Linear differentiation of cereal chromosomes

Summary The BSG test was used in a comparative study of the linear chromosome differentiation and the idiograms of T. Macha ssp. tubalicum v. letschchumicum Dek. et Men., T. georgicum Dek., T. timopheevi. Zhuk., T. carthlicum Nevski, T. dicoccum Schrank, v. rufum, T. durum Desf. v. Arnautka were compiled.The karyotype of each polyploid wheat species consists of two groups of chromosomes. The first is formed by ten pairs of constant chromosomes occurring almost in all species and the second by all the rest of the variable chromosomes that are either fully specific for the species in question or occur only in a few species. T. timopheevi largely differs from other species of polyploid wheats in the high level and specific localization of structural heterochromatin on chromosomes. The rols of introgression in wheat evolution and the necessity of establishing a General Cytological Nomenclature of Cereal Chromosomes are discussed.     

Energy Metabolism of Synaptosomal Subpopulations from Different Neuronal Systems of Rat Hippocampus: Effect of L-Acetylcarnitine Administration In Vivo

The maximum rate (V_max) of some enzyme activities related to glycolysis, Krebs' cycle, acetylcholine catabolism and amino acid metabolism were evaluated in different types of synaptosomes obtained from rat hippocampus. The enzyme characterization was performed on two synaptosomal populations defined as large and small synaptosomes, supposed to originate mainly from the granule cell glutamatergic mossy fiber endings and small cholinergic nerve endings mainly arising from septohippocampal fiber synapses, involved with cognitive processes. Thus, this is an unique model of pharmacological significance to study the selective action of drugs on energy metabolism of hippocampus and the sub-chronic i.p. treatement with L-acetylcarnitine at two different dose levels (30 and 60 mg · kg^–1, 5 day a week, for 4 weeks) was performed. In control animals, the results indicate that these two hippocampal synaptosomal populations differ for the potential catalytic activities of enzymes of the main metabolic pathways related to energy metabolism. This energetic micro-heterogeneity may cause their different behaviour during both physiopathological events and pharmacological treatment, because of different sensitivity of neurons. Therefore, the micro-heterogeneity of brain synaptosomes must be considered when the effect of a pharmacological treatment is to be evaluated. In fact, the in vivo administration of L-acetylcarnitine affects some specific enzyme activities, suggesting a specific molecular trigger mode of action on citrate synthase (Krebs' cycle) and glutamate-pyruvate-transaminase (glutamate metabolism), but mainly of small synaptosomal populations, suggesting a specific synaptic trigger site of action. These observations on various types of hippocampal synaptosomes confirm their different metabolic machinery and their different sensitivity to pharmacological treatment.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Is Ag(I) an adequate probe for Cu(I) in structural copper–metallothionein studies?

The binding abilities of silver(I) to mammalian MT 1 have been studied and compared with those of copper(I), recently reported [Bofill et al. (2001) J Biol Inorg Chem 6:408–417], with the aim of analyzing the suitability of Ag(I) as a Cu(I) probe in Cu–MT studies. The Zn/Ag replacement in recombinant mouse Zn₇–MT 1 and corresponding Zn₄-MT 1 and Zn₃-MT 1 fragments, as well as the stepwise incorporation of Ag(I) to the corresponding apo-MTs, have been followed in parallel by various spectroscopic techniques including electronic absorption (UV–vis), circular dichroism (CD) and electrospray mass spectrometry coupled to capillary zone electrophoresis (CZE-ESI-MS). A comparative analysis of the sets of data obtained in the titration of Zn₇–MT 1, Zn₄–MT 1 and Zn₃-MT 1 with AgClO₄ at pH 7.5 and 2.5 has led to the reaction pathways followed during the incorporation of silver to these proteins under these specific conditions, disclosing unprecedented stoichiometries and structural features for the species formed. Thus, the Zn/Ag replacement in Zn₇–MT 1 at pH 7.5 has revealed the subsequent formation of Ag₄Zn₅–MT, Ag₇Zn₃–MT, Ag₈Zn₃–MT, Ag₁₀Zn₂–MT, Ag₁₂Zn₁–MT, Ag_x–MT, x=14–19, whose structure consists of two additive domains only if Zn(II) remains coordinated to the protein. A second structural role for Zn(II) has been deduced from the different folding found for the Ag_x–MT species of the same stoichiometry formed at pH 7.5 or 2.5. Comparison of the binding features of Cu(I) and Ag(I) to the entire MT at pH 7.5 shows that, among all the _xZn_y–MT (0y<7) species found, only M^I₄Zn₅–MT [(Zn₄)(₄Zn₁)] and M^I₇Zn₃–MT [(₃Zn₂)(₄Zn₁)], which form during the first stages of the Zn(II)/M(I) metal replacement, show comparable 3D structures; thus, they are the only species where Ag(I) ions can be predicted to be an adequate probe for Cu(I).Electronic Supplementary Material Supplementary material is available in the online version of this article at .     

