No abstract

Generation of RNA dimeric form of the human immunodeficiency virus type 1 (HIV-1) genome is crucial for viral replication. The dimerization initiation site (DIS) has been identified as a primary sequence that can form a stem-loop structure with a self-complementary sequence in the loop and a bulge in the stem. It has been reported that HIV-1 RNA fragments containing the DIS form two types of dimers, loose dimers and tight dimers. The loose dimers are spontaneously generated at the physiological temperature and converted into tight dimers by the addition of nucleocapsid protein NCp7. To know the biochemical process in this two-step dimerization reaction, we chemically synthesized a 39-mer RNA covering the entire DIS sequence and also a 23-mer RNA covering the self-complementary loop and its flanking stem within the DIS. Electrophoretic dimerization assays demonstrated that the 39-mer RNA reproduced the two-step dimerization process, whereas the 23-mer RNA immediately formed the tight dimer. Furthermore, deletion of the bulge from the 39-mer RNA prevented the NCp7-assisted tight-dimer formation. Therefore, the whole DIS sequence is necessary and sufficient for the two-step dimerization. Our data suggested that the bulge region regulates the stability of the stem and guides the DIS to the two-step dimerization process.

Dimer formation of HIV-1 genomic RNA through its dimerization initiation site (DIS) is crucial to maintaining infectivity. Two types of dimers, the initially generated kissing-loop dimer and the subsequent product of the extended-duplex dimer, are formed in the stem-bulge-stem region with a loop including a self-complementary sequence. NMR chemical shift analysis of a 39mer RNA corresponding to DIS, DIS39, in the kissing-loop and extended-duplex dimers revealed that the three dimensional structures of the stem-bulge-stem region are extremely similar between the two types of dimers. Therefore, we designed two shorter RNA molecules, loop25 and bulge34, corresponding to the loop-stem region and the stem-bulge-stem region of DIS39, respectively. Based upon the chemical shift analysis, the conformation of the loop region of loop25 is identical to that of DIS39 for each of the two types of dimers. The conformation of bulge34 was also found to be the same as that of the corresponding region of DIS39. Thus, we determined the solution structures of loop25 in each of the two types of dimers as well as that of bulge34. Finally, the solution structures of DIS39 in the kissing-loop and extended-duplex dimers were determined by combining the parts of the structures. The solution structures of the two types of dimers were similar to each other in general with a difference found only in the A16 residue. The elucidation of the structures of DIS39 is important to understanding the molecular mechanism of the conformational dynamics of viral RNA molecules.

Excited states in 38,40,42 Si nuclei have been studied via in-beam γ-ray spectroscopy with multinucleon removal reactions. Intense radioactive beams of 40 S and 44 S provided at the new facility of the RIKEN Radioactive Isotope Beam Factory enabled γ-γ coincidence measurements. A prominent γ line observed with an energy of 742 (8) 23.20.Lv, 27.40.+z, 29.38.Db Shell closures and collectivity are important properties that characterize the atomic nucleus. Interchange of their dominance along isotopic or isotonic chains has attracted much attention. The recent extension of the research frontier to nuclei far away from the valley of stability has revealed several new phenomena for neutronor proton-number dependent nuclear structure. For example, a weakening or even disappearance of shell closures occur in several neutron-rich nuclei at N = 8 [1][2][3] and N = 20 [4][5][6]. A well known example in the case of N = 20 is the so-called 'island of inversion ' [7] located around the neutron-rich nucleus 32 Mg. The low excitation energy of the first 2 + state E x (2 + 1 ) and large E2 transition probability [4][5][6] clearly indicate shell quenching in 32 Mg despite the fact that N = 20 is traditionally a magic number. The next magic number, N = 28, which appears due to the f 7/2 -f 5/2 spin-orbit splitting, has also been explored [8][9][10][11][12][13]. Weakening of the shell closure is seen by the decrease of the 2 With proton number Z = 14 and neutron number N = 28, the nuclear structure of 42 Si is of special interest. A simple but important question that arises is whether the weakening of the N = 28 shell closure continues, causing an enhancement of nuclear collectivity, or if shell stability is restored owing to a possible doubly magic structure. A study on 42 Si was made by a two-proton removal reaction experiment with radioactive 44 S beams at the NSCL [15]. The small two-proton removal cross sec-

We have attempted to generate embryonic stem (ES) cell-derived hepatocytes expressing liver-specific functional properties by use of ES cell technology. It was found that ES cells are allowed to differentiate into hepatocytes possessing high metabolic activities when hepatocyte nuclear factor (HNF)-3beta-transfected ES cells are cultured in alpha-MEM medium supplemented with 10% fetal bovine serum (FBS) and fibroblast growth factor (FGF)-2 in the three-dimensional cell culture system at 5% CO2. The differentiated cells induced albumin, triacylglycerol, urea, and glycogen synthesis as well as further expression of metabolic proteins and serum factors as markers of hepatocytic differentiation for at least 4 months. The cells differentiated from HNF-3beta-transfected ES cells also had hepatocyte-like ultrastructural characteristics, including several endoplasmic reticula, mitochondrion, and glycogen. Our findings indicate that generation of hepatocytes maintaining high metabolic functions developed from mouse ES cells will facilitate the study of the basic mechanism for hepatogenesis and will certainly provide new opportunities for tissue transplantation.