Showing papers in "Carlsberg Research Communications in 1978"

Somatic hybrid plants of potato and tomato regenerated from fused protoplasts

Abstract: Mesophyll protoplasts of Lycopersicon esculentum Mill. var. cerasiforme (Dunal) Alef, mutant yellow green 6, Rick and protoplasts of a liquid callus culture of the dihaploid strain HH258 of Solanum tuberosum L. were prepared and many fusion products were visible after the protoplasts were incubated together first in the presence of polyethylene glycol and then with a high Ca2+ ion concentration. The protoplasts were transferred to a rich medium and the resultant calli were cultured. Some calli regenerated normal green shoots which were transferred to soil or grafted onto a tomato stock. The subunit polypeptide pattern of ribulose 1,5-bisphosphate carboxylase prepared from leaf material of four regenerated plants was analyzed by isoelectric focusing. The ribulose bisphosphate carboxylase enzyme oligomer in the four plants contained the small subunit products resulting from the expression of both tomato and potato nuclear genes proving these plants to be somatic hybrids between tomato and potato. In three of the four plants the large subunit polypeptides and hence the functional chloroplast DNA were from tomato whereas in the fourth the large subunit and therefore the chloroplast DNA was derived from potato. The plant material was insufficient to establish the chromosome numbers precisely, however counts close to 50 which is near to the expected 48 were obtained for three of the hybrids whereas in the fourth a number close to 72 was observed. In the absence of a selection system against the potato parent, the analysis of ribulose bisphosphate carboxylase provides a convenient marker to demonstrate the hybrid nature of the plants.

Human meiosis II. Chromosome pairing and recombination nodules in human spermatocytes

Abstract: Moiosis in human spermatocytes has been analyzed by three dimensional reconstructions of 4 leptotene, 4 earlymid zygotene, 10 late zygotene, and 21 early pachytene nuclei. At leptotene, a lateral component is organized along each chromosome and the telomeres attach to the nuclear envelope. At early zygotene, the attachment sites aggregate and a chromosome bouquet is formed. Pairing and synaptonemal complex formation are initiated from the telomeres by binding of precursor material for the central region to the lateral components of the aligned homologues. In the 10 late zygotene nuclei, on the average 72% of the autosomal complement had been paired. Synaptonemal complex formation is in most cases initiated from both ends of the homologues and only in 5 cases was initiation of complex formation interstitial. Pairing of the short arms of the acrocentric bivalents and the X and Y chromosomes is delayed compared to the remainder of the genome. Irregularities such as interlockings and breaks of the lateral components of chromosomes or breaks of the synaptonemal complexes of bivalents are observed in 8 out of the 10 nuclei. Most of the breaks appeared to be the result of a resolution of interlockings. At early pachytene, all bivalents are fully paired the only exception being the secondary constrictions on bivalents present between the X and Y chromosomes. Only two interlockings and three breaks of lateral components of chromosomes were found at this stage. Recombination nodules are present in or at the central region of the synaptonemal complex from early zygotene and devidence has been obtained in favor of an attachment of nodules to precursor material for the central region at the pairing fork. Nodules can, however, also attach to a fully formed synaptonemal complex. At late zygotene, an average number of 101 nodules per nucleus is present. Assuming that all regions of the complex have equal probabilities of receiving a nodule about 144 nodules are expected to be present at themoment of complete pairing. At early pachytene, the mean number of nodules is 75. Generally, nodules appear to be distributed evenly along the bivalent arms. Nodules are present, however, in excess in the telomere regions and in the XY bivalent while the centromere regions, the secondary constrictions, and the short arms of the acrocentric bivalents are relatively depleted of nodules. Measurements of the distances between adjacent nodules and a comparison with a theoretical distribution of such distances assuming a random positioning of nodules have demonstrated that at late zygotene nodules are more frequently clustered than would be expected if they were distributed independently. At early pachytene, the reverse pattern is observed.

