Evolutionary implications of permanent odd polyploidy in the stable sexual, pentaploid of Rosa canina L.

In Rosa canina (2n = 5x = 35), the pollen and ovular parents contribute, respectively, seven and 28 chromosomes to the zygote. At meiosis I, 14 chromosomes form seven bivalents and 21 chromosomes remain as univalents. Fluorescent in situ hybridization to mitotic and pollen mother cells (PMC) of R. canina showed that 10 chromosomes (two per genome) carry ribosomal DNA (rDNA) loci. Five chromosomes carry terminal 18S-5.8S-26S rDNA loci; three of these also carry paracentric 5S rDNA loci and were designated as marker chromosomes 1. Five chromosomes carry only 5S rDNA loci and three of these were designated as marker chromosomes 2. The remaining four of the 10 chromosomes with rDNA loci were individually identifiable by the type and relative sizes of their rDNA loci and were numbered separately. At PMC meiosis, two marker chromosomes 1 and two marker chromosomes 2 formed bivalents, whereas the others were unpaired. In a gynogenetic haploid of R. canina (n = 4x = 28), obtained after pollination with gamma-irradiated pollen, chromosomes at meiosis I in PMC remained predominantly unpaired. The data indicate only one pair of truly homologous genomes in R. canina. The 21 unpaired chromosomes probably remain as univalents through multiple generations and do not recombine. The long-term evolutionary consequence for the univalents is likely to be genetic degradation through accumulated mutational change as in the mammalian Y chromosome and chromosomes of asexual species. But there is no indication that univalents carry degenerate 5S rDNA families. This may point to a recent evolution of the R. canina meiotic system.

Oryzalin-induced chromosome doubling in Rosa and its effect on plant morphology and pollen viability.

Shoot tips of the diploid rose Thérèse Bugnet were treated in vitro to oryzalin at concentrations of 5 and 15 microM. Tetraploid shoots were obtained in highest frequencies (40%) after exposure to 5 microM oryzalin for 14 days. Thin (1 mm) nodal sections were treated with 5 microM oryzalin and the highest frequency of tetraploids (66%) was obtained after exposure for only 1 day. The shorter exposure times required to induce chromosome doubling in thin nodal sections is attributed to the more efficient delivery of oryzalin to the meristem. Tetraploids were obtained from four diploid roses and hexaploids from two triploid roses. Chromosome doubling was accompanied by increases in thickness and a darker green colouration of the leaves and, in all diploid to tetraploid and one triploid to hexaploid conversion, the breadth/length ratio of leaflets was significantly increased. Internodes were longer in tetraploids than diploids but significantly shorter in hexaploids than triploids. The number of petals per flower in the tetraploid form of Thérèse Bugnet was double that of the diploid. Significant increases in pollen viability accompanied chromosome doubling of all four diploids and one of the two triploids.

In-vitro selection of highly stabilized protein variants with optimized surface.

Thermostable proteins are of prime importance in protein science, but it has remained difficult to develop general strategies for stabilizing a protein. Site-directed mutagenesis based on comparisons with thermophilic homologs is rarely successful because the sequence differences are too numerous and dominated by neutral mutations. Here we used a method of directed evolution to increase the stability of a mesophilic protein, the cold shock protein Bs-CspB from Bacillus subtilis. It differs from its thermophilic counterpart Bc-Csp from Bacillus caldolyticus at 12 surface-exposed positions. To elucidate the stabilizing potential of exposed amino acid residues, six of these variant positions were randomized by saturation mutagenesis, the corresponding library of sequences was inserted into the gene-3-protein of the filamentous phage fd, and stabilized variants were selected by the Proside technique. Proside links the increased protease resistance of stabilized protein variants with the infectivity of the phage. Many strongly stabilized variants of Bs-CspB were identified in two selections, one in the presence of a denaturant and the other at elevated temperature. Several of them are significantly more stable than the naturally thermostable homolog Bc-Csp, and the best variant reaches Tm-Csp (the homolog from the hyperthermophile Thermotoga maritima) in stability. Remarkably, this variant differs from Tm-Csp at five and from Bc-Csp at all six randomized positions. This indicates that proteins can be strongly stabilized by many different sets of surface mutations, and Proside selects them efficiently from large libraries. The course of the selection could be directed by the conditions. In an ionic denaturant non-polar surface interactions were optimized, whereas at elevated temperature variants with improved electrostatics were selected, pointing to two different strategies for stabilization at protein surfaces.

