Selective protection of in vitro synthesized cDNA against nucleases by incorporation of phosphorothioate-analogues.

The conditions for the stepwise synthesis of single- (ss) and double-stranded (ds) cDNA using thio-analogues instead of dNTPs are described in this paper. RNA of paramyxovirus Sendai (strain 6/94) serves as template in these experiments. The increased resistance of this alpha S-modified cDNA against several nucleases, like S1-Nuclease, DNase I, Exonuclease III, snake venom Phosphodiesterase (PDE) and the combination of DNase I and PDE is demonstrated.     

Detection of rapid induction kinetics with a new type of high-frequency modulated chlorophyll fluorometer

A newly developed modulation fluorometer is described which operates with 1 sec light pulses from a light-emitting diode (LED) at 100 KHz. Special amplification circuits assure a highly selective recording of pulse fluorescence signals against a vast background of non-modulated light. The system tolerates ratios of up to 1:10⁷ between measuring light and actinic light. Thus it is possible to measure the dark fluorescence yield and record the kinetics of light-induced changes. A high time resolution allows the recording of the rapid relaxation kinetic following a saturating single turnover flash. Examples of system performance are given. It is shown that following a flash the reoxidation kinetics of photosystem II acceptors are slowed down not only by the inhibitor DCMU, but by a number of other treatments as well. From a light intensity dependency of the induction kinetics the existence of two saturated intermediate levels (I₁ and I₂) is apparent, which indicates the removal of three distinct types of fluorescence quenching in the overall fluorescence rise from F₀ to F_max.Abbreviations Q_A and Q_B consecutive electron acceptors of photosystem II - PS II photosystem II - P 680 reaction center chlorophyll of photosystem II - F₀ minimum fluorescence yield following dark adaptation - F_max maximum fluorescence yield - DCMU 3-(3, 4-dichlorophenyl)-1, 1-dimethyl-urea - DCCD N,N-dicyclohexylcarbodiimide - PQ plastoquinone - DAD diaminodurene Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement.     

Choline stimulates synthesis of extracellular proteins in Trichoderma reesei QM 9414

A correlation between intracellular phospholipid levels and the rate of exoprotein synthesis was investigated in the filamentous fungus Trichoderma reesei QM 9414 during growth on cellulose. When the incubation temperature was varied between 20 and 37°C, the exoprotein synthesis rate correlated with the total cellular amount of phospholipids, but not with an individual phospholipid component. In contrast, when phospholipid bases were added exogenuously, a significant stimulation of exoprotein synthesis was observed with choline. The addition of the surfactant Tween 80—which also stimulates exoprotein secretion in T. reesei QM 9414—prevented choline stimulation. Optimal stimulation occurred around 20 mM choline. Choline stimulated exoprotein synthesis in general as shown by increased activities of several extracellular enzymes. Mycelia required preincubation for at least 20 h before stimulation of choline could be seen. Mycelia pregrown in the absence or presence of choline were equally effective in formation of -glucosidase upon induction with methyl--d-glucoside, and the addition of choline to the induction medium had no effect. Choline did not alter the osmotic stability of protoplasts of T. reesei. Electron microscopic examinations and analysis of chemical constituents as well as marker enzymes from choline grown and non-choline grown mycelia revealed higher contents of mitochondria and endoplasmic reticula in choline grown mycelia. The possibility is discussed that choline may stimulate exoprotein synthesis by increasing the cellular content of endoplasmic reticula.     

Antibodies to spiroperidol and their anti-idiotypes as probes for studying dopamine receptors

Spiroperidol was covalently conjugated to bovine serum albumin (BSA). Conjugated spiroperidol was almost as efficient as free spiroperidol in its binding capacity to dopamine receptor. Antibodies to spiroperidol were produced in rabbits following repeated immunizations with the conjugate of spiroperidol and BSA. The obtained antibodies have an apparent K_D of 0.02 nM for [³H]-spiroperidol. These antibodies bind also to other butyrophenones with IC₅₀ values three to four orders of magnitude higher than the IC₅₀ obtained with unlabeled spiroperidol. Antibodies were purified from anti-spiroperidol sera by affinity chromatography. Anti-idiotypic antibodies were raised in rabbits by immunization with the purified anti-spiroperidol antibodies. Some rabbits produced anti-idiotypic antibodies which bind to rat and calf striatum.     