Biosynthesis of Δ-aminolevulinate in greening barley leaves: Glutamate 1-semialdehyde aminotransferase

Abstract: L-Glutamate-1-semialdehyde was synthesized by catalytic hydrogenation of N-carbobenzoxy-L-glutamyl-1-chloride-5-benzyl ester. Soluble protein extracts of chloroplasts isolated from greening barley leaves enzymically converted L-glutamate-1-semialdehyde to δ-aminolevulinate. The enzyme was partially purified by gel filtration on a Biogel column excluding proteins larger than 500,000 daltons. The enzyme had a broad pH optimum around 8.0 and required no specific cofactors for activity. Aminooxyacetate (20mM), cycloserine (20mM), ρ-chloromercuribenzoate (0.1mM), glyoxylate (20mM) and pyridoxal phosphate (5mM) inhibited δ-aminolevulinate formation from L-glutamate-1-semialdehyde. However, β- hydroxyglutamate (1mM) a potent inhibitor of L-glutamate-U-14C conversion to δ-aminolevulinate, had no effect on L-glutamate-1-semialdehyde aminotransferase. The aminotransferase activity was eluted from the Biogel column together with the enzyme activity that converted L-glutamate-U-14C into δ-aminolevulinate. Soluble proteins prepared from etiolated plastids and mature chloroplasts of barley had a low specific activity of L-glutamate-1-semialdehyde aminotransferase compared to soluble proteins from greening plastids. It is proposed that L-glutamate-1-semialdehyde aminotransferase catalyses a part reaction in the conversion of L-glutamate to δ-aminolevulinate in greening barley plastids.

Freeze-fracture studies on barley plastid membranes II. Wild-type chloroplast

Abstract: The ultrastructure of the thylakoids of barley chloroplasts was examined by freeze-fracturing and freeze-etching. Thylakoid fracture faces were objectively characterised in terms of the numbers of particles per square micron, average particle size and size distribution. Chloroplast envelope membranes were similarly examined and characterised. The changes in freeze-fracture appearance that occurred during greening are discussed in relation to current theories regarding thylakoid membrane structure. A procedure was developed that enables the induction of large numbers of particle arrays on the EF and PF faces and ES and PS surfaces of barley thylakoids. Measurements of the particles in these arrays revealed the asymmetric nature of EFs particles and confirmed that they span the thylakoid membrane. *** DIRECT SUPPORT *** A00GC017 00004

A mutant of Saccharomyces cerevisiae temperature sensitive for flocculation. Influence of oxygen and respiratory deficiency on flocculence

Abstract: A flocculent strain of Saccharomyces cerevisiae, containing the dominant gene for flocculenceFL04, was mutagenized with N-methyl-N′-nitro-N-nitrosoguanidine. Non-flocculent mutants were isolated with a selection procedure based on the slower sedimentation of non-flocculent cells. One closer studied mutant was due to an unlinked suppressor mutation forFL04. This suppressor gene is designatedsufl. The genesufl is neither centromere linked nor linked tohis4 or the mating type locus. The genesufl behaves as a recessive in some diploids and as a dominant in others, illustrating the genetic complexity of the flocculation phenomenon.

Isolation and translation of hordein messenger RNA from wild type and mutant endosperms in barley

Abstract: Messenger RNA has been isolated from developing barley endosperms by sucrose gradient sedimentation, Sepharose 4B gel filtration and preparative gel electrophoresis. Hordein messenger RNA was found to be a major constituent of the total messenger RNA population of the endosperm cell. Polyadenylated hordein messenger RNA sedimented at 11S in sucrose gradients and electrophoretic analysis reveals the presence of at least three RNA species with apparent molecular weights of 0.45, 0.36 and 0.30 megadaltons. The 11S messenger RNA was translated in vitro into hordein precursor polypeptides which are 2–4 kilodaltons larger than the native hordein polypeptides. The endosperm cell of mutant No. 1508 contained twice as much RNA as the wild type endosperm cell but the same amount of polyadenylated 11S RNA. The template activity of the latter was 10% of that found for the 11S hordein messenger RNA from the wild type and was limited to translation into one hordein precursor polypeptide.

Freeze-fracture evidence for the isolation of inside-out spinach thylakoid vesicles

Abstract: An aqueous dextran/polyethylene glycol two phase system is used to separate spinach chloroplast thylakoid vesicles obtained by Yeda press treatment of a grana-enriched fraction. It is shown by freeze-fracturing and freezeetching that the majority of the vesicles in the dextran-rich bottom phase are turned inside-out with respect to the surfaces of the thylakoid in the chloroplast. Most of the vesicles in the top phase had the normal orientation of the two thylakoid surfaces. These results confirm the previous proposal that the bottom phase vesicles show light induced proton extrusion instead of uptake because their membranes are turned inside-out. The polypeptide pattern of the bottom phase thylakoid fraction is enriched in the light harvesting chlorophyll a/b protein complex and the reaction centre protein of photosystem II, which is consistent with the origin of these thylakoids from granal regions enriched in photosystem II. A mechanism is suggested to explain the formation of inside-out vesicles from grana.