Libraries of hybrid proteins from distantly related sequences.

We introduce a method for sequence homology-independent protein recombination (SHIPREC) that can create libraries of single-crossover hybrids of unrelated or distantly related proteins. The method maintains the proper sequence alignment between the parents and introduces crossovers mainly at structurally related sites distributed over the aligned sequences. We used SHIPREC to create a library of interspecies hybrids of a membrane-associated human cytochrome P450 (1A2) and the heme domain of a soluble bacterial P450 (BM3). By fusing the hybrid gene library to the gene for chloramphenicol acetyl transferase (CAT), we were able to select for soluble and properly folded protein variants. Screening for 1A2 activity (deethylation of 7-ethoxyresorufin) identified two functional P450 hybrids that were more soluble in the bacterial cytoplasm than the wild-type 1A2 enzyme.

Selecting proteins with improved stability by a phage-based method.

We describe a method for the stabilization of proteins that links the protease resistance of stabilized variants of a protein with the infectivity of a filamentous phage. A repertoire of variants of the protein to be stabilized is inserted between two domains (N2 and CT) of the gene-3-protein of the fd phage. The infectivity of fd phage is lost when the three domains are disconnected by the proteolytic cleavage of unstable protein inserts. Rounds of in vitro proteolysis, infection, and propagation can thus be performed to enrich those phage containing the most stable variants of the protein insert. This strategy discriminates between variants of a model protein (ribonuclease T1) differing in conformational stability and selects from a large repertoire variants that are only marginally more stable than others. Because fd phage are exceptionally stable and the proteolysis in the selection step takes place in vitro a wide range of solvent conditions can be used, tailored for the protein to be stabilized.

Sequence profile of the parallel beta helix in the pectate lyase superfamily.

The parallel beta helix structure found in the pectate lyase superfamily has been analyzed in detail. A comparative analysis of known structures has revealed a unique sequence profile, with a strong positional preference for specific amino acids oriented toward the interior of the parallel beta helix. Using the unique sequence profile, search patterns have been constructed and applied to the sequence databases to identify a subset of proteins that are likely to fold into the parallel beta helix. Of the 19 families identified, 39% are known to be carbohydrate-binding proteins, and 50% belong to a broad category of proteins with sequences containing leucine-rich repeats (LRRs). The most striking result is the sequence match between the search pattern and four contiguous segments of internalin A, a surface protein from the bacterial pathogen Listeria monocytogenes. A plausible model of the repetitive LRR sequences of internalin A has been constructed and favorable 3D-1D profile scores have been calculated. Moreover, spectroscopic features characteristic of the parallel beta helix topology in the pectate lyases are present in the circular dichroic spectrum of internalin A. Altogether, the data support the hypothesis that sequence search patterns can be used to identify proteins, including a subset of LRR proteins, that are likely to fold into the parallel beta helix.

Surface-exposed phenylalanines in the RNP1/RNP2 motif stabilize the cold-shock protein CspB from Bacillus subtilis.

In the cold-shock protein CspB from Bacillus subtilis three exposed Phe residues (F15, F17, and F27) are essential for its function in binding to single-stranded nucleic acids. Usually, the hydrophobic Phe side chains are buried in folded proteins. We asked here whether the exposition of the essential Phe residues could be a cause for the very low conformational stability of CspB. Urea-induced and heat-induced equilibrium unfolding transitions were measured for three mutants of CspB, where Phe 15, Phe 17, and Phe 27 were individually replaced by alanine. Unexpectedly, all three mutations strongly destabilized CspB. The aromatic side chains of Phe 15, Phe 17, and Phe 27 in the active site are thus important for both binding to nucleic acids and conformational stability. There is no compromise between function and stability in the active site. Model calculations indicate that, although they are partially exposed to solvent, all three Phe residues nevertheless lose accessible surface upon folding, and this should favor the native state. A different result is obtained with the F38A variant. Phe 38 is hyperexposed in native CspB, and its substitution by Ala is in fact stabilizing.

