HIV-1(Lai) genomic RNA: combined used of NMR and molecular dynamics simulation for studying the structure and internal dynamics of a mutated SL1 hairpin

The genome of all retroviruses consists of two identical copies of an RNA sequence associated in a non-covalent dimer. A region upstream from the splice donor (SL1) comprising a self-complementary sequence is responsible for the initiation of the dimerization. This region is able to dimerize in two conformations: a loop-loop complex or an extended duplex. Here, we solve by 2D NMR techniques the solution structure of a 23-nucleotide sequence corresponding to HIV-1 SL1(Lai) in which the mutation G12-->A12 is included to prevent dimerization. It is shown that this monomer adopts a stem-loop conformation with a seven base pairs stem and a nine nucleotide loop containing the G10 C11 A12 C13 G14 C15 sequence. The stem is well structured in an A-form duplex, while the loop is more flexible even though elements of structure are evident. We show that the structure adopted by the stem can be appreciably different from its relaxed structure when the adenines A8, A9 and A16 in the loop are mechanically constrained. This point could be important for the efficiency of the dimerization. This experimental study is complemented with a 10 ns molecular dynamics simulation in the presence of counterions and explicit water molecules. This simulation brings about information on the flexibility of the loop, such as a hinge motion between the stem and the loop and a labile lattice of hydrogen bonds in the loop. The bases of the nucleotides G10 to C15 were found outside of the loop during a part of the trajectory, which is certainly necessary to initiate the dimerization process of the genuine SL1(Lai) sequence.     

A Quantitative Study of Shoot Development in Vicia faba: III. The Dry Weights of the Plumular Internodes

The development of the bean shoot has been studied by cuttinga piece out of each internode and determining the dry weightper unit stem length, the ‘linear density’ of theintemode. The measurements of this quantity are used in the mathematicalanalysis of physiological age. It is shown that there is a drasticreduction in relative growth rate of the stem during the unfoldingof the ninth leaf and that there is evidence of an unknown factoraffecting many of the shoot-growth processes with a maximumintensity in the fifth internode. The bearing of these factson the conception of the plastochrone as a unit of shoot growthis discussed.     

On loop folding in nucleic acid hairpin-type structures

In a series of studies, combining NMR, optical melting and T-jump experiments, it was found that DNA hairpins display a maximum stability when the loop part of the molecule comprises four or five nucleotide residues. This is in contrast with the current notion based on RNA hairpin studies, from which it had been established that a maximum hairpin stability is obtained for six or seven residues in the loop. Here we present a structural model to rationalize these observations. This model is based on the notion that to a major extent base stacking interactions determine the stability of nucleic acid conformations. The model predicts that loop folding in RNA is characterized by an extension of the base stacking at the 5'-side of the double helix by five or six bases; the remaining gap can then easily be closed by two nucleotides. Conversely, loop folding in DNA is characterized by extending base stacking at the 3'-side of the double helical stem by two or three residues; again bridging of the remaining gap can then be achieved by one or two nucleotides. As an example of loop folding in RNA the anticodon loop of yeast tRNAPhe is discussed. For the DNA hairpin formed by d(ATCCTAT4TAGGAT) it is shown that the loop structure obtained from molecular mechanics calculations obeys the above worded loop folding principles.     

The correlative effect of the stem apex on the level of endogenous gibberellins inBryophyllum crenatum leaves

Following a one-side transverse incision made into the middle stem part ofBryophyllum crenatum below one of the two opposite leaves, the leaf is exposed to an enhanced effect of correlatively inhibitory substances passing out of the apical part of the stem. Within 28 days this results in a formation of adventitious roots above the incision. The flow of correlatively inhibiting substances interrupted by the incision induces, as early as within 24 h, a decrease in the level of endogenous gibberellins in the leaf above the inoision.     

