Homology modeling by the ICM method

Five models have been built by the ICM method for the Comparative Modeling section of the Meeting on the Critical Assessment of Techniques for Protein Structure Prediction. The targets have homologous proteins with known three-dimensional structure with sequence identity ranging from 25 to 77%. After alignment of the target sequence with the related three-dimensional structure, the modeling procedure consists of two subproblems: side-chain prediction and loop prediction. The ICM method approaches these problems with the following steps: (1) a starting model is created based on the homologous structure with the conserved portion fixed and the noncon-served portion having standard covalent geometry and free torsion angles; (2) the Biased Probability Monte Carlo (BPMC) procedure is applied to search the subspaces of either all the nonconservative side-chain torsion angles or torsion angles in a loop backbone and surrounding side chains. A special algorithm was designed to generate low-energy loop deformations. The BPMC procedure globally optimizes the energy function consisting of ECEPP/3 and solvation energy terms. Comparison of the predictions with the NMR or crystallographic solutions reveals a high proportion of correctly predicted side chains. The loops were not correctly predicted because imprinted distortions of the backbone increased the energy of the near-native conformation and thus made the solution unrecognizable. Interestingly, the energy terms were found to be reliable and the sampling of conformational space sufficient. The implications of this finding for the strategies of future comparative modeling are discussed. © 1995 Wiley-Liss, Inc.     

Methylation and expression of a metallothionein promoter ovine growth hormone fusion gene (MToGH1) in transgenic mice

We have examined transgene methylation in the DNA from the livers of a pedigree of mice carrying three copies of an integrated MToGH1 transgene. Utilizing the methylation-sensitive isoschizomersMsp I andHpa II, Southern blot analysis revealed that all second generation animals derived from a transgenic female had hypermethylated DNA, whereas first generation animals sired by a transgenic male displayed a range of methylation phenotypes ranging from no methylation to hypermethylation of the transgene sequences. Of the mice that exhibited hypermethylation of the transgene in CpG dinucleotides (CmCGG), a minority of these animals also exhibited apparent CpC methylation (i.e. inhibition ofMsp I cutting, presumably blocked by methylation of the outer C of CCGG). Methylation was also examined in the inner C of CC(A/T)GG sequences in the MToGH1 transgene using the isoschizomer pairBstN I andEcoR II. A minority of MToGH1 animals in the F₁ generation showed clear evidence of methylation in these sites as well as in the inner and outer Cs of CCGG sites. An examination of MToGH1 expression in terms of oGH levels in serum revealed that there was a high degree of variation in the levels of circulating oGH between animals of this pedigree. There was a weak inverse relationship between the serum level of oGH and the extent of methylation of the transgene. In particular, mice exhibiting CpC together with CpG methylation were found to have very low levels of circulating oGH. Our results highlight the nature and complexity of epigenetic factors associated with transgene sequences which may ultimately influence expression of introduced genes in the mammalian genome.     

Division synchrony and the dynamics of microbial populations: A size-specific model

A discrete, environmentally coupled, size-specific model of microbial population dynamics in continuous culture is presented. It is mathematically simpler than other models based on similar assumptions and lends itself to numerical and analytic solutions. It displays several phenomena which have been reported in the experimental literature but which are not well understood; specifically, a loose relationship between biomass and numbers (i.e., a time lag between mass growth and cell division) and a critical damping of biomass while numbers continue to oscillate. In addition, the model provides several new predictions: The stable biomass distribution is independent of the environmental factors considered in the model and uniformly distributes the biomass among the size classes. The rate of approach to stability and the frequency of waves through the size distributions are a function of the flow rate and the variance in rate of growth and size at division. The model should provide a useful basis for studying the effects of size specificity on the dynamics of microbial populations cultured in chemostats.     

Removal of insect basal laminae using elastase

We have used the enzyme elastase to remove the basal lamina of epithelia from two insects: the upper Malpighian tubules of Rhodnius prolixus and imaginai discs of Drosophila melanogaster. Removal of the basal lamina was confirmed using scanning and transmission electron microscopy. Use of the technique on the Malphighian tubules of Rhodnius reveals for the first time the three-dimensional organization of the circumferential folds of the basal plasma membrane. Elastase is much more effective in removing the basal lamina than are the enzymes hyaluronidase, collagenase, and chymotrypsin, either alone or in combination. Following elastase treatment, cells of the Malpighian tubules dissociate with only mild mechanical agitation into single, viable cells. Treatment with elastase removes the basal laminae of imaginai discs of Drosophila and accelerates evagination as has been previously described for trypsin. To obtain single cell preparations from elastase-treated imaginai discs, mechanical stirring in Ringer low in Ca²⁺ was required. In addition to its usefulness in cell isolation, elastase treatment allows examination of the effect of removal of basal laminae on the physiology and development of insect epithelia.     

