Dimethylformamide is a novel nitrilase inducer in Rhodococcus rhodochrous

Nitrilases are of commercial interest in the selective synthesis of carboxylic acids from nitriles. Nitrilase induction was achieved here in three bacterial strains through the incorporation of a previously unrecognised and inexpensive nitrilase inducer, dimethylformamide (DMF), during cultivation of two Rhodococcus rhodochrous strains (ATCC BAA-870 and PPPPB BD-1780), as well as a closely related organism (Pimelobacter simplex PPPPB BD-1781). Benzonitrile, a known nitrilase inducer, was ineffective in these strains. Biocatalytic product profiling, enzyme inhibition studies and protein sequencing were performed to distinguish the nitrilase activity from that of sequential nitrile hydratase-amidase activity. The expressed enzyme, a 40-kDa protein with high sequence similarity to nitrilase protein Uniprot Q-03217, hydrolyzed 3-cyanopyridine to produce nicotinic acid exclusively in strains BD-1780 and BD-1781. These strains were capable of synthesising both the vitamin nicotinic acid as well as β-amino acids, a compound class of pharmaceutical interest. The induced nitrilase demonstrated high enantioselectivity (> 99%) in the hydrolysis of 3-amino-3-phenylpropanenitrile to the corresponding carboxylic acid.

     

The second case of a t(17;22) in a family with neurofibromatosis type 1: sequence analysis of the breakpoint regions

A reciprocal t(17;22)(q11.2;q11.2) was found in a female patient with neurofibromatosis type 1 (NF1) and in her affected daughter. Sequence analysis of cloned junction fragments traversing the breakpoints allowed the identification of the structures involved in the rearrangement. Aberrant bands in Southern hybridizations of restriction enzyme-digested DNA of the patient pointed to the disruption of the NF1 gene in intron 31. Semispecific polymerase chain reaction analysis of the genomic DNA of the patient with the specific primer anchored at NF1 exon 31 was used to obtain the breakpoint-spanning fragment of the derivative chromosome 17. The intron 31 sequence turned out to be interrupted within a large irregular (AT) repeat. The chromosome 22-derived sequence of the der(17) junction fragment allowed us to identify cosmids of the corresponding region from a chromosome 22-specific cosmid library. With the support of the breakpoint-spanning cosmids, the chromosome 22 region upstream of the fragment carried by the der(17) was characterized. Primers deduced from the sequence of this upstream region were used in combination with a primer in NF1 intron 31 distal to the breakpoint on chromosome 17 to amplify the der(22) junction fragment. The structure of the junction sequences suggested that the translocation had arisen by unequal homologous recombination between (AT)-rich repeats on chromosome 22 and on chromosome 17 in intron 31 of the NF1 gene. However, our data support the assumption of additional rearrangements prior to, or in the course of, the recombination event, leading to a loss of the sequences between the involved (AT) repeats on chromosome 22. In the direct vicinity of these (AT) repeats, two members of a previously undescribed low-copy repetitive sequence have been found, copies of which are also present on human chromosome 13. Received: 27 August 1996 / Revised: 7 October 1996     

Helix bundles and coiled coils in alpha-spectrin and tropomyosin: a theoretical CD study

The dipole interaction model is used to investigate the effects of interactions between helices and supertwisting of helices by determining whether the predicted UV absorption and CD spectra for the three-helix bundle and coiled coil are significantly different from spectra for the single straight alpha-helix. Crystallographic data by Yan et al. for alpha-spectrin are used to construct a three-helix bundle of poly(L-alanine) modeling the protein. Backbone torsion angles represented by Fourier series are used to generate supertwisted helices and coiled coil models of poly(L-alanine) that have pitch, radius, and residue repeat similar to experimental crystallographic data on tropomyosin. Calculated CD spectra are compared with available experimental data. Theoretical spectra for the three-helix bundle and the supertwisted structures are quite similar to predictions for the straight alpha-helix of the same length with similar torsion angles, suggesting that CD is primarily dependent on the average backbone conformation and would not be a sensitive tool for distinguishing between single straight helices and closely packed or twisted alpha-helices.     