Seasonal waves of asthma: A possible botanical cause

The seasonal incidence of asthma and of shoot-growth in eucalypts in four Australian capital cities (Brisbane, Sydney, Adelaide and Melbourne) are compared. The incidence of asthma appears to be closely correlated with the initial stages of the seasonal flushes in shoot-growth of eucalypts, when rapid cell-division occurs. A spring to early summer growth flush invariably occurs in all capitals when the mean monthly temperature rises above 16–18°C; it is initiated progressively later with increasing latitude firstly in Brisbane, then in Sydney, then Adelaide and finally Melbourne. Although the correlation between asthma and eucalypt shoot-growth is high, there is not necessarily a cause-and-effect relationship. Volatile compounds evaporated from young and old leaves are probably not responsible, but the fine faecal pellets from the numerous small larvae and adult insects which attack the young eucalypt shoots may produce possible asthma-allergens. The concentration of these allergens in the atmosphere is reduced by heavy rainfall and by off-shore winds, and may accumulate during periods when temperature inversions occur at low levels in the atmosphere (e.g. during autumn in Brisbane).Supported by a grant from the Asthma Foundation of Queensland.     

Crown architecture of grafted Stone pine (Pinus pinea L.): shoot growth and bud differentiation

The singular umbrella-like crown shape of Stone pine can be interpreted as a consequence of primary shoot-growth patterns and posterior axis differentiation due to differential secondary growth and down-bending of branches. This paper centres on the first aspect, analysing the growth, branching and flowering behaviour of about 5,000 individual shoots on 27 grafted Stone pines. The data measurement on standing trees allowed to study correlations of topologic and geometric variables in the shoot and their ancestors. The only significant correlations were found with parameters of the mother shoot formed the previous year and with the number of cones born 3 years before by the respective ancestor. The fitted relationships between geometric and topologic shoot and branch variables are the first step of a structural model construction that can be completed with functional components like a radiation and a carbon allocation submodel, stressing the importance of the heavy Stone pine cones as carbon sinks, with a total annual allocation similar to stem wood. In conclusion, the Stone pine crown shape emerges as consequence of the lack of initial vigour differentiation between stem and main-branch apical meristems that favour the generalized sylleptic reiteration in the open-grown trees.     

Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E. coli methionyl-tRNA synthetases.

The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems. In this study, the tRNA determinants recognized by mammalian or E. coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined. Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element. However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E. coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase. Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E. coli. tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem. In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme. Moreover, enlarging the size of the anticodon loop of E. coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase. Finally, E. coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C. This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).     

Observations on continuously growing roots of the sloth and the K14-Eda transgenic mice indicate that epithelial stem cells can give rise to both the ameloblast and root epithelium cell lineage creating distinct tooth patterns

SUMMARY Root development is traditionally associated with the formation of Hertwig's epithelial root sheath (HERS), whose fragments give rise to the epithelial cell rests of Malassez (ERM). The HERS is formed by depletion of the core of stellate reticulum cells, the putative stem cells, in the cervical loop, leaving only a double layer of the basal epithelium with limited growth capacity. The continuously growing incisor of the rodent is subdivided into a crown analog half on the labial side, with a cervical loop containing a large core of stellate reticulum, and its progeny gives rise to enamel producing. The lingual side is known as the root analog and gives rise to ERM. We show that the lingual cervical loop contains a small core of stellate reticulum cells and suggest that it acts as a functional stem cell niche. Similarly we show that continuously growing roots represented by the sloth molar and K14-Eda transgenic incisor maintain a cervical loop with a small core of stellate reticulum cells around the entire circumference of the tooth and do not form a HERS, and still give rise to ERM. We propose that HERS is not a necessary structure to initiate root formation. Moreover, we conclude that crown vs. root formation, i.e. the production of enamel vs. cementum, and the differentiation of the epithelial cells into ameloblasts vs. ERM, can be regulated independently from the regulation of stem cell maintenance. This developmental flexibility may underlie the developmental and evolutionary diversity in tooth patterning.     

