Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: distinction of the 1-monodeutero-isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy.

The 1H NMR spectrum obtained with the alpha- and beta-anomers of D-[1-2H]fructose 6-phosphate generated from D-glucose 6-phosphate sequentially exposed in D2O to phosphoglucoisomerase, phosphofructokinase and fructose-1,6-diphosphatase differed from that recorded when the deuterated ketohexose phosphate was produced from D-mannose 6-phosphate sequentially exposed in D2O to phosphomannoisomerase, phosphofructokinase and fructose-1,6-diphosphatase. The identification of the 2 isotopomers of D-fructose 6-phosphate by 1H NMR spectroscopy provides a new tool to assess the relative extent of interconversion of hexose phosphates in the reactions catalyzed by phosphoglucoisomerase and phosphomannoisomerase, respectively.     

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates; comparison with phosphomannoisomerase

The isotopic discrimination, diastereotopic specificity and intramolecular hydrogen transfer characterizing the reaction catalyzed by phosphomannoisomerase are examined. During the monodirectional conversion of D-[2-3H]mannose 6-phosphate to D-fructose 6-phosphate and D-fructose 1,6-bisphosphate, the reaction velocity is one order of magnitude lower than with D-[U-14C]mannose 6-phosphate and little tritium (less than 6%) is transferred intramolecularly. Inorganic phosphate decreases the reaction velocity but favours the intramolecular transfer of tritium. Likewise, when D-[1-3H]fructose 6-phosphate prepared from D-[1-3H]glucose is exposed solely to phosphomannoisomerase, the generation of tritiated metabolites is virtually restricted to 3H2O and occurs at a much lower rate than the production of D-[U-14C]mannose 6-phosphate from D-[U-14C]fructose 6-phosphate. However, no 3H2O is formed when D-[1-3H]fructose 6-phosphate generated from D-[2-3H]glucose is exposed to phosphomannoisomerase, indicating that the diastereotopic specificity of the latter enzyme represents a mirror image of that of phosphoglucoisomerase. Advantage is taken of such a contrasting enzymic behaviour to assess the back-and-forth flow through the reaction catalyzed by phosphomannoisomerase in intact cells exposed to D-[1-3H]glucose, D-[5-3H]glucose or D-[6-3H]glucose. Relative to the rate of glycolysis, this back-and-forth flow amounted to approx. 4% in human erythrocytes and rat parotid cells, 9% in tumoral cells of the RINm5F line and 47% in rat pancreatic islets.     

Distinguishing allozymes and isozymes of phosphoglucoisomerases by electrophoretic comparisons of pollen and somatic tissues

The different electrophoretic patterns of dimeric phosphoglucoisomerases extracted from haploid pollen and diploid somatic tissues of plants may be used to distinguish allozymes and isozymes. The analysis depends on the presence of two alleles at each locus in somatic tissues but only one or the other allele in pollen grains. Consequently, in heterozygotes, heterodimeric allozymes can be identified because they are formed in stems and leaves but not in pollen. The procedure is described in enzymes extracted from the diploid annual plant Clarkia dudleyana, which possesses three gene loci for PGI subunits. Comparison of the electrophoretic patterns of stem and pollen extracts makes it possible in many cases to identify allelic state without breeding tests. The technique also is likely to be useful in the interpretation of zymograms of other multimeric enzymes coded by more than one gene locus.     

Dual anomeric specificity of phosphomannoisomerase assessed by 2D phase sensitive 13C EXSY NMR

The reversible conversion between D-mannose 6-phosphate and D-fructose 6-phosphate catalyzed by yeast phosphomannoisomerase was studied by phase sensitive 2D ¹³C-¹H EXSY NMR spectroscopy at 100.623 MHz, using ¹³C enriched substrates in the C₂ position of the D-hexose 6-phosphates. The unique pair of isomerization cross-peaks observed in the 2D EXSY map correlates the ¹³C₂ resonances of the -anomers of both D-[2-¹³C]-mannose-6-phosphate and D-[2¹³C]-fructose 6-phosphate. This indicates that phosphomannoisomerase specifically catalyzes the reversible conversion between -D-mannose 6-phosphate and -D-fructose 6-phosphate. Since phosphoglucoisomerase was recently found to catalyze specifically the interconversion of -D-glucose 6-phosphate and -D-fructose 6-phosphate, the -anomer of the ketohexose ester could be directly channeled in a multi-enzyme system involving phosphoglucoisomerase, phosphomannoisomerase and phosphofructokinase.     

