Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

The coupling of biogeochemical cycles of nutrients

For any element which is incorporated into biomass, the biogeochemical cycle of that element in a given ecosystem will be coupled to that of any other element similarly incorporated. The mutual interaction of two such cycles is examined using a simple model in which each cycle is constrained into four compartments. In each cycle the assimilation rate (primary productivity) is related in a non-linear fashion to the two nutrients and to biomass. The interactions are represented by combining a hyperbolic dependence for each nutrient (involving a "Michaelis constant") with a logistic equation governing the dependence of rate on biomass (involving a "carrying capacity"). The response of the model to perturbation (e.g. mobilization of an abiotic reserve) is strongly governed by the values assigned to these constants. The coupled cycles can exhibit positive feed-back with anomalous responses of the steady state and time-dependent solutions may exhibit complex oscillatory behaviour. Both the steady-state sensitivity and the kinetic behaviour of such coupled systems are simplified if the range of atomic ratios permitted by the assimilation process is restricted. It will therefore be of importance to determine under what conditions the assimilation rates for different elements are governed by mass-action effects (Liebig's Law) or by stoichiometric constraints (Redfield ratios).     

A stable carbon isotope study of dissolved inorganic carbon cycling in a softwater lake

The dissolved inorganic carbon (DIC) cycle in a softwater lake was studied using natural variations of the stable isotopes of carbon,¹²C and¹³C. During summer stratification there was a progressive decrease in epilimnion DIC concentration with a concomitant increase in ¹³C_DIC), due to preferential uptake of¹²C by phytoplankton and a change in the dominant CO₂ source from inflow andin situ oxidation to invasion from the atmosphere. There was an increase in hypolimnion DIC concentration throughout summer with a concomitant general decrease in ¹³C_DIC from oxidation of the isotopically light particulate organic carbon that sank down through the thermocline from the epilimnion.Mass balance calculations of DI¹²C and DI¹³C in the epilimnion for the summer (June 23–September 25) yield a mean rate of net conversion of DIC to organic carbon (C_org) of 430 ± 150 moles d^-1 (6.5 ± 1.8 m moles m^-2 d^-1. Net CO₂ invasion from the atmosphere was 420 ± 120 moles d^-1 (6.2 ± 1.8 m moles m^-2 d^-1) with an exchange coefficient of 0.6 ± 0.3m d^-1. These results imply that at least for the summer months the phytoplankton obtained about 90% of their carbon from atmosphere CO₂. About 50% of CO₂ invasion and conversion to C_org for the summer occurred during a two week interval in mid-summer.DIC concentration increased in the hypolimnion at a rate of 350 ± 70 moles DIC d^-1 during summer stratification. The amount of DIC added to the hypolimnion was equivalent to 75 ± 20% of net conversion of DIC to C_org in the euphotic zone over spring and summer implying rapid degradation of POC in the hypolimnion. The ¹³C of DIC added to the deep water (-22.) was too heavy to have been derived from oxidation of particulate organic carbon alone. About 20% of the added DIC must have diffused from hypolimnetic sediments where relatively heavy CO₂ (-7) was produced by a combination of POC oxidation and as a by-product of methanogenesis.     

Retardation of cell cycle progression in yeast cells recovering from DNA damage: A study at the single cell level

Summary Pedigree analyses of individual yeast cells recovering from DNA damage were performed and time intervals between morphological landmark events during the cell cycle (bud emergence and cell separation), were recorded for three generations. The associated nuclear behavior was monitored with the aid of DAPI staining. The following observations were made: (1) All agents tested (X-rays, MMS, EMS, MNNG, nitrous acid) delayed the first bud emergence after treatment, which indicates inhibition of the initiation of DNA replication. (2) Cells that survived X-irradiation progressed further through the cell cycle in a similar way to control cells. (3) Progress of chemically treated cells became extremely asynchronous because surviving cells stayed undivided for periods of varying length. (4) Prolongation of the time between bud emergence and cell separation was most pronounced for cells treated with the alkylating agents MMS and EMS. This is interpreted as retardation of ongoing DNA synthesis by persisting DNA adducts. (5) Cell cycle prolongation in the second and third generation after treatment was observed only with MMS treated cells. (6) In all experiments, individual cells of uniformly treated populations exhibited highly variable responses.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - EMS ethyl methanesulfonate - MMS methyl methanesulfonate - MNNG N-methyl-N-nitro-N-nitrosoguanidine     

