A chemical synthesis of LNA-2,6-diaminopurine riboside, and the influence of 2'-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes

Modified nucleotides are useful tools to study the structures, biological functions and chemical and thermodynamic stabilities of nucleic acids. Derivatives of 2,6-diaminopurine riboside (D) are one type of modified nucleotide. The presence of an additional amino group at position 2 relative to adenine results in formation of a third hydrogen bond when interacting with uridine. New method for chemical synthesis of protected 3′-O-phosphoramidite of LNA-2,6-diaminopurine riboside is described. The derivatives of 2′-O-methyl-2,6-diaminopurine and LNA-2,6-diaminopurine ribosides were used to prepare complete 2′-O-methyl RNA and LNA-2′-O-methyl RNA chimeric oligonucleotides to pair with RNA oligonucleotides. Thermodynamic stabilities of these duplexes demonstrated that replacement of a single internal 2′-O-methyladenosine with 2′-O-methyl-2,6-diaminopurine riboside (D^M) or LNA-2,6-diaminopurine riboside (D^L) increases the thermodynamic stability (ΔΔG°₃₇) on average by 0.9 and 2.3 kcal/mol, respectively. Moreover, the results fit a nearest neighbor model for predicting duplex stability at 37°C. D-A and D-G but not D-C mismatches formed by D^M or D^L generally destabilize 2′-O-methyl RNA/RNA and LNA-2′-O-methyl RNA/RNA duplexes relative to the same type of mismatches formed by 2′-O-methyladenosine and LNA-adenosine, respectively. The enhanced thermodynamic stability of fully complementary duplexes and decreased thermodynamic stability of some mismatched duplexes are useful for many RNA studies, including those involving microarrays.     

Simple molecular engineering of glycol nucleic acid: Progression from self-pairing to cross-pairing with cDNA and RNA

The acyclic chiral nucleic acid analogue, Glycol Nucleic Acid (GNA), displayed exceptional structural simplicity and atom economy while forming self-paired duplexes, using canonical Watson–Crick base pairing. We disclose here that the replacement of phosphodiester linker in GNA with somewhat rigid and shorter carbamate linker in Glycol Carbamate Nucleic Acid (GCNA) backbone allows unprecedented stability to the antiparallel self-paired duplexes. The R-GCNA oligomers were further found to form cross-paired antiparallel duplexes with cDNA and RNA following Watson–Crick base pairing. The stability of cross-paired GCNA:DNA and GCNA:RNA duplexes was higher than the corresponding DNA:DNA and DNA:RNA duplexes. The chiral (R) and (S) precursors were easily accessible from naturally occurring l-serine.     

Tripodal amine catechol ligands: a fascinating class of chelators for aluminium(III)

Complexation constants based on potentiometric titrations and spectrophotometric measurements in an aqueous medium of 0.1 M KCl at 25 ± 1 °C for the complexes of Al(III) with multidentate tripodal polycatechol-amine ligands, cis,cis-1,3,5-tris[(2,3-dihydroxybenzylamino)aminomethyl]cyclohexane (TMACHCAT, L¹) and N¹,N³,N⁵-tris(2-(2,3-dihydroxybenzylamino)ethyl)cyclohexane-1,3,5-tricarboxamide (CYCOENCAT, L²) have been summarized in this paper. Both the ligands released six protons to form various monomeric complexes of the types AlLH₃, AlLH₂, AlLH and AlL (L = L¹ and L²). The first species AlLH₃ depicted at low pH for which a monocapped type geometry was suggested, where the ligands were coordinated through three catecholic oxygens at ortho. Other species are formed subsequently from the species AlLH₃ in steps upon deprotonation and coordination of the catecholic oxygens at meta to give encapsulated tris(catechol) type complexes. The probable structures of the metal complexes formed in solution were proposed through molecular modeling calculations. The pAl values calculated for AlL¹ and AlL² are appreciably higher than transferrin. The ligand L² showed higher affinity towards Al(III) than L¹ and desferrioxamine (DFO), the only approved drug for the treatment of aluminium intoxication.     

