Sinapic acid ester metabolism in wild type and a sinapoylglucose-accumulating mutant of arabidopsis.

Sinapoylmalate is one of the major phenylpropanoid metabolites that is accumulated in the vegetative tissue of Arabidopsis thaliana. A thin-layer chromatography-based mutant screen identified two allelic mutant lines that accumulated sinapoylglucose in their leaves in place of sinapoylmalate. Both mutations were found to be recessive and segregated as single Mendelian genes. These mutants define a new locus called SNG1 for sinapoylglucose accumulator. Plants that are homozygous for the sng1 mutation accumulate normal levels of malate in their leaves but lack detectable levels of the final enzyme in sinapate ester biosynthesis, sinapoylglucose:malate sinapoyltransferase. A study of wild-type and sng1 seedlings found that sinapic acid ester biosynthesis in Arabidopsis is developmentally regulated and that the accumulation of sinapate esters is delayed in sng1 mutant seedlings.     

Germ-Line Mutations in the von Hippel–Lindau Tumor-Suppressor Gene Are Similar to Somatic von Hippel–Lindau Aberrations in Sporadic Renal Cell Carcinoma

von Hippel–Lindau (VHL) disease is a hereditary tumor syndrome predisposing to multifocal bilateral renal cell carcinomas (RCCs), pheochromocytomas, and pancreatic tumors, as well as angiomas and hemangioblastomas of the CNS. A candidate gene for VHL was recently identified, which led to the isolation of a partial cDNA clone with extended open reading frame, without significant homology to known genes or obvious functional motifs, except for an acidic pentamer repeat domain. To further characterize the functional domains of the VHL gene and assess its involvement in hereditary and nonhereditary tumors, we performed mutation analyses and studied its expression in normal and tumor tissue. We identified germ-line mutations in 39% of VHL disease families. Moreover, 33% of sporadic RCCs and all (6/6) sporadic RCC cell lines analyzed showed mutations within the VHL gene. Both germ-line and somatic mutations included deletions, insertions, splice-site mutations, and missense and nonsense mutations, all of which clustered at the 3' end of the corresponding partial VHL cDNA open reading frame, including an alternatively spliced exon 123 nt in length, suggesting functionally important domains encoded by the VHL gene in this region. Over 180 sporadic tumors of other types have shown no detectable base changes within the presumed coding sequence of the VHL gene to date. We conclude that the gene causing VHL has an important and specific role in the etiology of sporadic RCCs, acts as a recessive tumor-suppressor gene, and appears to encode important functional domains within the 3' end of the known open reading frame.     

Quantitative phytogeography of the genus Allium in Siberia and Mongolia

The phytogeography of the genus Allium in Siberia and Mongolia is described, based on the numerical classification of a matrix of 56 species and 769 Operational Geographic Uniis (OGUs). Two main diversity centers can be detected, the Altai-Tuva region and southeastern Siberia, which can be further subdivided into 4 subcenters: Altai Mts., Tuva Mts., southern Baikal and Dahuria. The first three subcenters. located in southern Siberia, are rich in endemic species, which are mostly bound to semi-arid environments such as montane steppes and alpine vegetation. These old, isolated mountain ranges constitute the main refugial centers for the Allium flora of Siberia and Mongolia. The Tuva subcenter, rich in endemics and poor in polyploid species, seems to be the most conservative area; the south Baikal region, much richer in polyploid species, appears as an important center af speciation.     

Metropolis Monte Carlo calculations of DNA structure using internal coordinates and NMR distance restraints: An alternative method for generating a high-resolution solution structure