Enzyme diversity in pearl millet (Pennisetum glaucum)

Summary Polymorphism in twelve genes coding for eight enzymes in pearl millet (Pennisetum glaucum (L.) R. Br.): alcohol dehydrogenases (ADH), catalases (CAT), -esterases (EST), glutamate oxaloacetate transaminases (GOT), malate dehydrogenases (MDH), 6-Phosphogluconate dehydrogenases (PGD), phosphoglucoisomerases (PGI) and phosphoglucomutases (PGM), was observed by electrophoresis on 74 cultivated samples and 8 wild samples from West Africa. Six genes: Est A, Adh A, Pgm A, Cat A, Pgi A, Pgd A contain 95% of the total variation. Principal component analyses and discriminant analyses of the 82 samples described by 46 allelic frequencies showed an almost complete separation into 3 groups: wilds, early maturing cultivars and late maturing cultivars. The early group has the highest enzyme diversity, with cultivated millets from Niger showing the most diversity. The high diversity of the early group and its extensive divergence from West-African wild millets suggest, firstly, the existence, elsewhere in Africa of other enzymatically different sources of wild millet, and secondly, the occurrence, prehistorically, of several different domestications. The late group of cultivars has the lowest variability and a relatively low coefficient of differentiation. This relatively homogeneous enzyme structure does not seem to be associated to ecology. A hypothesis is advanced suggesting that West African late-cultivars were derived from a common cultivated early complex. This complex must have been distributed across the Sudanian zone and must have been later sumitted to modifications by limited gene flow with local early maturing cultivars.     

Synthesis of 2-methylthio-cis- and trans-ribosylzeatin and their isolation from Pisum tRNA

The cis isomer of 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthiopurine and its 9-β- and 9-α-d-ribofuranosyl derivatives have been synthesized and their physical and spectroscopic properties are described. The biological activities of these compounds have been determined in the tobacco bioassay and are compared with those of 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-2-methylthiopurine and its β-ribofuranoside. The 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d-ribofuranosylpurine (ms-ribosylzeatin) isolated from a Pisum tRNA preparation was shown to consist of both isomers, which were separated by TLC and identified by comparisons of UV and MS with those of the synthetic compounds.     

Caespitenone,a new cyclopropanoid pseudoguaiane and ent-sesquiterpenes from Porella species

Six Porella species and one Macvicaria species have been investigated and a new cyclopropane pseudoguaiane was isolated and its structure elucidated by chemical and spectral evidence. Macvicaria ulophylla and the Porella species, except P. caespitans ssp. setigera, contain the diterpene dialdehyde, perrottetianal A. (+)-Aristolone, (?)-α-eudesmol, and related sesquiterpene hydrocarbons and alcohols, enantiomeric to those found in higher plant, have been isolated from the Porella species.     

Sequence analysis and evolution of sea urchin (Lytechinus pictus and Strongylocentrotus purpuratus) histone H4 messenger RNAs

The putative histone H4 (F2a₁) mRNA has been isolated from early blastula Strongylocentrotus purpuratus sea urchin embryos. Nucleotide sequences of oligonucleotides obtained by digestion of this RNA with T₁ ribonuclease have been obtained and many are found to be colinear with the amino acid sequence of histone H4 protein. The sequences obtained from the H4 mRNAs of S. pnrpuratus have been compared with those obtained from Lytechinus pictus (Grunstein & Schedl, 1976). The two mRNAs for this highly conserved protein have undergone considerable divergence of the sort that would be predicted from the degeneracy of the genetic code. 11.5% of the bases have undergone substitution at a rate calculated to be 3 × 10^?9 base changes · codon^?1 · year^?1.     

Complexes formed between the restriction endonuclease EcoK and heteroduplex DNA

The complexes between the Escherichia coli K restriction endonuclease and heteroduplex DNA (one strand methylated and one unmethylated) have been characterized and shown to have different properties from those formed with unmodified DNA. The nature of the heteroduplex complex appears to commit the enzyme to its methylase mode.     

