryo development. Embryo aggregates and pre-globular stage embryo masses were given various treatments to further ontologic development. A 2- to 4-wk mild dehydration of the embryo aggregates suppressed recurrent embryogenesis, promoted development of globular embryos into cotyledonary stage embryos, and enhanced plant development beyond germination. Fine embryogenic tissue masses filtered from suspension formed cotyledonary-staged embryos when the collection filters were plated on solified medium. The embryogenic capacity of preglobular stage embryo masses was compared between media supplemented with varying concentrations of polyethylene glycol (molecular weight 8 000) vs. filter overlays. The filter paper overlays were not necessary for embryo development. An inverse relationship was found between the number of embryos that developed and the concentration of polyethylene glycol in the medium. However, this relationship was reversed for ability of embryos to germinate and develop into a plant.     

Soil P resources,plant growth and rooting characteristics in nutrient poor upland grasslands

A field study was undertaken to establish the demand for P by mixed herbage, manipulated by cutting regimes, and the extent to which orthophosphate alone in soil solution could meet this demand from three cambisols derived from different parent materials. Differences in soil types were sufficient to produce significantly different rooting patterns at each site. Yields for 7-and 10-cm treatments generally exceeded those for swards cut to 2-and 4-cm. The highest yields were from plots cut once at the end of the season, or when herbage was cut in June and October only. Yields fell in the second season by an average of 30%. Two cuts in the season resulted in almost twice the P uptake compared with other treatments, leading to the view that a silage cut stimulated root growth. Rooting was deepest in Tarves Association soil (Dystric cambisol), densest in Insch Association soil (Eutric cambisol) and intermediate in Foudland Association soil (Dystric cambisol) but herbage yield at each site was similar. Whole season mean P and N content in roots ranged from 1.0 to 3.4 and from 8.1 to 27.9 mg g^–1 dry weight, respectively. The lowest values were in once cut herbage and were half those in herbage cut in June and October only. Data for the total P resources of the soils, extractable P, and shoot and root P at each site are presented together with data for P in soil solution (principally organic) from an associated soil solution study. There was a disparity between daily uptake and orthophosphate in soil solution. These findings suggested that it was probable that soluble organic forms of P are important for P nutrition in these nutrient poor soils, and could account for the excess of observed P uptake (from soils low in P) over that predicted by mechanistic mathematical models.     

The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites

The plasmid-partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent PI and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species-specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA-binding domain of the P7 auto-repressor lies in the amino-terminal end of the P7 ParA protein. This region includes a helix-turn-helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter-operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.     

Large DNA inversions,deletions, and TaqI site mutations in severe haemophilia A

Large DNA inversions caused by an intrachromosomal recombination between homologous regions located in intron 22 and 5 of the factor VIII gene have recently been identified in patients with severe haemophilia A. To evaluate better the prevalence of this large inversion and to estimate the overall sensitivity of the Southern blot/hybridization method we analysed the factor VIII gene of 49 unrelated patients with severe haemophilia A. All patients were screened for the inversion mutation, TaqI site mutations, and deletions. Mutations were identified in 31 (63%) patients, and comprised 24 large inversions, 4 partial deletions, and 3 point mutations. Three different haplotypes were characterised in the patients presenting the inversion mutation, confirming its independent origin. Two novel deletions are reported: a large one spanning from intron 14 to intron 22 and a deletion of 86 bp comprising the 3 region of exon 1 and 39–41 bp of intron 1. DNA sequencing of the deletion junction showed no significant homology between normal 5 and 3 sequences around the breakpoints. A novel missense mutation is also reported: CGAGGA, Arg-2209 to Gly. These results confirm that the inversion mutation is the most common cause of severe haemophilia A and indicate that the Southern blot/hybridization assay should be used as the first method for screening of mutations in severe haemophilia A.     

