Rifampicin-induced protein synthesis: A pre-requisite for increased expression of the ββ′ operon in Escherichia coli

Summary In a rif ^S/rif^R heterodiploid strain of E. coli, a 4 minute pulse of rifampicin can induce a prolonged (>60 min) increase in the rate of synthesis of the RNA polymerase subunits, and . The application of a constraint on the fidelity of protein synthesis during, but not after, the rifampicin pulse partially arrests the development of this capacity for subunit synthesis. I discuss the implications of these findings in relation to the control of the operon in E. coli.     

Partial mispairing and crossing-over between β0 and δ genes as the origin of the δ β0 thalassemia gene

Summary Two double heterozygous ⁰/⁰ thalassemic sibs of Mexican descent were studied. The father had a ⁰/⁰ genotype, while the mother, one sib and several maternal relatives were ⁰/⁰ heterozygotes. Parental consanguinity and an apparently low frequency of thalassemia among Mexicans suggested a possible common origin of both ⁰ and ⁰ genes. A hypothesis to explain such a possibility is proposed on the basis of a partial mispairing between ⁰ and genes followed by a crossing-over which would results in a ⁰ recombinant gene. This hypothesis could also be extended to explain the 22 gluala, 22 alaglu and 116 arghis Hb variants as recombinants from double crossing-over between and mispaired genes for which the name interstitial-Lepore is proposed.     

Proliferation and germination of somatic embryos from embryogenic suspension cultures in <Emphasis Type="Italic">Phœnix dactylifera</Emphasis>

The present study demonstrates a procedure for the rapid development of a high number of somatic embryos from embryogenic suspension culture. This method might be efficient for mass propagation of Phnix dactylifera L. Embryogenic callus placed in liquid medium with 10^–5M ABA yielded an average 72 embryos per 100ml of culture medium within 2months, while those placed on solid medium yielded an average of 33, 20 and 16 embryos per 100ml of culture medium respectively for 10^–7, 10^–6 and 10^–5 M ABA after 4months. The combination of 2,4-DIchlorophenoxyacetic acid (2,4-D) (4.5×10^–7M), glutamine (6.7×10^–4M), and ABA (10^–5M) (L8 liquid medium) showed a beneficial effect on somatic embryos production compared to 2,4-D and glutamine alone, while this combination significantly (p<0.05) increased the accumulation of storage proteins (144 and 138mgg^–1 DW respectively for Jihel and Bousthami noir cultivars) in somatic embryos. The somatic embryos which underwent maturation on medium containing only 4.5×10^–7M 2,4-D and 10^–5M ABA (L6 liquid medium) accumulated more sugars (292 and 265mgg^–1 DW respectively for Jihel and Bousthami noir) than those matured on any other liquid medium. Histological studies revealed that somatic embryos (developed in L6 and L8 liquid media) accumulated less reserve compounds (proteins and sugars) than zygotic embryos. The addition of activated charcoal (0.25 and 0.5gl^–1) and phytagel^® (2.5gl^–1) to the germination medium may be useful for enhancing the germination of Phnix dactyliferasomatic embryos.     

Solution conformations of proline rings in proteins studied by NMR spectroscopy

Summary Three different conformations of proline rings in a protein in solution, Up, Down and Twist, have been distinguished, and stereospecific assignments of the pyrrolidine -, - and -hydrogens have been made on the basis of ¹H-¹H vicinal coupling constant patterns and intraresidue NOEs. For all three conformations, interhydrogen distances in the pairs -³, ³-³, ²-², ²-², and ³-³ (2.3 Å) are shorter than those in the pairs -², ²-³, ³-², ²-³, and ³-² (2.7–3.0 Å), resulting in stronger NOESY cross peaks. For the Up conformation, the ³-² and ²-³ spin-spin coupling constants are small (<3 Hz), and weak cross peaks are obtained in a short-mixing-time (10 ms) TOCSY spectrum; all other vicinal coupling constants are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. For the Down form, the -², ²-³, and ³-² vicinal coupling constants are small, leading to weak TOCSY cross peaks; all other couplings again are in the range 5–12 Hz, and result in medium to strong TOCSY cross peaks. In the case of a Twist conformation, dynamically averaged coupling constants are anticipated. The procedure has been applied to bovine pancreatic trypsin inhibitor and Cucurbita maxima trypsin inhibitor-V, and ring conformations of all prolines in the two proteins have been determined.     

