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PubMedDate not supplied

Prospects for the precise engineering of plant genomes by homologous recombination.

The targeting of chromosomal genes via homologous recombination (HR) is an essential tool of reverse genetics as applied for the functional assay of genes within complex genomes. However, in higher plants, foreign DNA integrates almost exclusively at random, non-homologous sites. A variety of environmental parameters known to influence levels of HR do not increase targeting frequencies when combined in gene-targeting experiments. The identification of cellular factors that may control the level of chromosomal HR in plant somatic cells is required. Plant genes encoding proteins similar to those involved in HR in other organisms can be found in the expanding sequence databases. Evidence for evolutionary conservation should help to decipher mechanisms of plant HR and possibly detect limiting factors. At present, however, only one genetic locus influencing levels of chromosomal recombination in plants has been well defined. Here we summarise current knowledge of HR and the status of gene targeting (GT) in plants, focusing on genetic approaches to molecular factors regulating HR levels.

DNA Damage↗
PubMed2003

Efficient cloning and engineering of entire mitochondrial genomes in Escherichia coli and transfer into transcriptionally active mitochondria.

We have devised an efficient method for replicating and stably maintaining entire mitochondrial genomes in Escherichia coli and have shown that we can engineer these mitochondrial DNA (mtDNA) genome clones using standard molecular biological techniques. In general, we accomplish this by inserting an E.coli replication origin and selectable marker into isolated, circular mtDNA at random locations using an in vitro transposition reaction and then transforming the modified genomes into E.coli. We tested this approach by cloning the 16.3 kb mouse mitochondrial genome and found that the resulting clones could be engineered and faithfully maintained when we used E.coli hosts that replicated them at moderately low copy numbers. When these recombinant mtDNAs were replicated at high copy numbers, however, mtDNA sequences were partially or fully deleted from the original clone. We successfully electroporated recombinant mouse mitochondrial genomes into isolated mouse mitochondria devoid of their own DNA and detected robust in organello RNA synthesis by RT-PCR. This approach for modifying mtDNA and subsequent in organello analysis of the recombinant genomes offers an attractive experimental system for studying many aspects of vertebrate mitochondrial gene expression and is a first step towards true in vivo engineering of mammalian mitochondrial genomes.

Animals↗
PubMed2006

Thematic review series: systems biology approaches to metabolic and cardiovascular disorders. Multi-organ whole-genome measurements and reverse engineering to uncover gene networks underlying complex traits.

Together with computational analysis and modeling, the development of whole-genome measurement technologies holds the potential to fundamentally change research on complex disorders such as coronary artery disease. With these tools, the stage has been set to reveal the full repertoire of biological components (genes, proteins, and metabolites) in complex diseases and their interplay in modules and networks. Here we review how network identification based on reverse engineering, as applied to whole-genome datasets from simpler organisms, is now being adapted to more complex settings such as datasets from human cell lines and organs in relation to physiological and pathological states. Our focus is on the use of a systems biological approach to identify gene networks in coronary atherosclerosis. We also address how gene networks will probably play a key role in the development of early diagnostics and treatments for complex disorders in the coming era of individualized medicine.

Animals↗
PubMed2005

Integrating genomics and metabolomics for engineering plant metabolic pathways.

Plant metabolites are characterized by an enormous chemical diversity, every plant having its own complex set of metabolites. This variety poses analytical challenges, both for profiling multiple metabolites in parallel and for the quantitative analysis of selected metabolites. We are only just starting to understand the roles of these metabolites, many of them being involved in adaptations to specific ecological niches and some finding beneficial use (e.g. as pharmaceuticals). Spectacular advances in plant metabolomics offer new possibilities, together with the aid of systems biology, to explore the extraordinary complexity of the plant biochemical capacity. State-of-the art genomics tools can be combined with metabolic profiling to identify key genes that could be engineered for the production of improved crop plants.

Arabidopsis↗
PubMedDate not supplied

Genetics, genomics and molecular medicine.

Genomics and the Human Genome Project are creating important changes in medical care and radiologic practice. Readers of this article will: Recall that deoxyribonucleic acid (DNA) is the information-containing molecule. Understand the relationship between DNA and protein synthesis. Learn that changes in DNA-encoded proteins produce pathogenesis. Distinguish between genomics and proteomics. Describe the Human Genome Project. Define and give examples of molecular medicine and its imaging applications. Discuss how genomics, genetic engineering and the Human Genome Project may influence radiologic practice.

Genetic Engineering↗
PubMed2003

Effects of a point mutation in the 3' end of the S genome segment of naturally occurring and engineered Bunyamwera viruses.

