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.     

A soluble interleukin 2 receptor produced by a normal alloreactive human T cell clone binds interleukin 2 with low affinity

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.     

Isolation and Characterization of Thermophilic Bacterial Strains with Inulinase Activity

Nine bacterial strains growing on inulin as the sole carbon and energy source were isolated from soil samples by enrichment culture on a mineral medium. Four of the strains were thermophilic and belong to the genus Bacillus. The thermophilic strains synthesized a β-fructosidase that was active on both inulin and sucrose. The presence of inulin in the culture medium is necessary for enzyme synthesis. Most of the activity on inulin was recovered in the culture medium, and the enzyme was synthesized during cell growth.     

Glucose and amino acid metabolism in chick telencephalon slices: Changes with incubation conditions and animals' development

Glucose and amino acid metabolism in 1- and 30-day-old chick telencephalon slices was studied in two incubation media in the presence or in the absence of a continuous oxygenation. Medium 1 has a composition and a tonicity similar to cerebrospinal fluid, medium 2 is hypertonic and does not contain any K⁺ ions. The incorporation of glucose carbon into amino acids and the distribution of radioactivity between the different amino acids are close to the ones observed in the chick brain in vivo only when the slices are incubated in medium 1, with oxygen at 30 days and without oxygen for the 1-day-old chick. It also appears that if oxygenation is necessary for incubation of mature brain tissue in vitro, the absence of the medium oxygenation is more suitable for the study of glucose metabolism in 1-day-old chick brain slices.     

The protein kinases tightly bound to DNA are present in normal tissues and in regenerating liver, but strongly decreased in hepatomas

We have previously described in rat liver two protein kinases tightly bound to DNA, one is serine-specific, the other arginine-specific. In this work we show that both enzymes are present in various rat tissues and in liver from various species. Both kinase specific activities are strongly decreased in methyl-DBA-induced hepatomas and in HTC cells but not in regenerating liver after hepatectomy. This decrease is then not related to cell proliferation.     

Thyroid hormone modulation of TRH precursor levels in rat hypothalamus, pituitary, thyroid and blood

In the present study we have examined the in vivo effects of thyroid hormones and TRH on tissue and blood levels of TRH and TRH-Gly (pGlu-His-Pro-Gly), a TRH precursor. Using specific radioimmunoassays (RIAs), we measured TRH immunoreactivity (TRH-IR) and TRH-Gly-IR concentrations in blood, hypothalamus, anterior and posterior pituitary, and thyroid in euthyroid, hypothyroid and thyroxine (T4)-treated 250 g male Sprague-Dawley rats. TRH-Gly-IR and TRH-IR were detected in all of these tissues. Highly significant positive correlations between whole blood TRH-Gly-IR levels and the corresponding serum TSH values (p less than 0.01), whole blood TRH-IR versus serum TSH (p less than 0.01) and whole blood TRH-Gly-IR versus whole blood TRH-IR (p less than 0.01) are consistent with cosecretion of TRH and TRH precursor peptides into the circulation. Euthyroid rats injected with TRH IP (1 microgram/100 g b.wt.) and hypothyroid rats had 4-fold higher whole blood TRH-Gly-IR levels compared to euthyroid controls (p less than 0.0005). Injection of TRH into euthyroid rats significantly increased the TRH-Gly-IR concentration in the hypothalamus, anterior and posterior pituitary and thyroid. The increase in blood TRH-Gly-IR following intravenous TRH may be due, in part, to partial saturation of TRH-degrading enzymes in blood and cell membranes. The ratio of TRH-Gly to TRH was significantly increased in the anterior pituitary by hypothyroidism and TRH injection, suggesting that thyroid hormones and TRH regulate the alpha-amidation of TRH-Gly to form TRH in this tissue. TRH-Gly levels of pooled pituitary and thyroid extracts quantitated by a combination of TRH-Gly RIA and high performance liquid chromatography (HPLC) revealed several-fold increases following incubation at 60 degrees C. Heating at this temperature may block the alpha-amidation activity in extra-hypothalamic tissues but not the "trypsin-like" enzymes which cleave prepro-TRH into TRH-Gly-immunoreactive peptides.