The tRNATyr multigene family of Nicotiana rustica: genome organization,sequence analyses and expression in vitro

Tobacco tRNA^Tyr genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA^Tyr-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector gtWES·B and in vitro packaging. The phage library was screened with a 5-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA^Tyr. Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA^Tyr genes code for the known tobacco tRNA₁ ^Tyr (GA) and seven code for tRNA₂ ^Tyr (GA). The two tRNA species differ in one nucleotide pair at the basis of the TC stem. Only one tRNA^Tyr gene (pNtY5) contains a point mutation (T54A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs^Tyr-with one exception-are processed and spliced in both extracts. The tRNA^Tyr gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.     

Theoretical stability diagram of solvent-containing black lipid films

Recently, we have developed an analytical, semi-microscopic theory for the macroscopic behavior of a solvent-containing black lipid film subjected to an electric cross film voltage, . Here we employ the theoretical expressions derived for the disjoining pressure, _D, the film elasticity, _F, and the film tension, _F, to construct the stability diagram of the film, in the _D-. Depending on its state (_D, ), the film is stable or is prone to squeezing or bending deformations. For a monooleate film we show how the destruction of the plane film due to a periodic thickness fluctuation (squeezing) is facilitated by two mechanisms: i) lowering of _D at fixed ; ii) lowering of at fixed _D, provided that the film is in a stable state characterized by _D<–7.03×10³ dyne/cm² and >0 mV. Bending of a low tension film (single interface tension _s 0.025 dyne/cm¹) can be achieved only for >170 mV and _D > –8.7 × 10⁴ dyne/cm². Finally, we demonstrate the existence of a marginal state ( _D ⁰ , ⁰) where the film is predicted to exhibit strong fluctuations both in the squeezing and in the bending mode.     

Protocol-dependence of equivalent circuit parameters of toad urinary bladder

Summary Determinations of current-voltage relationships are widely employed in the characterization of epithelial sodium transport. In order to determine the protocol dependence of transport parameters in the toad urinary bladder, studies were carried out in the presence and absence of amiloride, an inhibitor of active sodium transport. With symmetric positive and negative perturbations of the transepithelial electrical potential difference (0±100 mV) for 30 sec, the amiloride-sensitive current-voltage (i ^a -) relationship was near linear over the range –75+100 mV, indicating constancy of the conductance ^a and the apparent electromotive force E _Na, lumped parameters of the standard electrical equivalent circuit model of the active transport system. With a reverse protocol (±1000 mV) or 15 min perturbations thei ^a - relationships were highly nonlinear. Nonlinearity reflected voltage dependence of parameters: perturbations that increased active transport decreased E _Na and increased ^a, as evaluated from 10 sec perturbations of ; slowing of active transport produced the converse changes. These effects are usefully analyzed in both quasi-steady states and true steady states by means of a detailed equivalent circuit incorporating the significant ionic currents across each plasma membrane. Precise understanding of the significance of ^a and E _Na will require characterization of the partial ionic conductances on perturbation of .     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Light and electron microscopic study of adenosine triphosphatase activity of anuran tadpole musculature

Summary The histochemical activities of succinic dehydrogenase (SDH) and Ca⁺⁺-activated ATPase (pHs 7.4 and 9.4) were studied in the larval tail musculature of Rana japonica, Rana catesbeiana and Rana ornativentris. The ATPase reaction product was detected by both light and electron microscopy. Red and white muscle fibres, as distinguished by SDH, showed high and low Ca⁺⁺-ATPase reaction, respectively, at pHs 7.4, 9.4 and following preincubation in cold K₂-EDTA solution. The ultrastructural investigation of CA⁺⁺-ATPase reaction at pH 7.4 by the Ca⁺⁺-citrophosphate technique demonstrated electron-dense reaction product in association with A, I and Z bands, intermyofibrillar (SR) compartment and the mitochondrial inner chamber. However, Pb⁺⁺ precipitation technique demonstrated Mg⁺⁺-activated myosin ATPase activity at pH 9.2 ultrastructurally. The present histochemical data suggest that the anuran larval tail red muscle fibres are possible slow, and emphasize a possible lack of correlation between the speed of contraction with their ATPase activity. Moreover, red muscle fibres of the anuran tail musculature are not equivalent to Type I fibres of higher chordates.     

