Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences

Summary With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T⁺), also had acquired non-selected calf thymus carrier DNA sequences (C⁺), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas⁺), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated (transforming) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C⁺T⁺. Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C⁺T⁺:C^-T⁺: C⁺T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.     

Metabolic control of the cell cycle

Cell division is a metabolically demanding process, requiring the production of large amounts of energy and biomass. Not surprisingly therefore, a cell''s decision to initiate division is co-determined by its metabolic status and the availability of nutrients. Emerging evidence reveals that metabolism is not only undergoing substantial changes during the cell cycle, but it is becoming equally clear that metabolism regulates cell cycle progression. Here, we overview the emerging role of those metabolic pathways that have been best characterized to change during or influence cell cycle progression. We then studied how Notch signaling, a key angiogenic pathway that inhibits endothelial cell (EC) proliferation, controls EC metabolism (glycolysis) during the cell cycle.     

Influence of pH shocks on trace metal dynamics and performance of methanol fed granular sludge bioreactors

The influence of pH shocks on the trace metal dynamics and performance of methanol fed upflow anaerobic granular sludge bed (UASB) reactors was investigated. For this purpose, two UASB reactors were operated with metal pre-loaded granular sludge (1mM Co, Ni and Fe; 30°C; 96h) at an organic loading rate (OLR) of 5gCOD l reactor^–1d^–1. One UASB reactor (R1) was inoculated with sludge that originated from a full scale reactor treating alcohol distillery wastewater, while the other reactor (R2) was inoculated with sludge from a full scale reactor treating paper mill wastewater. A 30h pH shock (pH 5) strongly affected the metal retention dynamics within the granular sludge bed in both reactors. Iron losses in soluble form with the effluent were considerable: 2.3 and 2.9% for R1 and R2, respectively, based on initial iron content in the reactors, while losses of cobalt and nickel in soluble form were limited. Sequential extraction of the metals from the sludge showed that cobalt, nickel, iron and sulfur were translocated from the residual to the organic/sulfide fraction during the pH shock in R2, increasing 34, 47, 109 and 41% in the organic/sulfide fraction, respectively. This is likely due to the modification of the iron sulfide precipitate stability, which influences the extractability of iron and trace metals. Such a translocation was not observed for the R1 sludge during the first 30h pH shock, but a second 4day pH shock induced significant losses of cobalt (18%), iron (29%) and sulfur (29%) from the organic/sulfide fraction, likely due to iron sulfide dissolution and concomitant release of cobalt. After the 30h pH shock, VFA accumulated in the R2 effluent, whereas both VFA and methanol accumulated in R1 after the 4day pH shock. The formed VFA, mainly acetate, were not converted to methane due to the loss of methanogenic activity of the sludge on acetate. The VFA accumulation gradually disappeared, which is likely to be related to out-competition of acetogens by methanogens. Zinc, copper and manganese supply did not have a clear effect on the acetate removal and methanol conversion, but zinc may have induced the onset of methanol degradation after day 152 in R1.     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

A chromosomal linkage map of Agrobacterium tumefaciens and a comparison with the maps of Rhizobium spp

Summary Plasmid R68.45 was found to mobilize the Agrobacterium tumefaciens chromosome apolarly. With the aid of this R plasmid the linkage between 26 markers was determined, from which a circular genetic map of the A. tumefaciens chromosome could be constructed. The recombinants obtained were stable i.e. they did not segregate strains, with the parental phenotype upon purification. A system for the polar transfer of chromosomal material from a fixed origin was developed for A. tumefaciens. It was found that R plasmid pRL189, which carries a copy of transposon Tn5, is able to mobilize the chromosome polarly from chromosomal Tn5-insertion sites. A. tumefaciens phe-1 and trp-22 auxotrophs became prototrophic after the introduction of R primes pAJ21JI and pAJ73JI, respectively, which carry the corresponding phe and trp genes of Rhizobium meliloti. This result enabled a preliminary comparison of the gene orders in A. tumefaciens and Rhizobium spp. which suggested that the chromosome maps of these organisms are quite similar.     

T-DNA rearrangements due to tissue culture: somaclonal variation in crown gall tissues

After three years of apparent stability in tissue culture, the single cell derived shooty crown gall line sNT1.013 produced a revertant shoot which had switched from non-rooting (Rod⁺) and octopine synthesizing (Ocs⁺) to Rod^- Ocs^-, indicating that in this revertant TL-DNA genes 4 (causing the Rod⁺ trait) and gene 3 (causing the Ocs⁺ trait) had been inactivated. Southern blots revealed that the inactivation of these T-DNA genes was the result of a considerable rearrangement of DNA sequences, accompanied by deletions and possibly also by DNA amplifications. This study for the first time unambiguously proves that foreign genes which have been introduced via Agrobacterium tumefaciens can, at a low frequency, be inactivated after T-DNA integration because of reorganization of T-DNA sequences during tissue culture. This can be considered as an event of somaclonal variation.