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Pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in Neurospora crassa.
Neurospora crassa is a self-sterile filamentous fungus with two mating types, mat A and mat a. Its mating involves chemotropic polarized growth of female-specific hyphae (trichogynes) toward male cells of the opposite mating type in a process involving pheromones and receptors. mat A cells express the ccg-4 pheromone and the pre-1 receptor, while mat a strains produce mRNA for the pheromone mfa-1 and the pre-2 receptor; MFA-1 and CCG-4 are the predicted ligands for PRE-1 and PRE-2, respectively. In this study, we generated Deltaccg-4 and Deltamfa-1 mutants and engineered a mat a strain to coexpress ccg-4 and its receptor, pre-2. As males, Deltaccg-4 mat A and Deltamfa-1 mat a mutants were unable to attract mat a and mat A trichogynes, respectively, and consequently failed to initiate fruiting body (perithecial) development or produce meiotic spores (ascospores). In contrast, Deltaccg-4 mat a and Deltamfa-1 mat A mutants exhibited normal chemotropic attraction and male fertility. Deltaccg-4 Deltamfa-1 double mutants displayed defective chemotropism and male sterility in both mating types. Heterologous expression of ccg-4 enabled mat a males to attract mat a trichogynes, although subsequent perithecial differentiation did not occur. Expression of ccg-4 and pre-2 in the same strain triggered self-stimulation, resulting in formation of barren perithecia with no ascospores. Our results indicate that CCG-4 and MFA-1 are required for mating-type-specific male fertility and that pheromones (and receptors) are initial determinants for sexual identity during mate recognition. Furthermore, a self-attraction signal can be transmitted within a strain that expresses a pheromone and its cognate receptor.
Genetic interaction of flavivirus nonstructural proteins NS1 and NS4A as a determinant of replicase function.
Nonstructural protein 1 (NS1) of yellow fever virus (YF) is a glycoprotein localized to extracytoplasmic compartments within infected cells. We have previously shown that NS1 can be supplied in trans and is required for viral RNA replication, a process thought to occur in membrane-bound cytoplasmic complexes. Here we report that the NS1 gene from a related virus, dengue virus (DEN), is unable to function in the process of YF RNA replication. This virus-specific incompatibility leads to a lack of initial minus-strand accumulation, suggesting that DEN NS1 is unable to productively interact with the YF replicase. Based on a YF deletion mutant that requires NS1 in trans, a genetic screen for suppressor mutants was used to select virus variants able to utilize DEN NS1. In three independent selections, a single mutation was mapped to the NS4A gene, which encodes a putative transmembrane replicase component. This mutation, as well as several additional mutations, was engineered into the NS1-deficient genome and confirmed a genetic interaction between NS1 and NS4A. These findings suggest a potential mechanism for integrating NS1 into the cytoplasmic process of RNA replication.
Identification of six Autographa californica multicapsid nucleopolyhedrovirus early genes that mediate nuclear localization of G-actin.
Nuclear filamentous actin (F-actin) is required for nucleopolyhedrovirus (NPV) progeny production in NPV-infected, cultured lepidopteran cells. We have determined that monomeric G-actin is localized within the nuclei of host cells during the early stage of infection by Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). With a library of cloned AcMNPV genomic fragments, along with a plasmid engineered to express enhanced green fluorescent protein-Bombyx mori G-actin in transient transfection experiments, we identified six AcMNPV early genes that mediate nuclear localization of G-actin in TN-368 cells: ie-1, pe38, he65, Ac004, Ac102, and Ac152. Within this subset, ie-1 and pe38 encode immediate-early transcriptional transactivators, he65 encodes a delayed-early product, and the products encoded by Ac004, Ac102, and Ac152 have not been characterized. We found that when driven by foreign promoters, ie-1, pe38, and Ac004 had to be expressed prior to Ac102 or he65 for nuclear G-actin to accumulate and that expression of Ac152 was no longer required. These results and others suggested that the product of Ac152 was a transactivator (directly or indirectly) of both Ac102 and he65 and that recruitment of G-actin to the nucleus was a temporally regulated process. Determining the functions of each of the six AcMNPV gene products with respect to our assay should provide valuable clues to basic cellular mechanisms of actin regulation and how AcMNPV infection affects them.
Creating hybrid genes by homologous recombination.
Recombination of homologous genes is a powerful mechanism for generating sequence diversity, and can be applied to protein analysis and directed evolution. In vitro recombination methods such as DNA shuffling are very flexible and can give hybrid genes with multiple crossovers; they have been used extensively to evolve proteins with improved and novel properties. In vivo recombination in both E. coli and yeast is greatly enhanced by double-strand breaks; for E. coli, mutant strains are often necessary to obtain high efficiency. Intra- and inter-molecular recombination in vivo have distinct features; both give hybrids with one or two crossovers, and have been used to study structure-function relationships of many proteins. Recently in vivo recombination has been used to generate diversity for directed evolution, creating a large phage display antibody library. Recombination methods will become increasingly useful in light of the explosion in genomic sequence data and potential for engineered proteins.