Independent immunodominant and immunorecessive tumor-specific antigens on a malignant tumor: antigenic dissection with cytolytic T cell clones

We have analyzed the complexity of a unique tumor-specific transplantation antigen expressed by the murine ultraviolet light-induced fibrosarcoma 1591-RE. This tumor is highly immunogenic and is regularly rejected by normal mice. We have derived a cloned cytolytic T cell line showing a reactivity pattern representative of the cytolytic response of the host rejecting this regressor tumor. Using this T cell line (anti-A), variants of 1591-RE (1591-A-) were selected in vitro that had lost the same antigen as progressor variants of 1591-RE selected by the host in vivo. The in vitro derived variant was then used to generate a second T cell clone (anti-B) that recognized an antigen on the parental tumor that had been retained by the variants derived in vitro. Host-selected progressor variants were also found to have retained this antigen. By selecting for variants in vitro from the parental tumor with the anti-B T cell line, it was shown that the two different antigens (A and B) present on the parental tumor were lost independently of each other. Despite the independence of these two antigens, the host T cell response to the parental regressor tumor was invariably restricted to only the "immunodominant" A antigen.     

Isolation and Characterization of an Escherichia coli ruv Mutant Which Forms Nonseptate Filaments After Low Doses of Ultraviolet Light Irradiation

Two ultraviolet light (UV)-sensitive mutants have been isolated from Escherichia coli K-12. These mutants, designated RuvA(-) and RuvB(-), were controlled by a gene located close to the his gene on the chromosome map. They were sensitive to UV (10- to 20-fold increase) and slightly sensitive to gamma rays (3-fold increase). Host cell reactivation, UV reactivation and genetic recombination were normal in these mutants. Irradiation of the mutants with UV resulted in the production of single-strand breaks in deoxyribonucleic acid, which was repaired upon incubation in a growth medium. After UV irradiation, these mutants resumed deoxyribonucleic acid synthesis at a normal rate, as did the parent wild-type bacteria, and formed nonseptate, multinucleate filaments. From these results we concluded that the mutants have some defect in cell division after low doses of UV irradiation, similar to the lon(-) or fil(+) mutant of E. coli. The ruv locus was divided further into ruvA and ruvB with respect to nalidixic acid sensitivity and the effect of minimal agar or pantoyl lactone on survival of the UV-irradiated cell. The ruvB(-)mutant was more sensitive to nalidixic acid than were ruvA(-) and the parent strain. There was a great increase in the surviving fraction of the UV-irradiated ruvB(-) mutant when it was plated on minimal agar or L agar containing pantoyl lactone. No such increase in survival was observed in the ruvA(-) mutant.     

Genetic control of murine hemagglutinin formation to H-2.5

When B10.D2 (H-2^d) mice are immunized with lymphoid cells from C57B1/10 (H-2 ^d ) and their antisera tested against B10.A (H-2 ^a ) target cells, only antibodies to H-2.5 are measured. The same is true for immunization of DBA/2 (H-2 ^d ) mice when their antisera are absorbed with B10.D2 cells prior to testing. Irrespective of the dose of immunogen administered, the primary hemagglutinin response of B10.D2 mice is significantly lower than that of DBA/2 mice and (B10.D2 × DBA/2)F₁ hybrids, but the secondary responses are similar. The low responsiveness of B10.D2 mice appears to be determined by a single dominant gene with incomplete penetrance; the gene is not linked to eitherH- 2, Hc, or the immunoglobulin allotype loci. In addition, the H-2.5 hemagglutinin response is susceptible to nongenetic influences. When antisera from B10.D2, devoid of H-2.5 hemagglutinins, were assayed in a complement-mediated cytotoxic test, they contained almost as much anti-H-2.5 activity as did the antisera from DBA/2 mice or (B10.D2 × DBA/2)F₁ hybrids. The possibility is discussed that the locus responsible for the deficient primary hemagglutinin response of B 10.D2 may not be determinant-specific but may affect hemagglutinin responses in general.