Integration of multiple repeats of geminiviral DNA into the nuclear genome of tobacco during evolution.

Integration of viral DNA into the host nuclear genome, although not unusual in bacterial and animal systems, has surprisingly not been reported for plants. We have discovered geminvirus-related DNA (GRD) sequences, in the form of distinct sets of multiple direct repeats comprising three related repeat classes, situated in a unique locus in the Nicotiana tabacum (tobacco) nuclear genome. The organization of these sequences is similar or identical in eight different tobacco cultivars we have examined. DNA sequence analysis reveals that each repeat has sequences most resembling those of the New World geminiviral DNA replication origin plus the adjacent AL1 gene, encoding the viral replication protein. We believe these GRD sequences originated quite recently in Nicotiana evolution through integration of geminiviral DNA by some combination of the processes of illegitimate recombination, amplification, deletions, and rearrangements. These events must have occurred in plant tissue that was subsequently able to contribute to meristematic tissue yielding gametes. GRD may have been retained in tobacco by selection or by random fixation in a small evolving population. Although we cannot detect transcription of these sequences, this does not exclude the possibility that they may originally have been expressed.

Incorporation of glutamine repeats makes protein oligomerize: implications for neurodegenerative diseases.

Many transcription factors and some other proteins contain glutamine repeats; their abnormal expansion has been linked to several dominantly inherited neuro-degenerative diseases. Having found that poly(L-glutamine) alone forms beta-strands held together by hydrogen bonds between their amide groups, we surmised that glutamine repeats may form polar zippers, an unusual motif for protein-protein interactions. To test this hypothesis, we have engineered a Gly-Gln10-Gly peptide into the inhibitory loop of truncated chymotrypsin inhibitor 2 (CI2), a small protein from barley seeds, by both insertion and replacement. Gel filtration resolved both mutant inhibitors into at least three fractions, which analytical ultracentrifugation identified as monomers, dimers, and trimers of the recombinant protein; the truncated wild-type CI2 formed only monomers. CD difference spectra of the dimers and trimers versus wild type indicated that their glutamine repeats formed beta-pleated sheets, while those of the monomers versus wild type were more suggestive of type I beta-turns. The CD spectra of all three fractions remained unchanged even after incubation at 70 degrees C; neither the dimers nor the trimers dissociated at this temperature. We argue that the stability of all three fractions is due to the multiplicity of hydrogen bonds between extended strands of glutamine repeats in the oligomers or within a beta-hairpin formed by the single glutamine repeat of each monomer. Pathological effects may arise when expanded glutamine repeats cause proteins to acquire excessively high affinities for each other or for other proteins with glutamine repeats.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

On the time to reach a critical number of infections in epidemic models with infective and susceptible immigrants.

In this paper we examine the time T to reach a critical number K0 of infections during an outbreak in an epidemic model with infective and susceptible immigrants. The underlying process X, which was first introduced by Ridler-Rowe (1967), is related to recurrent diseases and it appears to be analytically intractable. We present an approximating model inspired from the use of extreme values, and we derive formulae for the Laplace-Stieltjes transform of T and its moments, which are evaluated by using an iterative procedure. Numerical examples are presented to illustrate the effects of the contact and removal rates on the expected values of T and the threshold K0, when the initial time instant corresponds to an invasion time. We also study the exact reproduction number Rexact,0 and the population transmission number Rp, which are random versions of the basic reproduction number R0.

Number of discernible colors for color-deficient observers estimated from the MacAdam limits

We estimated the number of colors perceived by color normal and color-deficient observers when looking at the theoretic limits of object-color stimuli. These limits, the optimal color stimuli, were computed for a color normal observer and CIE standard illuminant D65, and the resultant colors were expressed in the CIELAB and DIN99d color spaces. The corresponding color volumes for abnormal color vision were computed using models simulating for normal trichromatic observers the appearance for dichromats and anomalous trichomats. The number of colors perceived in each case was then computed from the color volumes enclosed by the optimal colors also known as MacAdam limits. It was estimated that dichromats perceive less than 1% of the colors perceived by normal trichromats and that anomalous trichromats perceive 50%–60% for anomalies in the medium-wavelength-sensitive and 60%–70% for anomalies in the long-wavelength-sensitive cones. Complementary estimates obtained similarly for the spectral locus of monochromatic stimuli suggest less impairment for color-deficient observers, a fact that is explained by the two-dimensional nature of the locus.

Optimizing the number of stations in arrays measurements: experimental outcomes for different array geometries and the f-k method

Array measurements have become a valuable tool for site response characterization in a non-invasive way. The array design, i.e. size, geometry and number of stations, has a great influence in the quality of the obtained results. From the previous parameters, the number of available stations uses to be the main limitation for the field experiments, because of the economical and logistical constraints that it involves. Sometimes, from the initially planned array layout, carefully designed before the fieldwork campaign, one or more stations do not work properly, modifying the prearranged geometry. Whereas other times, there is not possible to set up the desired array layout, because of the lack of stations. Therefore, for a planned array layout, the number of operative stations and their arrangement in the array become a crucial point in the acquisition stage and subsequently in the dispersion curve estimation. In this paper we carry out an experimental work to analyze which is the minimum number of stations that would provide reliable dispersion curves for three prearranged array configurations (triangular, circular with central station and polygonal geometries). For the optimization study, we analyze together the theoretical array responses and the experimental dispersion curves obtained through the f-k method. In the case of the f-k method, we compare the dispersion curves obtained for the original or prearranged arrays with the ones obtained for the modified arrays, i.e. the dispersion curves obtained when a certain number of stations n is removed, each time, from the original layout of X geophones. The comparison is evaluated by means of a misfit function, which helps us to determine how constrained are the studied geometries by stations removing and which station or combination of stations affect more to the array capability when they are not available. All this information might be crucial to improve future array designs, determining when it is possible to optimize the number of arranged stations without losing the reliability of the obtained results.