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At least 289 records · Page 16Linked to original sources
PubMed2002

Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions.

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.

Bacteria↗
PubMed2006

Potato in the age of biotechnology.

Biotechnology-based tools are now widely used to enhance and expand the traditional remit of potato in food production. By modifying its functionality, the capacity of the potato to produce, for example, therapeutic or industrial compounds is now a reality, and its ability to resist disease can also be radically improved. Two developments have been crucial to expanding the role of potato: the recent advances in the fields of structural and functional potato genomics and the ability to integrate genes of interest into the potato genome. In this review we discuss how both developments have diversified the remit of this crop.

Biotechnology↗
PubMed2003

ssDNA-dependent colocalization of adeno-associated virus Rep and herpes simplex virus ICP8 in nuclear replication domains.

The subnuclear distribution of replication complex proteins is being recognized as an important factor for the control of DNA replication. Herpes simplex virus (HSV) single-strand (ss)DNA-binding protein, ICP8 (infected cell protein 8) accumulates in nuclear replication domains. ICP8 also serves as helper function for the replication of adeno-associated virus (AAV). Using quantitative 3D colocalization analysis we show that upon coinfection of AAV and HSV the AAV replication protein Rep and ICP8 co-reside in HSV replication domains. In contrast, Rep expressed by a recombinant HSV, in the absence of AAV DNA, displayed a nuclear distribution pattern distinct from that of ICP8. Colocal ization of Rep and ICP8 was restored by the reintroduction of single-stranded AAV vector genomes. In vitro, ICP8 displayed direct binding to Rep78. Single-stranded recombinant AAV DNA strongly stimulated this interaction, whereas double-stranded DNA was ineffective. Our findings suggest that ICP8 by its strong ssDNA-binding activity exploits the unique single-strandedness of the AAV genome to form a tripartite complex with Rep78 and AAV ssDNA. This novel mechanism for recruiting components of a functional replication complex directs AAV to subnuclear HSV replication compartments where the HSV replication complex can replicate the AAV genome.

Active Transport, Cell Nucleus↗
PubMed1995

Limitations to in vivo import of hydrophobic proteins into yeast mitochondria. The case of a cytoplasmically synthesized apocytochrome b.

The apocytochrome b gene, exclusively encoded by the mitochondrial genome, was engineered so that it could be expressed in the yeast cytoplasm. Different combinations of the apocytochrome b transmembrane domains were produced in the form of hybrid proteins fused to both the N-terminal mitochondrial targeting sequence of the ATPase subunit 9 from Neurospora crassa and to a cytoplasmic version of the bI4 RNA maturase, localised on the N-terminal and C-terminal sides, respectively, of the hydrophobic stretches. The bI4 RNA maturase, which can complement mitochondrial mutations, was used as an in vivo reporter to assess the mitochondrial import of the different groups of transmembrane helices. This new, reliable and sensitive reporter activity allowed us to experimentally determine the limitations to the mitochondrial import of hydrophobic proteins. All eight transmembrane helices of apocytochrome b could be imported into mitochondria, either alone or in combination, but no more than three to four transmembrane helices could be imported together at one time. This limit is close to that observed in the population of nuclear-encoded mitochondrial proteins. The hydrophobic characteristics of engineered and natural proteins targeted to the mitochondrial inner membrane revealed two factors important in the import process. These were (a) the local hydrophobicity of a transmembrane segment, and (b) the average regional hydrophobicity of the protein over an extended length of 60-80 residues. Such features may have played a major role in the evolution of mitochondrial genomes.

Amino Acid Sequence↗
PubMed2006

Impact of Aspergillus oryzae genomics on industrial production of metabolites.