The genome of Bunyamwera virus (BUN) consists of three segments of single-stranded RNA of negative polarity. The smallest segment, S, encodes the N protein and a nonstructural protein called NSs. We recently described a mutant virus (BUNdelNSs) that does not express NSs but overexpresses N and grows to lower titres than wild-type (wt) BUN. Here we report a BUNdelNSs variant that expresses lower levels of N protein and grows to higher titres. Sequencing of the 3' and 5' termini of the BUNdelNSs S RNA segment and analysis using a minireplicon system show that the N overexpressing phenotype results from a single nucleotide substitution at position 16 in the 3' terminus. This mutation could also be detected in wtBUN populations, and was isolated by plaquing a 'wt' variant carrying the mutation. This variant was found to express increased N and NSs levels, and grew to lower titres than wtBUN.

3' Untranslated Regions↗
PubMed2003

High-throughput engineering of the mouse genome coupled with high-resolution expression analysis.

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive-negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5-1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.

Animals↗
PubMed2004

The ascent of man?

Anyone who cares about the moral and social implications of genomics, genetic engineering and biotechnology should read Michael J. Sandel's article, 'The Case Against Perfection', in the April 2004 issue of The Atlantic Monthly.

Biotechnology↗
PubMed2004

Use of genome-scale microbial models for metabolic engineering.

Metabolic engineering serves as an integrated approach to design new cell factories by providing rational design procedures and valuable mathematical and experimental tools. Mathematical models have an important role for phenotypic analysis, but can also be used for the design of optimal metabolic network structures. The major challenge for metabolic engineering in the post-genomic era is to broaden its design methodologies to incorporate genome-scale biological data. Genome-scale stoichiometric models of microorganisms represent a first step in this direction.

Algorithms↗
PubMed2001

The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

The 5.67-megabase genome of the plant pathogen Agrobacterium tumefaciens C58 consists of a circular chromosome, a linear chromosome, and two plasmids. Extensive orthology and nucleotide colinearity between the genomes of A. tumefaciens and the plant symbiont Sinorhizobium meliloti suggest a recent evolutionary divergence. Their similarities include metabolic, transport, and regulatory systems that promote survival in the highly competitive rhizosphere; differences are apparent in their genome structure and virulence gene complement. Availability of the A. tumefaciens sequence will facilitate investigations into the molecular basis of pathogenesis and the evolutionary divergence of pathogenic and symbiotic lifestyles.

Agrobacterium tumefaciens↗
PubMed2003

Identification of functional lox sites in the plastid genome.

Our objective was to test whether or not cyclization recombination (CRE), the P1 phage site-specific recombinase, induces genome rearrangements in plastids. Testing was carried out in tobacco plants in which a DNA sequence, located between two inversely oriented locus of X-over of P1 (loxP) sites, underwent repeated cycles of inversions as a means of monitoring CRE activity. We report here that CRE mediates deletions between loxP sites and plastid DNA sequences in the 3'rps12 gene leader (lox-rps12) or in the psbA promoter core (lox-psbA). We also observed deletions between two directly oriented lox-psbA sites, but not between lox-rps12 sites. Deletion via duplicated rRNA operon promoter (Prrn) sequences was also frequent in CRE-active plants. However, CRE-mediated recombination is probably not directly involved, as no recombination junction between loxP and Prrn could be observed. Tobacco plants carrying deleted genomes as a minor fraction of the plastid genome population were fertile and phenotypically normal, suggesting that the absence of deleted genome segments was compensated by gene expression from wild-type copies. The deleted plastid genomes disappeared in the seed progeny lacking CRE. Observed plastid genome rearrangements are specific to engineered plastid genomes, which contain at least one loxP site or duplicated psbA promoter sequences. The wild-type plastid genome is expected to be stable, even if CRE is present in the plastid.

3' Untranslated Regions↗
PubMed1998

Containment of herbicide resistance through genetic engineering of the chloroplast genome.

Glyphosate is a potent herbicide. It works by competitive inhibition of the enzyme 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSPS), which catalyzes an essential step in the aromatic amino acid biosynthetic pathway. We report the genetic engineering of herbicide resistance by stable integration of the petunia EPSPS gene into the tobacco chloroplast genome using the tobacco or universal vector. Southern blot analysis confirms stable integration of the EPSPS gene into all of the chloroplast genomes (5000-10,000 copies per cell) of transgenic plants. Seeds obtained after the first self-cross of transgenic plants germinated and grew normally in the presence of the selectable marker, whereas the control seedlings were bleached. While control plants were extremely sensitive to glyphosate, transgenic plants survived sprays of high concentrations of glyphosate. Chloroplast transformation provides containment of foreign genes because plastid transgenes are not transmitted by pollen. The escape of foreign genes via pollen is a serious environmental concern in nuclear transgenic plants because of the high rates of gene flow from crops to wild weedy relatives.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2000

Conservation and Evolution of the "Core Apoptotic Engine" Lesson from the Genome Comparison of Drosophila.

