Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.     

Two-Electron Reactions S2QB → S0QB and S3QB → S1QB are Involved in Deactivation of Higher S States of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S₀-S₄), and dark incubation leads to 25% S₀ and 75% S₁. This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q_B⁻. We measured flash-induced oxygen evolution to understand how S₃ and S₂ are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q_B⁻. Thylakoid samples preconditioned to make the concentration of the S₁ state 100% and to oxidize tyrosine Y_D were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S₂ and S₃ are converted to lower S states via recombination from S₂Q_B⁻ and S₃Q_B⁻ and by a slow change of the state of oxygen-evolving complex from S₃ and S₂ to S₁ and S₀ in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S₂Q_B → S₀Q_B, and S₃Q_B → S₁Q_B. The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S₃Q_B → S₂Q_B → S₁Q_B in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H₂O₂ acts as an electron donor for the reaction S₂Q_B → S₀Q_B but added H₂O₂ did not enhance this reaction.     

A nhaD Na/Hantiporter and a pcd homologues are among the Rhodothermus marinus complex I genes

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo₁ to nqo₇ (nqo_A) and nqo₁₀ to nqo₁₄ (nqo_B)] and two independent genes (nqo₈ and nqo₉). Unexpectedly, two genes encoding homologues of a NhaD Na⁺/H⁺ antiporter (NhaD) and of a pterin-4α-carbinolamine dehydratase (PCD) were identified within nqo_B, flanked by nqo₁₃ and nqo₁₄. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqo_B. The possible role of these two genes in R. marinus complex I is discussed.     

S-allele diversity in Prunus L. Cerasus subgenus from Iran

In this study, S-allele diversity of eight wild and two commercial species of the Cerasus subgenus in Iran was investigated using two primer pairs. A high level of S-allele polymorphism was detected among and within the species evaluated. Furthermore, most of wild species showed 2–4 alleles based on S-allele primers and may be considered as tetraploid. Sweet cherry cultivars, Siah-Mashhad, Siah-Shabestar, Takdaneh-Mashhad, Siah-Daneshkadeh and Protiva showed S₃S₁₂, S₃S₁₂, S₃S₁₂, S₃S₅ and S₃S₄ combinations, respectively, allele S₃ showing the highest frequency. Three Iranian sweet cherry cultivars had the same allelic combination (S₃S₁₂) that the same ancestor in genealogy of these cultivars may explain the loss of diversity observed at the S-locus. Wild cherry (mazzard) accessions showed wide range of alleles such as S₁, S₂, S₇, S₁₄ and S₂₀ and unknown alleles, while sour cherries showed S₆, S₉, S₁₃ and S₂₇ alleles. In conclusion, the conservation of these highly diverse native species of Iranian wild Cerasus germplasm is recommended for future breeding activity.     

Oxidative addition of 3,6-di-tert-butyl-o-benzoquinone and 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone to SnCl2

Addition of 3,6-di-tert-butyl-o-benzoquinone (3,6-DBBQ) to SnCl₂ in THF leads to the oxidation of Sn(II) to Sn(IV) with formation of catecholate complex (3,6-DBCat)SnCl₂ · 2THF (1), where 3,6-DBCat is 3,6-di-tert-butyl-catecholate dianion. The reaction of 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone (IBQ-Prⁱ) also proceeds on the oxidative-addition mechanism yielding bis-iminosemiquinonato species (ISQ-Prⁱ)₂SnCl₂(2), where ISQ-Prⁱ is anion-radical 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzosemiquinolate. The complexes have been characterized by IR, X-band EPR, ¹H NMR (for 1) spectroscopy and magnetochemistry (for 2). X-ray analysis data show the distorted octahedral environment of tin(IV) for both complexes. Complex 1 is diamagnetic (ground state S = 0), while 2 has triplet ground state (S = 1, biradical). Catecholate complex 1 is able to be a spin trap for different organic radicals.     

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides

Engineering photosynthetic bacteria to utilize a heterologous reaction center that contains a different (bacterio) chlorophyll could improve solar energy conversion efficiency by allowing cells to absorb a broader range of the solar spectrum. One promising candidate is the homodimeric type I reaction center from Heliobacterium modesticaldum. It is the simplest known reaction center and uses bacteriochlorophyll (BChl) g, which absorbs in the near-infrared region of the spectrum. Like the more common BChls a and b, BChl g is a true bacteriochlorin. It carries characteristic C3-vinyl and C8-ethylidene groups, the latter shared with BChl b. The purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides was chosen as the platform into which the engineered production of BChl g_F, where F is farnesyl, was attempted. Using a strain of Rba. sphaeroides that produces BChl b_P, where P is phytyl, rather than the native BChl a_P, we deleted bchF, a gene that encodes an enzyme responsible for the hydration of the C3-vinyl group of a precursor of BChls. This led to the production of BChl g_P. Next, the crtE gene was deleted, thereby producing BChl g carrying a THF (tetrahydrofarnesol) moiety. Additionally, the bchG^Rs gene from Rba. sphaeroides was replaced with bchG^Hm from Hba. modesticaldum. To prevent reduction of the tail, bchP was deleted, which yielded BChl g_F. The construction of a strain producing BChl g_F validates the biosynthetic pathway established for its synthesis and satisfies a precondition for assembling the simplest reaction center in a heterologous organism, namely the biosynthesis of its native pigment, BChl g_F.     

