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Results for “Genome engineering”

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At least 343 records · Page 19Linked to original sources

PubMed2006

Identification of genome-scale metabolic network models using experimentally measured flux profiles.

Genome-scale metabolic network models can be reconstructed for well-characterized organisms using genomic annotation and literature information. However, there are many instances in which model predictions of metabolic fluxes are not entirely consistent with experimental data, indicating that the reactions in the model do not match the active reactions in the in vivo system. We introduce a method for determining the active reactions in a genome-scale metabolic network based on a limited number of experimentally measured fluxes. This method, called optimal metabolic network identification (OMNI), allows efficient identification of the set of reactions that results in the best agreement between in silico predicted and experimentally measured flux distributions. We applied the method to intracellular flux data for evolved Escherichia coli mutant strains with lower than predicted growth rates in order to identify reactions that act as flux bottlenecks in these strains. The expression of the genes corresponding to these bottleneck reactions was often found to be downregulated in the evolved strains relative to the wild-type strain. We also demonstrate the ability of the OMNI method to diagnose problems in E. coli strains engineered for metabolite overproduction that have not reached their predicted production potential. The OMNI method applied to flux data for evolved strains can be used to provide insights into mechanisms that limit the ability of microbial strains to evolve towards their predicted optimal growth phenotypes. When applied to industrial production strains, the OMNI method can also be used to suggest metabolic engineering strategies to improve byproduct secretion. In addition to these applications, the method should prove to be useful in general for reconstructing metabolic networks of ill-characterized microbial organisms based on limited amounts of experimental data.

Computational Biology↗

PubMed2000

Novel trypanosomatid small nucleolar RNAs that guide methylation: their genome organization, expression and potential use to direct specific methylation on target RNA molecules.

Trypanosomatids are the causative agent of several major parasitic diseases including African trypanosomiasis, American trypanosomiasis, and leishmaniasis. These parasites possess unique RNA-processing mechanisms including trans-splicing of pre-mRNA and RNA editing of mitochondrial transcripts. In this study, we identified a trypanosomatid novel group of small nucleolar RNAs that belong to the box C/D snoRNA, which were shown to guide ribose methylation on rRNA. Three snoRNA genes were identified; snoRNA-2 carrying a single snoRNA and g2 and b2 coding for a single or multiple snoRNAs, respectively. Mapping of the methylation sites guided by snoRNA-2 using two different approaches suggest that snoRNA-2 has the potential to guide methylation on both 5.8S and 18S rRNAs. The trypanosomes follow the same guide-methylation rule established for yeast and for mammals. As a first attempt to change the methylation pattern of target RNAs, we generated transgenic parasites carrying the B2 and snoRNA-2, which were engineered to shift the methylation site on rRNA. Despite efficient expression of these tagged snoRNAs, the novel methylation site was not generated. However, efficient expression of tagged snoRNAs in transgenic parasites opens the possibility of engineering novel methylation sites on different target RNAs in vivo.

Animals↗

PubMed2003

Manipulation of the mouse genome: a multiple impact resource for drug discovery and development.

Few would deny that the pharmaceutical industry's investment in genomics throughout the 1990s has yet to deliver in terms of drugs on the market. The reasons are complex and beyond the scope of this review. The unique ability to manipulate the mouse genome, however, has already had a positive impact on all stages of the drug discovery process and, increasingly, on the drug development process too. We give an overview of some recent applications of so-called 'transgenic' mouse technology in pharmaceutical research and development. We show how genetic manipulation in the mouse can be employed at multiple points in the drug discovery and development process, providing new solutions to old problems.

Animals↗

PubMed2005

Two-step method for constructing unmarked insertions, deletions and allele substitutions in the yeast genome.

We describe three extensions of the method of site-specific genomic (SSG) mutagenesis. These three extensions of SSG mutagenesis were used to generate precise insertion, deletion, and allele substitution mutations in the genome of the budding yeast, Saccharomyces cerevisiae. These mutations are termed precise because no attached sequences (e.g., marker genes or recombination sites) are retained once the method is complete. Because the method is PCR-based, neither DNA cloning nor synthesis of long oligonucleotides is required. We demonstrated the efficacy of these methods by deleting an ORF, inserting the tandem affinity purification (TAP) tag, and replacing a wild-type allele with a mutant allele.

Alleles↗

PubMed2002

The MetaCyc Database.