Transductional analysis of chromosome replication time

Summary Following transduction of exponentially growing cultures of Escherichia coli with phage P1, cells with recombinant phenotype begin to increase in number after an initial lag of about one generation time. We show that transductants for markers located at different positions on the chromosome begin to increse at different times, in reverse order to that in which they are replicated. The period over which this happens is equal in duration to the time taken to replicate the chromosome and we have used this relationship to calculate the C-period of E. coli K12 growing at 30°C. We exclude transduction-induced filamentation as the cause of the initial lag and suggest that the lag may result from the way in which donor DNA is inherited.     

The Chlamydomonas cell cycle is regulated by a light/dark-responsive cell-cycle switch

Cultures of the unicellular green alga Chlamydomonas reinhardii can be synchronized by light/dark cycling not only under photoautotrophic but also under mixotrophic growth conditions. We observed that cultures synchronized in the presence of acetate continue to divide synchronously for one cell-cycle period when transferred to heterotrophic growth conditions. This finding enabled us to investigate the differential effects of light on cell growth and cell division. When cells were exposed to continuous light at the beginning of the growth period they entered the division phase earlier than dark-grown cells as a consequence of an increased growth rate. Illumination at the end of the growth period, however, caused a considerable delay in cell division and an extended growth period. The light-induced delay in cell division was also observed in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), an inhibitor of photosystem II. This finding demonstrates that cell division is directly influenced by a light/dard-responsive cell-cycle switch rather than by light/dark-dependent changes in energy metabolism. The importance of this light/dark control to the regulation of the Chlamydomonas cell cycle was investigated in comparison with other control mechanisms (size control, time control). We found that the light/dard-responsive cell-cycle switch regulates the transition from G1-to S-phase. This control mechanism is effective in cells which have attained the commitment to at least one round of DNA replication and division but have not attained the maximal cell mass which initiates cell division in the light.Abbreviations dCTP deoxycytidine 5-triphosphate - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Chromosomal initiation in Bacillus subtilis may involve two closely linked origins

Summary When the dnaB37 initiation mutant of Bacillus subtilis is returned to a permissive temperature following a period at 45° C, a synchronous round of DNA replication immediately ensues. Using this system we have been able to analyse the first fragments to be replicated while avoiding the use of thymine starvation or inhibitors of DNA replication. Such treatments are necessary to achieve even modest synchrony in germinating spores. Our results showed that the first fragment to be replicated was a 4kb BamHI-SalI restriction fragment, BS6. In contrast, when the analysis was performed out in the presence of novobiocin, an inhibitor of DNA gyrase, replication from BS6 was inhibited and the first fragment to be replicated was BS5, a 5.6 kb fragment located 1.7 kb to the right of BS 6. Replication from both putative origins was suppressed by rifamycin and was dependent upon dnaB. The results are discussed in relation to previous attempts to identify the first replicating fragment in germinating spores. We also discuss the possibility that B. subtilis contains two origins and suggest that either can act as the primary origin under certain conditions, or alternatively that both origins may act in concert in normal bidirectional replication, each site being required for the leading strand in each direction.     

Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication

Summary A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the — 35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.     

Mutations that increase the mitotic stability of minichromosomes in yeast: characterization of RAR1

Summary In an attempt to identify proteins involved in the initiation of DNA replication, we have isolated a series of Saccharomyces cerevisiae mutants in which the function of putative replication origins is affected. The phenotype of these Rar^- (regulation of autonomous replication) mutants is to increase the mitotic stability of plasmids whose replication is dependent on weak ARS elements. These mutations are generally recessive and complementation analysis shows that mutations in several genes may improve the ability of weak ARS elements to function. One mutation (rar1-1) also confers temperature-sensitive growth, and thus an essential gene is affected. We have determined the DNA sequence of the RAR1 gene, which reveals an open reading frame for a 48.5 kDa protein. The RAR1 gene is linked to rna1 on chromosome XIII.