Comparison of radial increment and volume growth in stems and roots of Quercus petraea

Dendrochronological studies dealing with roots, stems and branches are very rare or often take the form of short notes. The difficulties of detecting rings and of quantifying the radial growth in roots have already been described for various species. In oak the anatomical root structure differs from stemwood. The roots are radial-porous or diffuse-porous, and there is often no clear distinction between individual rings. In our study visual and radiographic techniques were used to examine radial increment in roots of sessile oak (Quercus petraea L.) which was compared with radial growth in branches and along the stem. Coarse roots were cut from four 30- to 34-year-old trees that had been uprooted mechanically and disks were taken at different distances from the stem-root base. Ring widths were measured in the stem at height of 0.3 m, at breast height (1.3 m), beneath the crown, in branches of the crown, and in roots every 20 cm. The ring widths were cross-dated, and the heterogeneity of growth within a root and within the root system were analysed. Asymmetric growth frequently occurred in roots so that ovals, I-beam and T-beam shapes were developed. With the method used in our study the annual growth layers close to the central cylinder could be distinguished as well as beneath the bark. Pointer years were detected in all sections of the tree and permitted correction of ring widths in roots. Root system, stem and branch showed a basic similarity in their radial sequence of ring width. The annual biomass increment was weaker and more variable with several consecutive changes in the roots than in the stems. The root/shoot ratio reached a minimum rather early, beginning at the cambial age of 20 years. This revised version was published online in June 2006 with corrections to the Cover Date.     

THE PHYSIOLOGICAL BASIS OF MORPHOLOGICAL POLARITY IN REGENERATION. II

1. The experiments show that the mass of air roots formed in a stem increases with the mass of the leaf attached to the stem, though it has not been possible to establish an exact mathematical relation between the two masses, owing to unavoidable sources of error. 2. Darkened leaves do not increase the mass of roots formed. 3. In stems suspended horizontally air roots appear on the lower side of the stem, with the exception of the cut end where they usually appear around the whole circumference of the stem. When the lower half of a stem suspended horizontally is cut off, roots are formed on the upper side. It is shown by experiments on leaves suspended horizontally that the more rapidly growing roots and shoots on the lower side inhibit the root and shoot formation in the upper half of such a leaf; and likewise the more rapid formation of roots on the lower side of a horizontally suspended stem seems to account for the inhibition of root formation on the upper side of such a stem. Likewise the more rapid growth of shoots on the upper side of a stem suspended horizontally is likely to inhibit the growth of shoots on the lower side. 4. Each leaf contains in its axil a preformed bud capable of giving rise to a root, which never grows out in the normal stem on account of the inhibitory influence of the normal roots at the base of the plant. These dormant root buds are situated above (apically from) the dormant shoot bud. The apical root buds can be caused to develop into air roots when a piece of stem is cut out from a plant from which the leaves except those in the basal node of the piece are removed. The larger these basal leaves the better the experiments succeed. 5. These apical air roots grow out in a few days, while the roots at the basal end of the stem (which in our experiments dip into water) grow out about a week later. As soon as the basal roots grow out in water they cause the air roots in the more apical region of the stem to dry out and to disappear. 6. In addition to the basal roots, basal nodes have also an inhibitory effect on the growth of the dormant root buds in the apical region of a stem. This is indicated by the fact that a stem with one pair of leaves near the base will form apical air roots more readily when no node is situated on the stem basally from the leaf than if there is a node basally from the leaf.     

Auxin Stimulation of Cambial Activity in Pinus silvestris II. Dependence upon Basipetal Transport

Natural auxin content has been determined in the cambial region of large Pinus silvestris L. trees at various dates during the year. The tissue was collected from the stem of intact or ring-barked trees and from stumps remaining after the trees were cut down at breast height in early summer or late autumn. No seasonal decrease of concentration of the extractable auxin in the cambial region could be detected. Decapitation or ring-barking produced severe reduction in auxin content and arrested cambial division. In the next season the auxin level and the cambial activity remained completely depressed. It is concluded that without tissue continuity in the region external to xylem and without basipetal supply of substances, no mechanism operated by roots or remaining stem tissue near the tree base can ensure a high level of auxin in the cambial region or activate and maintain the cambial division. The activity of extracted pine auxin was found not to be identical with the stimulatory potential of authentic IAA determined by standard bioassays. The possibility of interaction with other extracted substances is discussed.     