Heterotachy in mammalian promoter evolution.
We have surveyed the evolutionary trends of mammalian promoters and upstream sequences, utilising large sets of experimentally supported transcription start sites (TSSs). With 30,969 well-defined TSSs from mouse and 26,341 from human, there are sufficient numbers to draw statistically meaningful conclusions and to consider differences between promoter types. Unlike previous smaller studies, we have considered the effects of insertions, deletions, and transposable elements as well as nucleotide substitutions. The rate of promoter evolution relative to that of control sequences has not been consistent between lineages nor within lineages over time. The most pronounced manifestation of this heterotachy is the increased rate of evolution in primate promoters. This increase is seen across different classes of mutation, including substitutions and micro-indel events. We investigated the relationship between promoter and coding sequence selective constraint and suggest that they are generally uncorrelated. This analysis also identified a small number of mouse promoters associated with the immune response that are under positive selection in rodents. We demonstrate significant differences in divergence between functional promoter categories and identify a category of promoters, not associated with conventional protein-coding genes, that has the highest rates of divergence across mammals. We find that evolutionary rates vary both on a fine scale within mammalian promoters and also between different functional classes of promoters. The discovery of heterotachy in promoter evolution, in particular the accelerated evolution of primate promoters, has important implications for our understanding of human evolution and for strategies to detect primate-specific regulatory elements.
Construction of a bacterial artificial chromosome library.
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pFOS-LA: a modified vector for production of random shear fosmid libraries.
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Separate-component-stabilization system for protein and DNA production without the use of antibiotics.
Plasmid instability is a significant concern in the industrial utilization of microorganisms for protein or DNA production. Here we report on the development of a new and highly effective stabilization system based on the use of the ccd antidote/poison genes. For the first time, we separated the antidote gene from the poison gene: localizing the former in the plasmid and integrating the latter in the bacterial chromosome. We show that this separate-component-stabilization (SCS) strategy: (i) allows for perfect stabilization without the use of antibiotics; (ii) increases three to five times the recombinant protein production levels; and (iii) does not require any specific modification of the protein production process or culture medium. We illustrate that point by using the classical T7 promotor (i.e., used in most expression systems). Finally, we demonstrate that the SCS system increases by five the yield in DNA production, a result especially important for the design and production of gene therapy constructs void of any antibiotic resistance gene.
[Reverse genetics of negative-strand RNA viruses].
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[Establishment of Sendai virus gene manipulation and its applications].
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Understanding the role of dietary components on atherosclerosis using genetic engineered mouse models.
The generation by genetic engineering of two murine models to investigate atherosclerosis, such as the apoE- and LDLr- deficient mice, is providing an extraordinaire knowledge of the effect of different nutrients on this complex disease. The present revision provides a comprehensive overview of the advances in this field that point to a remarkable complexity. While some controversies over puzzling results could be explained invoking potential nutrient interactions or different food sources of nutrients, it also appears that other factors such as sex, genetic background or immunological status are emerging as generators of differential responses to nutrients during the atherosclerotic process.
Primer on medical genomics. Part X: Gene therapy.
Gene therapy is defined as any therapeutic procedure in which genes are intentionally introduced into human somatic cells. Both preclinical and clinical gene therapy research have been progressing rapidly during the past 15 years; gene therapy is now a highly promising new modality for the treatment of numerous human disorders. Since the first clinical test of gene therapy in 1989, more than 600 gene therapy protocols have been approved, and more than 3000 patients have received gene therapy. However, at the time of writing this article, no gene therapy products have been approved for clinical use. This article explains the potential clinical scope of gene therapy and the underlying pharmacological principles, describes some of the major gene transfer systems (or vectors) that are used to deliver genes to their target sites, and discusses the various strategies for controlling expression of therapeutic transgenes. Safety issues regarding clinical use of gene therapy are explored, and the most important technical challenges facing this field of research are highlighted. This review should serve as an introduction to the subject of gene therapy for clinician investigators, physicians and medical scientists in training, practicing clinicians, and other students of medicine.
The magisterium and modern genetics.
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Genetics and theology: a complementarity?
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Raising the social yield of research: challenge facing biotechnology.
The biological revolution is the latest in a series of scientific revolutions that have ushered in a new era in physics, chemistry, industry, and composite materials. The latest instruments of life - bioengineering for and by the living - challenge mankind with a number of fundamental questions, while offering an unprecedented series of opportunities. Alternative biotechnologies, biological agriculture for the environment, genomics applied to preventive medicine, genetic engineering are just a few of the benefits mankind may reap from bioengineering. Modern society demands more knowledge, and it is up to public authorities to invest in outreach programs to make public the latest scientific activities and findings. In the case of biotechnologies, it is particularly true that the product of research is not complete until its benefits are returned to society.
[An exhaustive overproduction of bacterial proteins].
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[Experimental animals in the era of genomics].
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