Holling's “hungry mantid” model for the invertebrate functional response considered as a Markov process. Part II: Negligible handling time

In this paper we analyse a stochastic model for invertebrate predation taking account of the predator's satiation. This model approximates Holling's hungry mantid model when handling time is negligible (see Part I). For this model we derive equations from which we can calculate the functional response and the variance of the total catch. Moreover we study a number of approximations which can be used to calculate these quantities in practical cases in a relatively simple manner.List of Notation a rate constant of digestion - b maximum of rate constant of prey encounter in the mantid - c satiation threshold for search - c satiation threshold for pursuit in the mantid - c _i (w^1/2(N- N)ⁱ) - expectation operator - f rate of change of satiation during search - F functional response: mean number of prey eaten per unit of time - g rate constant of prey capture - h probability generating function of N conditional on S = s times p - H probability generating function of N - m_i ¹ - n, N number of prey caught - p probability density of S - p_n simultaneous probability (density) of N and S - q probability of strike success - r dummy variable in generating function - s, S satiation - T _s search time - T _d digestion time - v asymptotic rate of increase of var v - V asymptotic rate of increase of var N - w weight of edible part of prey - W standard Wiener process - x prey density - z (N{S = s}-N)p - rate constant of prey escape time maximum pursuit time - (v{S = + w ^1/2}-v) - present time as a fraction of the time from the start to the end of the experiment - hazard rate of T _s - mean time between (downward) passages of S through c - v w_–1/2(N-) - edible prey biomass density - probability density of , number pi - parameter of Weibull distribution of T _s = (1/2acx(-g(c)))_1/2 - w_–1/2(S -) - satiation in the guzzler approximation: solution to d/dt = f() + g(), (0)=S(0). - biomass functional response: wF - total biomass catch in the guzzler approximation: solution to d/dt = g(), (0) = 0     

Patterns and conformations of commonly occurring supersecondary structures (basic motifs) in protein data bank

Short peptides connecting-helices and-strands have been analyzed in 240 proteins refined at resolutions of 0.25 nm or better. Connecting peptides of lengths between one and five residues have been classified as part of supersecondary motifs of four types:, , , and. Careful consideration has been given to the definition of secondary structures on the basis of hydrogen bonds and main-chain conformational angles. Using five classes of residue conformation—a, b, e, l, t—in the nonregular structure regions of, space, 34 classes of supersecondary motifs occurring at least five times have been identified. Among these 34 classes, 11 classes that occur more than 25 times are commonly occurring supersecondary structure motifs. The patterns and conformations of the 11 commonly occurring supersecondary structure motifs have been characterized, demonstrating that patterns and conformations adopted by supersecondary structure motifs are limited. The results have relevance to structure prediction, comparative modeling, and protein folding.     

Silver nitrate and aminoethoxyvinylglycine affect <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated apple transformation

In Fuji, the production of ethylene was increased with the addition of AgNO₃ and inhibited with the addition of 10 M aminoethoxyvinylglycine (AVG). The addition of 80 M AgNO₃ to transformed explants of Fuji cultured on selection medium resulted in increased ethylene production (20 l l^–1) at 3 weeks. Under examining the effect of AgNO₃ in Fuji, the 40 M AgNO₃ showed with higher 33.8% and 6.5% in the efficiency of regeneration and transformation. However, ethylene production in Gala explants treated with 10M AgNO₃ (3 l l^–1) decreased after 2 weeks compared with the control (5 l l^–1). Although the regeneration efficiency of Gala with 10 M AgNO₃ was higher (41.1%) than the control (20.1%), there was no significant difference in the transformation efficiency at the same concentration. Shoot regeneration of Fuji and Gala was completely inhibited with 10 M AVG. These results suggest that the addition of AgNO₃ affects the efficiency of Agrobacterium-mediated gene transfer in Fuji.Eun Soo Seong, Ill Min Chung- These two Authors Contributed equally to this work     

<Emphasis Type="Italic">In vitro</Emphasis> screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1×10¹ to 1×10⁶ cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6weeks following inoculation with 1×10⁵ mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3months following inoculation and incubation at 26°C. No sori were produced at mixed sporidial concentrations lower than 1×10⁵cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1×105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.     

Factors influencing synthesis and activity of β-galactosidase inLactobacillus acidophilus

Summary In the type-strainLactobacillus acidophilus ATCC 4356 -galactosidase (-gal) was inducible; lactose, galactose, melibiose and probably maltose, but not glucose, fructose, mannose, sucrose and cellobiose, induced -gal synthesis. Glucose partially inhibited -gal-induction by lactose but not by isopropyl--D-thiogalactoside. -gal synthesis during cell growth was maximal at 0.4% lactose, stimulated by Ca²⁺ but inhibited by Mg²⁺ and Mn²⁺. -gal in the cell-free extract had optimum activity at pH 6.5 and at 45°C. The enzyme activity was stimulated by Mg²⁺, inhibited by Ca²⁺, destroyed by oxidizing agents and protected by reducing agents.     