13C magnetic resonance study of the ionization of N-acetyl-dl-proline in aqueous solution

NMR titration curves have been recorded for all the ¹³C resonances of cis and trans$\text{N-}\text{acetyl}\text{-}\text{dl}\text{-}\text{proline}$ in ²H₂O. the measured pK_2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and C_γ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p²H.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

Nδ-acetylornithine and S-methylcysteine in blood plasma

N^δ-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 ± 0.4 μmol/l (range 0.8–0.2 μmol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 μmol/day). Human blood plasma contains 3.9 ± 1.9 μmol/l (range 1.4–6.5 μmol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.     

Pro-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from Phaseolus aureus was photoinactivated in the presence of methylene blue or rose bengal. Pro and several imino acid analogues protected the enzyme against dye-mediated photoinactivation but ATP was ineffective. Together with kinetic data, this evidence suggested that a His-residue near the Pro-binding site was involved in the enzyme reaction. In the absence of methylene blue, Phaseolus enzyme was stable to light whilst that from Delonix was rapidly and reversibly photoinactivated. ATP as well as Pro, protected the Delonix enzyme against dye-independent photoinactivation. In the presence of methylene blue, the Delonix enzyme was more rapidly photoinactivated than in the absence of the dye. p-Chloromercuribenzoate (pCMB)-inhibited enzyme from both Phaseolus and Delonix was reactivated by sulphydryl reducing reagents. Reactivation of Delonix enzyme was markedly temperature-dependent whilst Phaseolus enzyme was reactivated equally efficiently at all temperatures tested. ATP, tRNA, Pro and several analogues of Pro, protected both the Phaseolus and Delonix enzymes against pCMB inhibition. The possible roles of the His-residue and SH group are discussed in relation to the known differences in substrate specificity between the Phaseolus and Delonix enzymes.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)     

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination

Discrimination of Leishmaniainfantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteineproteaseB (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3’ end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.     

Ni[TCNE]2 · zCH2Cl2 (Tc = 13 K) and VxNi1 − x[TCNE]y · zCH2Cl2 solid solution room temperature magnets

Magnetically ordered Ni[TCNE]₂ · zCH₂Cl₂ (T_c = 13 K) is reported for the first time from the reaction of Ni(CO)₄ with tetracyanoethylene (TCNE). A family of new solid solution room temperature magnets of V_xNi_1 − x[TCNE]_y · zCH₂Cl₂ (0 ? x < 1; y ? 2) composition has been characterized by IR spectrometry, elemental analysis, and magnetic measurements (ac and dc susceptibility). Substitution of Ni^II for V^II in V[TCNE]_y · zCH₂Cl₂ does not alter the T_c significantly for x ? 0.05 and does not alter H_cr significantly for x > 0; however, the magnitude of M increases with x, as does the broadness of the peaks in the χ′(T) and χ″(T) ac susceptibilities. Hence, the magnetic properties of the room temperature V[TCNE]_y · zCH₂Cl₂ magnet can be finely tuned via synthetic chemistry methodology, making this material more amenable in future technologies.     

Evolutionary divergence of the paralogs Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila

Juvenile hormone (JH) signaling underpins both regulatory and developmental pathways in insects. However, the JH receptor is poorly understood. Methoprene tolerant (Met) and germ cell expressed (gce) have been implicated in JH signaling in Drosophila. We investigated the evolution of Met and gce across 12 Drosophila species and found that these paralogs are conserved across at least 63 million years of dipteran evolution. Distinct patterns of selection found using estimates of dN/dS ratios across Drosophila Met and gce coding sequences, along with their incongruent temporal expression profiles in embryonic Drosophila melanogaster, illustrate avenues through which these genes have diverged within the Diptera. Additionally, we demonstrate that the annotated gene CG15032 is the 5′ terminus of gce.In mosquitoes and beetles, a single Met-like homolog displays structural similarity to both Met and gce, and the intron locations are conserved with those of gce. We found that Tribolium and mosquito Met orthologs are assembled from Met- and gce-specific domains in a modular fashion. Our results suggest that Drosophila Met and gce experienced divergent evolutionary pressures following the duplication of an ancestral gce-like gene found in less derived holometabolous insects.     