Interactions contributing to the formation of a beta-hairpin-like structure in a small peptide.

A 12 amino acid peptide, model BB, was designed to adopt a beta-hairpin tertiary structure in water that might be stabilized by a variety of local, nonlocal, polar, and nonpolar interactions. The conformational properties of model BB with and without an intramolecular disulfide bond (BB-O and BB-R, respectively) were characterized by NMR and CD spectroscopy. The set of observed short- and medium-range nOes were consistent with the formation of stable beta-hairpin-like structures by both BB-R and BB-O. BB-O adopts two distinct conformations that differ from each other in the designed reverse turn segment. A reasonably well-defined set of structures was obtained by using restraints from the NMR data in distance geometry calculations. None of the beta-hairpin-like structures contain a beta-sheet hydrogen bonding network. The distinctive feature of intrastrand and cross-strand pairing of threonine residues observed in all of the calculated structures suggests that hydrophobic interactions between the gamma-methyl groups of threonine residues may be the structure-determining interaction in model BB. The implications of these results for the formation of beta-sheets during protein folding, the aggregation of peptides as beta-sheets, and the de novo design of independently folding beta-hairpin-like peptides are considered.

Circular dichroism of the parallel beta helical proteins pectate lyase C and E.

The pectate lyases, PelC and PelE, have an unusual folding motif, known as a parallel beta-helix, in which the polypeptide chain is coiled into a larger helix composed of three parallel beta-sheets connected by loops having variable lengths and conformations. Since the regular secondary structure consists almost entirely of parallel beta-sheets these proteins provide a unique opportunity to study the effect of parallel beta-helical structure on circular dichroism (CD). We report here the CD spectra of PelC and PelE in the presence and absence of Ca2+, derive the parallel beta-helical components of the spectra, and compare these results with previous CD studies of parallel beta-sheet structure. The shape and intensity of the parallel beta-sheet spectrum is distinctive and may be useful in identifying other proteins that contain the parallel beta-helical folding motif.

Cytotoxicity of wound dressing materials assessed using cultured skin equivalents.

An in vitro system, based on the Bell model of cultured composite skin equivalents, was used to assess the effect of a number of wound dressing materials on DNA synthesis. DNA synthesis was quantified using immunocytohistochemical identification of incorporated bromodeoxy-uridine and the percentage of labelled cells measured, following 7 days' exposure to the dressing material. Differences in labelling index were observed from replica gels covered by different dressing materials and between dressings of the same type of material, but made by different manufacturers.

Time and dose-related changes in the thickness of pig skin after irradiation with single doses of 90Sr/90Y beta-rays.

Time-related changes in pig skin thickness have been evaluated using a non-invasive ultrasound technique after exposure to a range of single doses of 90Sr/90Y beta-rays. The reduction in relative skin thickness developed in two distinct phases: the first was between 12 and 20 weeks postirradiation. No further changes were then seen until 52 weeks postirradiation when a second phase of skin thinning was observed. This was complete after 76 weeks and no further changes in relative skin thickness were seen in the maximum follow up period of 129 weeks. The timings of these phases of damage were independent of the radiation dose, however, the severity of both phases of radiation-induced skin thinning were dose related.

Cryopreservation of cultured dermal fibroblast impregnated collagen gels.

The survival of cultured dermal fibroblasts was evaluated following manufacture, freezing and disaggregation of fibroblast-impregnated collagen gels. The concentration which gave optimal cell survival was determined for three cryoprotectants (glycerol, dimethyl, sulphoxide (DMSO) and ethanediol) and their efficacy compared. DMSO led to the highest cell viability after freezing and thawing. The effect of rate of freezing was also compared and 0.5 degree C/min (within the range 20 degrees C to -70 degrees C) was found to result in a significant enhancement of cell viability in comparison with freezing at 1.0 degree C/min or rapid freezing.

Dose- and source-size-related changes in the late response of pig skin to irradiation with single doses of beta radiation from sources of differing energy.