Aspergillus oryzae is used extensively for the production of the traditional Japanese fermented foods sake (rice wine), shoyu (soy sauce), and miso (soybean paste). In recent years, recombinant DNA technology has been used to enhance industrial enzyme production by A. oryzae. Recently completed genomic studies using expressed sequence tag (EST) analyses and whole-genome sequencing are quickly expanding the industrial potential of the fungus in biotechnology. Genes that have been newly discovered through genome research can be used for the production of novel valuable enzymes and chemicals, and are important for designing new industrial processes. This article describes recent progress of A . oryzae genomics and its impact on industrial production of enzymes, metabolites, and bioprocesses.

Aspergillus oryzae↗
PubMed2026

In vivo immune cell engineering from bench to clinical reality.

Adoptive immune cell therapies, exemplified by chimeric antigen receptor T cells, have transformed the treatment of hematological malignancies. However, their broader clinical application is limited by complex ex vivo manufacturing, high cost, and safety concerns. In vivo immune cell engineering has emerged as an alternative strategy that delivers genetic instructions directly to immune cells, thereby generating or modulating therapeutic immune cells within the body and reducing the reliance on individualized in vitro operations. These advances underscore the need for a systematic evaluation of this emerging field. Therefore, this review systematically summarizes the mechanistic principles and delivery strategies underlying in vivo immune cell engineering, with an emphasis on in vivo CAR-T cell generation and the engineering of other immune cells. We then discuss major viral and non-viral delivery platforms and clarify how these platforms influence cargo delivery, cell specificity, and functional immune-cell programming. We further discuss recent preclinical and emerging clinical advances across cancer, autoimmune diseases, and degenerative diseases, while examining key translational challenges, including delivery specificity, off-target effects, controllability, persistence, and manufacturing standardization. Overall, although the field of in vivo immune cell engineering is advancing rapidly, its clinical success will depend on coordinated improvements in delivery precision, therapeutic efficacy, safety, and controllable immune-cell programming.

Cancer immunotherapy↗
PubMed2006

Mammalian genome targeting using site-specific recombinases.

Nowadays, a wide array of procedures in mouse technology has been made available to researchers in order to establish valuable models for the study of gene function. The efficiency of gene transfer and gene targeting as methods for producing genetic changes in mice, in addition to continuous advances in molecular biology tools, has converted the mouse into the major experimental model for the study of mammalian physiology. In recent years, the emergence of site-specific recombinases as tools to engineer mammalian genomes has opened new avenues into the design of genetically modified mouse models. The original Cre and FLP recombinases have demonstrated their utility in developing conditional gene targeting, and now other analogous recombinases are also ready to be used, in the same way or in combined strategies, to achieve more sophisticated experimental schemes for addressing complex biological questions. The properties of site-specific recombinases in combination with other biotechnological tools (tet on/off system, siRNA mediated gene silencing, fluorescent proteins, et al.) make them useful instruments to induce precise mutations in specific cells or tissues in a time-controlled manner. This ability can be applied in functional genomics in several ways: from conditional and inducible gene targeting to controlled expression of transgenes and recombination-mediated cassette exchange in mouse models for the study of development or disease phenotypes. This review focuses on the use of site-specific recombinases for mouse genome manipulation. A historical perspective of site-specific recombinases is considered and a number of strategies for achieving inducible or conditional genomic manipulations are contemplated in the context of current techniques for producing genetically modified mice. Finally, several model generation approaches from recent examples in the literature are revised.

Alleles↗
PubMed2003

The Frog Prince: a reconstructed transposon from Rana pipiens with high transpositional activity in vertebrate cells.

Members of the Tc1/mariner superfamily of transposable elements isolated from vertebrates are transpositionally inactive due to the accumulation of mutations in their transposase genes. A novel open reading frame-trapping method was used to isolate uninterrupted transposase coding regions from the genome of the frog species Rana pipiens. The isolated clones were approximately 90% identical to a predicted transposase gene sequence from Xenopus laevis, but contained an unpredicted, approximately 180 bp region encoding the N-terminus of the putative transposase. None of these native genes was found to be active. Therefore, a consensus sequence of the transposase gene was derived. This engineered transposase and the transposon inverted repeats together constitute the components of a novel transposon system that we named Frog Prince (FP). FP has only approximately 50% sequence similarity to Sleeping Beauty (SB), and catalyzes efficient cut-and-paste transposition in fish, amphibian and mammalian cell lines. We demonstrate high-efficiency gene trapping in human cells using FP transposition. FP is the most efficient DNA-based transposon from vertebrates described to date, and shows approximately 70% higher activity in zebrafish cells than SB. Frog Prince can greatly extend our possibilities for genetic analyses in vertebrates.

