Restriction fragment analysis of the secalin loci of rye

Analyses of wheat/rye addition lines by Southern blotting confirmed the presence of sequences related to theSec 1, Sec 2, andSec 3 loci on chromosomes 1R and 2R. Comparison of the 1R and 2R addition lines allowed the identification of -secalin genes atSec 1 andSec 2, respectively, while -secalin and -secalin genes atSec 1 were discriminated by comparative hybridization with three probes: -secalin, total -secalin, and 3 -secalin. The high molecular weight (HMW) secalin genes atSec 3 were identified using a homologous HMW subunit probe from wheat. Gene copy numbers were estimated as about 40–60 for -secalins, 5–10 for -secalins, and 2 for HMW secalins. Comparison of individual plants of cv. Gazelle showed a high degree of polymorphism, particularly for sequences related to -secalins and HMW secalins.     

Electric field-induced breakdown of lipid bilayers and cell membranes: A thin viscoelastic film model

Summary A simple viscoelastic film model is presented, which predicts a breakdown electric potential having a dependence on the electric pulse length which approximates the available experimental data for the electric breakdown of lipid bilayers and cell membranes (summarized in the reviews of U. Zimmermann and J. Vienken, 1982,J. Membrane Biol. 67:165 and U. Zimmermann, 1982,Biochim. Biophys. Acta 694:227). The basic result is a formula for the time of membrane breakdown (up to the formation of pores): =(/C)/( _m ² ₀ ² U ⁴/24Gh ³+T ²/Gh–1), where is a proportionality coefficient approximately equal to ln(h/2₀),h being the membrane thickness and ₀ the amplitude of the initial membrane surface shape fluctuation ( is usually of the order of unity), represents the membrane shear viscosity,G the membranes shear elasticity modules, _m the membrane relative permittivity, ₀=8.85×10^–12 Fm,U the electric potential across the membrane, the membrane surface tension andT the membrane tension. This formula predicts a critical potentialU _c ;U _c =(24Gh ³/ _m ² ₀ ² )^1/4 (for = andT=0). It is proposed that the time course of the electric field-induced membrane breakdown can be divided into three stages: (i) growth of the membrane surface fluctuations, (ii) molecular rearrangements leading to membrane discontinuities, and (iii) expansion of the pores, resulting in the mechanical breakdown of the membrane.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

Hexose transport and membrane depolarization in Riccia fluitans

In the aquatic liverwort Riccia fluitans, the uptake of ¹⁴C-labeled 3-O-methyl glucose (3-OMG) and membrane depolarization ( _m ) caused by different hexoses has been studied as a function of time and concentration of hexose, K⁺ and H⁺, respectively. The rate of uptake of the non-metabolized 3-OMG shows two components: (A)A pH-dependent saturable uptake with a k_m value around 0.1 mM which saturates at 2.1 and 7.2 mol G _DW ^-1 h^-1 at pH 6.8 and 5.0, respectively; and (B) a pH-insensitive uptake component which increases linearly with the external 3-OMG concentration and does not saturate 4 mM. Hexoses rapidly depolarize the plasmalemma of the thallus cell and increase its electrical conductance. The maximal _m was 60±2 mV, the concentrations (mM) for half-maximal _m were 0.24 glucose, 0.32 galactose, 0.37 2-deoxy glucose, 0.38 3-OMG, 0.57 mannose, and 34 fructose. In terms of a hexose carrier model and an equivalent circuit for the hexose-induced depolarized state of the membrane, it is proposed that a hexose carrier operates either electrogenically in its protonated, pH-and voltage-sensitive state, or by transmembrane diffusion of its uncharged state.Symbols and Abbreviations _m membrane potential (mV) - g _m membrane (slope) conductance (Sm^-2) - 3-OMG 3-O-methyl glucose     

Species diversities analyzed by density and cover in an early volcanic succession

To evaluate alpha diversities, various variables such as density, cover, volume, and weight have been used. However, density is often a distinct variable from the remaining three. To clarify differences in diversity measured by those two kinds of variables, the data collected in fourteen 2×5 m permanently-marked plots on Mount Usu, Japan, which erupted during 1977 and 1978 in growing seasons from 1983 to 1989 was analyzed, using Shannon's species diversity (H) that is represented as a result of combination of species richness and evenness (J). H and J were evaluated by density (density H and J) and cover (cover H and J). Cover H and J were significantly lower than density H and J, indicating that cover H has different characteristics from density H. Those differences are due to differences in evenness, because species richness is the same. The rank orders of species density are different from those of cover. The predominance of a few perennial herbs greatly decreases cover evenness, while seedling establishment success influences density evenness. Therefore, I propose that, during the early stages of succession on harsh environments such as volcanoes, density diversity represents seedling establishment success rate while cover diversity expresses vegetative reproduction success rate.     

