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At least 721 records · Page 40Linked to original sources
PubMed2000

Cytochromes P450 for engineering herbicide tolerance.

In recent years, genome sequencing has revealed that cytochromes P450 (P450s) constitute the largest family of enzymatic proteins in higher plants. P450s are mono-oxygenases that insert one atom of oxygen into inert hydrophobic molecules to make them more reactive and hydrosoluble. Besides their physiological functions in the biosynthesis of hormones, lipids and secondary metabolites, P450s help plants to cope with harmful exogenous chemicals including pesticides and industrial pollutants, making them less phytotoxic. The recovery of an increasing number of plant P450 genes in recombinant form has enabled their use in experimentation, which has revealed their extraordinary potential for engineering herbicide tolerance, biosafening, bioremediation and green chemistry.

Cytochrome P-450 Enzyme System↗
PubMed2025

New retron systems from environmental bacteria identify triggers of anti-phage defense and expand tools for genome editing.

Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase (RT), a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase (RT), noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we identify seven new retron systems, each isolated from a different host bacterium. We characterize DNA production by these retrons and test their ability to defend against a panel of Escherichia coli phages. We find that two of these retrons are disrupted by other elements, in one case a group II intron and in another a separate defense system, yet both retrons still produce RT-DNA. For two other retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.

Gene Editing↗
PubMed1976

[Successes and prospects for genetic engineering].

The review of literature (1970-1976) on problems of gene engineering is given. Gene engineering is pointed out to be a new method of modern biology and a new page of modern molecular genetics. Gene engineering detected a real possibility of artificial creating living hybrid organisms, i.e. constructing functional recombinant DNA molecules according to a project of investigator, but not to possibilities of crossing. The determination of gene engineering (in contrast with genetical engineering) is given in the first division of the article. Genetical engineering is a construction of hybrid organisms on the basis of recombination between non-homologous chromosomes cy crossing. Genetical engineering is based on sex crossing, thus the application of this method is restricted by crossability (i.e. experiments in vivo), which possibilities are determined by taxonomical limits. Gene engineering is a new method of operating directly with genes. It permits constructing in vitro any hybrid genomes desirable. There is no limits of combining ability for gene engineering. Three main stages of constructing hybrid genomes should be taken into account for the proper determination of gene engineering as a method of genome constructing: 1) the gene isolation; 2) their cross-linking in vitro; 3) the transfer of hybrid DNA into recipient cell or its genome. The cardinal stage of gene engineering is the construction of hybrid DNA, cross-linking any initial DNAs from any remote animals, plants and bacteria. All the methods known of gene isolation are described. The chemical method of gene isolation is based on that case, when DNA of some gene differs in its physico-chemical characteristics from total DNA, for example, DNAs of genes coding ribosomal RNAs or sea urchine histone DNA. Isolation of promotors and operators using DNA dependent RNA polymerase, which recognizes promotors, repressor and operator DNA, should also be considered as the chemical method of gene isolation. Restrictase method, which is also well known, is convenuent because the restricts have long enough sticky ends, which is important for the following gene cross-linking. The method of total restriction, reported by Lederberg et al. and Debabov et al., is described. The phage method (in particular, Shimada method) is given, permitting the direct integration of lambda phage into a number of sites of Escherichia coli chromosome. Gene engineering method of gene isolation is mentioned, in particular, the data of Kameron et al. on hybrid phages carrying DNA ligase gene, and Clark a. Carbon on hybrid plasmids carrying triptophane and arabinose operons genes. These methods are called "shot gun". Methods of gene isolation from higher organisms are less developed. A method of gene isolation using so called colony hybridization (according to Grünstein and Hognes) is also given...

Bacteriophages↗
PubMed2004

Extensive alterations in DNA methylation and transcription in rice caused by introgression from Zizania latifolia.

It has been demonstrated that insertion of foreign DNA into mammalian genome can profoundly alter the patterns of DNA methylation and transcription of the host genome. Introgression of alien DNA into plant genomes through sexual crossing and genetic engineering are commonly used in breeding, but it is not known if plant genomes have similar responses to alien DNA introgression as those of animals. Two stable rice lines with introgression from wild rice, Zizania latifolia, were analyzed for patterns of cytosine DNA methylation and transcription of a set of selected sequences, including cellular genes and transposable element (TE)-related DNA segments. In 21 of the 30 studied sequences, marked changes in DNA methylation and/or transcription were observed compared with those of the rice parent. In all analyzed sequences, the absence of Zizania homologues in the introgression lines was confirmed. No change in DNA methylation and expression patterns was detected in randomly selected individuals of the rice parent nor in two sibling lines without introgressed Zizania DNA. The changed methylation patterns in both introgression lines were stably maintained in all five randomly sampled individuals of a given line, as well as in selfed progenies of the lines. Changed patterns in methylation and expression were also found in an independently produced asymmetric somatic nuclear hybrid (SH6) of rice and Z. latifolia that involves a different rice genotype but also contains a small amount of Z. latifolia DNA integrated into the rice genome. Thus, we have demonstrated that alien DNA introgression into a plant genome can induce extensive alterations in DNA methylation and transcription of both cellular genes and TE-related DNA segments in a genotype-independent manner.

