The crystal structures of the full-length human eukaryotic initiation factor (eIF) 4E complexed with two mRNA cap analogues [7-methylguanosine 5'-triphosphate (m(7)GTP) and P(1)-7-methylguanosine-P(3)-adenosine-5',5'-triphosphate (m(7)GpppA)] were determined at 2.0 A resolution (where 1 A=0.1 nm). The flexibility of the C-terminal loop region of eIF4E complexed with m(7)GTP was significantly reduced when complexed with m(7)GpppA, suggesting the importance of the second nucleotide in the mRNA cap structure for the biological function of eIF4E, especially the fixation and orientation of the C-terminal loop region, including the eIF4E phosphorylation residue. The present results provide the structural basis for the biological function of both N- and C-terminal mobile regions of eIF4E in translation initiation, especially the regulatory function through the switch-on/off of eIF4E-binding protein-eIF4E phosphorylation.

No abstract

No abstract

An unnatural base pair of 2-amino-6-(N,N-dimethylamino)purine (designated as x) and pyridin-2-one (designated as y) has been developed for specific transcription. The ribonucleoside triphosphates of y and a modified y, 5-methylpyridin-2-one, are selectively incorporated into RNA opposite x in the templates by T7 RNA polymerase. In addition, the sequences of the DNA templates containing x can be confirmed by a dideoxynucleotide chainterminator method supplemented with the deoxynucleoside triphosphate of y. The bulky dimethylamino group of x in the templates effectively eliminates noncognate pairing with the natural bases. These results enable RNA biosynthesis for the specific incorporation of unnatural nucleotides at the desired positions.T ranscription involving specific, unnatural base pairs, in addition to the A⅐U and G⅐C pairs, would be a method for the site-directed incorporation of unnatural bases into RNAs, to achieve new functionality as ligands or catalysts (1-5) or novel codon-anticodon interactions between transfer and messenger RNAs in translation (6-9). Compared with the chemical synthesis of RNA, transcription is more efficient to produce and amplify RNAs with long chain lengths from DNA templates, which can be constructed by chemical synthesis in combination with enzymatic ligation. Thus, many efforts have been made to develop unnatural bases that are recognized by RNA polymerases as substrates and are specifically incorporated into RNA opposite the pairing bases in the DNA templates.Previous efforts to create unnatural base pairs for replication and transcription have relied on nonstandard hydrogen-bonding schemes that differ from those of the Watson-Crick base pairs (2, 10 -16). Unnatural pairs of nucleotides, such as isoguanosine⅐isocytidine and xanthosine⅐2,4-diaminopyrimidine nucleoside, are complementarily incorporated into DNA and RNA by polymerases with moderate selectivity (11-15). In addition, in vitro translation for the site-specific incorporation of an unnatural amino acid into a peptide has been demonstrated by using an extra codon-anticodon interaction between isoguanine and isocytosine (6, 7). These studies indicate that the unnatural, nonstandard hydrogen-bonded base pairs can also function in replication and transcription. However, some misincorporations resulting in noncognate pairings with natural bases cannot be ignored for the practical usage of these unnatural base pairs. For instance, a 14% misincorporation of adenosine into RNA opposite 2,4-diaminopyrimidine is observed at a xanthosine triphosphate-to-ATP ratio of 1:1 in transcription by T7 RNA polymerase (2). Isoguanine tautomerization also causes the misincorporation of T or U opposite this base (11,12). In addition, the isocytidine and 5-methylisocytidine nucleoside derivatives are chemically unstable, and gradually decompose in solution (11,16). Furthermore, nucleoside triphosphates of 2-aminopyrimidines, such as isocytosine and 2,4-diaminopyrimidine, are not recognized as substrates by some polymerases (11,14,15) becau...