Influence of photoperiod and medium formulation on peanut somatic embryogenesis

Somatic embryos were produced from peanut (Arachis hypogaea L.) immature zygotic cotyledons. Comparisons were made of the level of -naphthaleneacetic acid during induction, nitrogen formulation of the medium, and photoperiod. Over 70% embryogenesis was obtained regardless of NAA level used. Percent embryogenesis and number of embryos were markedly lower in explants induced on NAA compared to 2,4-D. Embryo production was not greatly affected by either the use of Murashige & Skoog versus Finer & Nagasawa salts or light versus dark culture conditions. However, embryo morphology was noticeably affected by photoperiod. Embryos produced under a 16 h photoperiod were tough, woody and difficult to separate for subsequent germination and conversion. Those produced under a 0-h photoperiod were succulent and pliable.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - MS Murashige & Skoog (1962) - B5 Gamborg et al. (1968) - picloram 4-amino-3,5,6-trichloropicolinic acid - FN Finer & Nagasawa (1988) - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

How does the G protein,Gi2, transduce mitogenic signals?

Serpentine receptors coupled to the heterotrimeric G protein, G_i2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the G_i2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the G_i2-coupled thrombin and acetylcholine muscarinic M₂ receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. G_i2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of α_i2 inhibits both the Raf-dependent and-independent pathways activated by G_i2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by G_i2-coupled receptors as well as other receptor systems, including the tyrosine kinases.     

The role of tyrosine-114 in the enzymatic activity of the Shiga-like toxin I A-chain

Shiga-like toxin I (SLT-I), the potent cytotoxin produced by certain pathogenic strains of Escherichia coli, is a member of a burgeoning family of ribosome-inactivating proteins (RIPS), which share common structural and mechanistic features. The prototype of the group is the plant toxin ricin. Recently we proposed a structural model for the Slt-IA active site, based in part on the known geometry of the enzymatic subunit of the ricin toxin. The model places three aromatic residues within the putative Slt-IA active site cleft: tyrosine 77, tyrosine 114, and tryptophan 203. Here we present biochemical and biophysical data regarding, the phenotypes of conservative point mutants of Slt-IA in which tyrosine 114 is altered. We used oligonucleotide-directed mutagenesis to replace tyrosine 114 with either phenylalanine (Y114F) or serine (Y114S). Periplasmic extracts of E. coli containing wild-type or mutant Slt-IA were tested for their ability to inhibit protein synthesis in vitro. Relative to wild-type, the activity of mutant Y1 14F was attenuated about 30-fold, while the mutant Y114S was attenuated about 500 to 1000-fold. In order to address the possibility that differential activation of the mutants rather than local effects at the active site might account for their diminished activity, we engineered the same mutations into a truncated slt-IA cassette that directs expression of a product corresponding to the activated A₁ form of Slt-IA (wild-type-). The same general relationships held: relative to wild type-, Y114F- was attenuated about 7-fold, and Y114S- about 300-fold. Tryptic digestion profiles of the mutant proteins were similar to those of the corresponding wild-type, indicating that the amino acid substitutions had not caused major alterations in conformation. We conclude that Y114 plays a significant role in the activity of Slt-IA, one which is quantitatively similar to that of Y77, and one which is predicated on the presence of both its weakly acidic phenolic hydroxyl and its aromatic ring.     

Increased shikonin production by hairy roots of Lithospermum erythrorhizon in two phase bubble column reactor

Summary Plant hairy root cultures of Lithospermum erythrorhizon were carried out to produce shikonin derivatives by employing in situ extraction with n-hexadecane in a shake flask and a bubble column bioreactor. Over 95 % shikonin produced was recovered in the n-hexadecane layer. In flask cultures the maximum concentration of shikonin with n-hexadecane extraction was 3 times higher than that obtained without extraction. In the two phase bubble column reactor, 572.6 mg/L of shikonin and 15.6 g/L of dry cell mass were obtained after 54 days. Shikonin was produced at a constant level of 10.6 mg/L day during this period.