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

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

Stable carbon and oxygen isotopic compositions of recent charophyte oosporangia and water from Malham Tarn,U.K.: palaeontological implications

Charophyte oosporangia and water samples from a highly calcareous lake were measured for stable carbon and oxygen isotopic composition. The time period over which the oosporangia calcify is short, thus any biochemical relationship between the water and oosporangia"s calcite represents only one time window (late Summer in Malham Tam). This important temporal restraint must also apply to interpretations of all fossil material measured. The ¹⁸O_c of the charophyte oosporangia is deduced to be in equilibrium with the ¹⁸O of the water for a given temperature. The ¹³ C_c of the charophyte oosporangia was approximately 2.5 per mil lower than the ¹³C_DIC in the water we measured. With the release Of CO₂ with phosphoric acid from the charophyte oosporangia, there was no significant difference in the ¹⁸O_c values obtained, regardless of whether or not the carbonate was separated from the organic center, however ¹³C_c values were marginally lower for carbonate plus organic center measurements. Our results indicate that fossil charophyte gyrogonites can be used to elucidate the geochemistry of the ancient water body in which they lived.     

One-bond carbon-proton coupling constants: Angular dependence in β-linked oligosaccharides

Summary The angular dependence of¹J_C,H in model compounds related to -linked oligosaccharides has been established by FPT INDO quantum chemical calculations. Values calculated for models of (1 1)-, (1 2)-, (1 3)- and (1 4)-linked disaccharides were compared, and the effect of the orientation of HO-2 elucidated. The angular dependence of¹J_C,H on the torsional angles ^H and ^H and the solvent dielectric constant (s) was characterized in the form:¹J_C,H = A cos2+B cos + C sin2 + D since + E + Fe. The¹J_C,H values, measured by DEPT methods for C-1-H-1 and C-X-H-X in cellobiose, cyclic trisaccharide and hexopyranoses were used to adjust the calculated angular dependences. Based on the occurrence of the conformers for agarobiose, neoagarobiose, mannobiose and methyl -xylobioside, the thermodynamically averaged <¹J_C,H > values were calculated. The results obtained (<¹J_C-1,H-1 > 162.4, <¹J_{C-4, H-4} > 147.6 Hz for methyl -xylobioside; <¹J_C-1,H-1 > 162.4 and <¹J_C-4,H-4] > 147.6 Hz for mannobiose; <¹J_C-1,H-1 > 162.8 Hz for neo agarobiose and <¹J_C-1,H-1 > 163.2 Hz for agarobiose) agree well with the experimental values of 162.7, 147.5, 160.4, 147.2, 160.9 and 165.7 Hz, respectively.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Conformational dynamics of the anticodon loop in yeast tRNAPhe as sensed by the fluorescence of wybutine

Conformational and dynamic properties of the anticodon loop of yeast tRNA^Phe were investigated by analyzing the time resolved fluorescence of wybutine serving as a local structural probe adjacent to the anticodon GmAA on its 3 side. The influence of Mg²⁺, important for stabilizing the tertiary structure of tRNA, and of the complementary anticodon s²UUC of E. coli tRNA ₂ ^Glu were investigated.Fluorescence lifetimes and anisotropies were measured with ps time resolution using time correlated single photon counting and a mode locked synchronously pumped and frequency doubled dye laser as excitation source. From the analysis of lifetimes () and rotational relaxation times ( _R ) we conclude that wybutine occurs in various structural states: (i) one stacked conformation where the base has no free mobility and the only rotational motion reflects the mobility of the whole tRNA molecule (=6 ns, _R =19 ns), (ii) an unstacked conformation where the base can freely rotate (=100 ps, _R = 370 ps) and (iii) an intermediary state (=2 ns, _R = 1.6 ns).Under biological conditions, i. e. in the presence of Mg²⁺ and neutral salts, wybutine is found in a stacked and immobile state which is consistent with the crystallographic picture. In the presence of the complementary codon however, as exemplified by the E. coli-tRNA ₂ ^Glu anticodon, our analysis indicates that the codon-anticodon complex exists in an equilibrium of structural states with different rotational mobility of wybutine. The conformation with wybutine freely mobile is the predominant one and suggests that this conformation of the codon-anticodon structure differs from the canonical 3–5 stack.     

