Agrobacterium rhizogenes T-DNA genes capable of inducing hairy root phenotype

Summary Segments of the TL-DNA of the agropine type Ri plasmid pRi 1855 encompassing single and groups of open-reading frames were cloned in the Ti plasmid-derived binary vector system Bin 19. Leaf disc infections on Nicotiana tabacum led to transformed plants, some of which showed typical hairy root phenotypes, such as the wrinkled leaf morphology, excessive and partially non geotropic root systems and the ability of leaf explants to differentiate roots in a hormone-free culture medium. Particularly interestingly, most of these traits were shown by plants transformed with a TL-DNA segment encompassing the single ORF 11, corresponding to the rolB locus. Hairy root can be induced by this latter T-DNA segment on wounded stems of tobacco plants; hairy root induction on carrot discs requires, on the contrary, a more complex complement of TL-DNA genes.Abbreviations YMB yeast mannitol broth - MS Murashige and Skoog medium - 6-BAP 6-benzylaminopurine - NAA naphthalene acetic acid - Km kanamycin - Cb carbenicillin     

Induction and growth properties of carrot roots with different complements of Agrobacterium rhizogenes T-DNA

Single and multiple infections of carrot discs were carried out with Agrobacterium strains harbouring different segments of pRi1855 TL-DNA cloned in the binary vector Bin 19 and with a strain carrying the TR-DNA from the same Ri plasmid. Roots induced by the various co-inoculations were cultured and their growth patterns were followed. Abundant roots could be induced by TL-DNA rol genes A, B and C as a single insert (rolA+B+C) and by rolB alone provided an extended segment beyond its 5 noncoding region was included in the construction. A depression of rooting capability was caused by the inclusion of rolC together with rolB (rolB+C). In all cases co-inoculation with the Agrobacterium carrying TR-DNA-borne auxin genes was necessary for root induction since none of the rol constructions was in itself capable of eliciting any response; an exceeding majority of these roots were however shown to contain rol genes but no TR-DNA. Rooting was also elicited if rol constructions were co-inoculated with a strain carrying TL-DNA genes 13 and 14 (ORF13+14) instead of the TR-DNA strain. These roots were shown to contain both rol genes and ORF13+14. Striking differences in growth properties were shown by roots containing different complements of TL-DNA genes. Typical hairy root traits, high growth rate, branching and, most noticeably, absence of geotropism, were shown by roots containing rolB alone, while roots with rolA+B+C were geotropic as normal carrot roots. Hairy root traits were conferred to rolA+B+C roots by the concomitant presence of ORF13+14 and by the addition of auxin to the culture medium. A model is presented which attempts to rationalize the growth patterns by assigning interplaying roles to the various TL-DNA genes involved.     

Synthesis of the herpes simplex virus type 1 trans-activator Vmw65 in insect cells using a baculovirus vector

Vmw65, the Herpes Simplex Virus trans-activator of immediate-early genes, was expressed in insect cells using a recombinant baculovirus expression vector and partially purified. Insect cell-derived Vmw65 was shown to be indistinguishable from authentic Vmw65 present in purified HSV-1 virions based on electrophoretic mobility, immunoreactivity with a monoclonal antibody, and ability to interact with cellular factors to form a protein/DNA complex with oligonucleotides containing a TAATGARAT element.Abbreviations AcNPV Autographica californica nuclear polyhedrosis virus - HSV Herpes Simplex Virus - IE Immediate Early - moi multiplicity of infection - Sf9 Spodoptera frugiperda cells     

Growth,ammonia accumulation and glutamine synthetase activity in alfalfa (Medicago sativa L.) shoots and cell cultures treated with phosphinothricin

Summary Phosphinothricin is a non-selective herbicide which inhibits glutamine synthetase (EC 6.3.1.2) activity causing an overaccumulation of ammonia in higher plants. Alfalfa (Medicago sativa L) shoot tissue and petiole-derived callus exposed to phosphinothricin show 50 and 70% reductions, respectively, in glutamine synthetase activity with a concomitant rise of 10 and 20 fold, respectively, in endogenous ammonia. The diffusibility of ammonia may limit the use of a detoxifying gene, phosphinothricin acetyltransferase, as a selectable marker for alfalfa transformation. However, the addition of up to 40 times the standard levels of ammonium nitrate to the culture media used in this study had no effect on callus growth, although glutamine synthetase activity was inhibited by 50% and endogenous ammonia increased 27 fold. Therefore, ammonia accumulation may not be the primary cause of cell death in alfalfa after exposure to phosphinothricin. It follows that diffusion of ammonia from cell to cell would not restrict the selection for phosphinothricin acetyltransferase transformed cells, thereby indicating that this enzyme could be used as a selectable marker in transformation experiments.Abbreviations PPT Phosphinothricin - PAT Phosphinothricin acetyltransferase     