八肋游仆虫大核基因组中组蛋白基因 H4A和H4B的克隆及分析

组蛋白作为核小体的基本组分,是染色质的结构和功能必需的。组蛋白的变体和修饰共同参与染色质修饰及基因的表达调控。真核生物细胞中的5种组蛋白在进化中高度保守,然而纤毛虫的组蛋白H4与其他真核生物相比有较大的差异。本实验应用PCR技术从八肋游仆虫(Euplotes octocarinatus)中获得了2种组蛋白H4基因,分别为H4A和H4B,GenBank登录号为:JN715068和JN715069。序列分析表明,H4A基因开放阅读框324 bp,预测编码107个氨基酸,分子量为11.6 ku,等电点为10.99。而H4B基因编码框384 bp,编码127个氨基酸,分子量为14.4 ku,等电点为9.93。Blast结果显示,H4A序列与其他生物中H4的一致性相对较高,达81%～94%,而H4B的一致性为36%～70%。H4A和H4B的一致性仅为44.7%。实时荧光定量PCR表明,H4A的转录本高于H4B。结果提示:在进化过程中八肋游仆虫可能进化出特殊的组蛋白H4基因,不同的组蛋白H4可能发挥不同的功能。     

New preparations and molecular structures of cis-MCl2(Ph2PCH2PPh2)2, M = Ru,Os and trans-RuCl2(Ph2PCH2PPh2)2

The title compounds were made by reacting bis(diphenylphosphino)methane (dppm) with reduced solutions of OsCl₆^4? and Ru₂OCl₁₀^4?. The crystal and molecular structures of these compounds have been determined form three-dimensional X-ray study. The cis-isomers crystallize with one CHCl₃ per molecule of the complex. All three compounds crystallize in the monoclinic space group P2₁/n with unit cell dimensions as follows: Cis-OsCl₂(dppm)₂·CHCl₃: a = 13.415(4) Å, b = 22.859(4) Å, c = 16.693(3) Å, β = 105.77(3)°, V = 4926(3) ų, Z = 4. cis-RuCl₂(dppm)₂·CHCl₃: a = 13.442(3) Å, b = 22.833(7) Å, c = 16.750(4) Å, β = 105.53(2)°, V = 4953(3) ų, Z = 4. trans-RuCl₂(dppm)₂: a = 11.368(7) Å, b = 10.656(6) Å, c = 18.832(12) Å; β = 103.90(6)°, V = 2213(7) ų; Z = 2. The structures were refined to R = 0.044 (R_w = 0.055) for cis-OsCl₂(dppm)₂·CHCl₃; R = 0.065 (R_w = 0.079) for cis-RuCl₂(dppm)₂·CHCl₃ and R = 0.028 (R_w = 0.038) for trans-RuCl₂(dppm)₂. The complexes are six coordinate with stable four-membered chelate rings. The PMP angle in the chelate rings is ca. 71° in each case.     

Probing the local lipid environment of the Rhodobacter sphaeroides cytochrome bc1 and Synechocystis sp. PCC 6803 cytochrome b6f complexes with styrene maleic acid

Intracytoplasmic vesicles (chromatophores) in the photosynthetic bacterium Rhodobacter sphaeroides represent a minimal structural and functional unit for absorbing photons and utilising their energy for the generation of ATP. The cytochrome bc₁ complex (cytbc₁) is one of the four major components of the chromatophore alongside the reaction centre-light harvesting 1-PufX core complex (RC-LH1-PufX), the light-harvesting 2 complex (LH2), and ATP synthase. Although the membrane organisation of these complexes is known, their local lipid environments have not been investigated. Here we utilise poly(styrene-alt-maleic acid) (SMA) co-polymers as a tool to simultaneously determine the local lipid environments of the RC-LH1-PufX, LH2 and cytbc₁ complexes. SMA has previously been reported to effectively solubilise complexes in lipid-rich membrane regions whilst leaving lipid-poor ordered protein arrays intact. Here we show that SMA solubilises cytbc₁ complexes with an efficiency of nearly 70%, whereas solubilisation of RC-LH1-PufX and LH2 was only 10% and 22% respectively. This high susceptibility of cytbc₁ to SMA solubilisation is consistent with this complex residing in a locally lipid-rich region. SMA solubilised cytbc₁ complexes retain their native dimeric structure and co-purify with 56 ± 6 phospholipids from the chromatophore membrane. We extended this approach to the model cyanobacterium Synechocystis sp. PCC 6803, and show that the cytochrome b₆f complex (cytb₆f) and Photosystem II (PSII) complexes are susceptible to SMA solubilisation, suggesting they also reside in lipid-rich environments. Thus, lipid-rich membrane regions could be a general requirement for cytbc₁/cytb₆f complexes, providing a favourable local solvent to promote rapid quinol/quinone binding and release at the Q₀ and Q_i sites.     

Oxidation-reduction potentials of respiratory chain components in ThiobacillusA2

(1) Cells of ThiobacillusA₂ grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a₃-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (E_m,7.0) and spectral maxima of c-, b-, a- and a₃-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a₃ complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.