Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

Summary Post-mitotic epidermal cells of barley leaves were found to contain, in addition to cortical microtubules (CMTs), distinct arrays of endoplasmic microtubules (EMTs). These encircle nuclei and continuously merge into the CMT arrays that underly the plasmalemma. Detailed three-dimensional reconstruction of both types of MTs during fungal infection showed that profound and very rapid MT rearrangements occurred especially in the case of incompatible (resistant) barley-powdery mildew genotype combination. The most early MT responses, followed by their subsequent complete disintegration, were recorded around nuclei. These events might be relevant for the induction of such nuclear processes as onset of DNA synthesis and nuclear chromatin condensation. Observed pattern of early infection events, as well as less prominent responses in the case of compatible (susceptible) barley-powdery mildew genotype combination, both findings suggest that rapid reorganization of the MT cytoskeleton could be involved in recognition of the fungus by host cells and in the initiation of resistance responses in barley leaves. We hypothesize that the integrity and dynamics of the MT cytoskeleton, especially of its perinuclear part, might participate in control mechanisms involved in activation of resistance genes.Abbreviations CMTs cortical microtubules - EMTs endoplasmic microtubules - MT microtubules - PI propidium iodide - SC sensitive combination - RC resistant combination     

Nuclear counterparts of the cytoplasmic mitochondrial 12S rRNA gene: A problem of ancient DNA and molecular phylogenies

Monkey mummy bones and teeth originating from the North Saqqara Baboon Galleries (Egypt), soft tissue from a mummified baboon in a museum collection, and nineteenth/twentieth-century skin fragments from mangabeys were used for DNA extraction and PCR amplification of part of the mitochondrial 12S rRNA gene. Sequences aligning with the 12S rRNA gene were recovered but were only distantly related to contemporary monkey mitochondrial 12S rRNA sequences. However, many of these sequences were identical or closely related to human nuclear DNA sequences resembling mitochondrial 12S rRNA (isolated from a cell line depleted in mitochondria) and therefore have to be considered contamination. Subsequently in a separate study we were able to recover genuine mitochondrial 12S rRNA sequences from many extant species of nonhuman Old World primates and sequences closely resembling the human nuclear integrations. Analysis of all sequences by the neighbor-joining (NJ) method indicated that mitochondrial DNA sequences and their nuclear counterparts can be divided into two distinct clusters. One cluster contained all temporary cytoplasmic mitochondrial DNA sequences and approximately half of the monkey nuclear mitochondriallike sequences. A second cluster contained most human nuclear sequences and the other half of monkey nuclear sequences with a separate branch leading to human and gorilla mitochondrial and nuclear sequences. Sequences recovered from ancient materials were equally divided between the two clusters. These results constitute a warning for when working with ancient DNA or performing phylogenetic analysis using mitochondrial DNA as a target sequence: Nuclear counterparts of mitochondrial genes may lead to faulty interpretation of results.Correspondence to: A.C. van der Kuyl     

Field Evaluation of a Commercial Formulation of the Spodoptera exigua (Lepidoptera: Noctuidae) Nuclear Polyhedrosis Virus for Control of Beet Armyworm on Vegetable Crops in Thailand

Small - scale field trials were conducted in the Kanchanaburi and Ratchaburi provinces of Thailand between December 1994 and March 1996 to gather efficacy data to support a registration dossier for a commercial formulation of a nuclear polyhedrois virus of beet armyworm , Spodoptera exigua, under the tradename Spod - X . A liquid concentrate formula tion of Spod - X was compared with two commercial standards , Bacillus thuringiensis subsp . aizawai (CenTuri 3 . 5 WDG) and the insect growth regulator tebufenozide (Mimic 20F , a benzoylhydrazine ecdysone agonist) , for control of beet armyworm on garden pea , Chinese kale , shallot and table grape . Spod - X applied at 4 - day intervals at 3 . 1 - 12 . 5 1011 occlusion bodies (OBs) ha 1 in a spray volume of 625 - 1000 l ha 1 provided excellent control of beet armyworm on garden pea and grape (75 - 100 % reductions) after the second application . On Chinese kale and shallot , rates of Spod - X in the rate 5 - 60 1011 OBs ha 1 sprayed every 4 days in a spray volume of 1000 l ha 1 provided significant protection from infestation of beet armyworm over the growing season; however , efficacy was poor to moderate . Tank - mixing Spod - X with the pyrethroid , deltamethrin (Decis3EC) , did not improveefficacy on shallot;however , combinations of virus and B. thuringiensis or tebufenozide achieved commercially acceptable levels of crop protection on Chinese kale compared with virus alone . Lower levels of control and crop pro tection observed with all materials on Chinese kale and shallot reflect , in part , the tendency of S. exigua larvae to feed within sheltered sites and directly damage marketable plant parts . In general , all rates of Spod - X performed as well as , or better than , the B. thuringiensis standard (56 . 3 - 105 g ha 1 a.i.) on all crops tested . In general , reductions of beet armyworm larval populations and plant damage provided by all labeled rates of the chemical standard , tebufenozide (125 - 250 g ha 1 a . i .), were marginally greater than all rates of Spod - X tested . On crops such as pea and grape , where beet armyworm predominately feed on exposed leaf surfaces , our data unequivocally support the use of Spod - X for cost - effective , season - long pest management . Strategies to improve the efficacy of virus on shallot and kale are discussed .     