Genome comparison is the main approach to deduce regulatory network from genome sequence. Apoptotic network is one kind of typical regulatory networks. EGL1, CED3, CED4, CED9 and their homologous proteins play essential roles in apoptosis of C.elegans and mammals, and were regarded as the components of the"core apoptotic engine". But in fruit fly, Drosophila melanogaster, this network is incomplete. A series of bioinformatic analyses found the lost chains of "core apoptotic engine" by discovering two homologues of BCL2/CED9 and one of EGL1 in fruit fly genome sequences. These findings proved that the "core apoptotic engine" is indeed widely conserved among multicellular organisms and the evolutionary complexity of this network of Drosophila is between that of C.elegans and mammals.

Journal Article↗
PubMed1999

In vivo addition of poly(A) tail and AU-rich sequences to the 3' terminus of the Sindbis virus RNA genome: a novel 3'-end repair pathway.

Alphaviruses are mosquito-transmitted RNA viruses that cause important diseases in both humans and livestock. Sindbis virus (SIN), the type species of the alphavirus genus, carries a 11.7-kb positive-sense RNA genome which is capped at its 5' end and polyadenylated at its 3' end. The 3' nontranslated region (3'NTR) of the SIN genome carries many AU-rich motifs, including a 19-nucleotide (nt) conserved element (3'CSE) and a poly(A) tail. This 3'CSE and the adjoining poly(A) tail are believed to regulate the synthesis of negative-sense RNA and genome replication in vivo. We have recently demonstrated that the SIN genome lacking the poly(A) tail was infectious and that de novo polyadenylation could occur in vivo (K. R. Hill, M. Hajjou, J. Hu, and R. Raju, J. Virol. 71:2693-2704, 1997). Here, we demonstrate that the 3'-terminal 29-nt region of the SIN genome carries a signal for possible cytoplasmic polyadenylation. To further investigate the polyadenylation signals within the 3'NTR, we generated a battery of mutant genomes with mutations in the 3'NTR and tested their ability to generate infectious virus and undergo 3' polyadenylation in vivo. Engineered SIN genomes with terminal deletions within the 19-nt 3'CSE were infectious and regained their poly(A) tail. Also, a SIN genome carrying the poly(A) tail but lacking a part or the entire 19-nt 3'CSE was also infectious. Sequence analysis of viruses generated from these engineered SIN genomes demonstrated the addition of a variety of AU-rich sequence motifs just adjacent to the poly(A) tail. The addition of AU-rich motifs to the mutant SIN genomes appears to require the presence of a significant portion of the 3'NTR. These results indicate the ability of alphavirus RNAs to undergo 3' repair and the existence of a pathway for the addition of AU-rich sequences and a poly(A) tail to their 3' end in the infected host cell. Most importantly, these results indicate the ability of alphavirus replication machinery to use a multitude of AU-rich RNA sequences abutted by a poly(A) motif as promoters for negative-sense RNA synthesis and genome replication in vivo. The possible roles of cytoplasmic polyadenylation machinery, terminal transferase-like enzymes, and the viral polymerase in the terminal repair processes are discussed.

3' Untranslated Regions↗
PubMed2026

Distinct YY dinucleotide periodicity in adeno-associated virus DNA.

Dinucleotide periodicity is a hallmark of genome organization, yet its role in single-stranded (ss)DNA viruses remains poorly understood. Here, we systematically analyzed dinucleotide spacing patterns in adeno-associated virus (AAV) genomes and other viruses. Across 13 primate AAV serotypes, we identified a pronounced and highly conserved ∼15-bp periodicity specific to pyrimidine-pyrimidine (YY) dinucleotides and their reverse complements (RR). Comparative analyses across >25,000 viral sequences demonstrate that this 15-bp YY/RR periodicity is unique to the genus Dependoparvovirus and absent from other ssDNA viruses, satellite viruses, and helper viruses, which predominantly exhibit canonical ∼10- to 11-bp periodicities. Upon disruption of the YY/RR pattern using DNA family shuffling of AAV capsid genes, and subsequent iterative selection for viral production or cell entry, we found that the pattern is under positive selection. Selected sequences display increased periodicity alongside reduced sequence diversity, supporting a functional role for this genomic feature. Finally, engineered recombinant AAV genomes containing YY periodic motifs exhibit enhanced production and, for some designs, improved transduction efficiency, demonstrating that YY periodicity can modulate viral replication and infectivity. Our findings uncover a unique DNA-encoded signal in dependoparvoviruses that contributes to AAV fitness, expands our knowledge of virus biology, and has implications for vector engineering.

Dependovirus↗