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Results for “Genome engineering”

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At least 271 records · Page 15Linked to original sources

PubMed1993

Genetic manipulation of negative-strand RNA virus genomes.

Negative-strand RNA viruses have been refractory to genetic manipulation using recombinant DNA techniques. Recently, new techniques were developed that allowed the rescue of synthetic RNA molecules into influenza A viruses and, subsequently, into other negative-strand RNA viruses. These techniques are presently being used to study the molecular biology of these viruses. Questions concerning cis- and trans-acting elements that are involved in transcription and replication of negative-sense RNA viral genomes can now be addressed with reverse genetic approaches. Further development of this methodology has enabled the construction--by recombinant DNA techniques--of influenza A viruses that contain altered genomes. The phenotypic characteristics and possible applications of these novel transfectant viruses are also discussed.

Base Sequence↗

PubMed2006

Targeted gene mutation in Phytophthora spp.

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosisinducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.

Algal Proteins↗

PubMed2000

Site-specific integration of targeted DNA into animal cell genomes.

Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells. The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element. When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette. These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units.

Animals↗

PubMed2003

Myxobacteria: proficient producers of novel natural products with various biological activities--past and future biotechnological aspects with the focus on the genus Sorangium.

Myxobacteria are gram-negative bacteria which are most noted for their ability to form fruiting bodies upon starvation. Within the last two decades, they increasingly gained attention as producers of natural products with biological activity. Here, recent and future biotechnological research on certain key myxobacteria and on their ability to produce natural products is reviewed with the focus on the production of myxovirescin, soraphen and epothilone. Aspects of product improvement and yield as well as statistics regarding secondary metabolite formation are discussed. Future research will deal with the exploitation of the biosynthetic potential of the myxobacteria, for example via the isolation of new myxobacterial species with different physiological properties. Additionally, the genetic potential of myxobacteria to form natural products can be exploited by the identification and activation of biosynthetic gene clusters. These can be found frequently within their genomes, which is shown by the analysis of the unfinished genomes of Myxococcus xanthus and Sorangium cellulosum. The current status of the S. cellulosum functional genome project with model strain So ce56 is discussed.

Biotechnology↗

PubMed2005

Biotechnology and the domestication of forest trees.

Wood is one of the major renewable materials. To compensate for the ever-increasing demand for wood and to reduce pressure on native forests, more wood of higher quality will need to be produced on less land by planting highly productive trees. Biotechnology has shown great promise for forest tree improvement and over the past 10 years this field has flourished. Not only has the potential of transgenic trees with optimized yield and quality traits been demonstrated in field trials, but progress in genetical genomics and association genetics promise quantum leaps forward for tree improvement.

Alleles↗

PubMed2005

A novel insertion site inside the potyvirus P1 cistron allows expression of heterologous proteins and suggests some P1 functions.

The P1 cistron encodes the first and most variable part of the polyprotein of potyviruses. A site tolerant to a pentapeptide insertion at the N-terminus of Potato virus A P1 (Genome Res. 12, 584-594) was used to express heterologous proteins (insertions up to 783 nucleotides) with or without flanking new proteolytic sites. Aequorea victoria green fluorescent protein (GFP) accumulated to high levels when proteolytically released from P1 and showed strong fluorescence in leaves systemically infected with vector virus. Deletions in GFP and adjacent viral sequences emerged 2-4 weeks after infection, revealing putative recombination hot spots. The inserts in P1 diminished infectivity host-specifically, reduced virus accumulation in protoplasts and systemically infected leaves, alleviated symptoms and reduced accumulation of mRNA and HCpro in cis in a virus-free system. This heterologous protein expression site is the first within a protein-encoding cistron and the third in the genome of potyviruses.

Amino Acid Sequence↗

PubMed2006

Deletion of BCY1 from the Saccharomyces cerevisiae genome is semidominant and induces autolytic phenotypes suitable for improvement of sparkling wines.