MetaCyc is a metabolic-pathway database that describes 445 pathways and 1115 enzymes occurring in 158 organisms. MetaCyc is a review-level database in that a given entry in MetaCyc often integrates information from multiple literature sources. The pathways in MetaCyc were determined experimentally, and are labeled with the species in which they are known to occur based on literature references examined to date. MetaCyc contains extensive commentary and literature citations. Applications of MetaCyc include pathway analysis of genomes, metabolic engineering and biochemistry education. MetaCyc is queried using the Pathway Tools graphical user interface, which provides a wide variety of query operations and visualization tools. MetaCyc is available via the World Wide Web at http://ecocyc.org/ecocyc/metacyc.html, and is available for local installation as a binary program for the PC and the Sun workstation, and as a set of flatfiles. Contact metacyc-info@ai.sri.com for information on obtaining a local copy of MetaCyc.

Database Management Systems↗

PubMed2000

Group II introns designed to insert into therapeutically relevant DNA target sites in human cells.

Mobile group II intron RNAs insert directly into DNA target sites and are then reverse-transcribed into genomic DNA by the associated intron-encoded protein. Target site recognition involves modifiable base-pairing interactions between the intron RNA and a >14-nucleotide region of the DNA target site, as well as fixed interactions between the protein and flanking regions. Here, we developed a highly efficient Escherichia coli genetic assay to determine detailed target site recognition rules for the Lactococcus lactis group II intron Ll.LtrB and to select introns that insert into desired target sites. Using human immunodeficiency virus-type 1 (HIV-1) proviral DNA and the human CCR5 gene as examples, we show that group II introns can be retargeted to insert efficiently into virtually any target DNA and that the retargeted introns retain activity in human cells. This work provides the practical basis for potential applications of targeted group II introns in genetic engineering, functional genomics, and gene therapy.

Base Pairing↗

PubMed2005

Mouse phenogenomics: the fast track to "systems metabolism".

With the completion of the many genomes, genetics is positioned to meet physiology. In this review, we summarize the coming of "systems metabolism" in mammals through the use of the mouse, as a model system to study metabolism. Building on mouse genetics with increasingly sophisticated clinical and molecular phenotyping strategies has enabled scientists to now tackle complex biomedical questions, such as those related to the pathogenesis of the common metabolic disorders. The ultimate goal of such strategies will be to mimic human metabolism with the click of a mouse.

Animals↗

PubMed2006

Stable knock-down of vomeronasal receptor genes in transgenic Xenopus tadpoles.

Xenopus V2R (xV2R), a family of G-protein-coupled receptors with seven transmembrane domains, is expressed in the Xenopus vomeronasal organ (VNO). There are six subgroups of xV2R, one of which, xV2RE, is predominantly expressed in the VNO. To understand the function of xV2R during VNO development, we developed a new method to achieve stable siRNA-suppression of the V2RE genes by introducing siRNA expression transgenes into the genomes of unfertilized eggs. We found that some of the derived transgenic tadpoles lacked VNOs and that their olfactory epithelium was fused. With the exception of one tadpole, expression of xV2RE was not detected in morphologically abnormal mutant tadpoles, although the olfactory marker protein and the olfactory receptors were expressed. These results suggest that we successfully produced transgenic tadpoles in which xV2RE expression was stably suppressed by siRNA, and that xV2RE plays a role in the morphogenesis of olfactory organs.

Animals↗

PubMed2006

Genome-wide transcriptional dependence on TAF1 functional domains.

Transcription factor IID (TFIID) plays a central role in regulating the expression of most eukaryotic genes. Of the 14 TBP-associated factor (TAF) subunits that compose TFIID, TAF1 is one of the largest and most functionally diverse. Yeast TAF1 can be divided into four regions including a putative histone acetyltransferase domain and TBP, TAF, and promoter binding domains. Establishing the importance of each region in gene expression through deletion analysis has been hampered by the cellular requirement of TAF1 for viability. To circumvent this limitation we introduced galactose-inducible deletion derivatives of previously defined functional regions of TAF1 into a temperature-sensitive taf1ts2 yeast strain. After galactose induction of the TAF1 mutants and temperature-induced elimination of the resident Taf1ts2 protein, we examined the properties and phenotypes of the mutants, including their impact on genome-wide transcription. Virtually all TAF1-dependent genes, which comprise approximately 90% of the yeast genome, displayed a strong dependence upon all regions of TAF1 that were tested. This finding might reflect the need for each region of TAF1 to stabilize TAF1 against degradation or may indicate that all TAF1-dependent genes require the many activities of TAF1. Paradoxically, deletion of the region of TAF1 that is important for promoter binding interfered with the expression of many genes that are normally TFIID-independent/SAGA (Spt-Ada-Gcn5-acetyltransferase)-dominated, suggesting that this region normally prevents TAF1 (TFIID) from interfering with the expression of SAGA-regulated genes.