ent-Kaurene synthase is located in proplastids of meristematic shoot tissues

Previous studies have indicated that ent-kaurene synthase (KS) is located in the proplastid stroma of rapidly dividing plant tissues. Here we present further and more direct evidence for this hypothesis and follow the activity of KS throughout the entire vegetative growth period of wheat plants. During germination of wheat caryopses, KS activity was maximal for a short period culminating on the third day in the scutellum and on the forth day in the meristematic shoot base. Throughout further development of the wheat plant, KS was found in the nodes but not in internodes or leaves. The activity of KS in each node increased when the internode above it was elongating and decreased again when this internode had almost reached its final size. The correlation of KS activity with growth was particularly striking in the case of tiller development from the forth node: here KS activity had already declined, but was restored when the tiller began elongating. Electron micrographs of wheat seedling tissue with high KS activity (shoot base) showed the presence of proplastids, whereas electron micrographs of tissue without such activity (primary leaves) showed only developing or mature chloroplasts. On density-gradient centrifugation, the plastids that yielded stroma preparations with KS activity became distributed over a greater density range and also had a lower NADP⁺-glyceraldehyde 3-phosphate dehydrogenase:shikimate oxidoreductase ratio than plastids yielding KS-inactive stroma preparations. Pea shoot apices contain both proplastids and mature chloroplasts. Here also, KS activity was associated with the stroma of plastids with characteristics similar to those of the wheat proplastids, indicating that KS is associated with proplastids in pea shoot apices as well. We conclude that the stromal location of KS may be a general feature of proplastids in rapidly dividing tissue. Received: 31 July 1996 / Accepted: 9 November 1996     

Contribution of heterotrophic plankton to nitrogen regeneration in the upwelling ecosystem of A Coruna (NW Spain)

The contribution of heterotrophic plankton to nitrogen (N) regenerationin the water column, and its significance for the requirementsof phytoplankton, were studied at the seasonal scale in thecoastal upwelling ecosystem of A Coruña (Galicia, NWSpain). During 1995–1997, monthly measurements were takenof hydrographic conditions, dissolved nutrients, and abundanceand biomass of microplanktonic heterotrophs (bacteria, flagellatesand ciliates), phytoplankton and mesozooplankton (>200 µm).Additionally, series of experiments were conducted to quantifyN fluxes, including primary production (¹⁴C method), phytoplanktonuptake of nitrate, ammonium and urea (¹⁵N-labelling techniques),microheterotrophic regeneration of ammonium, mesozooplanktongrazing (chlorophyll gut-content method) and excretion of ammoniumby mesozooplankton. Two N budgets were built for the averagesituations of high (>100 mg C m^-2 h^-1) and low (<100 mgC m^-2 h^-1) primary production. The results revealed that phytoplanktonrelied strongly on regenerated ammonium all year round (33 and43% of total N uptake in high and low production situations,respectively). This demand for ammonium was closely matchedby regeneration rates of microplankton (0.14–0.25 mmolN m^-2 h^-1), whereas zooplankton contributed on average <10%to N regeneration. Likewise, zooplankton grazing had littledirect control on phytoplanktonic biomass. The results obtainedindicate that in the A Coruña upwelling system, N biomassof heterotrophic plankton is generally higher than phytoplanktonN biomass. The high rates of N regeneration measured also suggestthat a large proportion of the organic matter produced afteran upwelling pulse is recycled in the water column through themicrobial food web.     

Condensation of DNA and chromatin by histones H1, H5 and protamines. The influence of a protein phosphorylation

A mutual displacement of the DNA crosslinking protiens, protamines, histones H1 and H5, is observed if cell nuclei are incubated in solutions containing one of these species. While their competition is in accord with their specific positive charge, a displacement of the core histones does not occur. Crosslinking proteins give rise to the formation of macrocomplexes from DNA and core particles, respectively which in the present study are investigated by stray-light and centrifugation methods. It is shown than the reduction of electrostatic affinities, which in vivo is achieved by a protein phosphorylation, serves to enhance complex sizes, probably by permitting at thermodynamically controlled, ordered association of DNA segments.     

DNA break repair: refined rules of an already complicated game.

Of the many types of DNA-damage repair, this review concentrates on the aspects of DNA single- and double-strand break repair. Originally considered to represent separate routes based on distinct enzymatic machineries, it has recently been shown that these pathways converge and are interlinked at a number of points. Poly(ADP-ribose) polymerase-1 (PARP-1) is a central player in this complicated game. We present new data and our view on the mechanisms by which PARP-1 is guided to its respective interaction partners to coordinate or participate in repair or apoptosis.     

Micrococcal nuclease treatment of bacteriophage heads alters the right-hand cohesive end of lambda deoxyribonucleic Acid

Isolated bacteriophage lambda heads were exposed to micrococcal nuclease prior to addition of the phage tail. The deoxyribonucleic acid (DNA) was extracted from the heads and sheared to half-molecules whose cohesive ends were annealed to normal lambda DNA half-molecules. Melting curves of each of the cohered halves indicated that only the right-hand termini are altered by nuclease treatment.