Allometric shape change and heterochrony in the freeliving coral Trachyphyllia bilobata (Duncan)

Regression analysis has been used to study the relationship between age, size, shape, and surface area in two ancestral-descendant populations of the Neogene Caribbean coral Trachyphyllia bilobata. Analyses of the relationship between size and age show that the relationship is isometric and that little difference occurs between populations in mean corallite length or height and in their rates of growth. Onset of columella growth is significantly earlier, however, in the descendant population. Studies of the relationship between size and shape show that growth is allometric, with shape change occurring in both corallum elongation and pinching of the corallite wall during ontogeny. In the descendant population, pinching and elongation initiate earlier in the ontogeny of the coral. These results suggest that the evolutionary development of the meandroid form in freeliving corals has been accomplished by heterochrony, involving a complex set of disassociated peramorphic changes in ontogeny accompanied by paedomorphic changes in astogeny. Further analyses show that the observed heterochronic changes serve to decrease corallum surface area which may in turn enhance sediment removal and nutrition in unstable habitats.     

Amber, ochre and opal suppressor tRNA genes derived from a human serine tRNA gene.

Amber, ochre and opal suppressor tRNA genes have been generated by using oligonucleotide directed site-specific mutagenesis to change one or two nucleotides in a human serine tRNA gene. The amber and ochre suppressor (Su+) tRNA genes are efficiently expressed in CV-1 cells when introduced as part of a SV40 recombinant. The expressed amber and ochre Su+ tRNAs are functional as suppressors as demonstrated by readthrough of the amber codon which terminates the NS1 gene of an influenza virus or the ochre codon which terminates the hexon gene of adenovirus, respectively. Interestingly, several attempts to obtain the equivalent virus stock of an SV40 recombinant containing the opal suppressor tRNA gene yielded virus lacking the opal suppressor tRNA gene. This suggests that expression of an efficient opal suppressor derived from a human serine tRNA gene is highly detrimental to either cellular or viral processes.     

Effects of Metals on Methanogenesis, Sulfate Reduction, Carbon Dioxide Evolution, and Microbial Biomass in Anoxic Salt Marsh Sediments

The effects of several metals on microbial methane, carbon dioxide, and sulfide production and microbial ATP were examined in sediments from Spartina alterniflora communities. Anaerobically homogenized sediments were amended with 1,000 ppm (ratio of weight of metal to dry weight of sediment) of various metals. Time courses in controls were similar for CH₄, H₂S, and CO₂, with short initial lags (0 to 4 h) followed by periods of constant gas production (1 to 2 days) and declining rates thereafter. Comparisons were made between control and experimental assays with respect to initial rates of production (after lag) and overall production. Methane evolution was inhibited both initially and overall by CH₃HgCl, HgS, and NaAsO₂. A period of initial inhibition was followed by a period of overall stimulation with Hg, Pb, Ni, Cd, and Cu, all as chlorides, and with ZnSO₄, K₂CrO₄, and K₂Cr₂O₇. Production of CO₂ was generally less affected by the addition of metals. Inhibition was noted with NaAsO₂, CH₃HgCl, and Na₂MoO₄. Minor stimulation of CO₂ production occurred over the long term with chlorides of Hg, Pb, and Fe. Sulfate reduction was inhibited in the short term by all metals tested and over the long term by all but FeCl₂ and NiCl₂. Microbial biomass was decreased by FeCl₂, K₂Cr₂O₇, ZnSO₄, CdCl₂, and CuCl₂ but remained generally unaffected by PbCl₂, HgCl₂, and NiCl₂. Although the majority of metals produced an immediate inhibition of methanogenesis, for several metals this was only a transient phenomenon followed by an overall stimulation. The initial suppression of methanogenesis may be relieved by precipitation, complexation, or transformation of the metal (possibly by methylation), with the subsequent stimulation resulting from a sustained inhibition of competing organisms (e.g., sulfate-reducing bacteria). For several environmentally significant metals, severe metal pollution may substantially alter the flow of carbon in sediments.