Parameters controlling the gene-targeting frequency at the Sphingomonas species rrn site and expression of the methyl parathion hydrolase gene

AIMS: To investigate the key parameters controlling the exogenous methyl parathion hydrolase (MPH) gene mpd-targeting frequency at the ribosomal RNA operon (rrn) site of Sphingomonas species which has a wide range of biotechnological applications. METHODS AND RESULTS: Targeting vectors with different homology lengths and recipient target DNA with different homology identities were used to investigate the parameters controlling the targeting frequency at the Sphingomonas species rrn site. Targeting frequency decreased with the reduction of homology length, and the minimal size for normal homologous recombination was >100 bp. Homologous recombination could succeed even if there were 3-4% mismatches; however, targeting frequency decreased with increasing sequence divergence. The Red recombination system could increase the targeting frequency to some extent. Targeting of the mpd gene to the rrn site did not affect cell viability and resulted in an increase of MPH-specific activity in recombinants. CONCLUSIONS: Targeting frequency was affected by homology length, identity and the Red recombination system. The rrn site is a good target site for the expression of exogenous genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This work is useful as a foundation for a better understanding of recombination events involving homologous sequences and for the improved manipulation of Sphingomonas genes in biotechnological applications.     

MareyMap: an R-based tool with graphical interface for estimating recombination rates

Comparing genetic and physical maps (the so-called Marey map approach) is still the most widely used approach to estimate genome-wide recombination rates. Remarkably, there is no available bioinformatics tool specifically devoted to Marey map approach. Here, we developed such a tool called MareyMap based on GNU R and Tcl/Tk. MareyMap offers a user-friendly graphical interface and includes useful features, such as data cleaning process, sophisticated interpolation methods to estimate local rates, possibility of complex queries, various range of import-export files. Moreover, MareyMap comes with ready-to-use maps for human, Drosophila, Caenorhabditis elegans and Arabidopsis. MareyMap has been made so that it can be easily upgraded with new data and interpolation methods. Availability: http://pbil.univ-lyon1.fr/software/mareymap/.     

Structured to reduce the mitogenicity of anti-CD3 antibody based on computer-guided molecular design

The mouse anti-human CD3 monoclonal antibody such as OKT3 is a potent immunosuppressive agent used in clinical transplantation to manipulate T-cell functions and prevent acute allograft rejection. However, the broad use of anti-CD3 antibody in clinical treatment was severely limited by the side effects of human anti-mouse antibody response and cytokine release syndrome. In this study, on the basis of a murine anti-human CD3 antibody yCD3 obtained in our previous work, a novel engineered anti-human CD3 antibody fragment (i.e. V(H)-Linker-V(L)-Hinge-CH(3)) was constructed with computer-guided molecular design method to avoid the clinical side effects. According to the distance geometry and intra-molecular interaction, the hinge region was re-designed and different from the parental hinge region in human IgG1. With the novel hinge region, the cysteine residues in hinge were exposure and prone to form the disulfide bond. Therefore, a novel bivalent antibody fragment named as mini-yCD3 was obtained. Mini-yCD3 displayed similar antigen-binding affinity and specificity to yCD3. Importantly, mini-yCD3 was shown to be much less potent in the induction of T-cell proliferation, cytokine release (interferon-gamma and interleukin-2) and early activation marker expression on the cell surface (CD69 and CD25) than parental yCD3. Furthermore, mini-yCD3 was effective in modulating T-cell receptor/CD3 and inhibiting mixed lymphocyte reaction with similarity as yCD3. In conclusion, the constructed mini-yCD3 was much less mitogenic to T cells but retained potent immunosuppression, suggesting it might be an alternative to yCD3 as an immunosuppressive drug with less immunogenicity and toxicity for clinical application.     

Functional interactions between coexisting toxin-antitoxin systems of the ccd family in Escherichia coli O157:H7

Toxin-antitoxin (TA) systems are widely represented on mobile genetic elements as well as in bacterial chromosomes. TA systems encode a toxin and an antitoxin neutralizing it. We have characterized a homolog of the ccd TA system of the F plasmid (ccd(F)) located in the chromosomal backbone of the pathogenic O157:H7 Escherichia coli strain (ccd(O157)). The ccd(F) and the ccd(O157) systems coexist in O157:H7 isolates, as these pathogenic strains contain an F-related virulence plasmid carrying the ccd(F) system. We have shown that the chromosomal ccd(O157) system encodes functional toxin and antitoxin proteins that share properties with their plasmidic homologs: the CcdB(O157) toxin targets the DNA gyrase, and the CcdA(O157) antitoxin is degraded by the Lon protease. The ccd(O157) chromosomal system is expressed in its natural context, although promoter activity analyses revealed that its expression is weaker than that of ccd(F). ccd(O157) is unable to mediate postsegregational killing when cloned in an unstable plasmid, supporting the idea that chromosomal TA systems play a role(s) other than stabilization in bacterial physiology. Our cross-interaction experiments revealed that the chromosomal toxin is neutralized by the plasmidic antitoxin while the plasmidic toxin is not neutralized by the chromosomal antitoxin, whether expressed ectopically or from its natural context. Moreover, the ccd(F) system is able to mediate postsegregational killing in an E. coli strain harboring the ccd(O157) system in its chromosome. This shows that the plasmidic ccd(F) system is functional in the presence of its chromosomal counterpart.     