Amino Acid Sequence↗
PubMed2002

A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques.

To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.

Animals↗
PubMed2001

Alteration of Cre recombinase site specificity by substrate-linked protein evolution.

Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence. Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site. This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region. Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification. We term this procedure substrate-linked protein evolution (SLiPE). The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy.

3T3 Cells↗
PubMed2003

Harvesting the genomic promise: recombineering sequences for phenotypes.

The past decade has witnessed the construction of linkage and physical maps defining quantitative trait loci (QTL) in various domesticated species. Targeted chromosomal regions are being further characterized through the construction of bacterial artificial chromosome (BAC) contigs in order to isolate and characterize genes contributing towards phenotypic variation. Whole-genome BAC contigs are also being constructed that will serve as the tiling path for genomic sequencing. Harvesting this genetic information for biological gain requires either genetic selection or the production of genetically modified animals. This later approach when coupled with nuclear transfer technology (NT) provides "clones" of genetically modified animals. However, to date, the production of genetically modified animals has been limited to either microinjection of small gene constructs into embryos with random insertion or complex gene constructs designed to knock-out targeted gene expression. Neither of these approaches provides for introducing directed genetic manipulation allowing for allelic substitution [knock-in], subsequent analyses of gene expression, and cloning. An alternative approach utilizing genomic sequence information and recombineering to direct gene targeting of specific porcine BACs is presented here.

Animals↗
PubMed2001

Studies on the genomic organization of recombinant Streptococcus gordonii and the development of a novel intergenic integration site for foreign gene expression.

The methods currently employed to produce recombinant Streptococcus gordonii strains for use as vaccines and/or protein expression vectors result in the insertion of foreign genes into an unknown integration site with no information on the transcriptional context or potential phenotypic consequences. Therefore, the genomic organization surrounding the insertion site of a recombinant strain of S. gordonii (GP1223) containing a portion of the emm6 gene of Streptococcus pyogenes was determined. The nucleotide sequence of chromosomal walks in both directions from the insertion site revealed that the insert was flanked by a duplicated 3061-bp ClaI fragment. A consensus gram-positive promoter and a factor-independent RNA polymerase terminator sequence could be deduced in the fragment immediately upstream of the insertion site. The ClaI fragment also encoded open reading frames (ORFs) with high homology and parallel structural organization to the leucine biosynthesis operon of Lactococcus lactis subsp. lactis. Chromosomal walks downstream of the identified promoter region on the non-recombinant parental strain, GP204, yielded the sequence of two ORFs which would be normal targets of the transcription derived from this promoter. Northern analyses detected a highly expressed M6-specific transcript in recombinant strain GP1223 consistent in size with the proposed transcription unit. Transcripts analogous in length to those observed for the leucine biosynthesis operon of L. lactis subsp. lactis were also detected encompassing the homologous ORFs of S. gordonii. This information has enabled the construction of a recombinant S. gordonii strain in which the emm6 gene from S. pyogenes was targeted to a distinct intergenic locus within the S. gordonii genome. This new recombination site allows for expression of foreign gene products with minimal perturbation of the genomic organization of the wild-type S. gordonii strain and has provided information essential for further optimization of foreign gene expression levels.

Base Sequence↗
PubMed1988

Proteins encoded by bovine viral diarrhea virus: the genomic organization of a pestivirus.

