A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5′ and 3′ splice sites

The genome of the Friend murine leukemia virus (Fr‐MLV) contains a 5′ splice site (5′ss) located at 205 nt and a 3′ss located at 5489 nt. In our previous studies, it was shown that if the HindIII–BglII (879–1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr‐MLV sequence, then cryptic splicing of env‐mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII–BglII fragment were constructed. The vector, in which a 38 bp fragment (1612–1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183–1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI–NdeI (5140–5400 bp) fragment just upstream of the 3′ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140–5400 nt region located just upstream of the 3′ss is required for the splicing function of the 38 nt fragment and its flanking sequences.     

Chronological Changes in Japanese Physicians' Attitude and Behavior Concerning Relationships with Pharmaceutical Representatives: A Qualitative Study

Background

Recent qualitative studies indicated that physicians interact with pharmaceutical representatives depending on the relative weight of the benefits to the risks and are also influenced by a variety of experiences and circumstances. However, these studies do not provide enough information about if, when, how and why their attitudes and behaviors change over time.

Methods and Findings

A qualitative study using semi-structured face-to-face individual interviews was conducted on 9 Japanese physicians who attended a symposium on conflicts of interest held in Tokyo. Interviews were designed to explore chronological changes in individual physicians'' attitude and behavior concerning relationships with pharmaceutical representatives and factors affecting such changes. Their early interaction with pharmaceutical representatives was passive as physicians were not explicitly aware of the meaning of such interaction. They began to think on their own about how to interact with pharmaceutical representatives as they progressed in their careers. Their attitude toward pharmaceutical representatives changed over time. Factors affecting attitudinal change included work environment (local regulations and job position), role models, views of patients and the public, acquisition of skills in information seeking and evidence-based medicine, and learning about the concepts of professionalism and conflict of interest. However, the change in attitude was not necessarily followed by behavioral change, apparently due to rationalization and conformity to social norms.

Conclusions

Physicians'' attitudes toward relationships with pharmaceutical representatives changed over time and factors affecting such changes were various. Paying attention to these factors and creating new social norms may be both necessary to produce change in behavior consistent with change in attitude.     

Suppressor of Cytokine Signaling 1 Counteracts Rhesus Macaque TRIM5α-Induced Inhibition of Human Immunodeficiency Virus Type-1 Production

Old world monkey TRIM5α is a host factor that restricts human immunodeficiency virus type-1 (HIV-1) infection. Previously, we reported that rhesus macaque TRIM5α (RhTRIM5α) restricts HIV-1 production by inducing degradation of precursor Gag. Since suppressor of cytokine signaling 1 (SOCS1) is known to enhance HIV-1 production by rescuing Gag from lysosomal degradation, we examined if SOCS1 is involved in RhTRIM5α-mediated late restriction. Over-expression of SOCS1 restored HIV-1 production in the presence of RhTRIM5α to a level comparable to that in the absence of RhTRIM5α in terms of titer and viral protein expression. Co-immunoprecipitation studies revealed that SOCS1 physically interacted with RhTRIM5α. Over-expression of SOCS1 affected RhTRIM5α expression in a dose-dependent manner, which was not reversed by proteasome inhibitors. In addition, SOCS1 and RhTRIM5α were detected in virus-like particles. These results suggest that SOCS1 alleviates RhTRIM5α-mediated regulation in the late phase of HIV-1 life cycle probably due to the destabilization of RhTRIM5α.     

A Conditional Knockout Toolkit for Caenorhabditis elegans Based on the Cre/loxP Recombination

Conditional knockout (cKO) based on site-specific recombination (SSR) technology is a powerful approach for estimating gene functions in a spatially and temporally specific manner in many model animals. In Caenorhabditis elegans (C. elegans), spatial- and temporal-specific gene functions have been largely determined by mosaic analyses, rescue experiments and feeding RNAi methods. To develop a systematic and stable cKO system in C. elegans, we generated Cre recombinase expression vectors that are driven by various tissue-specific or heat-shock promoters. Validation using Cre-mediated fluorescence protein inactivation or activation systems demonstrated successful Cre-dependent loxP excision. We established a collection of multi-copy Cre transgenic strains for each evaluated vector. To evaluate our Cre/loxP-based cKO system, we generated sid-1 deletion mutants harboring floxed sid-1 single-copy integration (SCI) using ultraviolet trimethylpsoralen (UV/TMP) methods. sid-1 mutants that were rescued by the floxed sid-1 SCI were then crossed with the Pdpy-7::Cre strain for cKO in the hypodermis. The sid-1 cKO animals were resistant to bli-3 RNAi, which causes the Bli-phenotyple in the hypodermis, but they were sensitive to unc-22 RNAi, which leads to twitching of the body wall muscle. Our system, which is based on the combination of a transgenic Cre collection, pre-existing deletion mutants, and UV/TMP SCI methods, provided a systematic approach for cKO in C. elegans.     

Mate choice and cannibalism in a natural population of a stream goby,Rhinogobius sp.

Breeding ecology of the stream goby,Rhinogobius sp. LD (Large Dark), was investigated under natural conditions. Males selectively courted females of similar size to lead them to the nests, whereas females followed courting males preferentially when the relative male size was greater. Male-male competition for a female was relatively infrequent and not severe. Developmental stages of eggs and egg numbers in one nest indicated that males receive 1–3 clutches during one breeding cycle. Males guarding multiple clutches frequently ate some of the eggs, but those guarding single clutches rarely did so. Gravid females in the nest also frequently cannibalized eggs laid by a previous female, probably in order to extend the area available for egg deposition. Mate choice in this species is discussed in relation to paternal ability, limitation of available spawning area and the female-biased sex ratio.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.