Effects of base sequence on the loop folding in DNA hairpins

High-resolution NMR and UV-melting experiments have been used to study the hairpin formation of partly self-complementary DNA fragments in an attempt to derive rules that describe the folding in these molecules. Earlier experiments on the hexadecanucleotide d(ATCCTA-TTTT-TAGGAT) had indicated that within the loop of four thymidines a wobble T-T pair is formed (Blommers et al., 1987). In the present paper it is shown that if the first and the last thymines of the intervening sequence are replaced by complementary bases, sometimes base pairs can be formed. Thus for the intervening sequences -CTTG- and -TTTA- with the pyrimidine in the 5'-position and the purine in the 3'-position, a base pair is formed leading to a loop consisting of two residues. For the intervening sequences -GTTC- and -ATTT- with the purine in the 5'-position and the pyrimidine in the 3'-position, this turns out not to be the case. It was found that it made no difference when the four-membered sequence was closed by a G-C base pair or an A-T base pair. Replacement of the two central thymidine residues by the more bulky adenine residues limits the hairpin to a four-membered loop scheme. Very surprisingly, it was found from 2D NOE experiments that the T-A base pair, formed in the loop consisting of the -TTTA- sequence, is a Hoogsteen pair. It is argued that the pairing of the bases in this scheme may facilitate the formation of a loop of two residues, since the distance of the C1' atoms in this base pair is 8.6 A instead of 10.4 A found in the canonical Watson-Crick base pair. Combination of the data obtained for the series of DNA fragments studied shows that the results can be explained by a simple, earlier proposed, loop folding principle which assumes that the folding of the four-membered loop is dictated by the stacking of the double-helical stem of the hairpin.     

THEORY OF REGENERATION BASED ON MASS ACTION

1. The writer''s older experiment, proving that equal masses of isolated sister leaves of Bryophyllum regenerate under equal conditions and in equal time equal masses (in dry weight) of shoots and roots, is confirmed. It is shown that in the dark this regeneration is reduced to a small fraction of that observed in light. 2. The writer''s former observation is confirmed, that when a piece of stem inhibits or diminishes the regeneration in a leaf, the dry weight of the stem increases by as much or more than the weight by which the regeneration in the leaf is diminished. It is shown that this is also true when the axillary bud in the stem is removed or when the regeneration occurs in the dark. 3. These facts show that the regeneration of an isolated leaf of Bryophyllum is determined by the mass of material available or formed in the leaf during the experiment and that such a growth does not occur in a leaf connected with a normal plant for the reason that in the latter case the material available or formed in the leaf flows into the stem where it is consumed for normal growth. 4. It is shown that the sap sent out by a leaf in the descending current of a stem is capable of increasing also the rate of growth of shoots in the basal parts of the leaf when the sap has an opportunity to reach the anlagen for such shoots. 5. The fact that a defoliated piece of stem forms normally no shoots in its basal part therefore demands an explanation of the polar character of regeneration which lays no or less emphasis on the chemical difference between ascending and descending sap than does Sachs'' theory of specific root- or shoot-forming substances (though such substances may in reality exist), but which uses as a basis the general mass relation as expressed in the first three statements of this summary. 6. It is suggested that the polar character of the regeneration in a stem of Bryophyllum is primarily due to the fact that the descending sap reaches normally only the root-forming tissues at the base of the stem, while the ascending sap reaches normally only the shoot-forming anlagen at the apex of the stem. 7. This suggestion is supported by the fact that when the anlagen for shoots and roots are close together as they are in the notch of a leaf, the sap of the leaf causes the growth of both roots and shoots from the same notch and the influence of the sap of the leaf on this growth increases for both roots and shoots in proportion with the mass of the leaf.     