Blotting, Southern↗
PubMed2006

A recombineering pipeline for functional genomics applied to Caenorhabditis elegans.

We present a new concept in DNA engineering based on a pipeline of serial recombineering steps in liquid culture. This approach is fast, straightforward and facilitates simultaneous processing of multiple samples in parallel. We validated the approach by generating green fluorescent protein (GFP)-tagged transgenes from Caenorhabditis briggsae genomic clones in a multistep pipeline that takes only 4 d. The transgenes were engineered with minimal disturbance to the natural genomic context so that the correct level and pattern of expression will be secured after transgenesis. An example transgene for the C. briggsae ortholog of lin-59 was used for ballistic transformation in Caenorhabditis elegans. We show that the cross-species transgene is correctly expressed and rescues RNA interference (RNAi)-mediated knockdown of the endogenous C. elegans gene. The strategy that we describe adapts the power of recombineering in Escherichia coli for fluent DNA engineering to a format that can be directly scaled up for genomic projects.

Animals↗
PubMed2002

Transcription and replication initiate at separate sites on the vesicular stomatitis virus genome.

The RNA-dependent RNA polymerase of the nonsegmented negative-strand RNA viruses carries out two distinct RNA synthetic processes: transcription of monocistronic, capped, and polyadenylated subgenomic messenger RNAs, and replication by means of the synthesis of a full-length positive-sense copy of the genome. The template for both processes is the negative-sense genomic RNA tightly encapsidated by the viral nucleocapsid protein. By applying UV transcriptional mapping to engineered variants of vesicular stomatitis virus, we discovered that, in infected cells, transcription and replication are controlled by initiation at different positions on the viral genome.

Animals↗
PubMed2005

Refactoring bacteriophage T7.

Natural biological systems are selected by evolution to continue to exist and evolve. Evolution likely gives rise to complicated systems that are difficult to understand and manipulate. Here, we redesign the genome of a natural biological system, bacteriophage T7, in order to specify an engineered surrogate that, if viable, would be easier to study and extend. Our initial design goals were to physically separate and enable unique manipulation of primary genetic elements. Implicit in our design are the hypotheses that overlapping genetic elements are, in aggregate, nonessential for T7 viability and that our models for the functions encoded by elements are sufficient. To test our initial design, we replaced the left 11,515 base pairs (bp) of the 39,937 bp wild-type genome with 12,179 bp of engineered DNA. The resulting chimeric genome encodes a viable bacteriophage that appears to maintain key features of the original while being simpler to model and easier to manipulate. The viability of our initial design suggests that the genomes encoding natural biological systems can be systematically redesigned and built anew in service of scientific understanding or human intention.

Algorithms↗
PubMed2002

Engineered viruses to select genes encoding secreted and membrane-bound proteins in mammalian cells.

We have developed a functional genomics tool to identify the subset of cDNAs encoding secreted and membrane-bound proteins within a library (the 'secretome'). A Sindbis virus replicon was engineered such that the envelope protein precursor no longer enters the secretory pathway. cDNA fragments were fused to the mutant precursor and expression screened for their ability to restore membrane localization of envelope proteins. In this way, recombinant replicons were released within infectious viral particles only if the cDNA fragment they contain encodes a secretory signal. By using engineered viral replicons to selectively export cDNAs of interest in the culture medium, the methodology reported here efficiently filters genetic information in mammalian cells without the need to select individual clones. This adaptation of the 'signal trap' strategy is highly sensitive (1/200 000) and efficient. Indeed, of the 2546 inserts that were retrieved after screening various libraries, more than 97% contained a putative signal peptide. These 2473 clones encoded 419 unique cDNAs, of which 77% were previously annotated. Of the 94 cDNAs encoding proteins of unknown function, 24% either had no match in databases or contained a secretory signal that could not be predicted from electronic data.