The pachytene karyotype of Schizophyllum commune analyzed by three dimensional reconstruction of synaptonemal complexes

Abstract: Three dimensional reconstructions of 15 pachytene nuclei of Schizophyllum commune have revealed in each nucleus 11 distinct synaptonemal complexes, demonstrating a haploid chromosome number of 11. Each bivalent contained one short region of condensed chromatin marking the position of the centromere. The synaptonemal complex of, each bivalent contained from 0 to six recombination nodules. A high correlation was found between the total length of the synaptonemal complexes in a nucleus and the number of recombination nodules. Total synaptonemal complex length correlated only weakly with the nuclear volume.

Human meiosis III. Electron microscopical analysis of chromosome pairing in an individual with a balanced translocation 46, XY, t(5p−;22p+)

Abstract: The meiotic behaviour of a balanced translocation involving the short arms of chromosomes 5 and 22 has been analyzed by reconstructions of lateral components and synaptonemal complexes at early and mid pachytene in human spermatocytes. At early pachytene, the normal chromosome 5 and the segment translocated onto chromosome 22 are paired with a synaptonemal complex in all 8 nuclei reconstructed, while pairing between the normal chromosome 22 and the translocation chromosome 5 was never observed. The pairing pattern was less regular at mid pachytene where a quadrivalent was observed in only 3 out of the 4 nuclei analyzed, while in the fourth nucleus, pairing was in part nonhomologous: the segment translocated onto chromosome 22 exhibited foldback pairing with itself and the short arm of the normal chromosome 5 was paired with the differential segment of the X chromosome. The short arms of translocation chromosome 5 and the normal chromosome 22 were paired in only one mid pachytene nucleus. Translocation chromosome 5 possessed in all 4 nuclei a 200 nm long terminal region of condensed chromatin resembling the heterochromatin of the short arms of the acrocentric chromosomes. This together with the observation that the telomere of the short arm of translocation chromosome 5 was attached to the nuclear envelope permits the conclusion that the translocation is reciprocal with transfer of the telomere region from chromosome 22 to chromosome 5 and that of the latter to chromosome 22. Recombination nodules were more frequent in bivalent 5 than in the remainder of the genome whereas a similar increase in nodule frequency was not observed for bivalent 22.

Fractionation and characterization of beer proteins

Abstract: A combination of large scale gel filtration with preparative isoelectric focusing and ion exchange chromatography has been used for fractionation of beer proteins. From gel filtration on Sephadex G-150, a high molecular weight fraction, eluting close to the void volume of the column, a fraction appearing corresponding to a molecular weight of 44,000 and a fraction containing rather low-molecular weight (about 10,000) components were obtained and used for preparative isoelectric focusing in the pH-range 3.5–10. The high-molecular weight fraction was rich in carbohydrate and cross-reacted with yeast antibodies. After preparative isoelectric focusing the amino acid composition of the isolated subfractions resembled that of yeast cell wall components. Preparative isoelectric focusing of the two fractions with molecular weight about 44,000 and 10,000 revealed the presence of two classes of components based on their amino acid composition. One class, which seemed to be present in low concentration in nearly all isolated subfraction, had an amino acid composition resembling that of barley glutelins. In the fraction with molecular weight about 44,000 another class of components—having an amino acid composition resembling barley albumins and globulins—were observed in the region with isoelectric points of about pH 4–5. They cross-reacted immunologically with antibodies against soluble barley proteins. This class of components could also be separated from the glutelin-like constituents by ion exchange chromatography. However, they could not be completely separated from carbohydrate by the fractionation procedures employed. Carbamylation expents demonstrated that approx, half of the e-amino groups of lysine residues of these proteins were blocked. Furthermore, partial amino acid sequence determination by the Edman procedure revealed a strong heterogeneity with respect to N-terminal amino acid residues. These observations are consistent with a suggestion that a large part of the beer proteins might be polypeptide chains crosslinked by carbohydrates.

