A role for Rev in the association of HIV-1 gag mRNA with cytoskeletal β-actin and viral protein expression

Human immunodeficiency virus type-1 (HIV-1) Rev acts by inducing the specific nucleocytoplasmic transport of a class of incompletely spliced RNAs that encodes the viral structural proteins. The transfection of HeLA cells with a rev-defective HIV-1 expression plasmid, however, resulted in the export of overexpressed, intron-containing species of viral RNAs, possibly through a default process of nuclear retention. Thus, this system enabled us to directly compare Rev⁺ and Rev⁻ cells as to the usage of RRE-containing mRNAs by the cellular translational machinery. Biochemical examination of the transfected cells revealed that although significant levels of gag and env mRNAs were detected in both the presence and absence of Rev, efficient production of viral proteins was strictly dependent on the presence of Rev. A fluoroscence in situ hybridisation assay confirmed these findings and provided further evidence that even in the presence of Rev, not all of the viral mRNA was equally translated. At the early phase of RNA export in Rev⁺ cells, gag mRNA was observed throughout both the cytoplasm and nucleoplasm as uniform fine stippling. In addition, the mRNA formed clusters mainly in the perinuclear region, which were not observed in Rev⁻ cells. In the presence of Rev, expression of the gag protein was limited to these perinuclear sites where the mRNA accumulated. Subsequent staining of the cytoskeletal proteins demonstrated that in Rev⁺ cells gag mRNA is colocalized with β-actin in the sites where the RNA formed clusters. In the absence of Rev, in contrast, the gag mRNA failed to associate with the cytoskeletal proteins. These results suggest that in addition to promoting the emergence of intron-containing RNA from the nucleus, Rev plays an important role in the compartmentation of translation by directing RRE-containing mRNAs to the β-actin to form the perinuclear clusters at which the synthesis of viral structural proteins begins.     

Cloning and antigenic expression of two genes from Chlamydia psittaci avian strain

Abstract: Genes from Chlamydia psittaci P-1041 were cloned into the Bam HI site of pUC19 and were transformed to host Escherichia coli JM109. Two recombinant plasmids that expressed protein antigens of Chlamydia were isolated. The sizes of the DNA fragments were 1350 and 1710 bp, and encoded for polypeptides of M _r 25 and 42 kilodaltons (kDa), respectively. The 25-kDa protein had cross-reactivity with antisera to ten C. psittaci strains and two C. trachomatis strains, whereas the 42-kDa protein reacted only with homologous antiserum to the C. psittaci P-1041 strain. Furthermore, in Southern hybridization analysis these two fragments as probes hybridized with DNA of ten C. psittaci strains and four C. trachomatis strains. These results indicated that the two fragments shared a DNA sequence common to the chlamydial genus.     

Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases.

Two tropinone reductases (TRs) constitute a key branch point in the biosynthetic pathway of tropane alkaloids, which are mainly produced in several solanaceous plants. The two TRs share 64% identical amino acid residues and reduce the 3-carbonyl group of a common substrate, tropinone, but they produce distinct alcohol products with different stereospecific configurations. Previous x-ray crystallographic analysis has revealed their highly conserved overall folding, and the modeling of tropinone within the putative substrate-binding sites has suggested that the different stereospecificities may be determined solely by the different binding orientations of tropinone to the enzymes. In this study, we have constructed various mutant TRs, in which putative substrate-binding residues from one TR were substituted with those found in the corresponding positions of the other TR. Substitution of five amino acid residues resulted in an almost complete reversal of stereospecificity, indicating that the different stereospecificities are indeed determined by the binding orientation of tropinone. Detailed kinetic analysis of the mutant enzymes has shown that TR stereospecificity is determined by varying the contributions from electrostatic and hydrophobic interactions and that the present TR structures represent highly evolved forms, in which strict stereospecificities and rapid turnover are accomplished together.     

Dynamical Systems Approach to Higher-level Heritability

To explain higher-level heritability, we propose a dynamical systems approach, based on simulations of the high-dimensional replicator equation with mutation dynamics. We assume that all variants are generated from within the groups of variants through mutations. Simulating the equation with a random interaction matrix and possible variants, we report that this system tends to have many attractors, of fixed point, chaotic and quasiperiodic type. In a chaotic attractor, special gene-like variants appear to control the heritability ofthe system, in the sense that removal of the variants would easily enable the system to depart from the attractor. Those variants do not predominate in thepopulation size, but have the lowest net reproduction and mutation rates on average. Because their rate of growth is small, they are named neutral phenotypes. Additionally, combinatorial effects of these neutral variants to the entire system are reported.     

Apterichtus keramanus, a new snake-eel from Okinawa, southern Japan (Ophichthinae, Ophichthidae)

A new snake-eel,Apterichtus keramanus, is described on the basis of a single 276-mm TL specimen trawled from the coast of Kerama Islands, Okinawa Prefecture, Japan. The species is unique in the genus in having the posterior nostril opening entirely inside the mouth and a dark band running from the anteroventral margin of the eye to the upper lip.     

Type V collagen in splenic reticular fibers of the macaque monkey

The distribution of type I, III and V collagens in the monkey spleen was examined by indirect immunofluorescent microscopy and immunoelectron microscopy, and compared with that of reticular fibers revealed by a silver impregnation method. Type I collagen was localized on reticular fibers in the white pulps and on coarse reticular fibers in the splenic cords. Type III collagen was localized on the reticular fibers in the white pulps, and on the coarse reticular fibers and a limited number of fine reticular fibers, in the splenic cords. The anti-type V collagen antibody reacted with annular reticular fibers around the splenic sinuses, as well as with the reticular fibers in the white pulps and with the coarse and fine reticular fibers in the splenic cords. Thus, the distribution pattern of fibers that reacted with the anti-type V collagen antibody was very similar to that of the reticular fibers revealed by the silver impregnation method. Electron-microscopically, the fine reticular fibers in the splenic cords were composed of collagen fibrils, 30-50 nm in diameter, and amorphous substances. They were covered by reticular cell processes. By immunoperoxidase labeling with the anti-type V collagen antibody, electron-dense reaction products were found over the collagen fibrils with a banding pattern. These results indicate that type V collagen is an indispensable component of the reticular fibers.