Summary A new method, a restrained Monte Carlo (rMC) calculation, is demonstrated for generating high-resolution structures of DNA oligonucleotides in solution from interproton distance restraints and bounds derived from complete relaxation matrix analysis of two-dimensional nuclear Overhauser effect (NOE) spectral peak intensities. As in the case of restrained molecular dynamics (rMD) refinement of structures, the experimental distance restraints and bounds are incorporated as a pseudo-energy term (or penalty function) into the mathematical expression for the molecular energy. However, the use of generalized helical parameters, rather than Cartesian coordinates, to define DNA conformation increases efficiency by decreasing by an order of magnitude the number of parameters needed to describe a conformation and by simplifying the potential energy profile. The Metropolis Monte Carlo method is employed to simulate an annealing process. The rMC method was applied to experimental 2D NOE data from the octamer duplex d(GTA-TAATG)·d(CATTATAC). Using starting structures from different locations in conformational space (e.g. A-DNA and B-DNA), the rMC calculations readily converged, with a root-mean-square deviation (RMSD) of <0.3 Å between structures generated using different protocols and starting structures. Theoretical 2D NOE peak intensities were calculated for the rMC-generated structures using the complete relaxation matrix program CORMA, enabling a comparison with experimental intensities via residual indices. Simulation of the vicinal proton coupling constants was carried out for the structures generated, enabling a comparison with the experimental deoxyribose ring coupling constants, which were not utilized in the structure determination in the case of the rMC simulations. Agreement with experimental 2D NOE and scalar coupling data was good in all cases. The rMC structures are quite similar to that refined by a traditional restrained MD approach (RMSD<0.5 Å) despite the different force fields used and despite the fact that MD refinement was conducted with additional restraints imposed on the endocyclic torsion angles of deoxyriboses. The computational time required for the rMC and rMD calculations is about the same. A comparison of structural parameters is made and some limitations of both methods are discussed with regard to the average nature of the experimental restraints used in the refinement.Abbreviations MC Monte Carlo - rMC restrained Monte Carlo - MD molecular dynamics - rMD restrained molecular dynamics - DG distance geometry - EM energy minimization - 2D NOE two-dimensional nuclear Overhauser effect - DQF-COSY double-quantum-filtered correlation spectroscopy - RMSD root-mean-square deviation To whom correspondence should be addressed.     

Interproton distance bounds from 2D NOE intensities: Effect of experimental noise and peak integration errors

Summary The effect of experimental and integration errors on the calculations in interproton distances from NOE intensities is examined. It is shown that NOE intensity errors can have a large impact on the distances determined. When multiple spin (spin diffusion) effects are significant, the calculated distances are often underestimated, even when using a complete relaxation matrix analysis. In this case, the bias of distances to smaller values is due to the random errors in the NOE intensities. We show here that accurate upper and lower bounds of the distances can be obtained if the intensity errors are properly accounted for in the complete relaxation matrix calculations, specifically the MARDIGRAS algorithm. The basic MARDIGRAS algorithm has been previously described [Borgias, B.A. and James, T.L. (1990) J. Magn. Reson., 87, 475–487]. It has been shown to provide reasonably good interproton distance bounds, but experimental errors can compromise the quality of the resulting restraints, especially for weak cross peaks. In a new approach introduced here, termed RANDMARDI (random error MARDIGRAS), errors due to random noise and integration errors are mimicked by the addition of random numbers from within a specified range to each input intensity. Interproton distances are then calculated for the modified intensity set using MARDIGRAS. The distribution of distances that define the upper and lower distance bounds is obtained by using N randomly modified intensity sets. RANDMARDI has been used in the solution structure determination of the interstrand cross-link (XL) formed between 4-hydroxymethyl-4,5,8-trimethylpsoralen (HMT) and the DNA oligomer d(5-GCGTACGC-3)₂ [Spielmann, H.P. et al. (1995) Biochemistry, 34, 12937–12953]. RANDMARDI generates accurate distance bounds from the experimental NOESY cross-peak intensities for the fixed (known) interproton distances in XL. This provides an independent internal check for the ability of RANDMARDI to accurately fit the experimental data. The XL structure determined using RANDMARDI-generated restrains is in good agreement with other biophysical data that indicate that there is no bend introduced into the DNA by the cross-link. In contrast, isolated spin-pair approximation calculations give distance restraints that, when applied in a restrained molecular dynamics protocol, produce a bent structure.Abbreviations NOE nuclear Overhauser effect - SD standard deviation - HMT 4-hydroxymethyl-4,5,8-trimethylpsoralen - XL psoralen-DNA interstrand cross-link     

Physical mapping of cleavage sites recognized by restriction endonucleases on the genome of bacteriophage T5