Transfer of in vitro synthesized barley endosperm proteins into the lumen of the endoplasmic reticulum

Abstract: Microsomes were prepared from 20 day old Bomi barley endosperm by sucrose density gradient centrifugation Electron microscopy revealed the presence of ribosome-studded vesicles in the material banding at the 175–226m sucrose interface The isolated microsomes were active in the wheat-germ cell-free protein synthesizing system Hordeins were present among the in vitro synthesized products and were identified by their solubility in 55% isopropanol and by their co-migration with native hordeins on SDS-polyacrylamide gels The microsome-and polysome-directed, in vitro synthesized hordeins were analysed before and after chymotrypsin treatment by SDS-polyacrylamide gel electrophoresis The hordeins were degraded when polysomes were used as a template However, hordeins synthesized on microsomes showed significant protection from proteolysis This protection could be abolished by treatment with membrane-solubilizing detergents The reported experiments show that hordeins synthesized on microsomes were discharged vectorially into the lumen of the microsomes

The effects of inhibitors on the biosynthesis of the long chain lipids with even carbon numbers in barley spike epicuticular wax

Abstract: The effects of inhibitors on the incorporation of label from [2-14C]-acetate into the epicuticular wax classes with even carbon numbers have been determined. They include the free fatty acids, the primary alcohols, the aldehydes, the ester acids and a newly identified wax class on barley spikes, the short chain esters. Dithiothreitol, which has no effect on β-diketone synthesis, inhibits decarboxylation of very long fatty acyl chains which are precursors of the alkanes. Mercaptoethanol, which greatly stimulates wax synthesis at low concentrations and has no effect on β-diketone synthesis, blocks further elongation of fatty acyl precursors having 20 carbons at higher concentrations. A relatively small inhibition of decarboxylation was also observed with mercaptoethanol. Arsenite, which inhibits β-diketone synthesis presumably by blocking the entrance of C16 precursors into the β-diketone elongation mechanism, was also found to prevent the C20–C22 elongation step in the sequence leading to the alkanes. Cyanide, which blocks β-diketone synthesis by an unknown mechanism, greatly stimulated synthesis of the other wax classes. At higher concentrations a block in the elongation sequence leading to the alkanes was also apparent between C26–C28. These results are incorporated into a composite figure illustrating the biosynthetic relationships among the wax classes. All identified chemical and genetic blocks have been included. The conclusion is reached that at least three elongation systems must be involved.

Peptide mapping of the major components of in vitro synthesized barley hordein: Evidence of structural homology

Abstract: Membrane bound polysomes from Bomi barley endosperm were used as a template for in vitro hordein synthesis. The (35S)-methionine-labelled hordein was isolated by extraction with 55% isopropanol and the individual component polypeptides were resolved by SDS-polyacrylamide gel electrophoresis with detection by autoradiography. Eight bands in the molecular weight range 28,000 to 67,000 were excised from the gel. Treatment of the gel slices with chymotrypsin released soluble peptide products from the polypeptides. The resultant peptides were analysed by two dimensional thin layer chromatography and autoradiography of the (35S)-methionine-containing peptides. The peptide maps had a number of features in common, suggesting a primary structural homology between the different polypeptides.

In vitro synthesis and transport of barley endosperm proteins: Reconstitution of functional rough microsomes from polyribosomes and stripped microsomes

Abstract: Synthesis of hordein, a hydrophobic storage protein of developing barley endosperms, and its transport into the lumen of the endoplasmic reticulum has been studied in vitro. Microsomes of the rough endoplasmic reticulum have been prepared from 20 day old Bomi barley endosperms and their ability to synthesize all the major hordein polypeptides during in vitro cell-free protein synthesis demonstrated. Some 50% of these hordein polypeptides are discharged into the lumen of the microsome and are consequently inaccessible to chymotrypsin digestion.

