Actin-binding domain of mouse plectin. Crystal structure and binding to vimentin.

Plectin, a large and widely expressed cytolinker protein, is composed of several subdomains that harbor binding sites for a variety of different interaction partners. A canonical actin-binding domain (ABD) comprising two calponin homology domains (CH1 and CH2) is located in proximity to its amino terminus. However, the ABD of plectin is unique among actin-binding proteins as it is expressed in the form of distinct, plectin isoform-specific versions. We have determined the three-dimensional structure of two distinct crystalline forms of one of its ABD versions (pleABD/2alpha) from mouse, to a resolution of 1.95 and 2.0 A. Comparison of pleABD/2alpha with the ABDs of fimbrin and utrophin revealed structural similarity between plectin and fimbrin, although the proteins share only low sequence identity. In fact, pleABD/2alpha has been found to have the same compact fold as the human plectin ABD and the fimbrin ABD, differing from the open conformation described for the ABDs of utrophin and dystrophin. Plectin harbors a specific binding site for intermediate filaments of various types within its carboxy-terminal R5 repeat domain. Our experiments revealed an additional vimentin-binding site of plectin, residing within the CH1 subdomain of its ABD. We show that vimentin binds to this site via the amino-terminal part of its rod domain. This additional amino-terminal intermediate filament protein binding site of plectin may have a function in intermediate filament dynamics and assembly, rather than in linking and stabilizing intermediate filament networks.     

Characterization of 12 microsatellite loci of the human MHC in a panel of reference cell lines

 The human genome contains a large number of interspersed microsatellite repeats which exhibit a high degree of polymorphism and are inherited in a Mendelian fashion, making them extremely useful genetic markers. Several microsatellites have been described in the HLA region, but allele nomenclature, a set of broadly distributed controls, and typing methods have not been standardized, which has resulted in discrepant microsatellite data between laboratories. In this report we present a detailed protocol for genotyping microsatellites using a semi-automated fluorescence-based method. Twelve microsatellites within or near the major histocompatibility complex (MHC) were typed in the 10th International Histocompatibility Workshop homozygous typing cell lines (HTCs) and alleles were designated based on size. All loci were sequenced in two HTCs providing some information on the level of complexity of the repeat sequence. A comparison of allele size obtained by genotyping versus that obtained by direct sequencing showed minor discrepancies in some cases, but these were not unexpected given the technical differences in the methodologies. Fluorescence-based typing of microsatellites in the MHC described herein is highly efficient, accurate, and reproducible, and will allow comparison of results between laboratories. Received: 10 May 1997 / Revised: 1 August 1997     

Effect of cytochrome oxidase inhibitors on the yeast thermotolerance

The investigation of the effect of the cytochrome oxidase inhibitors sodium cyanide and sodium azide on the thermotolerance of the yeasts Rhodotorula rubra, Debaryomyces vanriji, and Saccharomyces cerevisiae showed that these inhibitors diminish the thermotolerance of R. rubra and D. vanriji, but do not affect the thermotolerance of S. cerevisiae. Taking into account the fact that, unlike the latter yeast, R. rubra and D. vanriji are nonfermentative yeasts, the difference in the effects of the inhibitors on the yeast thermotolerance can be readily explained by the different types of glucose utilization (either oxidative or fermentative) in these yeasts. The data obtained also provide evidence that there is a correlation between the functional activity of mitochondria and the thermotolerance of yeast cells.     

Synthetic esters recognized by glucuronoyl esterase from <Emphasis Type="Italic">Schizophyllum commune</Emphasis>

