Measurements of genetic variability for symbiotic dinitrogen fixation in fieldgrwon fababean (Vicia faba L.) using a low level15N-tracer technique

Before starting a breeding program aimed at improving the nitrogen nutrition ofVicia faba, the authors tried an alternative technique to the acetylene reduction assay, to measure some genetic variability in the plant material. The quantity of dinitrogen fixed by several cultivars ofVicia faba was estimated using a low enrichment¹⁵N tracer method and high precision¹⁵N mass spectrometry. The fababeans were cultivated for two years in two different soils. The percentage of fixed dinitrogen in the seed varied between genotypes from 40 to 83% of the total nitrogen and was positively correlated with the total seed nitrogen (r=0.64 to 0.86). A highly significant positive correlation was also found between the total seed nitrogen and the quantity of fixed dinitrogen in the seed (r=0.95 to 0.99). The technique used to measure dinitrogen fixation proved to be useful and reliable enough to discriminate between various genotypes, grown over a period of two years in two different soils. However, several non-fixing control plants showed significant differences in their¹⁵N enrichment and the problem of choosing a good reference plant was raised and discussed.     

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of (3)H-thymidine and differential gating method

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, ³H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.     

Cortical microtubule involvement in bordered pit formation in secondary xylem vessel elements ofAesculus hippocastanum L. (Hippocastanaceae): A correlative study using electron microscopy and indirect immunofluorescence microscopy

Summary A correlative study, using indirect immunofluorescence microscopy (IIF) of anti--tubulin stained sections and transmission electron microscopy (TEM), gave details of the involvement of cortical microtubules (CMTs) in the development of bordered pits in secondary xylem vessel elements ofAesculus hippocastanum L. In addition, aspects of wall cytochemistry were studied during this process using the Thiéry (PATAg) test, immunolocalization with the monoclonal antibodies JIM5 and JIM7, and a range of other cytochemical procedures. IIF showed that the alternately-arranged pits are pre-figured as perforations within a reticulum of randomly-oriented CMTs before any secondary wall thickening is obvious. Each incipient pit border is subsequently delimited by a circle of CMTs whose diameter decreases as deposition of secondary wall takes place around the perforation. These IIF observations are corroborated by a parallel TEM study. During the period of bordered pit formation, the secondary walls of the cell are lignifying. At maturity, however, the pit membrane is unlignified and continues to stain strongly with the monoclonal antibody JIM5, a marker of primary, juvenile wall. The results are discussed in terms of the relationship of the CMT cytoskeleton with development of bordered pits.Abbrevations BSA bovine serum albumin - CMT(s) cortical micro-tubule(s) - EGTA ethylene glycol-bis-(ß-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - IIP indirect immunofluorescence - MAP microtubule-associated protein - mf(s) wall microfibril(s) - MTSB microtubule-stabilising buffer - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PBS Phosphate-buffered saline - PIPES piperazine-N,N-bis-[2-ethylsulphonic acid] - SVS secondary vascular system - TEM transmission electron microscopy     

DNA sequence analysis of spontaneous and γ-radiation (anoxic)-induced lacI mutations in Escherichia coliumuC122::Tn5: differential requirement for umuC at G · C vs. A · T sites and for the production of transversions vs. transitions

Escherihica coliumC122::Tn5 cells were γ-radiated (¹³⁷Cs, 750 Gy, under N₂), and lac-constitutive mutants were produced at 36% of the wild-type level (the umC strain was not deficient in spontaneous mutagenesis, and the mutational spectrum determined by sequencing 263 spontaneous lacI^d mutations was very similar to that for the wild-type strain). The specific nature of the umC strain's partial radiation was determined by sequencing 325 radiation-induced lacI^d mutations. The yields of radiation-induced mutation classes in the umC strain (as a percentage of the wild-type yield) were: 80% for A · T → G · C transitions, 70% for multi-base additions, 60% for single-base deletions, 53% for A · T → C · G transversions, 36% for G · C → A · T transitions, 25% for multi-base deletions, 21% for A · T → T · A transversions, 11% for G · C → C · G transversions, 9% for G · C → T · A transversions and 0% for multiple mutations. Based on these deficiencies and other factors, it is concluded that the umC strain is near-normal for A · T → G · C transitions, single-base deletions and possibly A · T → C · G transversions; is generally deficient for mutagenesis at G · C sites fro transversions, and is grossly deficient in multiple mutations. Damage at G · C sites seems more difficult for translesion DNA synthesis to bypass than damage at A · T sites, and especially when trying to produced a transversion. The yield of G · C → A · T transitions in the umC strain *36% of the wild-type level) argues that a basic sites are involved in no more than 64% of γ-radiation-induced base substitutions in the wild-type strain. Altogether, these data suggest that the UmuC and UmuD′ proteins facilitate, rather than being absolutely required for, translesion DNA synthesis; with the degree of facilitation being dependent both on the nature of the noncoding DNA damage, i.e., at G · C vs A · T sites, and on the nature of the misincorporated base, i.e., whether it induces transversions or transitions.     

A fast and simplein situ method for the determination of the absolute configuration of amino acid esters using the chiroptical properties of their Eu(FOD)3 complexes

Summary The NMR shift reagent, Europium(III)-tris-(1,1,1,2,2,3,3)-heptafluoro-7,7-dimethyl-4-6-octanedione [Eu(fod)₃], complexes efficiently with-amino acid esters in chloroform. These complexes exhibit characteristic circular dichroism (CD) spectral patterns in the 350-250 nm region. A fast and simple procedure (also in microscale) has been worked out which utilizes the signs of these CD bands for the determination of the absolute configuration at the-carbon atomin situ. In the L-series, a positive CD band is observed at around 310 nm and a negative one in the 290-280 nm region. The CD spectra of the Eu complexes of the D-isomers are mirror images of those of the L-configurations. An empirical rule is proposed.Presented in part at the 2nd International Congress on Amino Acids and Analogues, Vienna, Austria, August 5–9, 1991.