A distance geometry program for determining the structures of small proteins and other macromolecules from nuclear magnetic resonance measurements of intramolecular1H−1H proximities in solution

DISGEO is a new implementation of a distance geometry algorithm which has been specialized for the calculation of macromolecular conformation from distance measurements obtained by two-dimensional nuclear Overhauser enhancement spectroscopy. The improvements include (1) a decomposition of the complete embedding process into two successive, more tractable calculations by the use of “substructures”, (2) a compact data structure for storing incomplete distance information on a structure, (3) a more efficient shortest-path algorithm for computing the triangle inequality limits on all distances from this information, (4) a new algorithm for selecting random metric spaces from within these limits, (5) the use of chirality constraints to obtain good covalent geometry without the use ofad hoc weights or excessive optimization. The utility of the resultant program with nuclear magnetic resonance data is demonstrated by embedding complete spatial structures for the protein basic pancreatic trypsin inhibitor vs all 508 intramolecular, interresidue proton-proton contacts shorter than 4.0 Å that were present in its crystal structure. The crystal structure could be reproduced from this data set to within 1.3 Å minimum root mean square coordinate difference between the backbone atoms. We conclude that the information potentially available from nuclear magnetic resonance experiments in solution is sufficient to define the spatial structure of small proteins.     

Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.Abbreviations 9-HFCA 9-hydroxyfluorenecarboxylic acid - NPA naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - IAA indole-3-acetic acid     

The response of the atrium to direct mechanical wounding in the adult heart of the newt,Notophthalmus viridescens

Summary Wound repair and proliferation were examined in the injured newt atrium with light- and electron-microscopic techniques including autoradiography. Hearts were injured by removing a piece approximately 0.5 mm² of the atrial wall. The five-day wound was an endothelial and mesothelial-lined blood clot bordered by a 150-m necrotic zone. Repair progressed from the periphery inward with areas of macrophage activity replaced by fibroblasts and connective tissue. The wound at 25 days consisted of a scar with few myocytes. There was no difference in the proliferative behavior between the right and left atria. Proliferative cells were localized to a 500-m reactive zone surrounding the wound. The maximum mesothelial cell thymidine-labeling index of 20.5% and mitotic index of 1.4% were seen 5 days after injury. The peak connective tissue cell thymidine-labeling index of 10.2% and mitotic index of 0.4% were seen 10 days after wounding. The peak thymidine-labeling index of 9.8% for myocardial cells was recorded 10 days after injury with a mitotic index of 0.2%. Proliferation returned to control levels by 25 days post-injury. Electron microscopy demonstrated that myocytes engaged in DNA synthesis were indistinguishable from control myocytes. Z-band material was not observed in mitotic myocytes, but myofilaments and junctions were present.     

Auxin action on proton influx in corn roots and its correlation with growth

At concentrations inhibitory to the elongation of corn (Zea mays L.) roots, the auxins, indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (α-NAA), cause an increase in the pH of the bathing medium; this increase occurs with an average latent period shorter than the latent period for the inhibitory effect of these auxins on elongation. Indole-2-carboxylic acid, an inactive structural analogue of IAA, and β-naphthaleneacetic acid, an inactive analogue of α-NAA, affect neither growth nor the pH of the medium. Since acid pH is known to promote and basic pH to inhibit root elongation, the data are consistent with the hypothesis that hormone-induced modification of cell-wall pH plays a role in the control of elongation of roots, as has been proposed for elongation of stems and coleoptiles.     

Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium

Summary Conventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCl _i ) inNecturus gallbladder epithelium. As shown previously (Reuss, L., Weinman, S.A., 1979;J. Membrane Biol. 49:345), when the tissue was exposed to NaCl-Ringer on both sidesaCl _i was about 30mm, i.e., much higher than the activity predicted from equilibrium distribution (aCl_eq) across either membrane (5–9mm). Removal of Cl from the apical side caused a reversible decrease ofaCl _i towards the equilibrium value across the basolateral membrane. A new steady-stateaCl _i was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases ofaCl _i when Li, choline, tetramethylammonium or N-methyl-d-glucamine (NMDG) were employed to replace Na. During bilateral Na substitutions with choline the cells depolarized significantly. However, no change of cell potential was observed when NMDG was employed as Na substitute. Na replacements with choline or NMDG on the serosal side only did not changeaCl _i . When K substituted for mucosal Na, the cells depolarized andaCl _i rose significantly. Combinations of K for Na and Cl for SO₄ substitutions showed that net Cl entry during cell depolarization can take place across either membrane. The increase ofaCl _i in depolarized cells exposed to K₂SO₄-Ringer on the mucosal side indicates that the basolateral membrane Cl permeability, (P _Cl) increased. These results support the hypothesis that NaCl entry at the apical membrane occurs by an electroneutral mechanism, driven by the Na electrochemical gradient. In addition, we suggest that Cl entry during cell depolarization is downhill and involves an increase of basolateral membraneP _Cl.     

FLORAL PARAMETERS,BREEDING SYSTEM AND POLLINATOR TYPE IN TRICHOSTEMA (LABIATAE)

Diversity in flower size and degree of exsertion of anthers and stigma from the corolla in the California species of Trichostema appear to be correlated with breeding system and pollinator type (bee vs. bird). Autogamous (self-pollinating) species unlike xenogamous (cross-pollinating) species lack spatially separate anthers and stigma and nototribic pollination. The outcrossing species have significantly larger flowers, significantly larger nectar volumes and significantly higher pollen-ovule ratios than do selfing species. Because autogamous species are less reliant on flower visitors to facilitate pollination, there may be relaxed selection for large nectar producing flowers. Pollen-ovule ratios are correlated with breeding system and reflect the efficiency of pollen transport. Data on floral parameters suggest xenogamous species expend more energy per floral unit and are less efficient seed producers than closely related autogamous species.