Production and characterization of asymmetric somatic hybrids between Arabidopsis thaliana and Brassica napus

Summary Cell suspension-derived protoplasts of a chlorsulfuron-resistant (GH50) strain of Arabidopsis thaliana cv Columbia were X-irradiated at 60 or 90 krad, to facilitate the elimination of GH50 donor chromosomes in fusion products. Irradiated GH50 protoplasts were fused, with polyethylene glycol, to protoplasts derived from stem epidermal strips of Brassica napus cv Westar. Chlorsulfuron-resistant colonies were selected in vitro and then transferred to shoot and root regeneration medium. Seventeen hybrid lines were regenerated in vitro, and eight were successfully established in the greenhouse, where they flowered. These eight asymmetric hybrids were intermediate in vegetative morphology between Arabidopsis and Brassica. The flowers from these hybrids were male-sterile with abnormal petal and pistil structures. Zymograms for phosphoglucomutase, esterase, and peroxidase showed the presence of all parental isozymes in each of the hybrids tested. Nuclear hybridity was also confirmed for the ribosomal RNA genes using a wheat rDNA probe; however, the chloroplast genome in each of the hybrids was derived solely from the Brassica parent. All selected somatic hybrids were capable of rooting at levels of chlorsulfuron which were inhibitory to unfused Brassica plantlets. The degree of herbicide resistance in the hybrid shoots is presently being evaluated.Contribution No. 1428, Plant Research Centre, Agriculture Canada     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

Partitioning and separation of α-lactalbumin and β-lactoglobulin in polyethylene glycol/ammonium sulphate aqueous two-phase systems

The partition behaviour of -lactalbumin (la) and -lactoglobulin (lg) on PEG/(NH₄)₂SO₄ system was studied. For purified proteins, a partition coefficient of 12.8 for la and 0.34 for lg, with mass recovery yields of 96.7% for la in the upper phase and 83.8% for lg in the lower phase was obtained, in 18% (w/w) PEG 900/14% (w/w) (NH₄)₂SO₄ system, at pH 7. PEG/(NH₄)₂SO₄ system was an economical alternative for the recovery and separation of the two proteins in cheese whey, allowing a 50% reduction in costs. An efficient and inexpensive separation of both proteins in cheese whey could be achieved, by using 16% (w/w) PEG 900/15% (w/w) (NH₄)₂SO₄, at pH 7.5.     

Exploration of RNA 3′ terminal locations in rat ribosome

The locations of the 3 ends of RNAs in rat ribosome were studied by a fluorescencelabeling method combined with high hydrostatic pressure and agarose electrophoresis. Under physiological conditions, only the 3 ends of 28 S and 5.8 S RNA in 80 S ribosome could be labeled with a high sensitive fluorescent probe – fluorescein 5thiosemicarbazide (FTSC), indicating that the 3 termini of 28 S and 5.8 S RNA were located on or near the surface of 80 S ribosome. The 3 terminus of 5 S RNA could be attacked by FTSC only in the case of the dissociation of the 80 S ribosome into two subunits induced by high salt concentration (1 M KCl) or at high hydrostatic pressure, showing that the 3 end of 5 S RNA was located on the interface of two subunits. However, no fluorescencelabeled 18 S RNA could be detected under all the conditions studied, suggesting that the 3 end of 18 S RNA was either located deeply inside ribosome or on the surface but protected by proteins. It was interesting to note that modifications of the 3 ends of ribosomal RNAs including oxidation with NaIO₄, reduction with KBH₄ and labeling with fluorescent probe did not destroy the translation activity of ribosome, indicating that the 3 ends of RNAs were not involved in the translation activity of ribosome.     