Glucuronoyl esterase is a novel carbohydrate esterase recently discovered in the cellulolytic system of the wood-rotting fungus Schizophyllum commune on the basis of its ability to hydrolyze methyl ester of 4-O-methyl-d-glucuronic acid. This substrate was not fully corresponding to the anticipated function of the enzyme to hydrolyze esters between xylan-bound 4-O-methyl-d-glucuronic acid and lignin alcohols occurring in plant cell walls. In this work we showed that the enzyme was capable of hydrolyzing two synthetic compounds that mimic the ester linkages described in lignin-carbohydrate complexes, esters of 4-O-methyl-d-glucuronic and d-glucuronic acid with 3-(4-methoxyphenyl)propyl alcohol. A comparison of kinetics of hydrolysis of methyl and 3-(4-methoxyphenyl)propyl esters indicated that the glucuronoyl esterase recognizes the uronic acid part of the substrates better than the alcohol type. The catalytic efficiency of the enzyme was much higher with the ester of 4-O-methyl-d-glucuronic acid than with that of d-glucuronic acid. Examination of the action of glucuronoyl esterase on a series of methyl esters of 4-O-methyl-d-glucopyranuronosyl residues α-1,2-linked to xylose and several xylooligosaccharides suggested that the rate of deesterification is independent of the character of the carbohydrate part glycosylated by the 4-O-methyl-d-glucuronic acid.     

A field study of pollen-mediated gene flow from Mediterranean GM rice to conventional rice and the red rice weed

The objective of this study was to assess the frequency of pollen-mediated gene flow from a transgenic rice line, harbouring the gusA and the bar genes encoding respectively, -glucuronidase and phosphinothricin acetyl transferase as markers, to the red rice weed and conventional rice in the Spanish japonica cultivar Senia. A circular field trial design was set up to investigate the influence of the wind on the frequency of pollination of red rice and conventional rice recipient plants with the transgenic pollen. Frequencies of gene flow based on detection of herbicide resistant, GUS positive seedlings among seed progenies of recipient plants averaged over all wind directions were 0.036 ± 0.006% and 0.086 ± 0.007 for red rice and conventional rice, respectively. However, for both red rice and conventional rice, a clear asymmetric distribution was observed with pollination frequency favoured in plants placed under the local prevailing winds. Southern analyses confirmed the hemizygous status and the origin of the transgenes in progenies of surviving, GUS positive plants. Gene flow detected in conventional rice planted at 1, 2, 5 and 10 m distance revealed a clear decrease with increasing distance which was less dramatic under the prevailing wind direction. Consequences of these findings for containment of gene flow from transgenic rice crops to the red rice weed are discussed. The precise determination of the local wind conditions at flowering time and pollination day time appear to be of primary importance for setting up suitable isolation distances.     

Air method measurements of apple vessel length distributions with improved apparatus and theory

Studies showing that rootstock dwarfing potential is related to plant hydraulic conductance led to the hypothesis that xylem properties are also related. Vessel length distribution and other properties of apple wood from a series of varieties were measured using the 'air method' in order to test this hypothesis. Apparatus was built to measure and monitor conductivity to air of fresh wood segments of different lengths. Theory for determining vessel length distribution was improved to give a single parameter uni-modal vessel length probability density function. The function, derived by combining the exponential extinction (with extinction coefficient k) of wood conductivity to air (C) as wood length (x) increases (i.e. C=Co exp (-kx)) with the differential double difference formula, is Px=xxk2 exp (-kx), where Px is the fraction of vessels of length x. The main parameter of the distribution, k, was found to be the inverse of the mode of the distribution, i.e. the most common vessel length, Lo. Lo for ten apple rootstock and scion varieties varied from 5.6+/-0.1 cm (+/-SE) for MM.111 to 9.0+/-1.0 for Prunifolia (P <0.05). Average maximum vessel length was approximately 50 cm, and differences were not significant. Effective vessel radii ranged from 14 for Prunifolia to 24.3+/-0.7 micro m for M.26, with standard errors less than 12% of the mean. Specific conductivity of a 15 cm wood segment ranged from 2x109-4) to 1.6+/-0.2x10(-2) dm3 s(-1) kPa(-1) m(-1) for maruba and M.26, respectively, with standard errors up to 63% of the mean. Vessel density at the air entry point ranged from 18+/-3 to 42+/-6 vessels mm-2 for M.26 and MM.106, respectively, with standard errors as high as 89% of the mean. It was concluded that there is no general relationship between the wood properties investigated and rootstock size class, and that plasticity increases from vessel lengths to radii to specific conductivity and vessel densities.