Isolation and characterization of a polypeptide absent from non-flocculent mutants of Saccharomyces cerevisiae

Abstract: Alkaline cell extracts obtained from whole cells of a flocculent strain of Saccharomyces cerevisiae, containing the dominant gene for flocculenceFL04, and a nonflocculent mutant (FL04, fsul) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The mutant lacked a low molecular weight (13,000) polypeptide present in the extract from the parent strain. This polypeptide difference was also observed when five other independently isolated non-flocculent mutants of the parent strain were analyzed. One of these five mutants was characterized genetically and also in this case was non-flocculence shown to be due to an unlinked suppressor mutation ofFL04. This suppressor gene is designatedfsu2. Analysis of petites with different flocculation phenotypes further extended the correlation between non-flocculence and the absence of the polypeptide. The polypeptide was isolated by gel filtration in the presence of sodium dodecyl sulfate followed by ionexchange chromatography on DEAE-cellulose. By electrophoretic analysis the purity of the preparation was estimated to be 95%. From amino acid analysis the polypeptide was calculated to consist of 121 residues with a molecular weight of 12,900 daltons. Differential extraction of proteins iodinated in situ by lactoperoxidase suggested an external location of the polypeptide.

Light stimulated Δ-aminolevulinate accumulation in levulinate treated barley seedlings

Abstract: In dark-grown barley shoots treated with levulinate and briefly illuminated the protochlorophyllide which fails to reform is replaced by an excessive amount of δ-aminolevulinate (ALA). This accumulation of ALA is strongly inhibited by 100 μg·ml−1 cycloheximide but not chloramphenicol or actinomycin D. In darkness levulinate treated shoots form only little ALA; in light ALA forms at an increasing rate reaching a maximum after 2 to 3 hours. When returned to the dark the rate of ALA formation decays to a low level. Cycloheximide does not prevent this decay. Subsequent illumination results in a rise in the rate of ALA accumulation slightly inhibited by cycloheximide pretreatment. The rate of ALA formation during greening is not initially sensitive to DCMU (10−4-m) but is progressively inhibited up to 45% by 18 hours. It is concluded that light causes the synthesis of ALA by enzymes already present in dark-grown barley as well as the synthesis of the ALA forming enzymes on cytoplasmic ribosomes. In a dark period after formation of the enzymes during greening feedback inhibition is exerted by anabolic products of ALA although regulation of the enzymes through turnover also occurs. In later stages of greening light stimulates ALA formation through photosynthesis.

1,6-dihydro-NAD as an humidity-induced lactate dehydrogenase inhibitor in NADH preparations

Abstract: An authentic mixture of 1,2-, 1,4- and 1,6-dihydro-NAD was characterized by high pressure liquid chromatography. Pure 1,6-dihydro-NAD isolated from this mixture was shown to be a potent inhibitor of lactate dehydrogenase and to exhibit an UV spectrum identical to that of an extensively purified lactate dehydrogenase inhibitor generated by exposing NADH to moisture. The humidity induced inhibitor, which could not be separated from NADH by DEAE chromatography, cochromatographed with 1,6-dihydro-NAD during both DEAE and high pressure liquid chromatography and is therefore presumably identical to 1,6-dihydro-NAD.

Mutations affecting flavonoid synthesis in barley

Abstract: The anthocyanin, anthocyanidin and proanthocyanidin content of 52 induced barley mutants with altered anthocyanin pigmentation in different organs of the plant has been investigated. Comparing these mutants no correlation between the amount of anthocyanin in the plant and the amount of proanthocyanidin in the dry grains is found. The proanthocyanidins of the lemma, palea, pericarp, testa and aleurone of Bonus, Foma, Kristina, Herta, Balder and 37ant mutants yielded upon hydrolysis delphinidin and cyanidin. In 50 of the mutants belonging to at least 16 gene loci only anthocyanin synthesis is impaired. In mutantant 13–13 a step in the biosynthetic pathway leading to anthocyanins, catechins and proanthocyanidins is blocked.

Human meiosis IV. The elimination of synaptonemal complex fragments from metaphase I bivalents of human spermatocytes

Abstract: Three dimensional reconstructions of 3 prometaphase 1, 6 methaphase 1 and 2 anaphase 1 cells from 4 normal human males have been performed. At prometaphase I, 45, 36 and 32 fragments of synaptonemal complexes are present, of which 42, 22 and 23 were located within the bivalents. At metaphase I, the synaptonemal complex fragments are expelled from the bivalents, either as apparently intact fragments or as rearranged subunits of the synaptonemal complex which form small synaptonemal polycomplexes at the border of the bivalents or in the nucleoplasm. At early anaphase I, the bivalents were almost completely devoid of synaptonemal complex fragments. The possible relationship between these fragments and chiasmata is discussed. The changes in the ultrastructure of the bivalents during these stages is described. At prometaphase each of the four chromatids of a bivalent possesses a kinetochore.