Flagellar systems in the euglenoid flagellates

The flagellar apparatus of euglenoids consists of two functional basal bodies, three unequal microtubular roots subtending the reservoir, and a fourth band of microtubules nucleated from one of the flagellar roots and subtending the reservoir membrane. The flagellar apparatus of some euglenoids may contain additional basal bodies, striated roots ("rhizoplasts"), fibrous roots, striated connecting fibers between basal bodies, layered structures, or various electron-dense connective substances. With the possible exception of Petalomonas cantuscygni, nearly all euglenoids are biflagellate although the length of one flagellum may be highly reduced. The flagellar transition zone and number of basal bodies are highly variable among species. In recent years a cytoplasmic pocket that branches off from the reservoir has been discovered. The microtubules of the ventral flagellar root are continuous with the microtubules which line this pocket. Based on positional and structural similarities, this structure is believed to be homologous with the MTR/cytostome of bodonids. Coupled with other ultrastructural and biochemical data, the fine structure of the flagellar apparatus supports the belief that the euglenoid flagellates are descendant from bodonid ancestors.     

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.     

Conformational feasibility of a hairpin with two purines in the loop. 5'-d-GGTACIAGTACC-3'

Structural feasibility and conformational requirements for the sequence 5'-d-GGTACIAGTACC-3' to adopt a hairpin loop with I6 and A7 in the loop are studied. It is shown that a hairpin loop containing only two nucleotides can readily be formed without any unusual torsional angles. Stacking is continued on the 5'-side of the loop, with the I6 stacked upon C5. The base A7, on the 3'-side of the loop, can either be partially stacked with I6 or stick outside without stacking. Loop closure can be achieved for both syn and anti conformations of the glycosidic torsions for G8 while maintaining the normal Watson-Crick base pairing with the opposite C5. All torsional angles in the stem fall within the standard B-family of DNA helical structures. The phosphodiesters of the loop have trans,trans conformations. Loop formation might require the torsion about the C4'-C5' bond of G8 to be trans as opposed to the gauche+ observed in B-DNA. These results are discussed in relation to melting temperature studies [Howard et al. (1991) Biochemistry (preceding paper in this issue)] that suggest the formation of very stable hairpin structures for this sequence.     

Changes in hydraulic conductivity, mechanical properties, and density reflecting the fall in strain along the lateral roots of two species of tropical trees

Roots have been described as having larger vessels and so greater hydraulic efficiency than the stem. Differences in the strength and stiffness of the tissue within the root system itself are thought to be an adaptation to the loading conditions experienced by the roots and to be related to differences in density. It is not known how potential mechanical adaptations may affect the hydraulic properties of the roots. The change in strength, stiffness, conductivity, density, sapwood area, and second moment of area distally along the lateral roots of two tropical tree species in which the strain is known to decrease rapidly was studied and the values were compared with those of the trunk. It was found that as the strain fell distally along the roots, so did the strength and stiffness of the tissue, whereas the conductivity increased exponentially. These changes appeared to be related to differences in density. In contrast to the distal-most roots, the tissue of the proximal roots had a lower conductivity and higher strength than that of the trunk. This suggests that mechanical requirements on the structure rather than the water potential gradient from roots to branches are responsible for the general pattern that roots have larger vessels than the stem. In spite of their increased transectional area, the buttressed proximal roots were subjected to higher levels of stress and had a lower total conductivity than the rest of the root system.     

Underground system of Mandevilla atroviolacea (Stadelm.) Woodson (Apocynaceae,Apocynoideae) from the Brazilian high-altitude grassland

The underground systems of plants show the most varied structures adapted to survival in unfavorable environmental conditions. For instance, long-term droughts may induce the development of either water and reserve substances or vegetative propagation structures. Since Mandevilla atroviolacea is a species found at high altitudes on rocky outcrops, this study aimed to provide information on the morpho-anatomy of the underground system of this species that may assist in understanding the adaptive strategies at play. Samples from the underground system of three young and two mature plants collected in the field were sectioned and processed according to standard plant anatomical techniques. The upper area of the underground system corresponds to a xylopodium, while the lower is a tuberous stem organ. Tuberous roots account for the majority of the underground system. The increase in tuberous root diameter results from the action of a typical cambium. The high shoot-forming potential and storage of water and reserve substances observed in the underground system of this species are important strategies for it to survive in this habitat.