Galactose↗

PubMed2004

A complex network of RNA-RNA interactions controls subgenomic mRNA transcription in a tombusvirus.

Eukaryotic (+)-strand RNA viruses utilize a wide variety of gene expression strategies to achieve regulated production of their viral proteins. A common mechanism used by many is to transcribe viral subgenomic (sg) mRNAs. Transcription of sg mRNA2 in tombusviruses allows for expression of the p19 suppressor of gene silencing and p22 movement proteins. We have investigated the mechanism of transcription of this sg mRNA in Tomato bushy stunt virus and have determined that this process is facilitated by no less than three different RNA modules that are located throughout the viral genome. These RNA units perform distinct tasks and function via long-distance RNA-RNA interactions. Systematic deconstruction of the RNA network and analysis of related RNA promoter elements allowed us to identify fundamental properties necessary for productive sg mRNA2 transcription. Collectively, our results (i) establish specific roles for the different RNA components of a multipartite RNA-based control system, (ii) support a premature termination mechanism for tombusvirus sg mRNA transcription and (iii) reveal a close mechanistic relationship between sg mRNA transcription, viral RNA replication and RNA recombination.

Base Pairing↗

PubMed2005

Genomic segments cloning and analysis of Cotesia plutellae polydnavirus using plasmid capture system.

Cotesia plutellae polydnaviruses (CpBV) has a segmented genome consisting of multiple circular double stranded DNAs. Recently, we have developed an easy, simple, and convenient system based on Tn7 transposition in order to clone genomic segments of CpBV in Escherichia coli cell and designated plasmid capture system (PCS). The PCS donor-S transferred a pUC19 origin of replication and an ampicillin resistance marker into CpBV genomic DNA by in vitro transposition. Through PCS system, we were able to clone 53 genomic clones ranging from 0.1 to 25.5 kb and further they were classified into 29 segments by their sizes and restriction endonuclease patterns. Among them, a complete nucleotide sequence of CpBV-S28 segment was determined and 10 putative genes were predicted from this segment. Interestingly, 9 of 10 putative ORFs had high level of similarities with catalytic domain of protein tyrosine phosphatase. Also, ORF2807 showed similarity with EP1-like proteins of C. congregata polydnavirus.

Chromosome Mapping↗

PubMed2001

Attenuated lapinized chinese strain of classical swine fever virus: complete nucleotide sequence and character of 3'-noncoding region.

The complete nucleotide sequence including precise 5'- and 3'-terminal non-coding regions (NCRs) of the attenuated lapinized Chinese strain (HCLV) of Classical Swine Fever Virus (CSFV) was determined from overlapping cDNA clones constructed by separated RT-PCR and rapid amplification of cDNA ends (RACE) methods. The genomic RNA of the HCLV strain consists of 12.310 nucleotides (nts) including 374 nts and 242nts in the 5'- and 3'-NCRs, respectively. It contains one large open reading frame (ORF) encoding a polyprotein of 3,898 amino acids with a calculated molecular weight of 437.6 kDa. There is one notable insertion of 12 continuous nts, CTTTTTTCTTTT in the 3'-NCR of HCLV genomic cDNA when compared with its parental virulent Shimen strain. Sequence alignment of partial 3'-NCR reveals two groups of CSFV vaccine strains carrying similar T-rich insertions at different positions in this region. Computer-predicted secondary structures suggest that T-rich insertion greatly change the structures and thus decrease the promoter functions of 3'-NCRs during the replications of these two groups of CSFV vaccine strains.

3' Untranslated Regions↗

PubMedDate not supplied

The human genome and public policy: a nursing perspective.

The Human Genome Project has completed a rough draft of the sequence that comprises human DNA. For the first time, the scientific discoveries have been conducted in tandem with research exploring the social, legal, and ethical implications of the research. Several committees have studied the policy implications of the genetics information explosion. The Secretary's Advisory Committee on Genetic Testing's report was received by the Secretary of the U.S. Department of Health and Human Services in November 2000 and contains recommendations for the oversight of genetic testing. Public policy issues that affect nursing practice in the area of genetics must be explored by individual nurses and professional nursing organizations.

