Transfer RNA structure by carbon NMR: C2 of adenine, uracil and cytosine.

Fourier transform 13C NMR spectra of E. coli tRNA enriched on 13C in either position 2 of adenine (60 atom % 13C) or in position 2 of uracil (82%) and cytosine (63%) were taken at 25.16 MHz over the temperature range 10 degrees - 76 degrees. For C2 of adenine the peak as initially 5 ppm wide, but narrowed to 0.5 ppm as the molecule unfolded. C2 of uracil displayed behavior similar to that of adenine while the cytosine peak, initially relatively narrow at low temperature, sharpened less dramatically. Comparison of spectra at 26.16 MHz and 67.9 MHz showed that the peak widths for folded tRNA were determined largely by chemical shift non-equivalence. T2 T2 measurements suggested that intrinsic line widths of most cytosine C2 peaks were 4 Hz and 2-3 Hz for uracil. Adenine C2 with a directly bonded proton had resonances of about 40 Hz line width. T1 values were measured for C2 of adenine and the ribose carbons of tRNA. Consideration of dipolar relaxation and chemical shift anisotrophy led to a calculated rotational correlation time of 1.6 +/- 0.4 x 10(-8) sec for the adenines and 1.3 +/- 0.3 x 10(-8) sec for the ribose carbons.     

Mutations in two nonhomologous genes in a head-to-head configuration cause Ellis-van Creveld syndrome

Ellis-van Creveld syndrome (EvC) is an autosomal recessive skeletal dysplasia. Elsewhere, we described mutations in EVC in patients with this condition (Ruiz-Perez et al. 2000). We now report that mutations in EVC2 also cause EvC. These two genes lie in a head-to-head configuration that is conserved from fish to man. Affected individuals with mutations in EVC and EVC2 have the typical spectrum of features and are phenotypically indistinguishable.     

Structural and catalytic response to temperature and cosolvents of carboxylesterase EST1 from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1

The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

Evc2 is a positive modulator of Hedgehog signalling that interacts with Evc at the cilia membrane and is also found in the nucleus

Background  

Evc is essential for Indian Hedgehog (Hh) signalling in the cartilage growth plate. The gene encoding Evc2 is in close proximity in divergent orientation to Evc and mutations in both human genes lead to the chondrodysplasia Ellis-van Creveld syndrome.     

Sequencing <Emphasis Type="Italic">EVC</Emphasis> and <Emphasis Type="Italic">EVC2</Emphasis> identifies mutations in two-thirds of Ellis–van Creveld syndrome patients

Ellis–van Creveld syndrome (EvC) is caused by mutations in EVC and EVC2, genes in a divergent orientation separated by only 2.6 kb. We systematically sought mutations in both genes in a panel of 65 affected individuals to assess the proportion of cases resulting from mutations in each gene. We PCR amplified and sequenced the coding exons of both genes. We investigated mutations that could affect splicing by in vitro splicing assays and cDNA analysis. We have identified EVC mutations in 20 cases (31%); in all of these we have detected the mutation on each allele. We have identified EVC2 mutations in 25 cases (38%); in 22 of these we have isolated a mutation on each allele. The majority of the mutations introduce a premature termination codon. We sequenced the region between the two genes in 10 of the 20 cases in which we had not identified a mutation in either gene, revealing only one SNP that was not a common polymorphism. As we have not identified mutations in either gene in 20 cases (31%) it is possible that there is further genetic heterogeneity. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

A Recessive Skeletal Dysplasia, SEMD Aggrecan Type, Results from a Missense Mutation Affecting the C-Type Lectin Domain of Aggrecan

Analysis of a nuclear family with three affected offspring identified an autosomal-recessive form of spondyloepimetaphyseal dysplasia characterized by severe short stature and a unique constellation of radiographic findings. Homozygosity for a haplotype that was identical by descent between two of the affected individuals identified a locus for the disease gene within a 17.4 Mb interval on chromosome 15, a region containing 296 genes. These genes were assessed and ranked by cartilage selectivity with whole-genome microarray data, revealing only two genes, encoding aggrecan and chondroitin sulfate proteoglycan 4, that were selectively expressed in cartilage. Sequence analysis of aggrecan complementary DNA from an affected individual revealed homozygosity for a missense mutation (c.6799G → A) that predicts a p.D2267N amino acid substitution in the C-type lectin domain within the G3 domain of aggrecan. The D2267 residue is predicted to coordinate binding of a calcium ion, which influences the conformational binding loops of the C-type lectin domain that mediate interactions with tenascins and other extracellular-matrix proteins. Expression of the normal and mutant G3 domains in mammalian cells showed that the mutation created a functional N-glycosylation site but did not adversely affect protein trafficking and secretion. Surface-plasmon-resonance studies showed that the mutation influenced the binding and kinetics of the interactions between the aggrecan G3 domain and tenascin-C. These findings identify an autosomal-recessive skeletal dysplasia and a significant role for the aggrecan C-type lectin domain in regulating endochondral ossification and, thereby, height.