Epistatic interactions of three loci regulate flowering time under short and long daylengths in a backcross population of rice

The short-day plant rice varies greatly in photoperiod sensitivity (PS) for flowering. The hybrid F₁ from a cross between the day-neutral pure line EM93-1 and the weedy rice accession SS18-2 had stronger PS than SS18-2. Some BC₁ (EM93-1/F₁) segregates were even more sensitive to photoperiod than the F₁, as indicated by later flowering or no flowering after 250 days under a 14-h long daylength. A genome-wide scan identified the quantitative trait loci Se _7.1, Se _7.2 and Se ₈ for PS from the BC₁ population, with all alleles that inhibit flowering derived from SS18-2. These three loci regulate the time of flowering under long daylength through their main effects, and di- and trigenic epistases. Under a 10-h short daylength, the regulation is through Se _7.1 and Se ₈ main effects and digenic epistases involving all three loci. The short daylength not only nullified the main effect of Se _7.2, but also changed its epistatic effects from inhibiting flowering under long daylength to promoting flowering. The epistases indicate that genes underlying the three PS loci work in the same pathway for the control of flowering. Many non-flowered BC₁s were the trigenic heterozygote; this suggests that the three PS loci are also involved in genetic control of critical daylength.     

Effect of the luteinizing hormone on embryo production in superovulated rabbit does

For most domestic animals, the responses to superovulation treatments are not controlled as a consequence of the lack of knowledge on exogenous gonadotrophins effects on the ovarian function. The role of luteinizing hormone (LH) on the number and quality of embryos produced was evaluated on rabbit does superovulated with porcine FSH (pFSH). Parameters of embryos recovery, in vitro and in vivo embryo development rates after freezing/thawing were compared. We used three experimental groups: (1) control group without superovulation treatment, (2) "pFSH+pLH" and (3) "pFSH" groups where females were treated with pFSH, respectively, with (20%) or without (0%) porcine LH supplementation. The number of corpora lutea and the number of embryos produced were significantly higher (p<0.001) in superovulated does than in control group (27.1, 26.7 versus 11.9 corpora lutea and 20.3, 21.2 versus 9.6 embryos produced for pFSH+pLH, pFSH and control group, respectively). However, both gonadotrophins administrations (groups 2 and 3) led to defaults of ovulation when compared with untreated does. No significant difference was observed between the number and quality of the embryos produced by does treated with pFSH+pLH or with pFSH alone. Moreover, we observed no significant difference between results of in vivo and in vitro viability assays after thawing. We concluded that pFSH alone seems to be sufficient to stimulate the follicles growth and that exogenous pLH administrated has no effect on the quantity and quality of embryos. Further studies are needed to evaluate the hormonal patterns before and after the gonadotrophins injections in the rabbit species.     

Real-time imaging nuclear translocation of Akt1 in HCC cells

Akt is one of the critical mediators in cellular signaling, and overactivation of Akt related pathway frequently occurs in hepatocellular carcinoma (HCC). In this study, we presented that Akt was upregulated in HCC cell lines, and its active phosphorylated form was mainly located in the nucleus. Employing the laser confocal techniques for imaging intracellular protein dynamics, we monitored the transnuclear movement of GFP-tagged wild-type Akt1 (Akt1-WT-GFP) and its inactive mutant (Akt1-T308A/S473A-GFP) in live SMMC-7721 HCC cells, and both of fusion proteins were found to distribute over the cytoplasm and nucleus. Moreover, it was found that platelet derived growth factor (PDGF) was able to accelerate the nuclear translocation of wild-type Akt1 in HCC cells but failed to speed up the motion of the mutant. It was demonstrated that activation of phosphatidylinositol 3-kinase (PI3K) and Akt1 facilitated the nuclear translocation of Akt1, but the phosphorylation at threonine 308 and serine 473 was not prerequisite.     

Interspecific recombinant congenic strains between C57BL/6 and mice of the Mus spretus species: a powerful tool to dissect genetic control of complex traits

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.     

Genetic diversity of the natural populations of Arabidopsis thaliana in China

Although extensive studies have been conducted on the genetic structure of Arabidopsis thaliana (A. thaliana) populations worldwide, the populations from China have never been studied. In this study, we collected 560 individuals from 19 natural populations of A. thaliana distributed in East China along the lower reaches of the Yangtze River, and two populations from northwest China (Xinjiang Province). We adopted two kinds of molecular marker, inter-simple sequence repeats (ISSRs) and random amplified polymorphic DNA (RAPDs) to investigate the genetic diversity within and among populations, and the correlation between the genetic and geographic distances. Thirteen ISSR primers produced 165 polymorphic bands (PPB) (96%) and 11 RAPD primers produced 162 polymorphic bands (98%) in about 560 individuals. The two marker systems generated similar patterns of genetic diversity in these natural populations. The AMOVA analysis indicated about 42-45% of the total genetic variation existed within populations, and found possible geographic structure. The Mantel test revealed a significant correlation between the geographic distance and the genetic distance of these populations in general. A close genetic relationship was found among four populations in the Jiangxi Province, and these always appeared clustered together as a monophyletic group in unweighted pair-group method with arithmetic averages dendrograms based on both ISSR and RAPD data sets. Based on the observation of recolonization and extinction of naturally distributed populations of A. thaliana, and the pattern of their genetic differentiation, the distribution of this species in China might be a result of natural dispersal under the strong influence of human activity.