Simplified typing of mouse hemoglobin (Hbb) phenotypes using cystamine

Cellulose acetate electrophoresis of mouse hemoglobins modified with the disulfide reagent cystamine permits rapid, unequivocal discrimination of all combinations of the codominant mouse hemoglobin single (Hbb ^s ) and diffuse (Hbb ^d and Hbb ^p ) alleles. The single, diffuse major, diffuse d-minor, and diffuse p-minor adult hemoglobins are all resolved by this method, which depends on the presence of a cysteine in the chains of diffuse mice which is not found in the chain of single mice.This work was supported by research grants ACS-VC58 and NIH CA-01074. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

Uptake of sewage derived nitrogen by<Emphasis Type="Italic"> Ulva rigida</Emphasis> (Chlorophyceae) in Bahía Nueva (Golfo Nuevo,Patagonia, Argentine)

Uptake kinetics of nitrogen derived from sewage–seawater mixtures (2.5–20% v/v effluent) were determined in the laboratory for Ulva rigida (Chlorophyceae) native from Bahía Nueva (Golfo Nuevo, Patagonia, Argentine). In terms of nitrogen concentration, experimental enrichment levels varied between 53.7 and 362.3M of ammonium and between 0.77 and 6.21M of nitrate+nitrite. Uptake rates were fitted to the Michaelis–Menten equation, with the following kinetic parameters: ammonium: V_max = 591.2molg^–1h^–1, K _s=262.3M, nitrate+nitrite: V _max=12.9molg^–1h^–1, K _s=3.5M). Both nutrients were taken up simultaneously, but ammonium incorporation was faster in all cases. The results show a high capability of Ulva rigida to remove sewage-derived nitrogen from culture media. In the field, most of the nitrogen provided by the effluent would be tied up in algal biomass, supporting low nitrogen levels found at a short distance away from the source.     

On the role of the invariant glutamine at position 54 in the human choriogonadotropin β subunit

The twelve Cys and eight of the non-Cys residues are invariant in the glycoprotein hormone subunits from a variety of mammalian species. -Gin-54 of human lutropin (hLH) and choriogonadotropin (hCG) is one of these invariant amino acid residues. A single AG mutation in the LH gene of a patient presenting with hypogonadism resulted in the replacement of Gin-54 with Arg [1]. The authors also reported that an expressed mutant of hLH, with Arg replacing Gin-54, associated with the subunit, but there was no demonstrable binding of the mutant hormone to receptor. We have replaced Gin-54 in hCG with Glu and with Lys using site-directed mutagenesis. The expression plasmids pRSV-hCG (wild-type and mutants) were transiently transfected into CHO cells containing a stably integrated gene for bovine , and the media were analyzed for holoproteins, which were characterizedin vitro using competitive binding and steroidogenic assays with MA-10 cells. hCG(Glu-54) bound to almost as well as hCG wild-type, and the resulting heterodimer competed with [¹²⁵l]hCG binding to the LH/CG receptor and stimulated progesterone production to the same extent as the wild-type control. However, the apparent potencies, as judged by ED₅₀s, were less than those of the wild-type control, the effect being more pronounced in binding than in steroidogenesis. In contrast, hCG(Lys-54) associated very poorly with . Our results suggest that while Gin-54 in hCG participates in receptor binding, its major function appears to involve binding. Such dual functionality leads to interesting models for holoprotein formation and receptor binding.     

Different ζ globin gene deletions among Black Americans

Summary Four types of chromosomes with a deletion between the human embryonic and globin genes were identified among 2.8% of 321 Black Americans from Georgia. Two deletions of approximately 11 kb which differed by about 300 bp occurred on chromosomes with or without a polymorphic Xba I site 5 to the globin gene [(X⁺) or (X^-)]. The deletions are identifiable in Xba I digests of genomic DNA using an or a globin gene probe which yield fragments of 23 kb from (X⁺)–^* chromosomes or 27 kb from (X^–)–^* chromosomes. Digestion with other enzymes and probing with both and probes gave fragments typical of the two globin gene deletions previously identified in Polynesians. Among Black Americans, these globin gene deletions have been found in combination with globin gene deletions in trans but not in cis. Homozygotes have not been found. Hematologic data on carriers of the globin gene deletions in association with Hb AS, SS, and SC suggest that these deletions have no effect on the function of the adult globin genes.     