The relation of proton motive force,adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction inBacillus licheniformis under aerobic growth conditions

The magnitude of the proton motive force (p) and its constituents, the electrical () and chemical potential (-ZpH), were established for chemostat cultures of a protease-producing, relaxed (rel ^–) variant and a not protease-producing, stringent (rel ⁺) variant of an industrial strain ofBacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of p with the specific growth rate was found. The calculated intracellular pH (pH_in) was not constant but inversely related to . This change in pH_in might be related to regulatory functions of metabolism but a regulatory role for pH_in itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low and an increased value at higher . For both limitations, the ATP/ADP ratio was directly related to .The phosphorylation potential (G'p) was invariant with . From the values for G'p and p, a variable H⁺/ATP-stoichiometry was inferred: H⁺/ATP=1.83+0.52µ, so that at a given H⁺/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for =0 to _max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.Abbreviations CCCP carbonylcyanide-p-trichloromethoxyphenylhydrazone - DW dry weight of biomass - F Faraday's constant, 96.6 J/(mV × mol) - Fo chemostat outflow-rate (ml/h) - FCCP carbonylcyanide-p-trifluoromethoxyphenylhydrazone - G'p phosphorylation potential, the Gibbs energy change for ATP-synthesis from ADP and P_i - G'⁰p standard Gibbs energy change at specified conditions - H⁺/ATP number of protons translocated through - ATP synthase in synthesis of one ATP - H⁺/O protons translocated during transfer of 2 electrons from substrate to oxygen - specific growth rate (1/h) - _H+ transmembrane electrochemical proton potential, J/mol - M_b molar weight (147.6 g/mol) of bacteria with general cell formula C_6.0H_10.8O_3.0N_1.2 - pH_out,in extracellular, intracellular pH - P_i (intracellular) inorganic phosphate - p proton motive force, mV - pH transmembrane pH-difference - transmembrane electrical potential, mV - P/O number of ADP phosphorylated to ATP upon reduction of one O^2– to H₂O by two electrons transferred through the electron transfer chain - P/O (H⁺/O) × (H⁺/ATP)^–1 - P/O_F, P/O_N P/O with the two electrons donated by resp. (NADH + H⁺) and FADH - q specific rate of consumption or production (mol/g DW × h) - rel ⁺,rel ^– stringent, relaxed genotype - R universal gas constant, 8.36 J/(mol × degree) - T absolute temperature - TPMP⁺ triphenylmethylphosphonium ion - TPP⁺ tetraphenyl phosphonium ion - Y growth yield, g DW/mol - Z conversion constant=61.8 mV for 310 K (37 °C) - ZpH transmembrane proton potential or chemical potential, mV     

Protein kinase C isoforms in pituitary cells displaying differential sensitivity to phorbol ester

Investigations with protein kinase C (PKC) isoform-specific antisera, revealed distinct profiles of PKC isoform content amongst pituitary tissues. Western analysis revealed the and isoforms of PKC are present in rat anterior and posterior pituitary tissue as well as in the GH₃ somatomammotrophic cell line. AtT-20/D16-V corticotrophic and T3-1 gonadotrophic murine cell lines contained no PKC-. The or isoforms were undetected in any pituitary tissue. PKC activity measurements revealed Ca²⁺-independent PKCs in T3-1 and GH₃ cells which were more sensitive to activation by phorbol-dibutyrate (PDBu) than the corresponding PKC activity found in COS cells. However, Ca²⁺-dependent PKC activities were of similar sensitivity to PDBu in GH₃, T3-1 and COS cells, indicating that functional differences observed in PDBu-sensitivity in these cells may be due to differential activation of Ca²⁺-independent PKC isoforms. Moreover, substrate-specificity of these PKCs were also compared indicating that the amount of Ca²⁺-dependency of the observed PKC activity from the same pituitary tissue is dependent upon the substrate utilized by the PKC isotypes present. These findings explain differential sensitivities of PKC-mediated actions that have previously been observed in a range of pituitary cells. (Mol Cell Biochem 000-000, 1999)     

A directed alteration of ribonuclease specificity. Hydrolysis of polyribonucleotides containing modified cytosine bases