1H Nuclear magnetic resonance spectroscopy of yeast copper-zinc superoxide dismutase. Structural homology with the bovine enzyme

Abstract: High resolution nuclear magnetic resonance (n.m.r.) spectroscopy of the copper-zinc superoxide dismutase from Saccharomyces cerevisiae has revealed a substantial structural homology with the bovine enzyme. N.m.r. spectra of the apo enzyme and the holo-reduced and holo-oxidized enzymes are reported and assignments are made to the histidines in the active site and the single tyrosine residue. All the assignments are in agreement with the known amino-acid sequence and the geometry of the active site is virtually unchanged. Addition of halide ions to the reduced holo enzyme results in the perturbation of the chemical shift of three histidine C2 protons and the degree of perturbation is Cl−∼Br−>I−>F− for 1m solutions of the anions. The enzyme has also been shown to retain its structure at 75°C.

Peptide mapping of the ribulose bisphosphate carboxylase large subunit from the genus Oenothera

Abstract: A peptide mapping procedure for the peptides derived by chymotryptic digestion of the large subunit of ribulose-1,5-bisphosphate carboxylase from Oenothera is described. The peptides were characterized first by ion exchange chromatography and then by thin layer chromatography in a number of solvent systems, using ninhydrin and amino acid specific stains for detection. A comparison of the maps from different Oenothera species discussed in relation to the different chloroplast genomes present in Oenothera. *** DIRECT SUPPORT *** A00GC017 00005

Synchronous sporulation of Saccharomyces cerevisiae at high cell concentrations

Abstract: A procedure for the sporulation of yeast is described which combines good synchrony and high cell concentration, important features in biochemical studies of meiosis. Meiosis and ascospore formation were followed by a modified Feulgen staining procedure, which allowed the detection of chromosomes during meiotic prophase. The sporulation process was also characterized with respect to nucleic acid synthesis, commitment to meiosis, and the segregation of the mutant and wild type alleles at theade2 locus.

Mutants of barley heat-sensitive for chloroplast development

Abstract: Six nuclear gene mutants of barley, heat-sensitive for chloroplast development, are described. These conditional lethal mutants in six different genes can be grown as homozygous viable plants in the field, their mutant phenotype only being expressed with development at high temperature. Chlorophyll accumulation in the mutants but not in the wild type is inhibited by a growth temperature of 31°C. The degree of temperature sensitivity varies amongst the mutants. For example, partial inhibition of chlorophyll production is evident in the mutantvir-zf ts4 above 20° while inhibition is complete at a growth temperature of 29°. The mutantvir-zi ts49 remains green at 29° but is bleached at 31°. The reduction in chlorophyll content of the leaves at high growth temperatures is accompanied by the appearances of structural abnormalities in the chloroplasts and reduced photochemical activity per mg chlorophyll. The inhibitory effect of elevated temperatures is confined to some early stage of plastid development as the light-dependent conversion of etioplasts into chloroplasts in the mutants is not inhibited at 32°. Leaf elongation in the mutants compared with the wild type is not affected by high growth temperatures. When the mutants are grown at temperatures permitting normal chlorophyll accumulation, the chloroplast thylakoid membranes appear to be no less heat stable than in the wild type.

Purification of a fungal endo-β-glucanase with high activity on barley β-glucan

Abstract: An endo-β-glucanase was purified from a commercial enzyme preparation of fungal origin by ammonium sulphate fractionation, ion exchange chromatography, and gel filtration followed by preparative isoelectric focusing. The enzyme was homogeneous in sedimentation equilibrium analysis from which the molecular weight was determined to be 23.500 in agreement with the value 23.653 calculated on the basis of the amino acid composition. The enzyme did not contain carbohydrate and a molecular weight of 24.000 estimated by polyacrylamide gel electrophoresis in dodecyl sulphate after 2-mercaptoethanol treatment, indicated that it consisted of a single polypeptide chain. It had an isoelectric point of 4.47. The enzyme rapidly decreased the specific viscosity of barley β-glucan with a small concomitant increase in reducing sugar. Its activity toward carboxymethyl-cellulose, acid-swollen cellulose, and a mixture of cellodextrins was low. Laminarin, carboxymethyl-pachyman, cellobiose, and p-nitrophenyl-β-D-glucoside were not hydrolyzed. The Km for hydrolysis of barley β-glucan and carboxymethyl-cellulose was 1.8 and 11 mg/ml, respectively, the corresponding molecular activities being 7750 and 750 equivalents of glucosidic bonds hydrolyzed per min per mole of enzyme. The products of exhaustive hydrolysis of barley β-glucan were 4.3% glucose, 4.2% disaccharide, 72.7% trisaccharide, and 18.8% tetrasaccharide, higher oligomers and polymers were absent. The enzymic activity was completely destroyed by treatment with N-bromosuccinimide but was insensitive to EDTA, sulfhydryl modifying reagents, and glucono-1,5-lactone.