The formation pathway of i-motif tetramers

The i-motif is a four-stranded structure formed by two intercalated parallel duplexes containing hemiprotonated C•C⁺ pairs. In order to describe the sequence of reactions by which four C-rich strands associate, we measured the formation and dissociation rates of three [TC_n]₄ tetramers (n = 3, 4 and 5), their dissociation constant and the reaction order for tetramer formation by NMR. We find that TC_n association results in the formation of several tetramers differing by the number of intercalated C•C⁺ pairs. The formation rates of the fully and partially intercalated species are comparable but their lifetimes increase strongly with the number of intercalated C•C⁺ pairs, and for this reason the single tetramer detected at equilibrium is that with optimal intercalation. The tetramer half formation times vary as the power −2 of the oligonucleotide concentration indicating that the reaction order for i-motif formation is 3. This observation is inconsistent with a model supposing association of two preformed duplex and suggests that quadruplex formation proceeds via sequential strand association into duplex and triplex intermediate species and that triplex formation is rate limiting.     

Pseudonocardia hispaniensis sp. nov., a novel actinomycete isolated from industrial wastewater activated sludge

A novel actinomycete, designated PA3^T, was isolated from an oil refinery wastewater treatment plant, located in Palos de la Frontera, Huelva, Spain, and characterized taxonomically by using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate formed a distinct subclade in the Pseudonocardia tree together with Pseudonocardia asaccharolytica DSM 44247^T. The chemotaxonomic properties of the isolate, for example, the presence of MK-8 (H₄) as the predominant menaquinone and iso-C_16:0 as the major fatty acid, are consistent with its classification in the genus Pseudonocardia. DNA:DNA pairing experiments between the isolate and the type strain of P. asaccharolytica DSM 44247^T showed that they belonged to separate genomic species. The two strains were readily distinguished using a combination of phenotypic properties. Consequently, it is proposed that isolate PA3^T represents a novel species for which the name Pseudonocardia hispaniensis sp. nov. is proposed. The type strain is PA3^T (= CCM 8391^T = CECT 8030^T).     

Studies on Anticodon-anticodon Interactions: Hemi-protonation of Cytosines Induces Self-pairing Through the GCC Anticodon of E. Coli tRNA-Gly

Abstract

The temperature-jump method was used to compare the stability of anticodon-anticodon duplexes formed by the self-association of two tRNAs: yeast tRNA-Asp and Escherichia coli tRNA-Gly. Yeast tRNA-Asp duplexes contain a U/U mismatch while E. coli tRNA-Gly dimers have a C/C mismatch in the middle position of their quasi self-complementary anticodons GUC and GCC, respectively. At neutral pH, it is found that only tRNA-Asp duplexes exist whereas at pH 5.0 only tRNA-Gly duplexes are formed. This reflects the hemi- protonation of the N3 of the cytosines at pH 5.0 which induces a pairing between the two middle residues of the anticodon GCC in E. coli tRNA-Gly. This is the first evidence that a protonated C-C(+) base pair is compatible with the formation of a double helix with antiparallel strands in a natural RNA molecule.     

Potassium Ions are More Effective than Sodium Ions in Salt Induced Peptide Formation

Prebiotic peptide formation under aqueous conditions in the presence of metal ions is one of the plausible triggers of the emergence of life. The salt-induced peptide formation reaction has been suggested as being prebiotically relevant and was examined for the formation of peptides in NaCl solutions. In previous work we have argued that the first protocell could have emerged in KCl solution. Using HPLC-MS/MS analysis, we found that K⁺ is more than an order of magnitude more effective in the L-glutamic acid oligomerization with 1,1'-carbonyldiimidazole in aqueous solutions than the same concentration of Na⁺, which is consistent with the diffusion theory calculations. We anticipate that prebiotic peptides could have formed with K⁺ as the driving force, not Na⁺, as commonly believed.     

