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PubMed2000

Annuality, perenniality and cell death.

This essay considers annuality and perenniality as quantitative traits and discusses the application of established and new genetic tools to the analysis of plant life histories. Annual/perennial status is a function of meristem determinacy in combination with the processes of cell death and disposal employed by plants to generate well-adapted anatomies and morphologies. Creeping perennials, like clover or bracken, seem to move around in the environment. They do this by extending into unoccupied space while the oldest tissues behind the growing and mature regions senesce, die and decompose. Trees do essentially the same thing, except that they develop vertically and the old dead tissue does not disappear but instead persists as wood. A root system is a kind of upended vertical perennial. The balance between exploratory growth and the wave of tissue death that succeeds it is a major determinant of perenniality. So although perenniality and annuality may appear to be dramatically different traits, extremes of behaviour can arise by a relatively minor change in the relationship between growth and death. This conclusion is supported by evidence from genome dosage studies, from the practical experiences of breeding perennial-type traits into annual backgrounds and from molecular cladistics. Applications of methods for the genetic analysis of quantitative characters are described, including the exploitation of introgression mapping in Lolium-Festuca and quantitative trait locus mapping in cereals and other species.

Apoptosis↗
PubMed2026

Minimizing Off-Target Effects of CRISPR-Cas9 With Optimized sgRNA: Evaluation of Efficiency and Specificity in the Tumor Protein 53 (TP53) Region.

CRISPR-Cas9 is a widely used genetic tool with therapeutic potential in molecular biology. CRISPR-Cas9 enables precise genome editing by its ability to target specific DNA sequence. After off-target and on-target regions are identified, CRISPR-Cas9 is applied to these regions based on the match between the guide RNA (gRNA) and target DNA sequence. This study points to the off-target impact of mismatches between the gRNA and target DNA on exon regions of the TP53 gene, which are involved in regulating multiple genes and cellular functions. Off-target positions are typically evaluated using scoring methods. In this study, we have used latent class analysis to reveal subclasses of off-target positions. Thus, we have created the levels of off-target positions and evaluated the effects of mismatching positions within these classes using machine learning classifiers. The results revealed that mismatching positions could be categorized into three levels: low, middle, and high off-target positions. We have improved a computational framework to minimize off-target effects and to identify the PAM sequences in the gRNA design. Thus, carefully designed gRNAs will ensure that desired genetic edits are performed and target variants are achieved. This work will avail the future research aimed at optimizing genome editing by customizing CRISPR-Cas9 to target specific protospacer DNA through gRNA.

CRISPR-Cas Systems↗
PubMed2005

State-of-the-art modified RNAi compounds for therapeutics.

In recent years, the recognition of non-protein coding RNAs as a functional effector of genetic expression has been highlighted by the discovery of RNA interference (RNAi). RNAi is an intracellular phenomenon that enables the eukaryotic cell to utilize double-stranded RNA molecules to silence gene expression in a sequence-specific manner. The short interfering RNA (siRNA) pathway has been intensively investigated and continues to serve as the basis for the development of potent molecular genetic tools. The power of this technology is most clearly evidenced by the fact that siRNA effector molecules can be chemically synthesized and exogenously delivered to specifically target and silence any gene of choice. This capability enables not only basic research, but also opens the door to a new therapeutic modality. Furthermore, the introduction of certain chemical modifications to siRNA effectors can produce a more robust knockdown of gene expression, hence, optimizing serum stability and increasing target specificity yet limiting the induction of cellular stress response, which are key features for in vivo delivery and successful therapeutics. This article outlines the progress in the development of differentially modified siRNA duplexes and their potential role as human therapeutics.

Animals↗
PubMed1991

Humoral and cell-mediated immune responses to live recombinant BCG-HIV vaccines.

Several viral and bacterial live recombinant vaccine vehicles are being developed to produce a new generation of vaccines against a broad spectrum of infectious diseases. The human tuberculosis vaccine Mycobacterium bovis bacillus Calmette-Guerin (BCG) has features that make it a particularly attractive live recombinant vaccine vehicle. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been studied extensively. With nearly two billion immunizations, BCG has a long record of safe use in man. It is one of the few vaccines that can be given at birth, it engenders long-lived immune responses with only a single dose, and there is a worldwide distribution network with experience in BCG vaccination. Recently developed molecular genetic tools and methods for mycobacteria have provided the means to introduce foreign genes into BCG. Here we report that a variety of human immunodeficiency virus type 1 polypeptides can be expressed in BCG recombinants under the control of the mycobacterial hsp70 promoter and that the foreign polypeptides produced in BCG can induce antibody and T-cell responses. These results demonstrate that BCG can be used as a live recombinant vaccine vehicle to induce immune responses to pathogen proteins produced by the bacillus.

Animals↗
PubMed2004

Phylogenomics of the reproductive parasite Wolbachia pipientis wMel: a streamlined genome overrun by mobile genetic elements.