The genome of bovine viral diarrhea virus (BVDV) contains a single large open reading frame capable of encoding 449 kDa of protein. Short segments from along the length of the molecularly cloned BVDV genome were engineered so as to be expressed as bacterial fusion polypeptides in Escherichia coli. These BVDV analog fusion proteins were used as immunogens to generate a panel of sequence-specific antisera. These antiserum reagents were in turn employed in immunoprecipitation analyses to identify the authentic BVDV protein to which they were directed. The results allowed for the identification and positioning along the genome of BVDV gene products accounting for approximately 83% of the coding capacity of the virus. A preliminary map of the genetic organization of BVDV is presented and discussed.

Antibodies, Viral↗
PubMed1986

[Construction of the bacterial genome--the next goal of genetic engineering].

The article deals primarily with literature data on the organization and evolution of bacterial genome. An account of the conception of "current" bacterial evolution consisting of periodical inclusions into a bacterial genome of genes having exogenous origin is given. A hypothesis is proposed that rare inversions going through the oriC or terC chromosomal regions could serve as reparative rearrangements providing balanced distribution of the DNA segments of different origin within the circular genome. It is asserted that the progress in the study of the model microorganism Escherichia coli K-12 as well as in genetical methods give rise to a new experimental challenge--the creation of a "rational" bacterial genome on the basis of E. coli cell. It is proposed that a "rational" bacterial genome could be devoid of many genes which provide the evolutionary formed ecological potential of the species and its capacity for rapid adaptation to new environment.

Biological Evolution↗
PubMed1989

Sindbis virus: an efficient, broad host range vector for gene expression in animal cells.

Sindbis virus, an enveloped virus with a single-stranded RNA genome, was engineered to express a bacterial protein, chloramphenicol acetyltransferase (CAT), in cultured insect, avian, and mammalian cells. The vectors were self-replicating and gene expression was efficient and rapid; up to 10(8) CAT polypeptides were produced per infected cell in 16 to 20 hours. CAT expression could be made temperature-sensitive by means of a derivative that incorporated a temperature-sensitive mutation in viral RNA synthesis. Vector genomic RNAs were packaged into infectious particles when Sindbis helper virus was used to supply virion structural proteins. The vector RNAs were stable to at least seven cycles of infection. The expression of CAT increased about 10(3)-fold, despite a 10(15)-fold dilution during the passaging. Sindbis virus vectors should prove useful for expressing large quantities of gene products in a variety of animal cells.

Aedes↗
PubMed1989

Segregation of cauliflower mosaic virus symptom genetic determinants.

We have created a series of hybrid cauliflower mosaic virus (CaMV) genomes between a severe virus strain (Cabb BJI) and a mild strain (Bari 1) to map the virus genetic loci responsible for specific systemic symptom characters produced in infected turnip plants. Recombinants were generated in vivo by recombinational rescue and in vitro by restriction enzyme fragment exchange. On infection, hybrids induced either parental (wild-type) symptoms or segregated parental characters. Some of the engineered hybrid genomes produced novel symptomatic effects not observed in either of the parental strains whilst others reverted to express parental symptom characters following passaging. Determinants defining differences between the two CaMV strains in respect of four specific symptom characters were delimited to separate genome regions. A locus involved in determining the rate of spread of systemic vein clearing symptoms mapped to a region containing part of gene VII and gene I (nts 109-780). This phenomenon is consistent with the putative involvement of the CaMV gene I product in mediating virus movement within infected plants. Determinants influencing the degree of leaf chlorosis were located in a separate genome domain encompassing part of gene VI together with the large intergenic region and part of gene VII (nts 6103-90). Determinants controlling timing of initial systemic symptom appearance were mapped to a region between nts 2150 and 4438 containing part of gene III, gene IV, and part of gene V. Plant stunting was influenced by loci in at least two separate regions, one containing parts of gene I and II, and a second within the reverse transcriptase gene (V). We conclude that symptoms produced by CaMV infection can be subdivided into individual characters, the genetic determinants of which segregate to different virus genetic loci and are not restricted to a single gene product.

Brassica↗