Relationships between carbon and hydrogen isotope ratios and nitrogen levels in leaves ofClusia species and two other Clusiaceae genera at various sites and different altitudes in Venezuela

Samples of the Clusiaceae generaClusia, Oedematopus andDystovomita were collected at various sites and different altitudes in northern and south-western Venezuela. Analyses of stable isotopes of carbon and hydrogen and of leaf-nitrogen levels were performed on the dried samples. Correlations among these variables, i.e. carbon isotope discrimination (), hydrogen isotope ratio (D) and N-levels, and with altitude were assessed. In the samples, where values of above 15 indicate predominant performance of C₃ photosynthesis, there were slight tendencies of increasing , D and N-levels with increasing altitude and of increasing with increasing N. Although these correlations taken separately were not statistically significant, they support each other and indicate increasing transpiration and increased leaf-nutrient supply at increasing altitude. Performance of crassulacean acid metabolism (CAM) in species ofClusia appears to be restricted to altitudes below 1500 m a.s.l. There was a significant negative correlation of with altitude in the samples, where values of below 10 indicated predominant performance of CAM. This suggests that phases II and IV of CAM are progressively suppressed towards the upper altitudinal limit of CAM inClusia in northern Venezuela. It is concluded that among the large number of environmental factors and combinations thereof, which determine the expression of CAM inClusia and trigger C₃-CAM transitions in C₃/CAM intermediate species, low availability of water is the most important.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Formation ofα,ω-dodecanedioic acid andα,ω-tridecanedioic acid from different substrates by immobilized cells of a mutant ofCandida tropicalis

Summary The metabolic formation of either,-dodecanedioic acid or,-tridecanedioic acid from the individual n-alkane, n-alcohol, n-monoacid and,-diol with corresponding carbon chain length using K-carrageenan entrapped mutants S76 ofCandida tropicalis was studied. The immobilized cells of S76 could also directly produce-hydroxy acid and,-dioic acid from,-diol. With n-alcohol and n-monoacid as substrate, the amount of-hydroxy acid and,-dioic acid produced was also a function of the incubation time.The results demonstrated that in the immobilized cells of S76 the formation of,-dioic acid from n-alcohol can also run both via n-monoacid and via,-diol as well as in the normal cells of S76.     

The effect of the crossability loci Kr1 and Kr2 on fertilization frequency in hexaploid wheat x maize crosses

Summary Dominant alleles of the Kr1 and Kr2 genes reduce the crossability of hexaploid wheat with many alien species, including rye and Hordeum bulbosum, with Kr1 having the greater effect. However, a cytological study of wheat ovaries fixed 48 h after pollination showed that the wheat genotypes Highbury (kr1, Kr2) and Chinese Spring (Hope 5B) (kr1, kr2) were crossable with Seneca 60 maize, fertilization occurring in 14.4 and 30.7% of embryo sacs respectively. The latter figure was similar to the 29.7% fertilization found in Chinese Spring (kr1, kr2). Most embryo sacs in which fertilization occurred contained an embryo but lacked an endosperm and where an endosperm was formed it was usually highly aberrant. All three wheat x maize combinations were karyotypically unstable and rapidly eliminated maize chromosomes to produce haploid wheat embryos.     

Contrasting leaf and ‘ecosystem’ CO2 and H2O exchange in Avena fatua monoculture: Growth at ambient and elevated CO2

Elevated CO₂ (ambient + 35 Pa) increased shoot dry mass production in Avena fatua by 68% at maturity. This increase in shoot biomass was paralleled by an 81% increase in average net CO₂ uptake (A) per unit of leaf area and a 65% increase in average A at the ecosystem level per unit of ground area. Elevated CO₂ also increased ecosystem A per unit of biomass. However, the products of total leaf area and light-saturated leaf A divided by the ground surface area over time appeared to lie on a single response curve for both CO₂ treatments. The approximate slope of the response suggests that the integrated light saturated capacity for leaf photosynthesis is 10-fold greater than the ecosystem rate. Ecosystem respiration (night) per unit of ground area, which includes soil and plant respiration, ranged from-20 (at day 19) to-18 (at day 40) mol m^-2 s^-1 for both elevated and ambient CO₂ Avena. Ecosystem below-ground respiration at the time of seedling emergence was -10 mol m^-2 s^-1, while that occuring after shoot removal at the termination of the experiment ranged from -5 to-6 mol m^-2 s^-1. Hence, no significant differences between elevated and ambient CO₂ treatments were found in any respiration measure on a ground area basis, though ecosystem respiration on a shoot biomass basis was clearly reduced by elevated CO₂. Significant differences existed between leaf and ecosystem water flux. In general, leaf transpiration (E) decreased over the course of the experiment, possibly in response to leaf aging, while ecosystem rates of evapotranspiration (ET) remained constant, probably because falling leaf rates were offset by an increasing total leaf biomass. Transpiration was lower in plants grown at elevated CO₂, though variation was high because of variability in leaf age and ambient light conditions and differences were not significant. In contrast, ecosystem evapotranspiration (ET) was significantly decreased by elevated CO₂ on 5 out of 8 measurement dates. Photosynthetic water use efficiencies (A/E at the leaf level, A/ET at the ecosystem level) were increased by elevated CO₂. Increases were due to both increased A at leaf and ecosystem level and decreased leaf E and ecosystem ET.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