Production,characterization and gene cloning of the extracellular enzymes from the marine-derived yeasts and their potential applications

In this review article, the extracellular enzymes production, their properties and cloning of the genes encoding the enzymes from marine yeasts are overviewed. Several yeast strains which could produce different kinds of extracellular enzymes were selected from the culture collection of marine yeasts available in this laboratory. The strains selected belong to different genera such as Yarrowia, Aureobasidium, Pichia, Metschnikowia and Cryptococcus. The extracellular enzymes include cellulase, alkaline protease, aspartic protease, amylase, inulinase, lipase and phytase, as well as killer toxin. The conditions and media for the enzyme production by the marine yeasts have been optimized and the enzymes have been purified and characterized. Some genes encoding the extracellular enzymes from the marine yeast strains have been cloned, sequenced and expressed. It was found that some properties of the enzymes from the marine yeasts are unique compared to those of the homologous enzymes from terrestrial yeasts and the genes encoding the enzymes in marine yeasts are different from those in terrestrial yeasts. Therefore, it is of very importance to further study the enzymes and their genes from the marine yeasts. This is the first review on the extracellular enzymes and their genes from the marine yeasts.     

Palaeobiogeographic and metric analysis of the Mesozoic fern Weichselia

Eighty-three publications have been examined to recover information on the palaeogeography, the chronostratigraphy and the presence of the fossil fern genus Weichselia Stiehler in different depositional environments. This fossil fern foliage was reported in 72 localities worldwide. Its fossil record ranges from the Bathonian to the Cenomanian, predominating from the Berriasian to the Barremian, but still common during the Aptian to Cenomanian. Weichselia mostly occurred in continental depositional environments in Europe, whereas it has been mostly related to marginal marine depositional environments in Africa, North America, and India. Three species have been described: W. reticulata, W. peruviana, and W. negevensis. We analyse the measurements on specimens collected from sixteen localities as well as published data from twelve others. Our results suggest that: (i) the climate changes during the Aptian might have affected the pinnule size of Weichselia, as the Aptian-Cenomanian pinnules are larger than those from the Berriasian-Barremian; (ii) some differences might be explained by the metric variation of the frond parts; (iii) there are no clear differences in size between remains collected from different depositional environments; (iv) there is no sufficient evidence to determine how many species of Weichselia have existed.     

Ability of plant callus cultures to synthesize and accumulate lower terpenoids

Callus cultures derived from seven oil-producing plants have been maintained under different regimes of media, temperature and illumination, and have been assayed for ability both to synthesize and to accumulate mono- and sesqui-terpenes. Callus of Pinus radiata (the sole gymnosperm) accumulated (α- and β-pinenes at levels comparable with those in the parent stem and needles; and that of Jasminum officinale accumulated traces of several monoterpenes (< 0.1 % the amount in petals) but cultures of Rosmarinus officinalis, Lavandula angustifolia, Anethum graveolens, Ocimum basilicum and Tanacetum vulgare did not detectably accumulate the lower terpenoids or secrete them into the medium. However, all seven culture lines yielded cell-free extracts containing prenyltransferase and an isomerizing system (as assayed by conversion of IPP into GPP, NPP and FPP) with activities some fold greater than those extracted from the parent mature plants, or up to 90-fold the levels extractable from young seedlings of the various species. Callus of five of the species (A. graveolens and O. basilicum were not assayed) also contained MVA-kinase, MVAP-kinase, MVAPP-decarboxylase and IPP-DMAPP isomerase at levels comparable with those in the parent tissue. Hence the angiosperms yielded cultures that presumably contained the crucial enzyniic machinery necessary for the synthesis of the lower terpenoids, although accumulation of those compounds did not occur. Reasons for this unexpected situation are discussed. These results imply that callus cultures may be a convenient source of biomass for studies on the enzymes of terpenoid biosynthesis.     

Comparative study of cellulases and hemicellulases from four fungi: mesophiles Trichoderma reesei and Penicillium sp. and thermophiles Thielavia terrestris and Sporotrichum cellulophilum

The enzymes produced by two thermophilic fungi claimed to produce heat-stable cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] have been compared to those of two mesophilic fungi on the basis of the following criteria: polysaccharolytic spectrum, heat and pH effects on stability and on activity of the different enzymes, and the ability to hydrolyse raw natural substrates. The cellulases produced by one of the thermophiles, Sporotrichum cellulophilum, appeared to be as heat-labile as those from the mesophile Trichoderma reesei; moreover, the former enzyme preparation is the least efficient of the four tested. Thielavia terrestris enzymes are the most thermostable; on the basis of the other properties tested, T. terrestris enzymes are comparable to, or in some cases better than, those from mesophilic strains. However, the differences are not so great as to compensate for the much lower productivity of T. terrestris compared to the improved T. reesei and Penicillium sp. strains.     