Animals↗
PubMed2005

Deciphering transcriptional regulatory elements that encode specific cell cycle phasing by comparative genomics analysis.

Transcriptional regulation is a major tier in the periodic engine that mobilizes cell cycle progression. The availability of complete genome sequences of multiple organisms holds promise for significantly improving the specificity of computational identification of functional elements. Here, we applied a comparative genomics analysis to decipher transcriptional regulatory elements that control cell cycle phasing. We analyzed genome-wide promoter sequences from 12 organisms, including worm, fly, fish, rodents and human, and identified conserved transcriptional modules that determine the expression of genes in specific cell cycle phases. We demonstrate that a canonical E2F signal encodes for expression highly specific to the G1/S phase, and that a cis-regulatory module comprising CHR-NF-Y elements dictates expression that is restricted to the G2 and G2/M phases. B-Myb binding site signatures occur in many of the CHR-NF-Y target genes, suggesting a specific role for this triplet in the regulation of the cell cycle transcriptional program. Remarkably, E2F signals are conserved in promoters of G1/S genes in all organisms from worm to human. The CHR-NF-Y module is conserved in promoters of G2/M regulated genes in all analyzed vertebrates. Our results reveal novel modules that determine specific cell cycle phasing, and identify their respective putative target genes with remarkably high specificity.

Animals↗
PubMed2006

Construction of an infectious clone of canine herpesvirus genome as a bacterial artificial chromosome.

Canine herpesvirus (CHV) is an attractive candidate not only for use as a recombinant vaccine to protect dogs from a variety of canine pathogens but also as a viral vector for gene therapy in domestic animals. However, developments in this area have been impeded by the complicated techniques used for eukaryotic homologous recombination. To overcome these problems, we used bacterial artificial chromosomes (BACs) to generate infectious BACs. Our findings may be summarized as follows: (i) the CHV genome (pCHV/BAC), in which a BAC flanked by loxP sites was inserted into the thymidine kinase gene, was maintained in Escherichia coli; (ii) transfection of pCHV/BAC into A-72 cells resulted in the production of infectious virus; (iii) the BAC vector sequence was almost perfectly excisable from the genome of the reconstituted virus CHV/BAC by co-infection with CHV/BAC and a recombinant adenovirus that expressed the Cre recombinase; and (iv) a recombinant virus in which the glycoprotein C gene was deleted was generated by lambda recombination followed by Flp recombination, which resulted in a reduction in viral titer compared with that of the wild-type virus. The infectious clone pCHV/BAC is useful for the modification of the CHV genome using bacterial genetics, and CHV/BAC should have multiple applications in the rapid generation of genetically engineered CHV recombinants and the development of CHV vectors for vaccination and gene therapy in domestic animals.

Animals↗
PubMed2005

Unraveling Brucella genomics and pathogenesis in immunocompromised IRF-1-/- mice.

PROBLEM: Brucellosis causes abortion in domestic animals and Malta fever in humans. Comparison of Brucella species genomes may reveal potential virulence mechanisms. Engineering bioluminescent Brucella would permit monitoring bacterial dissemination. METHOD OF STUDY: Microarray of the B. melitensis genome allowed comparison of gene content from six Brucella species. Bioluminescent B. melitensis strains were developed using transposon mutagenesis permitting the study of pathogenic Brucella in mice. Monitoring bacterial dissemination as well as organ localization permits evaluating the role of genes and genomic islands in mutant bacteria. RESULTS: Comparative genomic analysis revealed 217 ORFs altered in five Brucella species and were often found in islands. Bioluminescent bacteria disseminated from the injection site to liver, spleen, inguinal lymph nodes, testes and submanibular region. CONCLUSIONS: Genomic islands contribute to Brucella pathogenicity. Biophotonic imaging suggests that Brucella dissemination in mice parallels acute and chronic infections of humans.

Animals↗
PubMed2005

Site-specific DNA recombinases as instruments for genomic surgery.

Site-specific DNA recombinases can "cut and paste" DNA. For example, they can promote excision of specific DNA segments or insertion of new DNA segments in specific places. However, natural recombinases act only at their cognate recombination sites, so current applications are limited to genetically modified organisms in which these sites have been introduced into the genome. Transposases also catalyze DNA rearrangements; they promote insertion of specific DNA sequences but at nonspecific locations. Applicability of site-specific recombinases and transposases in experimental genetics, biotechnology, and gene therapy would be much wider if they could be re-engineered so as to act specifically at chosen sequences within an organism's natural genome. This review will discuss progress towards the creation of such "designer" recombinases.

Base Sequence↗