Synonymous substitution rates in Drosophila: Mitochondrial versus nuclear genes

Synonymous substitution rates in mitochondrial and nuclear genes of Drosophila were compared. To make accurate comparisons, we considered the following: (1) relative synonymous rates, which do not require divergence time estimates, should be used; (2) methods estimating divergence should take into account base composition; (3) only very closely related species should be used to avoid effects of saturation; (4) the heterogeneity of rates should be examined. We modified the methods estimating synonymous substitution numbers to account for base composition bias. By using these methods, we found that mitochondrial genes have 1.7–3.4 times higher synonymous substitution rates than the fastest nuclear genes or 4.5–9.0 times higher rates than the average nuclear genes. The average rate of synonymous transversions was 2.7 (estimated from the melanogaster species subgroup) or 2.9 (estimated from the obscura group) times higher in mitochondrial genes than in nuclear genes. Synonymous transversions in mitochondrial genes occurred at an approximately equivalent rate to those in the fastest nuclear genes. This last result is not consistent with the hypothesis that the difference in turnover rates between mitochondrial and nuclear genomes is the major factor determining higher synonymous substitution rates in mtDNA. We conclude that the difference in synonymous substitution rates is due to a combination of two factors: a higher transitional mutation rate in mtDNA and constraints on nuclear genes due to selection for codon usage. Received: 27 November 1996 / Accepted: 8 May 1997     

利用非洲爪蟾精子染色质和卵提取物在体外重建细胞核

应用非洲爪蟾去膜精子染色质和卵提取物成功地进行了细胞核本外重建。当精子染色质加入卵提取物后,首先发生染色质去浓缩作用,染色质整体结构膨胀;膜泡在膨胀的染色质外周聚集并逐渐彼此融合成双层膜;核孔复合体以某种未知方式组装入双层膜而形成核膜结构,并逐渐完全覆盖膨大的染色质,最终形成典型的间期核结构。     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect     

Crankshaft motions of the polypeptide backbone in molecular dynamics simulations of human type-α transforming growth factor

Summary Order parameters for the backbone N–H and C–H bond vectors have been calculated from a 150 ps molecular dynamics (MD) simulation of human type- transforming growth factor in H₂O solvent. Two kinds of crankshaft motions of the polypeptide backbone are observed in this MD trajectory. The first involves small-amplitude rocking of the rigid peptide bond due to correlated changes in the backbone dihedral angles _i–1 and _i. These high-frequency librational crankshaft motions are correlated with systematically smaller values of motional order parameters for backbone N–H bond vectors compared to C–H bond vectors. In addition, infrequent crankshaft flips of the peptide bond from one local minimum to another are observed for several amino acid residues. These MD simulations demonstrate that comparisons of N–H and C–H order parameters provide a useful approach for identifying crank-shaft librational motions in proteins.     

Changes in intracellular pH and inorganic phosphate concentration during and after muscle contraction as studied by time-resolved 31P-NMR. Alkalinization by contraction

The morula and the mesenchyme blastula nuclei contained approx. 30 nuclear proteins which were preferentially released by limited digestion with DNase I, but no proteins were released from sperm nuclei. While most of the proteins released by DNase I digestion were common to the two embryonic stages, 2 and 6 proteins were specific or enriched in morulae and mesenchyme blastulae, respectively.