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Summary Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt⁶⁰,100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via gamma-fusion.     

Expression of Streptomyces genes encoding extracellular enzymes in Brevibacterium lactofermentum: secretion proceeds by removal of the same leader peptide as in Streptomyces lividans

The -amylase gene (amy) from Streptomyces griseus IMRU 3570 and the -galactosidase gene (lac) from S. lividans were subcloned into Brevibacterium lactofermentum or B. lactofermentum/Escherichia coli shuttle vectors. The amy gene was not expressed in B. lactofermentum from its own promoter but was efficiently expressed when the promoter of the kanamycin resistance gene (kan) was inserted upstream of the promoterless amylase gene. The lac gene from S. lividans was subcloned without its native promoter and was expressed when placed downstream of pBL1 promoters P₂ or P₃. The -amylase was secreted extracellularly by removal of the same 28-amino acid leader peptide as in S. lividans. The amy and lac genes provide useful markers for selection of transformants and will facilitate the study of protein secretion in B. lactofermentum. Correspondence to: J. F. Martín     

Activation of dynamin II by POPC in giant unilamellar vesicles: a two-photon fluorescence microscopy study

The interaction of dynamin II with giant unilamellar vesicles was studied using two-photon fluorescence microscopy. Dynamin II, labeled with fluorescein, was injected into a microscope chamber containing giant unilamellar vesicles, which were composed of either pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or a mixture of POPC and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂). Binding of the fluorescent dynamin II to giant unilamellar vesicles, in the presence and absence of PI(4,5)P₂, was directly observed using two-photon fluorescence microscopy. This binding was also visualized using the fluorescent N-methylanthraniloyl guanosine 5-[-thio]triphosphate analogue. The membrane probe 6-dodecanoyl-2-dimethylamine-naphthalene was used to monitor the physical state of the lipid in the giant unilamellar vesicles in the absence and presence of dynamin. A surprising finding was the fact that dynamin II bound to vesicles in the absence of PI(4,5)P₂. Activation of the GTPase activity of dynamin II by pure POPC was then shown.     

Pulse schemes for the measurement of3 JC′Cγ and3 JNCγ scalar couplings in 15N,13C uniformly labeled proteins

Pulse sequences are presented for the measurement of³J_CC and³J_NC scalar couplings for allC containing residues in¹⁵N,¹³C uniformly labeled proteins. The methodsdescribed are based on quantitative J correlation spectroscopy pioneered byBax and co-workers [Bax et al. (1994) Methods Enzymol., 239, 79–105].The combination of ³J_CC and³J_NC scalar coupling constants allows theassignment of discrete rotameric states about the ₁ torsion angle in cases where such states exist or, alternatively,facilitates the establishment of noncanonical ₁conformations or the presence of rotameric averaging. The methods areapplied to a 1.5 mM sample of staphylococcal nuclease.     

Charybdotoxin-sensitive K(Ca) channel is not involved in glucose-induced electrical activity in pancreatic β-cells

Summary The effects of charybdotoxin (CTX) on single [Ca²⁺] _i -activated potassium channel (K _(Ca)) activity and whole-cell K⁺ currents were examined in rat and mouse pancreatic -cells in culture using the patch-clamp method. The effects of CTX on glucose-induced electrical activity from both cultured -cells and -cells in intact islets were compared. K_(Ca) activity was very infrequent at negative patch potentials (–70<V _m <0 mV), channel activity appearing at highly depolarizedV _m . K_(Ca) open probability at these depolarizedV _m values was insensitive to glucose (10 and 20mm) and the metabolic uncoupler 2,4 dinitrophenol (DNP). However, DNP blocked glucose-evoked action potential firing and reversed glucose-induced inhibition of the activity of K⁺ channels of smaller conductance.The venom fromLeiurus quinquestriatus hebreus (LQV) and highly purified CTX inhibited K_(Ca) channel activity when applied to the outer aspect of the excised membrane patch. CTX (5.8 and 18nm) inhibited channel activity by 50 and 100%, respectively. Whole-cell outward K⁺ currents exhibited an early transient component which was blocked by CTX, and a delayed component which was insensitive to the toxin. The individual spikes evoked by glucose, recorded in the perforated-patch modality, were not affected by CTX (20nm). Moreover, the frequency of slow oscillations in membrane potential, the frequency of action potentials and the rate of repolarization of the action potentials recorded from pancreatic islet -cells in the presence of glucose were not affected by CTX.We conclude that the K_(Ca) does not participate in the steady-state glucose-induced electrical activity in rodent pancreatic islets.     

