Tomato yellow leaf curl virus from Sardinia is a whitefly-transmitted monopartite geminivirus.

The genome of an isolate of tomato yellow leaf curl virus from Sardinia, Italy (TYLCV-S), a geminivirus transmitted by the whitefly Bemisia tabaci, has been cloned and sequenced. The single circular DNA molecule comprises 2770 nucleotides. Genome organisation closely resembles that of the DNA A component of the whitefly-transmitted geminiviruses with a bipartite genome. A 1.8 mer of the TYLCV-S genome in a binary vector of Agrobacterium tumefaciens is infectious upon agroinoculation of tomato plants. Typical tomato yellow leaf curl disease symptoms developed about three weeks after inoculation. The disease was transmitted by the natural vector B.tabaci from agroinfected plants to test plants, reproducing in this way the full biological cycle and proving that the genome of TYLCV-S consists of only one circular single-stranded DNA molecule. Contrary to the other whitefly-transmitted geminiviruses described so far, there is no evidence for the existence nor the necessity of a second component (B DNA) in the TYLCV-S genome.     

Visualization of cAMP receptor protein-induced DNA kinking by electron microscopy

The effect of specific DNA binding of the cAMP . cAMP receptor protein complex to two DNA fragments (301 and 2685 base-pairs in length) containing the lac operon has been investigated by electron microscopy. It is shown that specific DNA binding of the cAMP . cAMP receptor protein complex induces a kink of 30 to 45 degrees in the DNA with the apex of the kink located at the site of protein attachment. These findings lend direct visual support for the kinking hypothesis based on the observation of anomalous electrophoretic mobility of DNA fragments containing specifically bound cAMP receptor protein.     

Internal mobility in a double-stranded B DNA hexamer and undecamer. A time-dependent proton-proton nuclear Overhauser enhancement study

The internal mobility of the deoxyribose H2'-H2" and base C(H5)-C(H6) and T(CH3)-T(H6) vectors has been investigated by means of time-dependent nuclear Overhauser enhancement (NOE) measurements in a B DNA hexamer and undecamer. Cross-relaxation rates between these proton pairs are determined from the initial slopes of the time development of the NOEs, and, as the interproton distances between these proton pairs are fixed, apparent correlation times for the 3 interproton vectors are calculated from the cross-relaxation rate data. It is shown that there is little residue to residue variation in the cross-relaxation rates of the interproton vectors within each oligonucleotide, that the mean apparent correlation times of the C(H5)-C(H6) and T(CH3)-T(H6) vectors are approximately equal and significantly greater than that of the H2'-H2" vectors, and that the data for the H2'-H2" vectors of both oligonucleotides and the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer cannot be accounted for by isotropic tumbling alone. The data are analysed in terms of a two motion model with isotropic tumbling and a single internal motion. The relaxation time of the internal motion at 23 degrees C is less than or equal to 1 ns for the H2'-H2" vectors of both oligonucleotides and less than or equal to 3 ns for the C(H5)-C(H6) and T(CH3)-T(H6) vectors of the undecamer. In the case of the H2'-H2" vectors, however, the amplitude of the internal motion is found to be too large to be compatible with the known stereochemistry of DNA. This finding can only be explained by invoking additional degrees of internal freedom with a larger number of internal motions of small amplitude of the type deduced from the analysis of crystallographic thermal factors [(1984) J. Mol. Biol. 173, 361-388].     

The conformations of hirudin in solution: a study using nuclear magnetic resonance, distance geometry and restrained molecular dynamics

The solution conformations of the protein hirudin have been investigated by the combined use of distance geometry and restained molecular dynamics calculations. The basis for the structure determination comprised 359 approximate inter-proton distance restrains and 10 phi backbone torsion angle restrains derived from n.m.r. measurements. It is shown that hirudin is composed of three domains: a central core made up of residues 3-30, 37-46 and 56-57; a protruding 'finger' (residues 31-36) consisting of the tip of an antiparallel beta sheet, and an exposed loop (residues 47-55). The structure of each individual domain is relatively well defined with average backbone atomic r.m.s. differences of <2 A between the final seven converged restrained dynamic structures and the mean structure obtained by averaging their coordinates. The orientation of the two minor domains relative to the central core, however, could not be determined as no long-range (i-h >5) interdomain proton-proton contacts could be observed in the two-dimensional nuclear Overhauser enhancement spectra. From the restrained molecular dynamics calculations it appears that the two minor domains exhibit large rigid-body motions relative to the central core.     

Molecular evidence for the rapid propagation of mouse t haplotypes from a single, recent, ancestral chromosome

Mouse t haplotypes are variant forms of chromosome 17 that exist at high frequencies in worldwide populations of two species of commensal mice. To determine both the relationship of t haplotypes to each other and the species within which they exist, 35 representative t haplotypes were analyzed by means of 10 independent molecular probes, including five DNA clones and five polypeptide spots identified by means of two- dimensional gel electrophoresis. All of the tested haplotypes were found to share restriction fragments and polypeptide spots that are absent in mice carrying wild-type forms of chromosome 17. This observation provides the first direct evidence that all of the known t haplotypes are descendents of a single ancestral chromosome. The absence of variation among t haplotypes could mean that this ancestral chromosome existed relatively recently, in which case it would be necessary to postulate introgressions of t haplotypes across species lines to explain their presence in both Mus domesticus and M. musculus. Alternatively, it is possible that the ancestral chromosome existed prior to the split between M. domesticus and M. musculus and that, by chance, our probes fail to detect polymorphisms that exist among the t haplotypes. A further result of our analysis is the characterization of a partial t haplotype in a wild population of Israeli mice.      