Ultrastructure and behaviour of the spindle pole body of the aphid-pathogenic fungusErynia neoaphidis

Summary A detailed account of the ultrastructure and behaviour of the spindle pole body (SPB) of the entomophthoraceous fungusErynia neoaphidis is presented for the first time.The SPB consists of extranuclear (ENC) and intranuclear (INC) components. The ENC is a saucepan-shaped structure which lies in a pocket of the nuclear envelope. It is composed of a forked, fibrillar handle and a shallow, cylindrical pan. The pan has a wall of two layers, both of which are thickened with a regular periodicity so that they appear to be beaded. It is postulated that the pan is formed from rough endoplasmic reticulum and that it synthesizes the amorphous, electron-dense material coating the ENC.The INC is a saucer-shaped, electron-dense plaque in which the ends of the spindle microtubules terminate. During metaphase, a clear zone separates the INC from the nuclear envelope and persists until telophase. The roles of the amorphous, electron-dense material and the clear zone as well as the method of SPB replication are discussed.     

Characteristics of βA chromosome haplotypes in Japanese

DNA polymorphism patterns linked to the ^A-globin gene were analyzed in healthy Japanese using four different restriction endonucleases. The chromosomes with the ^A-globin gene were mapped through an evaluation of the presence of seven different restriction sites (HincII 5 to ; HindIII in ^G and ^A; HincII in, and 3 to, ₁; AvaII in ; Bam-HI 3 to ). Among 36 chromosomes analyzed, 20 chromosomes had a haplotype of [+–––––+]. Among 55 individuals examined, 7 possessed a homozygous haplotye of [+–––––+]. All Japanese with the ^AT-globin gene had a subhaplotype of [–++–+] 5 to the -globin gene. Their major haplotypes were [–++–+–+] and [–++–++–]. It was expected that the presence of the ^AT-globin gene in Japanese may be deduced from subhaplotypes 5 to the -globin gene.     

Variations in the rate of synthesis of beta and beta'' RNA polymerase polypeptides under the influence of certain factors.

Summary In merodiploid cells containing a double dose of structural genes of RNA polymerase subunits-rpoBand rpoC-the rate of and subunits synthesis is 2 times higher than in haploidcells. Missence mutation rpoC1 (tsX) alters polypeptide and inducesthe and subunits synthesis at increased rate, particularly at a nonpermissive temperature. When rpoBCoperon carrying mutation rpoC1 is duplicated no dosage effect is observed. In the rpoC ⁺/rpoC1 heterodiploid the rpoC1 mutation does not significantly accelerate RNA polymerase subunits synthesis i.e. is recessive with respect to rpoC ⁺ Rifampicin causes 6-fold stimulation of RNA polymerase subunits synthesis in a sensitive wild-type strain. The rpoC1 mutation itself accelerates the synthesis of these subunits 3-fold. In the presence of rifampicin the mutant strain produces 13–22-fold faster as compared to wild-type strain without the drug. Thus, the effects of rifampicin and the mutation are multiplied suggesting that these factors act independently. Similar data have also been obtained with rifampicin-treated cells of rpoB22 (ts22) amber-mutant.After UV-irradiation of cells and synthesis is depressed much stronger than the total protein synthesis. Infection with a transducing phage rif ^d-47 which carries rpoB gene provokes a higher rate of synthesis. When pre-irradiated cells (500 erg/mm²) are infected with this phage, the rate of synthesis grows 20-fold compared to irradiated, non-infected cells and 6.5-fold compared to intact cells.The data are discussed in terms of the possible regulatory mechanisms of RNA polymerase subunit synthesis.     

β-Glucanases in developing cotton (Gossypium hirsutum L.) fibres

Cotton fibres possess several -glucanase activities which appear to be associated with the cell wall, but which can be partially solubilised in buffers. The main activity detected was that of an exo-(13)--d-glucanase (EC 3.2.1.58) but which also had the characteristics of a -glucosidase (EC 3.2.1.21). Endo-(13)--d-glucanase activity (EC 3.2.1.39) and much lower levels of (14)--d-glucanase activity were also detected. The exo-(13)--glucanase showed a maximum late on (40 days post-anthesis) in the development of the fibres, whereas the endo-(13)--glucanase activity remained constant throughout fibre development. The -glucanase complex associated with the cotton-fibre cell wall also functions as a transglucosylase introducing, inter alia, (16)--glucosyl linkages into the disaccharide cellobiose to give the trisaccharide 4-O--gentiobiosylglucose.Abbreviations CMC carboxymethylcellulose - ONPG o-nitrophenyl--d-glucopyranoside - TLC thin-layer chromatography Presented at the Third Cell Wall Meeting held in Fribourg in 1984     