Induction of Sexual Reproduction in Single A<Superscript>2</Superscript> Isolates of <Emphasis Type="Italic">Phytophthora</Emphasis> Species by <Emphasis Type="Italic">Trichoderma viride</Emphasis>

ABOUT half the species of Phytophthora, including many important plant pathogens such as P. palmivora, P. cinnamomi and P. infestans, are heterothallic. Consistent induction of sexual reproduction in agar cultures of heterothallic species requires the pairing of isolates of the two compatibility groups, A¹ and A² (refs. 1–3). Although sex organs are occasionally formed in cultures of single isolates, such isolates though intrinsically bisexual are normally self-sterile^2–3. In consequence it has been assumed that in nature the presence of both compatibility types is required for the formation of oospores.     

Solution of Levinthal’s paradox is possible at the level of the formation and assembly of protein secondary structures

The complete volume of a protein’s conformation space is smaller by many orders of magnitude at the level of secondary-structure elements as compared with the conformation of amino-acid residues. According to Levinthal’s estimate, the latter is ~10^2L, with L being the number of residues in the chain, while the former, at the level of secondary structures, increases no faster than ~L^N with N being the number of the secondary-structure elements. N is approximately L/15 according to the statistics of protein structures. This drastic decrease in the exponent (L/15 instead of 2L) considerably reduces the sampling space and explains the reason that sampling of the conformation space at the level of secondary-structure elements does not prevent the protein chain from finding its most stable structure.     

The effect of valinomycin on the electrical properties of solutions of red cell lipids in n-decane

This paper reports the electrical properties of thick lipid membranes in the absence and presence of valinomycin. The thick lipid membranes were formed by placing a solution of sheep red cell lipids in decane between two cellophane partitions which formed the interfaces between the membrane and the two aqueous bathing solutions. The DC electrical resistance of these structures was found to be directly proportional to the reciprocal of the concentration of lipids in the decane (C_L). The limiting resistance, as (C_L ^-1) approached zero, was 3 x 10⁸ ohm-cm². Resistance was also found to be linearly related to membrane thickness. The limiting resistance at zero thickness was again 1–3 x 10⁸ ohm-cm². These data are interpreted to indicate that the DC resistance of thick lipid membranes comprises two surface resistances (R_S) at each interface with the aqueous bathing solutions, and a bulk resistance (R_B) of the lipid-decane solution, arranged in series. Measurements of the effect of variations of area on resistance were consistent with this interpretation. Valinomycin reduced R_S but had no effect on R_B. Under certain conditions, thick lipid membranes containing valinomycin behaved like highly selective K⁺ electrodes.     

Persistence and stability of seed-dispersed species in a patchy environment

A mathematical model is developed for the dynamics of seed-dispersed tree species in a region of forest patches or islands. The relevant variables are the population numbers of individual species populations on each patch. A model that considers only one tree species (of the many present) in a region of N distinct patches is formally analogous to a model of an N-species mutualistic community. Using the theory of M-matrices, which has been successfully applied to mutualistic communities, criteria for persistence and stability of the species are derived. The model is then extended to a community of L species that interact competitively and mutualistically in an N-island region. The possibilities of obtaining numerical parameters for the models are discussed.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Morphology, sociality, and ecology: can morphology predict pairing behavior in coral reef fishes?

Morphology can contain valuable information about the ecological performance of reef fishes, but it has rarely been used in combination with social traits. Social behavior is known to influence the ecological role of fishes; however, the ecological basis for pairing in reef fishes is not well understood. Field observations of 2,753 individuals, in 47 species in six families of biting reef fishes (Acanthuridae, Chaetodontidae, Kyphosidae, Labridae, Pomacanthidae, Siganidae), were used in combination with six morphological measurements, to examine the morphology of fishes in different social systems. A principal components analysis of morphological traits segregated species with high proportions of pairing individuals from non-pairing species along principal component 1, explaining 40.8 % of the variation. Pairing species were characterized by large eyes, concave foreheads, pointed snouts, deep bodies, and small maximum sizes. There was a significant positive relationship between these morphological traits (i.e., scores on PC1) and the prevalence of pairing within the Chaetodontidae (r ² = 0.59; P = 0.026), Siganidae (r ² = 0.72; P = 0.004), and Acanthuridae (r ² = 0.82; P < 0.001). This was consistent when traits were corrected for phylogenetic effects. No pattern was evident in the scarine Labridae (r ² = 0.15; P = 0.17). The morphological characteristics found among pairing species suggest that pairing species share common ecological traits, including foraging for small prey items in micro-topographically complex environments such as reef crevices. These ecological traits may have played a role in the evolution of pairing behavior and subsequently led to the development of reproductive patterns based on monogamy.     