Purification and characterization of the DNA-binding protein Ner of bacteriophage Mu

The construction is described of a plasmid (pL-ner) which directs the high-level production of the bacteriophage Mu Ner protein in Escherichia coli. The protein, recovered in the soluble cellular fraction, was susceptible to in vivo proteolytic processing, in many host strains, but not in E. coli B, a natural lon- prototroph. A simple purification method is described which takes advantage of the basic nature of the protein. The purified protein was shown to be physically and chemically homogeneous and to have an amino acid sequence identical to that predicted for the authentic protein. The protein was also shown to have in vitro biological activity, as measured by specific binding to a DNA fragment containing the consensus Ner-binding sequence, and in vivo biological activity as the protein produced by the pL-ner plasmid allowed lysogenic-like maintenance of a Mu prophage c mutant unable to synthesise a functional Mu repressor.     

Refinement of the solution structure of the DNA dodecamer 5'd(CGCGPATTCGCG)2 containing a stable purine-thymine base pair: combined use of nuclear magnetic resonance and restrained molecular dynamics

The solution structure of the self-complementary dodecamer 5'd(CGCGPATTCGCG)2, containing a purine-thymine base pair within the hexameric canonical recognition site GAATTC for the restriction endonuclease EcoRI, is investigated by nuclear magnetic resonance spectroscopy and restrained molecular dynamics. Nonexchangeable and exchangeable protons are assigned in a sequential manner. A set of 228 approximate interproton distance restraints are derived from two-dimensional nuclear Overhauser enhancement spectra recorded at short mixing times. These distances are used as the basis for refinement using restrained molecular dynamics in which the interproton distance restraints are incorporated into the total energy function of the system in the form of effective potentials. Eight calculations are carried out, four starting from classical A-DNA and four from classical B-DNA. In all cases convergence to very similar B-type structures is achieved with an average atomic root mean square (rms) difference between the eight converged structures of 0.7 +/- 0.2 A, compared to a value of 6.5 A for that between the two starting structures. It is shown that the introduction of the purine-thymine mismatch does not result in any significant distortion of the structure. The variations in the helical parameters display a clear sequence dependence. The variation in helix twist and propeller twist follows Calladine's rules and can be attributed to the relief of interstrand purine-purine clash at adjacent base pairs. Overall the structure is straight. Closer examination, however, reveals that the central 5 base pair steps describe a smooth bend directed toward the major groove with a radius of curvature of approximately 38 A, which is compensated by two smaller kinks in the direction of the minor groove at base pair steps 3 and 9. These features can be explained in terms of the observed variation in roll and slide.     

The cost of ant attendance and melezitose secretion in the black bean aphid Aphis fabae

1. The aphid Aphis fabae (Scopoli) is facultatively tended by Lasius niger (Linnaeus) ants. Previously, we found that A. fabae colonies can be made up of several clones, and that clones display significant differences in the composition of their honeydew sugars, especially in the amount of the ant attractant sugar melezitose that they produce. 2. These clonal differences could greatly impact the strength of the mutualistic interaction with ants as well as the aphids' fitness. 3. Hence, the aim of this study was to compare the fitness of different A. fabae clones that differed in their melezitose secretion, and whether or not they were tended by ants. 4. Individual fitness indices, colony growth, and alate production of single‐clone aphid colonies were analysed. 5. The results demonstrate that the fitness consequences of ant attendance critically depend on an interaction between levels of melezitose production. In particular, we show that high‐melezitose secreting clones produce fewer alates and hence might have a lower dispersal ability in the presence of ants. 6. Furthermore, these data confirm previous evidence that ant attendance is costly and results in the production of fewer apterae.     

High-resolution three-dimensional structure of reduced recombinant human thioredoxin in solution

The solution structure of recombinant human thioredoxin (105 residues) has been determined by nuclear magnetic resonance (NMR) spectroscopy combined with hybrid distance geometry-dynamical simulated annealing calculations. Approximate interproton distance restraints were derived from nuclear Overhauser effect (NOE) measurements. In addition, a large number of stereospecific assignments for beta-methylene protons and torsion angle restraints for phi, psi, and chi 1 were obtained by using a conformational grid search on the basis of the intraresidue and sequential NOE data in conjunction with 3JHN alpha and 3J alpha beta coupling constants. The structure calculations were based on 1983 approximate interproton distance restraints, 52 hydrogen-bonding restraints for 26 hydrogen bonds, and 98 phi, 71 psi, and 72 chi 1 torsion angle restraints. The 33 final simulated annealing structures obtained had an average atomic rms distribution of the individual structures about the mean coordinate positions of 0.40 +/- 0.06 A for the backbone atoms and 0.78 +/- 0.05 A for all atoms. The solution structure of human thioredoxin consists of a five-stranded beta-sheet surrounded by four alpha-helices, with an active site protrusion containing the two redox-active cysteines. The overall structure is similar to the crystal and NMR structures of oxidized [Katti, S. K., LeMaster, D. M., & Eklund, H. (1990) J. Mol. Biol. 212, 167-184] and reduced [Dyson, J. H., Gippert, G. P., Case, D. A., Holmgren, A., & Wright, P. (1990) Biochemistry 29, 4129-4136] Escherichia coli thioredoxin, respectively, despite the moderate 25% amino acid sequence homology. Several differences, however, can be noted. The human alpha 1 helix is a full turn longer than the corresponding helix in E. coli thioredoxin and is characterized by a more regular helical geometry. The helix labeled alpha 3 in human thioredoxin has its counterpart in the 3(10) helix of the E. coli protein and is also longer in the human protein. In contrast to these structural differences, the conformation of the active site loop in both proteins is very similar, reflecting the perfect sequence identity for a stretch of eight amino acid residues around the redox-active cysteines.