Sequence-specific recognition of the major groove of DNA by oligodeoxynucleotides via triple helix formation. Footprinting studies.

Homopyrimidine oligodeoxynucleotides recognize the major groove of the DNA double helix at homopurine.homopyrimidine sequences by forming local triple helices. The oligonucleotide is bound parallel to the homopurine strand of the duplex. This binding can be revealed by a footprinting technique using copper-phenanthroline as a cleaving reagent. Oligonucleotide binding in the major groove prevents cleavage by copper-phenanthroline. The cleavage patterns on opposite strands of the duplex at the boundaries of the triple helix are asymmetric. They are shifted to the 3'-side, indicating that the copper-phenanthroline chelate binds in the minor groove of the duplex structure. Binding of the chelate at the junction between the triple and the double helix is not perturbed on the 5'-side of the bound homopyrimidine oligonucleotide. In contrast, a strong enhancement of cleavage is observed on the purine-containing strand at the triplex-duplex junction on the 3'-side of the homopyrimidine oligonucleotide.     

Hybridization specificity, enzymatic activity and biological (Ha-ras) activity of oligonucleotides containing 2,4-dideoxy-beta-D-erythro-hexopyranosyl nucleosides.

Antisense oligonucleotides with a 2,4-dideoxyhexopyranosyl nucleoside incorporated at the 3'-end and at a mutation site of the Ha-ras oncogene mRNA were synthesized. Melting temperature studies revealed that an A*-G mismatch is more stable than an A*-T mismatch with these hexopyranosyl nucleosides incorporated at the mutation site. The oligonucleotides are stable against enzymatic degradation. RNase H mediated cleavage studies revealed selective cleavage of mutated Ha-ras mRNA. The oligonucleotide containing two pyranose nucleosides at the penultimate position activates RNase H more strongly than natural oligonucleotides. No correlation, however, was found between DNA - DNA or RNA - DNA melting temperatures and RNase H mediated cleavage capacity. Although the A*-G mismatch gives more stable hybridization than the A*-T base pairing, only the oligonucleotides containing an A*-T base pair are recognized by RNase H. This modification is situated 3 base pairs upstream to the cleavage site. Finally, the double pyranose modified oligonucleotide was able to reduce the growth of T24 cells (bladder carcinoma) while the unmodified antisense oligonucleotide was not.     

Photochemically and chemically activatable antisense oligonucleotides: comparison of their reactivities towards DNA and RNA targets.

Dodecadeoxyribonucleotides derivatized with 1,10-phenanthroline or psoralen were targeted to the point mutation (G<-->U) in codon 12 of the Ha-ras mRNA. DNA and RNA fragments, 27 nucleotides in length, and containing the complementary sequence of the 12mers, were used to compare the reactivity of the activatable dodecamers (cleavage of the target by the phenanthroline-12mer conjugates; photo-induced cross-linking of psoralen-12mer conjugates to the target). The reactivity of the RNA with the dodecamers was weaker than that of the DNA target. With psoralen-substituted oligonucleotides, it was possible to obtain complete discrimination between the mutated target (which contained a psoralen-reactive T(U) in the 12th codon) and the normal target (which contained G at the same position). When longer Ha-ras RNA fragments were used as targets (120 and 820 nucleotides), very little reactivity was observed. Part of the reactivity could be recovered by using 'helper' oligonucleotides that hybridized to adjacent sites on the substrate. A 'helper' chain length greater than 13 was required to improve the reactivity of dodecamers. However, the dodecanucleotides induced RNase H cleavage of the target RNA in the absence of 'helper' oligonucleotide. Therefore, in the absence of the RNase H enzyme, long oligonucleotides are needed to compete with the secondary structures of the mRNA. In contrast, formation of a ternary complex oligonucleotide-mRNA-RNase H led to RNAT cleavage with shorter oligonucleotides.     

苜蓿叶片水杨酸含量与其蓟马抗性的关系

采用不同抗蓟马特性的苜蓿品系,于不同蓟马虫口密度下测定苜蓿水杨酸含量和虫害程度,以明确苜蓿叶片水杨酸含量与其抗蓟马特性的关系.结果表明,苜蓿叶片游离态、结合态水杨酸含量均与苜蓿抗蓟马特性密切相关;高抗品系(抗蓟马品系)的水杨酸初始含量(0.539 mg·g-1)高于低抗品系(0.403 mg·g-1);与较低抗品系相比,高抗品系受害器官(叶)水杨酸含量随虫口密度和危害点面积的增大而增加相对缓慢,而随危害指数的增大而增加较快(3.84倍).可见,水杨酸主要是间接增强苜蓿对蓟马的抗性;高抗蓟马苜蓿品系能通过快速获得较高水杨酸含量来阻碍被危害伤口的扩大,降低其受危害程度,促使自身产量和品质提高.     

Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis

Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.     

Inhibition of restriction endonuclease cleavage via triple helix formation by homopyrimidine oligonucleotides

A 17-mer homopyrimidine oligonucleotide was designed to bind to the major groove of SV40 DNA at a 17 base pair homopurine-homopyrimidine sequence via Hoogsteen base pairing. This sequence contains the recognition site for the class II-S restriction enzyme Ksp 632-I. The oligonucleotide was shown to inhibit enzymatic cleavage under conditions that allow for triple helix formation. Inhibition is sequence-specific and occurs in the micromolar concentration range. Triple helix formation by oligonucleotides opens new possibilities for sequence-specific regulation of gene expression.     

Phylogenetic significance of the kinethmoid‐associated Y‐shaped ligament and long intercostal ligaments in the Cypriniformes (Actinopterygii: Ostariophysi)

Liao, T.Y. & Kullander, S.O. (2012). Phylogenetic significance of the kinethmoid‐associated Y‐shaped ligament and long intercostal ligaments in the Cypriniformes (Actinopterygii: Ostariophysi). —Zoologica Scripta, 42, 71–87. The phylogenetic significance of the Y‐shaped and long intercostal ligaments in the Cypriniformes is examined using character optimization in 184 species representing 20 non‐ostariophysan teleost species, five ostariophysan orders, seven cypriniform families and 14 cyprinid subfamilies. Character states were optimized on the phylogenetic trees of previous studies. Given the topology of Saitoh et al. (2011) , the Y‐shaped ligament, connecting the kinethmoid to the ethmoid complex, is shown to be a synapomorphy for the Cyprinidae, with reversals observed in the Cyprininae, Danioninae, Gobioninae and Psilorhynchinae. The condition of the Y‐shaped ligament is consistent within most subfamilies with a few exceptions. Despite the exceptions, the Y‐shaped ligament may be considered as a diagnostic character distinguishing cyprinid subfamilies with otherwise similar morphology, that is, the Danioninae and Opsariichthyinae. The long intercostal ligament, connecting five to eight ribs and ascending from the subdistal end of the fifth rib, is present in the Catostomidae and all cyprinid subfamilies, except for the Psilorhynchinae and two developmentally truncated genera, Danionella and Paedocypris. In addition to these two cypriniforme families, the long intercostal ligament is homoplastically present in some catfishes. Given the topology of Saitoh et al. (2011) , presence of the long intercostal ligament is a synapomorphy of Cyprinidae+Catostomidae. Some shorter ligaments are also present in the Cypriniformes and Chilodus gracilis (Characiformes), near the base of the anterior ribs and only occurring anterodorsally to the putative line of the long intercostal ligament even when it is absent.     

Rejecting strictly allopatric speciation on a continental island: prolonged postdivergence gene flow between Taiwan (Leucodioptron taewanus,Passeriformes Timaliidae) and Chinese (L. canorum canorum) hwameis

Allopatry is conventionally considered the geographical mode of speciation for continental island organisms. However, strictly allopatric speciation models that assume the lack of postdivergence gene flow seem oversimplified given the recurrence of land bridges during glacial periods since the late Pliocene. Here, to evaluate whether a continental island endemic, the Taiwan hwamei (Leucodioptron taewanus, Passeriformes Timaliidae) speciated in strict allopatry, we used weighted‐regression‐based approximate Bayesian computation (ABC) to analyse the genetic polymorphism of 18 neutral nuclear loci (total length: 8500 bp) in Taiwan hwamei and its continental sister species, the Chinese hwamei (L. canorum canorum). The nonallopatry model was found to fit better with observed genetic polymorphism of the two hwamei species (posterior possibility = 0.82). We also recovered unambiguous signals of nontrivial bidirectional postdivergence gene flow (N_em » 1) between Chinese hwamei and Taiwan hwamei until 0.5 Ma. Divergence time was estimated to be 3.5 to 2 million years earlier than that estimated from mitochondrial cytochrome b sequences. Finally, using the inferred nonallopatry model to simulate genetic variation at 24 nuclear genes examined showed that the adiponectin receptor 1 gene may be under divergent adaptation. Our findings imply that the role of geographical barrier may be less prominent for the speciation of continental island endemics, and suggest a shift in speciation studies from simply correlating geographical barrier and genetic divergence to examining factors that facilitate and maintain divergence, e.g. differential selection and sexual selection, especially in the face of interpopulation gene flow.