Autolysis of Saccharomyces cerevisiae is the main source of molecules that contribute to the quality of sparkling wines made by the traditional method. In this work the possibility of accelerating this slow process in order to improve the quality of sparkling wines by using genetically engineered wine yeast strains was explored. The effect of partial or total deletion of BCY1 (which encodes a regulatory subunit of cAMP-dependent protein kinase A) in haploid and diploid (heterozygous and homozygous) yeast strains was studied. We proved that heterozygous strains having partial or complete BCY1 deletions have a semidominant phenotype for several of the properties studied, including autolysis under simulated second-fermentation conditions, in contrast to previously published reports describing mutations in BCY1 as recessive. Considering the degree of autolysis, ethanol tolerance, and technical feasibility, we propose that deletion of the 3' end of the open reading frame of a single copy of BCY1 is a way to improve the quality of sparkling wines.

Cyclic AMP↗

PubMed2006

Targeted engineering of the Caenorhabditis elegans genome following Mos1-triggered chromosomal breaks.

The Drosophila element Mos1 is a class II transposon, which moves by a 'cut-and-paste' mechanism and can be experimentally mobilized in the Caenorhabditis elegans germ line. Here, we triggered the excision of identified Mos1 insertions to create chromosomal breaks at given sites and further manipulate the broken loci. Double-strand break (DSB) repair could be achieved by gene conversion using a transgene containing sequences homologous to the broken chromosomal region as a repair template. Consequently, mutations engineered in the transgene could be copied to a specific locus at high frequency. This pathway was further characterized to develop an efficient tool--called MosTIC--to manipulate the C. elegans genome. Analysis of DSB repair during MosTIC experiments demonstrated that DSBs could also be sealed by end-joining in the germ line, independently from the evolutionarily conserved Ku80 and ligase IV factors. In conjunction with a publicly available Mos1 insertion library currently being generated, MosTIC will provide a general tool to customize the C. elegans genome.

Animals↗

PubMed2006

Systems biology as a foundation for genome-scale synthetic biology.

As the ambitions of synthetic biology approach genome-scale engineering, comprehensive characterization of cellular systems is required, as well as a means to accurately model cell-scale molecular interactions. These requirements are coincident with the goals of systems biology and, thus, systems biology will become the foundation for genome-scale synthetic biology. Systems biology will form this foundation through its efforts to reconstruct and integrate cellular systems, develop the mathematics, theory and software tools for the accurate modeling of these integrated systems, and through evolutionary mechanisms. As genome-scale synthetic biology is so enabled, it will prove to be a positive feedback driver of systems biology by exposing and forcing researchers to confront those aspects of systems biology which are inadequately understood.

Biology↗

PubMed2002

Phytochemicals in plants: genomics-assisted plant improvement for nutritional and health benefits.

Plants are an important source of essential nutrients and health-beneficial components that are crucial for human life. Because the intake of these phytochemicals is not always adequate, the resources of plant biotechnology are being used to enhance the nutritional quality of our plant-based food supply. Various improvement strategies are feasible, depending on whether the phytochemical target is a major or minor constituent. Recent efforts in gene discovery and functional genomics are providing the necessary understanding to develop and evaluate different approaches to manipulate phytochemical composition.

Diet↗

PubMed2000

Genetic modification of mammalian genome at chromosome level.

The review is concerned with a progress in genetic modification of a mammalian genome in vitro and in vivo at chromosomal level. Recently three new approaches for the chromosome biotechnology have been developed: Using Cre/loxP-system a researcher is able to produce targeted rearrangements of whole chromosomes or their segments or particular genes within the genome, and therefore to modify the set, position and copy number of the endogenous elements of the genome. Mammalian artificial chromosomes (MACs) provide a possibility to introduce into genome relatively large segments of alien chromosome material, either artificially constructed or derived from the genome of different species. Using ES-somatic cell hybrids allows to transfer whole chromosomes or their fragments between different genomes within and between species. Advantages and limitations of these approaches are discussed.

Animals↗

PubMed2006

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Biopharmaceutics↗

PubMed2000

Milking the genome for profit.

Despite recent accomplishments, significant challenges remain before cloning can become a routine part of transgenic animal production.

Animals↗

PubMed2000

Genomes and form. The case for teleomorphic recursivity.