The action of ribonucleases on poly and oligoribonucleotides containing cytosine bases modified by methoxyamine and bisulphite was examined. Resistance of phosphodiester bonds in (Cp) _n Xp (where n 1 and X stands for A, G or U) to T₂ RNase hydrolysis was observed if substrates were modified chemically. The phenomenon formed the basis for isolation of (Cp) _n Xp blocks as an additional tool in sequence investigations. After modification of cytosine pancreatic RNase was unable to hydrolyse (Cp) _n Up blocks. Therefore the specificity of pyrimidyl RNase may be narrowed to uridyl RNase.Abbreviations cytidine modified with methoxyamine and bisulphite (5, 6-dihydro-6-sulpho-N⁴-methoxycytidine) - cytidine modified with methoxyamine (N⁴-methoxycytidine)     

Comparative Study of Effects of Various Sterols and Triterpenoids on Permeability of Model Lipid Membranes

Using permeability to labeled glucose as a criterion of stability for liposomal membranes, a comparative study on stabilizing properties of different sterols and triterpenes in phospholipid bilayer has been carried out as well as on structural peculiarities of sterols responsible for membranolytic properties of cucumarioside G₁ from the cucumaria Eupentacta fraudatris. Stabilizing action of the studied sterols and triterpenoides incorporated in the bilayer decreases in the following order: cholesterol sulfate > cholesterol > ⁵-sterols > -sitosterol > ergosterols > ⁷-sterols > epicholesterol > pregnane > androstane > coprosterol > 14-methylcholest-9(11)-en-3-ol > 4, 14-dimethylcholest-9(11)-en-3-ol > holothurinogenin A₁ > glucoside of cholesterol > -xylosidase of ⁷-sterols > betulin > protopanaxatriol > phosphatidylcholine liposomes without sterol > protopanaxadiol > oleanolic acid. Sterol-dependent membranolytic cucumarioside G₁ practically loses its ability to increase permeability of phospholipid membranes containing sterols obtained from this holothuria as well as coprosterol, epicholestrol, sulfated and glycosylated forms of sterols. The obtained results confirm the sterol hypothesis of the mechanism of membranotropic action of holothuria glycosides and of resistance to them of holothuria cell membranes.     

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     

Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly 13C-labeled deoxyribose ring

Summary [¹³C₅]-2-Deoxy-d-ribose, synthesized from [¹³C₆]-d-glucose (98% ¹³C), was coupled with thymine to give [1,2,3,4,5-¹³C₅]-thymidine (T) in an 18% overall yield. The thymidine was converted to the 3-phosphoramidite derivative and was then incorporated into a dodecamer 5-d(CGCGAATTCGCG)-3 by solid-phase DNA synthesis. Preparation of 0.24 mole of the labeled dodecamer, which is sufficient for a single NMR sample, consumed only 25 mg of glucose. By virtue of the ¹³C labels, all of the ¹H-¹H vicinal coupling constants in the sugar moieties were accurately determined by HCCH-E.COSY.     

The levels of ζ, γ, and δ chains in patients with Hb H disease

Summary Details are given of a study of blood samples from 24 patients with Hb H disease from different Mediterranean countries and from the Far East. Four different types of -thal-1 (--) were observed, namely-() ( 20.5-kb deletion);--MED-I ( 17.5-kb deletion);--MED-II (>26.5-kb deletion); and--SEA ( 18-kb deletion, in Orientals only). The -thal-2 was mainly of the deletion type (16 with the 3.7-kb deletion; 1 with the 4.2-kb deletion), while 4 of the 7 patients with a nondeletional type had the five-nucleotide deletion at the donor splice site of the first intron of the 2 gene. All patients had a mild-to-moderate hemolytic anemia; no significant differences in hematology were observed between the groups. Hb A₂ was decreased to about one-third of the normal level. The Hb H formation varied considerably and its quantitation was not always satisfactory. Patients with Hb H disease due to any -thal-1 combined with a nondeletional -thal-2 had the highest Hb H levels and a more marked anemia. The chain production was small and absent in patients with the MED-II type of -thal-1 because this deletion included the and genes. The highest chain levels were present in the four patients with the SEA type of -thal-1. The chain production was increased, particularly in patients with a mutation of C T at position-158 to the ^G globin gene. This chain was primarily present as Hb Bart's (or ₄) and only about 15% was recovered as Hb F or ₂₂. The evaluation of the rate of chains produced in these patients was greatly facilitated by data from one patient who had Hb H disease and a heterozygosity for the ^A-⁺. The low levels of Hb A₂ and of Hb F (relative to Hb Bart's) can be explained by a decreased affinity of chains for and chains as compared with chains in conditions of severe chain deficiency.