The DNA of bacteriophage T5 has been treated with restriction endonucleases EcoRi, HindIII, BamI, SmaI, PstI, SalI, KpnI and the electrophoretic pattern obtained in agarose gel has been analyzed in order to localize the specific cleavage sites on the T5 DNA. The localization of cleavage sites has been deduced from the electrophoretic pattern of double and partial digests, the digests of isolated restriction fragments and the digests of deletion mutant T5st(o) DNA.Four BamI cleavage sites have been found and localized on the physical map of T5 DNA at 0.21, 0.225, 0.685 and 0.725 fractional length. Endonuclease SmaI cleaves at 0.39, 0.59 and 0.69 fractional length. Endonuclease PstI cuts T5 DNA at 11 sites: 0.090, 0.210, 0.320, 0.510, 0.635, 0.670, 0.705, 0.770, 0.815, 0.840, 0.875 fractional length. Six KpnI cleavage sites have been mapped at 0.170, 0.215, 0.525, 0.755, 0.830, 0.850 fractional length. A complete cleavage map of the phage genome is presented for seven restriction enzymes.     

Origin,destination and mapping of tritocerebral neurons of locust

Summary The connectivities of the tritocerebrum of locust (Locusta migratoria L., Schistocerca gregaria (Forsk.)) were studied histologically and by means of cobalt chloride infusion. Its neuropil consists partly of fibers which traverse the tritocerebrum and areas consisting of neuropilar agglomerizations (glomeruli). The following direct connections between the tritocerebrum and other regions were observed: connections to 1) dorsal and lateral brain regions (mushroom body, optic lobe), 2) the ventral nerve cord, 3) the stomatogastric nervous system (here the protocerebrum and the subesophageal ganglion are also involved in these connections), 4) the retro-cerebral glands (corpora cardiaca, corpora allata), and 5) muscles of the foregut. Acknowledgements. This study was supported by grants from the Deutsche Forschungsgemeinschaft to N.K. The authors wish to thank Dr. H.-W. Honegger (Fachbereich Biologie, Universität Konstanz) and Dr. N.J. Strausfeld (E.M.B.L., Heidelberg) for helpful comments and for assisting with the English text     

Purification and characterization of a human recombinant T-cell protein-tyrosine-phosphatase from a baculovirus expression system.

A 48-kDa human T-cell protein-tyrosine-phosphatase (TC.PTPase) and a truncated form missing an 11-kDa C-terminal segment (TC delta C11.PTPase) were expressed by using the baculovirus system and characterized after extensive purification. The full-length PTPase was restricted to the particulate fraction of the cells from which it could be released by a combination of salt and detergent. The enzyme was entirely specific for phosphotyrosine residues. It displayed a low level of activity toward phosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme (RCML), but was 12 times more active toward phosphorylated myelin basic protein (MBP). By contrast, the 37-kDa form localized in the soluble fraction, and its activity toward RCML was 5 times higher than that observed with MBP. The autophosphorylated cytoplasmic domain of the EGF receptor served as substrate for both enzymes. Limited proteolysis of either protein gave rise to a 33-kDa fragment displaying the substrate specificity of the truncated form. These data lend further support to the view that the C-terminal segment of the T-cell PTPase serves a regulatory function, playing an important role in the localization and substrate specificity of the enzyme.     

CHLOROPHYLL-SPECIFIC PHOTOSYNTHESIS AND QUANTUM EFFICIENCY AT SUBSATURATING LIGHT INTENSITIES12

Light-limited rates of photosynthesis normalized for chlorophyll a, (α), and actual photon absorption (quantum efficiency, Ф) were determined for six eponentially growing algal species grown under identical conditions. The same parameters, α and Ф, were also monitored for a single diatom species, Thalassiosira pseudonana Hasle & Heimdal, through its growth cycle in batch culture. Statistical differences in α could be demonstrated among the six different exponentially growing species while no differences could be shown for Ф. Statistical differences among the six species were minimized when photosynthetic rates were normalized for in vivo fluorescence rather than extracted chlorophyll a. Both α and Ф were constant while T. pseudonana was in the exponential phase of growth, but both declined as the culture entered stationary phase. While cells were in exponential growth, differences in a were attributed to varying rates of in vivo light absorption per chlorophyll a, thus providing experimental evidence that the in vivo chlorophyll a extinction coefficient, k_c (m²· mg Chl a^?1), cannot be assumed constant.