The genotype-phenotype (genome-form) distinction is considered by many to be fundamental to modern evolutionary thinking. Indeed, the premises that: DNA solely constitutes the genotype; that the phenotype is a transient product of the genotype, with the latter not only describing, but also implementing the construction of the former; and that the constructed materials and systems of the cell have no impact on the genotype, have become dogmas. Yet a vast body of data from molecular genetics reveals that cellular systems, directly and indirectly, alter the genome. Some of these data are reviewed. Proteins can influence mutations along the chromosomes, heritably modify the information content of DNA sequences, and, in some instances, reorganize the germline or somatic genome via DNA engineering pathways. These data suggest that the constructed (proteins, chromatin arrays, and metabolic pathways) has an important role in shaping the descriptor. Insofar as it is biochemically possible for states adopted by cellular structures to be stabilized and eventually memorized by engineering chromosomes, semantic closure can be transcended--meaning can be transferred from the domain of form to the genome, and this presumably ongoing process is termed teleomorphic recursivity. Throughout the paper, I implicitly argue that the genome-form partition is strictly a formal one, with no deeply material basis.

Biological Evolution↗

PubMed2004

Transgene inheritance in plants.

The patterns of transgene inheritance in plants and the possible explanations for non-Mendelian transmission are reviewed. The non-Mendelian inheritance of a transgene has been recorded with a frequency between 10% and 50% in transgenic plants produced either by Agrobacterium-mediated transformation or through particle bombardment. Different effects such as deletion, duplication, rearrangement, repeated sequence recombination as well as gene interaction have been observed for transgenic loci. The nature of the recipient genome, nature of the transgene and the interactions between them seem to contribute to the non-Mendelian segregation of transgenes.

Gene Deletion↗

PubMed2003

Out of the woods: forest biotechnology enters the genomic era.

Trees represent a unique life form of upmost importance for mankind, as these organisms have developed a perennial lifestyle that produces the majority of terrestrial biomass. The difference between trees and annual plants is one of the main arguments behind the effort to sequence the entire genome of the poplar tree. This initiative is being backed up with a full-scale functional genomics effort on trees that will set a completely new agenda for forest research.

Agriculture↗

PubMed2001

Genetic methods and strategies for secondary metabolite yield improvement in actinomycetes.

The foundation for any strain improvement program is efficient random chemically-induced mutagenesis coupled with highly reproducible fermentation and product assays. The broad spectrum of spontaneous mutations can be leveraged in some cases by direct selection of mutants with desired traits. Transposons containing outward-reading promoter activity might be used to enhance yields by inducing promoter fusions, disrupting negative regulatory elements, or disrupting genes involved in competing pathways. Transposons might also be used to identify and clone positive regulatory genes. As knowledge of the key elements in the fermentation process and secondary metabolite biosynthesis grows, gene cloning and targeted gene duplication becomes an important tool. Duplication of genes involved in rate limiting steps can be achieved to improve product yields by inserting the desired gene(s) into neutral sites in the chromosome by homologous recombination or by site-specific integration. The probabilities and frequencies of success of the molecular genetic approaches should increase with an increasing knowledge of key factors influencing product yields. This knowledge can be broadened dramatically by a combination of structural and functional genomics, gene disruption analysis and metabolic modeling. Protoplast fusion can be used to recombine beneficial traits from any of the other approaches.

Actinomycetales↗

PubMed2003

New scientific paradigms for probiotics and prebiotics.

The inaugural meeting of the International Scientific Association for Probiotics and Prebiotics (ISAPP) was held May 3 to May 5 2002 in London, Ontario, Canada. A group of 63 academic and industrial scientists from around the world convened to discuss current issues in the science of probiotics and prebiotics. ISAPP is a non-profit organization comprised of international scientists whose intent is to strongly support and improve the levels of scientific integrity and due diligence associated with the study, use, and application of probiotics and prebiotics. In addition, ISAPP values its role in facilitating communication with the public and healthcare providers and among scientists in related fields on all topics pertinent to probiotics and prebiotics. It is anticipated that such efforts will lead to development of approaches and products that are optimally designed for the improvement of human and animal health and well being. This article is a summary of the discussions, conclusions, and recommendations made by 8 working groups convened during the first ISAPP workshop focusing on the topics of: definitions, intestinal flora, extra-intestinal sites, immune function, intestinal disease, cancer, genetics and genomics, and second generation prebiotics.

Animals↗