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N⁶-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N⁶-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.     

X-4 Translocations and Meiotic Drive in Drosophila melanogaster Males: Role of Sex Chromosome Pairing

Males carrying certain X-4 translocations exhibit strongly skewed sperm recovery ratios. The X^P4^D half of the translocation disjoins regularly from the Y chromosome and the 4^PX^D half disjoins regularly from the normal 4. Yet the smaller member of each bivalent is recovered in excess of its pairing partner, apparently due to differential gametic lethality. Chromosome recovery probabilities are multiplicative; the viability of each genotype is the product of the recovery probability of its component chromosomes. Meiotic drive can also be caused by deficiency for X heterochromatin. In(1)sc^4Lsc^8R males show the same size dependent chromosome recoveries and multiplicative recovery probabilities found in T(1;4)B^S males. Meiotic drive in In(1)sc^4Lsc^8R males has been shown to be due to X-Y pairing failure. Although pairing is regular in the T(X;4) males, the striking phenotypic parallels suggest a common explanation. The experiments described below show that the two phenomena are, in fact, one and the same. X-4 translocations are shown to have the same effect on recovery of independently assorting chromosomes as does In(1)sc^4Lsc^8R. Addition of pairing sites to the 4^PX^D half of the translocation eliminates drive. A common explanation—failure of the distal euchromatic portion of the X chromosome to participate in X:Y meiotic pairing—is suggested as the cause for drive. The effect of X chromosome breakpoint on X-4 translocation induced meiotic drive is investigated. It is found that translocations with breakpoints distal to 13C on the salivary map do not cause drive while translocations broken proximal to 13C cause drive. The level of drive is related to the position of the breakpoint—the more proximal the breakpoint the greater the drive.     

Structural and energetic aspects of spin pairing in lanthanides

Numerical Hartree-Fock calculations on the lanthanide ions confirm the earlier results of Marcantonatos and coworkers, by showing that the excitation from the ground state of a given fⁿ- configuration to a state with lower spin multiplicity is accompanied by a change in size and shape of the orbitals. This orbital change is also shown to be at the basis of a new interpretation of the spin pairing phenomenon in lanthanides. This interpretation is significantly different from the picture offered by conventional multiplet theory; indeed, the spin pairing energy, calculated on the basis of Hartree- Fock theory is accompanied by a decrease in interelectronic repulsion.Although the relevant energetic (repulsion) effects are obviously quite large, the underlying size effects are very much smaller. Therefore, it seems highly unlikely that they are responsible for the change in coordination number of Gd(H₂O)₈³⁺ upon excitation, as suggested by Marcantonatos.     

Preferential transposition of aDrosophila P element to the corresponding region of the homologous chromosome

Genetic and physical analyses have demonstrated an intimate interaction or pairing of homologous chromosomes in the nuclei of manyDrosophila cell types. Experiments were performed to determine whether P elements transposing from a given chromosome to its homolog would preferentially insert in the region corresponding to the donor site, perhaps due to such a proximity. AP[lacZ;ry ⁺] element at thecactus locus (35F) on the second chromosome was mobilized and 96 insertions on the homolog were recovered. The distribution of these new insertions was determined by recombination mapping and molecular analysis, and compared with a control set of 93 second-chromosome insertions originating from theX chromosome. A nearly threefold preference was observed for re-insertion in a region of two to three number divisions aroundcactus on the homolog. However, none of these “local” insertions was actually within ～ 50 kb of the site atcactus corresponding to the starting site. This is in marked contrast to the previously described phenomenon of intrachromosomal local transposition, where the majority of local transpositions are within 10 kb. The data suggest that the mechanisms for intrachromosomal and interchromosomal local transposition are distinct, and are consistent with a model for interchromosomal local transposition involving proximity of homologous chromosomal regions in the nuclei of the germline cells.     