Laboratory culture studies of Trichodesmium isolated from the Great Barrier Reef Lagoon, Australia

Cultures of Trichodesmium from the Northern and Southern Great Barrier Reef Lagoon (GBRL) have been established in enriched seawater and artificial seawater media. Some cultures have been maintained with active growth for over 6years. Actively growing cultures in an artificial seawater medium containing organic phosphorus (glycerophosphate) as the principal source of phosphorus have also been established. Key factors that contributed to the successful establishment of cultures were firstly, the seed samples were collected from depth, secondly, samples were thoroughly washed and thirdly, incubations were conducted under relatively low light intensities (PAR 40–50molquantam^–2s^–1). N₂ fixation rates of the cultured Trichodesmium were found to be similar to those measured in the GBRL. Specific growth rates of the cultures during the exponential growth phase in all enriched media were in the range 0.2–0.3day^–1 and growth during this phase was characterised by individual trichomes (filaments) or small aggregations of two to three trichomes. Characteristic bundle formation tended to occur following the exponential growth phase, which suggests that the bundle formation was induced by a lack of a necessary nutrient e.g. Fe. Results from some exploratory studies showed that filament-dominated cultures of Trichodesmium grew over a range of relatively low irradiances (PAR 5–120molquantam^–2s^–1) with the maximum growth occurring at 40–50molquantam^–2s^–1. These results suggest that filaments of the tested strain are well adapted for growth at depth in marine waters. Other studies showed that growth yields were dependent on salinity, with maximum growth occurring between 30 and 37psu. Also the cell yields decreased by an order of magnitude with the reduction of Fe additions from 450 to 45nM. No active growth was observed with the 4.5nM Fe addition.     

Bacterial toxicity of cyclodextrins: Luminuous Escherichia coli as a model

The effect of various concentrations of natural and chemically modified cyclodextrins on the luminescence of an Escherichia coli suspension was investigated. All cyclodextrins were found to reduce, albeit to a varying degree, the luminescence level of the bacterial cells, thus suggesting a direct interaction between the cyclodextrins and cells. The inhibitory concentrations IC₂₀ and IC₅₀ of the various cyclodextrins were determined and taken to represent their toxicity effects upon the bacterial cells. Among the natural cyclodextrins, - and -CD interfered minimally with the bacterial luminescence and consequently were essentially non-toxic. The following descending order of toxicity was observed: -CD -CD > -CD. Among the chemically modified cyclodextrins, Dimeb was clearly toxic while Trimeb and the hydroxylated derivatives (hydroxypropyl-ga-CD, HPACD;--CD, HPBCD;--CD, HPGCD) were essentially non-toxic. The following descending order of toxicity was observed: Dimeb HPBCD > Trimeb > HPACD > HPGCD.     

T-cell receptor gene rearrangements and expression in normal human large granular lymphocytes (LGL) and their pathological expansions

The lineage to which normal large granular lymphocytes/natural killer (LGL/NK) cells belong is controversial; in fact they share some surface markers and functional activities with monocytes, but also with T lymphocytes. The relationship of LGL to the T cell lineage by analysis with the T cell receptor (T-rec) gene has been investigated. Pure preparations of human LGL and their CD11⁺ CD8^- and CD11^- CD8⁺ subsets had the T gene in its unrearranged germline configuration. Expression of T and T genes was not detectable. The organization of T gene, which is of particular importance because it occurs early in T cell ontogeny, was also found in its germline configuration.A rare type of lymphoproliferative disorder, termed T-LPD, is characterized by expansion of cells very similar to LGL for morphology, phenotype, and functional activity. Of 17 patients with T-LPD studied for T-rec rearrangement, 15 displayed rearrangement of T and T loci and were CD3+ (14/15 had monoclonal rearrangement), while 2 cases were in germline configuration and were CD3–. Similarly to very small subsets of CD3+ LGL recently described, most T-LPD cases are CD3+ and have T-rec genes rearranged. These data suggest that either a subset of LGL or a particular step of differentiation may be related to the T cell lineage; they also demonstrate that, in contrast to previous views, most TLPD are monoclonal, presumably neoplastic, lymphoproliferative disorders.Abbreviations LGL large granular lymphocytes - NK natural killer - T-rec T-cell receptor - TLPD T lymphoproliferative disease