Regio- and stereo-selective synthesis of vinyl glucose ester catalyzed by an alkaline protease of Bacillus subtilis

The transesterification of -d-glucose with divinylsuccinate, divinyladipate and divinylsebacate in pyridine at 55 °C for 3 days was catalyzed by an alkaline protease from Bacillus subtilis to give corresponding 6-O-vinyl glucose esters at 30%, 53% and 35% yield, respectively. The stereo-selectivity of the alkaline protease toward the -anomer was affected by the acyl donor chain length. 6-O-Vinylsuccinyl-d-glucose was mixture of - and -anomers (/=44/56), the other two products were the pure -d-glucose derivatives.     

Cloning and comparison of Aα mating-type alleles of the basidiomycete Schizophyllum commune

Summary An A mating-type allele (A4) was isolated by walking the chromosome from the closely linked PAB1 gene. A cosmid clone containing the A1 allele isolated from the walk was used as a probe to recover the A1 allele from another cosmid library. Cosmids encoding mating-type activity were identified by transforming Schizophyllum cells and screening for activation of A-regulated development. Putative mating-type transformants were confirmed in mating tests and genetic analyses of progeny. The identity of the specific alleles isolated was demonstrated by showing that their effectiveness in transforming for mating type is limited to recipient strains possessing an A allele different from the one encoded by the cloned sequences. Transforming DNA is active in trans, suggesting that A encodes a diffusible product. Restriction mapping shows that A1 and A4 are coded in the same physical region of the genome, but within a subregion that contains extensive sequence divergence. In addition, Southern analyses show that there is only one copy of A1 or A4 per haploid genome, and that they do not cross-hybridize to one another or to any of the other A alleles. A1 and A4 were subcloned as 2.8 and 1.2 kb fragments, respectively, retaining in transformation all the mating-type activity demonstrated of the original cosmids.     

Interaction of non-allelic loci in expression of the extra-sexcomb phenotype inDrosophila melanogaster

Summary The expression of the extra-sexcomb phenotype is enhanced in males that are doubly heterozygous for mutants from two non-allelic loci,extra-sexcomb (Polycomb, Extra-sexcomb) andbithorax (bithorax, bithoraxoid, Ultrabithorax) as compared to the expression of the extra-sexcomb mutants when no bithorax mutants are present in the genotype. If the bithorax mutants are recessive, the expression is usually greater when the non-allelic mutants are cis-hererozygotes than when they are trans-heterozygotes, but there is no difference between cis-and trans-heterozygotes in extra-sexcomb andUltrabithorax compounds.This work, begun while the author was a member of the staff of the Zoology Dept. of the University of California at Berkeley, was completed, with the assistance of a National Institutes of Health research grant (RG-6754), in the Dept. of Genetics of the University of Turku.     

Evolutionary analysis ofα andβ hemoglobin genes by reh theory under the assumption of the equiprobability of genetic events

Summary It is shown how REH theory in conjunction with mRNA or gene sequence data can be used to obtain estimates of the fixation intensity, the number of varions, and the total mutations fixed between homologous pairs of nucleic acids. These estimates are more accurate than those that can be derived from amino acid sequence data. The method is illustrated for and hemoglobin genes and these improved estimates are compared with those made from the amino acid sequences for which those genes code. Significant differences are found between the estimates made by these two methods. For the hemoglobin gene sequences examined here, the fixation intensity is some-what less than the protein data had suggested, and the number of rations is considerably greater. Depending on the gene sequences examined, between 62 and 83% of the codons appear able to fix mutations during the divergences considered. This reflects the constraints of natural selection on acceptable mutations. The total number of base replacements separating the genes for human, mouse, and rabbit hemoglobin varies from 61 to 105 depending on the pair examined. Rabbit and hemoglobin are separated by at least 290 fixed mutations. For such distantly related sequences estimates made from protein and mRNA data differ less, reflecting the higher quality of information from the many observed changes in primary structure. The effects of nonrandom gene structure on these evolutionary estimates and the fact that various genetic events are not equiprobable are discussed.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.