Immobilization of β-glucanase and studies on its degradation of barley β-glucan

Abstract: A commercial fungal β-glucanase preparation was immobilized by various methods including adsorption to DEAE-cellulose, cross-linking with glutaraldehyde, adsorption to a phenol-formaldehyde resin (Duolite) and to perlite followed by fixation with glutaraldehyde, covalent binding to glutaraldehyde-treated, partially hydrolyzed or partially aminolyzed nylon, and covalent binding in the Ugi-reaction to polyisonitrile-nylon. The recovery of enzymatic activity varied from 0.02 to 4.5% and the immobilized enzyme preparations showed specific activities from 1 to 25% of that of the starting material.

High mobility group proteins in yeast

Abstract: A class of low molecular weight proteins was extracted from isolated yeast nuclei by 5% (w/v) perchloric acid followed by fractional acetone precipitation. Their solubility properties, electrophoretic mobility and the amino acid composition were similar to those of the high mobility group (HMG) chromosomal proteins of higher eukaryotes.

Heat synchronization of cell division in Tetrahymena thermophila and a mutant, NP1, with a temperature sensitive defect for oral development

Abstract: Optimal conditions for induction of synchronous cell division in Tetrahymena thermophila have been investigated. Wild type cells and a mutant, NP1, with temperature sensitive oral development have been used. NP1 forms no food vacuoles after long exposures to 37°C or higher temperatures. According to one temperature regime both clones were grown at 30°C and exposed to five 30 min heat shocks at 42.3°C spaced 30 min apart. This treatment resulted in 80% and in 50% increase in cell number over 15 min intervals placed at the time of the first and the second synchronized cell division, respectively. NP1 formed food vacuoles at the same rate as wild type cells. According to a second temperature regime the cells were grown at 30°C and heat shocks at 41.8°C were spaced one cell generation apart. This treatment resulted in division of about 95% of the cells within a one hour period between the sixth and seventh shock. Heat shocks were also applied to cells grown at 37°C. Five shocks at 43.1°C spaced 30 min apart synchronized cell divisions of wild type cells but not of mutant cells.

Buoyant titration of ovalbumin in four alkali halides. Hydration and ion binding

Abstract: The buoyant titrations of ovalbumin in CsCl, RbCl, CsBr and RbBr were measured between pH 2 and 13. The buoyant densities were found to depend on the salt employed to generate the density gradient and the solution pH. At low pH, nearly identical buoyant densities were observed in solutions having a common anion while at high pH, salt solutions having the same cation produced nearly indentical buoyant densities. The buoyant density of ovalbumin in RbBr was found to decrease as the pH was increased from 2 to 6. This is the first demonstration of a drop in the buoyant titration curve for a biopolymer.

The cell cycle and glycolytic activity of Schizosaccharomyces pombe synchronized in defined medium

Abstract: Schizosaccharomyces pombe was grown in a chemically defined medium (EMM 2). Cell cycle synchrony was induced with 5 or 6 heat shocks (60min, 41°C) spaced a normal cell generation apart (140 min, 32°C). The sequence of nuclear division, cell plate formation, DNA synthesis and cell division is normal, and both DNA and cell number double in each cycle.

Brewing performance of a yeast after prolonged growth on a synthetic medium

Abstract: Two clones of brewers yeast, grown for more than 300 cell generations on a synthetic minimal medium were propagated on wort and used in pilot brewing experiments. Parallel brews with wort cultured yeast were carried out for comparison. Analytical data showed no differences between beer brewed with yeast grown on minimal medium and wort cultured yeast. One out of the two clones grown on minimal medium gave a beer with a somewhat inferior taste.