Chromosome and isoenzyme studies on cells derived from protoplast fusion of Nicotiana glauca with glycine max-Nicotiana glauca cell hybrids

Summary The somatic hybrids of Glycine max (L)Merr.-Nicotiana glauca Grah. exhibited a preferential loss of N. glauca chromosomes. When protoplasts from such hybrid cells were back fused twice to N. glauca protoplasts, a considerable increase in stability of the N. glauca chromosomes was observed. Gel electrophoresis studies of aspartate aminotransferase showed that the chromosome(s) responsible for this enzyme was stabilized in the back fused hybrid cell lines. The data suggest that the back fusion technique described in this study might aid in stabilizing somatic hybrids.NRCC No. 18040     

Formation of nucleoside 5′-phosphoramidates under potentially prebiological conditions

Summary Adenosine 5-phosphoramidates form when solutions containing adenosine 5-polyphosphates p_nA (n 3) or P₁, P₂-diadenosine 5-diphosphate and amines are allowed to dry out. Mg ions catalyze these reactions. We have studied systems containing ammonia, imidazole, glycine, ethylenediamine and histamine. The yields of adenosine 5-phosphoramidates range from 10–50 % based on the nucleotide. The prebiotic significance of the reactions is discussed.Abbreviations Im imidazole - hist histamine - gly glycine - en ethylenediamine - CDI 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride - EDTA ethylenediaminetetraacetic acid - A adenosine - P_n (n = 1, 2 ) linear polyphosphate containing n phosphate residues - p_nA adenosine 5-polyphosphate containing n phosphate residues - ADP adenosine 5-diphosphate - ATP adenosine 5-triphosphate - AppA P₁, P₂-diadenosine 5-diphosphate - gly-pA adenylyl-(5N)-glycine - ImpA adenosine 5-phosphorimidazolide - NH₂-pA adenosine 5-phosphoramidate - en-pA adenylyl-(5N)-ethylenediamine - hist (NH) - pA adenosine 5-phospho-[2-(4-imidazolyl)-ethylamide] - hist(Im)-pA adenosine 5-phospho-[4-(2-aminoethyl)-imidazolide] - enP_1,2 phosphoramidates of ethylenediamine derived from H₃PO₄ and H₄P₂O₇     

Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences

Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically demonstrable sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)_n/(gaca)_m are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)₄ probe. The (GATA)₄ oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In contrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)₄. Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)₄ probe.by E.R. SchmidtDedicated to Professor Karl Sperling on the occasion of his 50th birthday     

Induction of de novo synthesis of phenylalanine ammonia-lyase by L-α-aminooxy-β-phenylpropionic acid in suspension cultures ofDaucus carota L.

Application of L--aminooxy--phenylpropionic acid (L-AOPP), a potent and specific competitive inhibitor of L-phenylalanine ammonia-lyase (PAL), to an anthocyanin-producing cell suspension culture ofDaucus carota results in a dramatic increase in extractable PAL activity and an accumulation of phenylalanine (Noé et al., 1980, Planta149, 283–287). Using an immunoprecipitation technique, evidence was obtained that the increase in PAL activity the result of de-novo synthesis. The activity of the other enzymes of the general phenylpropanoid metabolism, e.g., trans-cinnamate 4-hydroxylase and hydroxycinnamate: CoA ligase, were not affected by L-AOPP. This result strongly supports the view that PAL is regulated independently.Abbreviations CAH trans-cinnamate 4-hydroxylase - L-AOPP L--aminooxy--phenylpropionic acid - PAL L-phenylalanine ammonia-lyase