The composition and properties of gum exudates from subspecies of Acacia tortilis

Gum specimens from Acacia tortilis ssp. spirocarpa, ssp. raddiana var. pubescens (two specimens) and ssp. heteracantha (three specimens) have been analysed. The results are of chemotaxonomic interest. Although the gum from ssp. raddiana var. pubescens appears to be intermediate between those from ssp. spirocarpa and ssp. heteracantha in terms of some of the analytical parameters, the overall impression is that ssp. raddiana is more similar chemically to ssp. spirocarpa than to ssp. heterocantha. The latter yields a viscous, proteinaceous polysaccharide that differs from those from both ssp. spirocarpa and ssp. raddiana by having a much higher ratio of arabinose to galactose, higher nitrogen and methoxyl contents, and much higher intrinsic viscosity and molecular weight; preliminary experiments have shown this gum to consist of two components.     

Investigation into the distribution and properties of histones and other basic proteins in bacteria

1. Methods have been developed for the extraction and purification of bacterial basic proteins. These proteins were initially examined by a micro-method of starch-gel electrophoresis and were then characterized more fully. 2. Although it was found that most of the DNA in both Bacillus megaterium and Escherichia coli must be free from combination with basic protein, there was some evidence that a small portion might be in the form of a typical nucleohistone complex. 3. The ribosomes of both B. megaterium and E. coli were shown to contain approximately 2% of basic protein. On the basis of ultraviolet-absorption curves, partial amino acid analyses and their behaviour on electrophoresis in polyacrylamide gels, it was concluded that some of these ribosomal basic proteins may be extremely similar to typical histones. 4. These results are discussed in relation to those of other authors, and the possible functions of basic proteins present in micro-organisms are considered with reference to those that have already been proposed for the histones of higher organisms.     

A Chemical and Physical Characterization of Echinoid RNA during Early Embryogenesis

The properties of the major RNA components prepared from Arbacia punctulata have been characterized by sedimentation in sucrose gradients and analysis of base composition. Comparison of the components found in the unfertilized egg with those observed subsequent to fertilization revealed no differences in any of the embryonic stages examined. The base composition and sedimentation profiles of RNA from unfertilized nuclear egg fragments and from 105,000 g pellet were similar to those of the intact egg. It is concluded that the early stages of embryogenesis are not accompanied by detectable alteration of the physical or chemical characteristics of the major RNA components found in the unfertilized egg.     

Maximum likelihood estimation of linkage and interference from tetrad data

Maximum likelihood equations have been derived for estimation of map distance and interference from two-point and ranked tetrad data. The estimators have been applied to data from Saccharomyces cerevisiae and Schizosaccharomyces pombe. S. cerevisiae consistently shows quite strong interference over the mapped genome. In striking contrast, S. pombe consistently shows much weaker interference and many crosses exhibit negative interference. In neither species was there a conspicuous tendency for intervals spanning a centromere to show less interference than those that did not. Since the amount of recombination per microgram of DNA in the two species is similar, the difference in interference characteristics seems to be a reflection of some fundamental difference in the recombination process of the two species.     

Iridoids from Viburnum betulifolium

Two iridoid glycosides have been isolated from Viburnum betulifolium. Viburnalloside, the major leaf glycoside, is composed of an iridoid aglucone acylated at C-1 with isovaleric acid and with a di-O-acetyl-β-D-allopyranosyl moiety attached through a glycosidic bond to C-11. Decapetaloside (10-hydroxyiridodial glucoside) has been isolated from the bark. The structure and absolute configuration of viburnalloside have been established by spectroscopic means, and those of decapetaloside by chemical correlation with adoxoside.     

Gel mobility shift scanning of the acetate-inducible promoters fromNeurospora crassa reveals a common co-inducible DNA-binding protein

The promoter regions of four acetate-inducible genes ofNeurospora crassa, acu-3, acu-5, acu-8 andacu-9, have been sequenced. Using a scanning gel mobility shift assay particular DNA regions in each promoter have been shown specifically to bind partially purified protein extracted from acetate-induced mycelia. The protein-binding regions so defined have common sequence motifs, elements of which are similar to those required for acetate induction inAspergillus nidulans.