Late radiation damage to pig skin has been assessed at 104 weeks after exposure to sources of 90Sr/90Y (Emax 2.2 MeV) and 170Tm (Emax 0.9 MeV). Damage was assessed from measurements of dermal thickness in histological sections of irradiated skin and was compared with that of unirradiated skin to establish the relative reduction in dermal thickness. The size of the source varied from 0.1 to 40.0 mm in diameter; this covered the range of source sizes designed to simulate exposure to radioactive "hot" particles (< or = 1.0 mm diameter) up to the lower range of field size that patients may be exposed to in radiotherapy treatments. Radiation doses were measured using an extrapolation chamber with a collecting electrode of 1.1 mm2, and thus the quoted doses represent an average dose over this area. For the larger 90Sr/90Y sources of > or = 5 mm diameter and the larger 170Tm sources of > or = 2 mm diameter there was no evidence, based on levels of damage consistent with a > or = 10, > or = 20, > or = 30, and > or = 40% reduction in relative dermal thickness, for any effect of source size on the ED50 value for each of these specified levels of effect. However, there was a marked effect of beta-particle energy; the skin surface doses associated with the ED50 values (+/- SE) for a > or = 20% reduction in relative dermal thickness were approximately 12 and approximately 40 Gy for 90Sr/90Y and 170Tm, respectively. This difference in skin surface dose for an equivalent level of dermal injury reflects the variation in the depth dose from these two beta-particle emitters. These skin surface doses, for what was considered to be a clinically detectable dermal effect, were below the ED10 for the early skin response of moist desquamation. This supports the selection of late dermal thinning as the effect on which to base dose limits in radiation protection for more generalized larger area skin exposures. In comparison, single exposures to a small area, from sources designed to simulate those from hot particles, reinforced the view that acute ulceration should be the effect on which dose limitation is based. Either the isoeffect doses for a clinically detectable reduction in relative dermal thickness of > or = 20%, following a single exposure to a small area, were higher than the ED10 for acute ulceration or the area of skin showing dermal thinning was so small that it was not considered to be detrimental.

Quantification of radiation-induced epilation in the pig: a biological indicator of radiation dose to the skin.

Epilation in the pig has been quantified following single exposures of X-rays in the range 1.0-25.0 Gy. The number of hairs in each field was determined by counting hairs from photographic negatives weekly for 10 weeks following irradiation, and the percentage hair loss was calculated for each individual field from an initial unirradiated control value. Hair loss was dose-dependent for exposures between 1.0 and 15.0 Gy and this response was linearly related to dose. No further increase in hair loss was observed for doses > or = 15.0 Gy, as 20-30% of the hairs remained. It was assumed that these hairs were not actively growing at the time of irradiation and did, therefore, not express damage. The ED50 for the loss of > or = 30% of hairs was 3.8 Gy whilst that for the loss of > or = 50% of hairs was 6.8 Gy and for the loss of > or = 67% of hairs was 12.5 Gy. There was +/- 7% hair loss per Gy exposure for doses between 0.0 and 15.0 Gy. Quantification of hair loss provided a more sensitive assay than the use of visual scoring systems. Hair loss was detectable within 4 weeks of irradiation. The system is simple, non-invasive and appears to have considerable potential for use as a biological dosimeter.

Reduction in the diameter of human hairs following irradiation.

A measurable reduction in hair diameter was observed in human hairs following single exposures to gamma-rays, 250 KeV X-rays, 8 MV photons and 10 MV electrons in the range 2.9-14.0 Gy. The data from the different types of radiation were pooled and fitted by linear regression with a slope of 2.34 +/- 0.42% Gy-1. There was approximately 2.4% reduction in hair diameter per Gy exposure. It would appear that the measurement of damage to the matrix cells of growing human hairs exhibits potential for use in biological dosimetry, especially in cases of non-uniform overexposure.

Morphological characteristics of cells derived from plucked human hair in vitro.

Cells of various morphologies have been cultured from plucked hairs in vitro. These include keratinocytes, large polygonal cells, large and small spindle-shaped cells and endothelial cells.