Telomere attachment, meiotic chromosome condensation, pairing, and bouquet stage duration are modified in spermatocytes lacking axial elements

During the extended prophase to the meiosis I division, chromosomes assemble axial elements (AE) along replicated sister chromatids whose ends attach to the inner nuclear membrane (NM) via a specialized conical thickening. Here, we show at the EM level that in Sycp3^-/- spermatocyte chromosomes lack the AE and the conical end thickening, but still they attach their telomeres to the inner NM with an electron-dense plate that contains T₂AG₃ repeats. Immunofluorescence detected telomere proteins, SCP2, and the meiosis-specific cohesin STAG3 at the Sycp3^-/- telomere. Bouquet stage spermatocytes were approximately threefold enriched, and the number of telomere but not centromere signals was reduced to the haploid in advanced Sycp3^-/- spermatocytes, which indicates a special mode of homolog pairing at the mammalian telomere. Fluorescence in situ hybridization with mouse chromosome 8- and 12-specific subsatellite probes uncovered reduced levels of regional homolog pairing, whereas painting of chromosomes 13 revealed partial or complete juxtapositioning of homologs; however, condensation of Sycp3^-/- bivalents was defective. Electron microscopic analysis of AE-deficient spermatocytes revealed that transverse filaments formed short structures reminiscent of the synaptonemal complex central region, which likely mediate stable homolog pairing. It appears that the AE is required for chromosome condensation, rapid exit from the bouquet stage, and fine-tuning of homolog pairing.     

Most of the homoeologous pairing at metaphase I in wheat-rye hybrids is not chiasmatic

The use of telomeric C-bands in wheat-rye hybrids has made it possible to distinguish three types of wheat-wheat (1B^L) and wheat-rye associations (a, end-to-end extremely distal; b, end-to-ed distal; and c, interstitial) between homoeologous chromosomes at different metaphase I stages (early, middle and late) and also to estimate the actual recombination frequencies for such associations at anaphase I. There was a decrease of the a and b association frequencies during the different metaphase I stages, whereas the c type remained without variation in all stages. A good fit between the frequencies of c associations at metaphase I and the number of recombinant chromosomes at anaphase I, assuming a maximum of one chiasma per bond, was found; however, there was no correspondence between metaphase I and anaphase I data when all associations (a + b + c) were considered. In addition, rye-rye homologous pairing was observed at metaphase I, but no evidence for rye-rye recombination was found at anaphase I. The results indicate that most of end-to-end (a and b) homoeologous and nonhomologous associations are actually nonchiasmatic and are a remnant of prophase pairing.     

Copy Number Variation of CCL3-like Genes Affects Rate of Progression to Simian-AIDS in Rhesus Macaques (Macaca mulatta)

Variation in genes underlying host immunity can lead to marked differences in susceptibility to HIV infection among humans. Despite heavy reliance on non-human primates as models for HIV/AIDS, little is known about which host factors are shared and which are unique to a given primate lineage. Here, we investigate whether copy number variation (CNV) at CCL3-like genes (CCL3L), a key genetic host factor for HIV/AIDS susceptibility and cell-mediated immune response in humans, is also a determinant of time until onset of simian-AIDS in rhesus macaques. Using a retrospective study of 57 rhesus macaques experimentally infected with SIVmac, we find that CCL3L CNV explains approximately 18% of the variance in time to simian-AIDS (p<0.001) with lower CCL3L copy number associating with more rapid disease course. We also find that CCL3L copy number varies significantly (p<10⁻⁶) among rhesus subpopulations, with Indian-origin macaques having, on average, half as many CCL3L gene copies as Chinese-origin macaques. Lastly, we confirm that CCL3L shows variable copy number in humans and chimpanzees and report on CCL3L CNV within and among three additional primate species. On the basis of our findings we suggest that (1) the difference in population level copy number may explain previously reported observations of longer post-infection survivorship of Chinese-origin rhesus macaques, (2) stratification by CCL3L copy number in rhesus SIV vaccine trials will increase power and reduce noise due to non-vaccine-related differences in survival, and (3) CCL3L CNV is an ancestral component of the primate immune response and, therefore, copy number variation has not been driven by HIV or SIV per se.