The sperm-activating and -attracting factor released from the eggs of the ascidians Ciona intestinalis and Ciona savignyi requires extracellular Ca 2؉ for activating sperm motility and eliciting chemotactic behavior of the activated sperm toward the egg. Here, we show that modulators of the store-operated Ca 2؉ channel, SK&F96365, Ni 2؉ , 2-aminoethoxydiphenylborane, and thapsigargin inhibit the chemotactic behavior of the ascidian sperm; on the other hand, blockers of voltage-dependent Ca 2؉ channels did not inhibit the chemotaxis, even though they inhibited the sperm activation operated by voltage-dependent Ca 2؉ channels. The blockers of store-operated Ca 2؉ channels also inhibited the asymmetrical flagellar beating and turning movements of the ascidian sperm, which are typical signs of sperm chemotaxis. Depletion of internal Ca 2؉ stores by thapsigargin induced capacitative Ca 2؉ entry into the sperm, which was blocked by SK&F96365. These results suggest that the intracellular Ca 2؉ concentration increase through the store-operated Ca 2؉ channels induces asymmetrical flagellar movements to establish the chemotactic behavior of the sperm. I t is now well known that many cells, e.g., leukocytes, starved amoebae of Dictyostelium discoideum, bacteria, and migrating axon cells during development, can detect an extracellular chemical gradient and migrate toward the source of the chemicals, that is, exhibit chemotactic behavior. There is a variety of factors inducing chemotaxis of cells, e.g., food, pathogens, partner cells, etc., and chemotactic behavior of cells is one of the important communication systems between separated cells. Chemotactic behavior of the sperm toward an egg during fertilization in animal species is a typical case, first reported in hydrozoans (1). This phenomenon is now widely known to occur, from sponges to humans (2-4), and much effort has been devoted to clarifying the mechanisms underlying sperm chemotaxis.Extracellular Ca 2ϩ is generally accepted as being an important factor in the induction of chemotactic behavior of sperm in many plant and animal species (5-8). In the case of the sea urchin, Arbacia punctulata, a peptide named resact in the jelly layer of the egg has been shown to have sperm-activating and -attracting activities (7). The peptide increases the intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) via cAMP-gated Ca 2ϩ channels (9, 10), which, in turn, induces asymmetrical flagellum movements (5, 11, 12) that result in reorientation of the sperm swimming direction (13). The role of extracellular Ca 2ϩ -induced asymmetrical flagellar movement in inducing sperm chemotaxis has also been reported in hydrozoan species (14). Ascidian sperm chemotactic behavior is also characterized by abrupt turning movements of the sperm (15, 16). Furthermore, it has been shown that the sperm-attracting activity in the ascidians Ciona intestinalis and Ciona savignyi does not originate from the overall surface of the egg coats, such as follicle cells, but from the vegetal pole of the egg (17). We al...

The present study was designed to compare the cycle characteristics of in-vitro fertilization (IVF) and the chromosomal normality of oocytes in patients with polycystic ovarian syndrome (PCOS) with those of patients with tubal factor infertility. In all, 28 cycles of 24 PCOS patients and 55 cycles of 31 patients with tubal factor infertility (control) were investigated. Although a significantly greater number of oocytes were retrieved from PCOS patients (mean +/- SD: 15.6 +/- 6.4 versus 9.0 +/- 4.0, PCOS versus control group, P < 0.05), the percentage of fertilized oocytes was significantly lower in the PCOS group compared with controls (40.1 versus 73.8%, P < 0.01). The pregnancy rate per embryo transfer did not differ between the two groups. Cytogenetic analysis was performed on 74 oocytes from PCOS patients and 73 oocytes from control patients. In the PCOS group, 10 of the 74 oocytes (13.5%) demonstrated aneuploidy, four (5.4%) oocytes were diploid and six (8.1%) oocytes were metaphase II with a prematurely condensed sperm chromosome (PCC). In the tubal infertility group, nine of the 73 (12.3%) oocytes showed aneuploidy, four (5.5%) oocytes were diploid and five (6.8%) oocytes were found to have PCC. There was no significant difference in the aneuploidy, diploidy and PCC rates between the two groups. These results suggest that the reduced fertilization observed in PCOS is not attributable to chromosomal aberrations or immaturity of oocytes recruited from patients with PCOS.

No abstract

No abstract