Genetic Engineering↗

PubMed2004

Infectious cDNA clones of porcine reproductive and respiratory syndrome virus and their potential as vaccine vectors.

Full-length infectious cDNA clones have recently become available for both European and North American genotypes of porcine reproductive and respiratory syndrome virus (PRRSV), and it is now possible to alter the PRRSV genome and create genetically defined mutant viruses. Among many possible applications of the PRRSV infectious cDNA clones, development of genetically modified vaccines is of particular interest. Using infectious clones, the PRRSV genome has been manipulated by changing individual amino acids, deleting coding regions, inserting foreign sequences, and generating arterivirus chimeras. The limited available data suggest that all structural proteins of PRRSV are essential for replication of the virus, and that PRRSV infectivity is relatively intolerant of subtle changes within the structural proteins. The major tasks in PRRSV research are to identify virulence factors and pathogenic mechanisms, and to understand the structure-function relationships of individual viral proteins. Utilizing these infectious clones as tools, a new generation of safe and efficacious PRRS vaccines may be constructed.

Animals↗

PubMed2003

From plant genomics to breeding practice.

New alleles are constantly accumulated during intentional crop selection. The molecular understanding of these alleles has stimulated new genomic approaches to mapping quantitative trait loci (QTL) and haplotype multiplicity of the genes concerned. A limited number of quantitative trait nucleotides responsible for QTL variation have been described, but an acceleration in their rate of discovery is expected with the adoption of linkage disequilibrium and candidate gene strategies for QTL fine mapping and cloning. Additional layers of regulatory variation have been studied that could also contribute to the molecular basis of quantitative genetics of crop traits. Despite this progress, the role of marker-assisted selection in plant breeding will ultimately depend on the genetic model underlying quantitative variation.

Breeding↗

PubMed1992

[The construction of rich genomic library of bovine Y chromosome].

According to the fundamentals of DNA renatured kinetics, we made a study in which genomic DNA from male cattle was completely digested with Sau 3A, while female DNA was sheared to an average size of 500 bp fragments. The Y derived DNA sequence rich library of bovine has been contracted with pUC19 plasmid as vector and JM109 as host bacteria by reassociation of male and female DNA. Male and female DNA probes, were used. The result of colony hybridization showed that out of 1000 recombinants analysed, 6 were found to be stronger signal with male probe. Analysis of Pvu' I digested DNA fragment revealed that most of the inserts were in the 90-300 bp.

Animals↗

PubMed2026

Base editing for precision therapeutics.

Base editing (BE), the precise installation of single-nucleotide changes in DNA or RNA without inducing double-strand breaks, holds substantial therapeutic promise for correcting single-nucleotide variants, which constitute more than half of the known pathogenic genetic variants. Recent advances have improved base editor specificity, efficiency, and delivery, enabling clinically oriented procedures. Clinically, BE has shown early success or strong translational promise in sickle cell disease, β-thalassemia, leukemia (via CAR T and epitope engineering), hypercholesterolemia (PCSK9 and ANGPTL3), alpha-1-antitrypsin deficiency, and glycogen storage disease type Ia. Key remaining challenges include bystander editing within the activity window, residual off-target DNA and RNA editing, delivery constraints (payload size, tissue targeting, and redosing limits), immunogenicity, and the need for durable long-term safety evidence across relevant cell types and disease contexts. Continued technological refinements, careful preclinical validation, and rigorous clinical assessment will be essential to fully realize BE's transformative potential in precision medicine.

Humans↗

PubMed2006

Integrating metabolomics into a systems biology framework to exploit metabolic complexity: strategies and applications in microorganisms.

As an important functional genomic tool, metabolomics has been illustrated in detail in recent years, especially in plant science. However, the microbial category also has the potential to benefit from integration of metabolomics into system frameworks. In this article, we first examine the concepts and brief history of metabolomics. Next, we summarize metabolomic research processes and analytical platforms in strain improvements. The application cases of metabolomics in microorganisms answer what the metabolomics can do in strain improvements. The position of metabolomics in this systems biology framework and the real cases of integrating metabolomics into a system framework to explore the microbial metabolic complexity are also illustrated in this paper.

Bacteria↗