The complete sequence of the 1,267,782 bp genome of Wolbachia pipientis wMel, an obligate intracellular bacteria of Drosophila melanogaster, has been determined. Wolbachia, which are found in a variety of invertebrate species, are of great interest due to their diverse interactions with different hosts, which range from many forms of reproductive parasitism to mutualistic symbioses. Analysis of the wMel genome, in particular phylogenomic comparisons with other intracellular bacteria, has revealed many insights into the biology and evolution of wMel and Wolbachia in general. For example, the wMel genome is unique among sequenced obligate intracellular species in both being highly streamlined and containing very high levels of repetitive DNA and mobile DNA elements. This observation, coupled with multiple evolutionary reconstructions, suggests that natural selection is somewhat inefficient in wMel, most likely owing to the occurrence of repeated population bottlenecks. Genome analysis predicts many metabolic differences with the closely related Rickettsia species, including the presence of intact glycolysis and purine synthesis, which may compensate for an inability to obtain ATP directly from its host, as Rickettsia can. Other discoveries include the apparent inability of wMel to synthesize lipopolysaccharide and the presence of the most genes encoding proteins with ankyrin repeat domains of any prokaryotic genome yet sequenced. Despite the ability of wMel to infect the germline of its host, we find no evidence for either recent lateral gene transfer between wMel and D. melanogaster or older transfers between Wolbachia and any host. Evolutionary analysis further supports the hypothesis that mitochondria share a common ancestor with the alpha-Proteobacteria, but shows little support for the grouping of mitochondria with species in the order Rickettsiales. With the availability of the complete genomes of both species and excellent genetic tools for the host, the wMel-D. melanogaster symbiosis is now an ideal system for studying the biology and evolution of Wolbachia infections.

Adenosine Triphosphate↗
PubMed2024

Clocking out and letting go to unleash green biotech applications in a photosynthetic host.

Cyanobacteria are photosynthetic bacteria whose gene expression patterns are globally regulated by their circadian (daily) clocks. Due to their ability to use sunlight as their energy source, they are also attractive hosts for "green" production of pharmaceuticals, renewable fuels, and chemicals. However, despite the application of traditional genetic tools such as the identification of strong promoters to enhance the expression of heterologous genes, cyanobacteria have lagged behind other microorganisms such as Escherichia coli and yeast as economically efficient cell factories. The previous approaches have ignored large-scale constraints within cyanobacterial metabolic networks on transcription, predominantly the pervasive control of gene expression by the circadian (daily) clock. Here, we show that reprogramming gene expression by releasing circadian repressor elements in the transcriptional regulatory pathways coupled with inactivation of the central oscillating mechanism enables a dramatic enhancement of expression in cyanobacteria of heterologous genes encoding both catalytically active enzymes and polypeptides of biomedical significance.

Photosynthesis↗
PubMed2001

Cre stoplight: a red/green fluorescent reporter of Cre recombinase expression in living cells.

The Cre/lox system is a powerful genetic tool with which to manipulate the genome. Here, we describe the development of a simple reporter system for Cre recombinase, called the Cre Stoplight. In the absence of Cre, the red fluorescent protein is expressed; when Cre catalyzes a recombination event, the green fluorescent protein is produced. Testing this system in transiently transfected cells showed that it produced robust signals (90% of the cells converted from red to green) when equal amounts of the plasmids encoding Cre recombinase and the Cre Stoplight were used. A 1:100 ratio of enzyme to reporter plasmid produced similar results, and a 1:10000 ratio was necessary to significantly reduce the number of cells converting to green (1%).

Cell Line↗
PubMed2006

Regional distribution of mutations of the ATP7B gene in patients with Wilson disease: impact on genetic testing.

Wilson disease is an autosomal recessive inherited disorder of copper metabolism. The Wilson disease gene codes for a copper transporting P-type ATPase (ATP7B). Molecular genetic analysis reveals at least 300 distinct mutations. While most reported mutations occur in single families, a few are more common. The most common mutation in patients from Central, Eastern, and Northern Europe is the point mutation H1069Q (exon 14). About 50-80% of Wilson disease (WD) patients from these countries carry at least one allele with this mutation with an allele frequency ranging between 30 and 70%. Other common mutations in Central and Eastern Europe are located on exon 8 (2299insC, G710S), exon 15 (3400delC) and exon 13 (R969Q). The allele frequency of these mutations is lower than 10%. In Mediterranean countries there is a wide range of mutations, the frequency of each of them varies considerably from country to country. In Sardinia, a unique deletion in the 5' UTR (-441/-427 del) is very frequent. In mainland Spain the missense mutation M645R in exon 6 is particularly common. Data from non-European countries are scarce. Most data from Asia are from Far Eastern areas (China, South Korea and Japan) where the R778L missense mutation in exon 8 is found with an allele frequency of 14-49%. In summary, given the constant improvement of analytic tools genetic testing will become an integral part for the diagnosis of WD. Knowledge of the differences in the worldwide distribution of particular mutations will help to design shortcuts for genetic diagnosis of WD.

Adenosine Triphosphatases↗
PubMed2001

A common functional C-T substitution polymorphism in the promoter region of the human catalase gene influences transcription factor binding, reporter gene transcription and is correlated to blood catalase levels.

Oxidative stress is implicated in disease and aging. In order to obtain molecular genetic tools that can be used to determine the potential impact of oxidative stress we examined the human catalase gene promoter for possible variation. Genomic DNA isolated from 10 individuals was screened for polymorphisms in the 5'-flanking region by direct sequence analysis of PCR products (nt -307 to -46 from the transcription start site). A common C/T polymorphism -262 base pairs from the transcription start site was detected. Computer analysis indicated that the two variants bound different transcription factors. Indeed, gel retardation analysis revealed different protein binding patterns to the two variants. Expression studies with reporter constructs showed significantly higher transcriptional activity of the T variant in HepG2 and K562 cells (1.5-fold,p <.05 Wilcoxon test). Thus a higher expression in human liver and blood cells is possible. In order to test this hypothesis, catalase levels in red blood cells were determined in 29 donors. The corresponding genotype was determined with a restriction enzyme-based assay. It was found that catalase levels were significantly higher in donors carrying the T allele in comparison to donors homozygous for the C allele (p <.03). In conclusion, we report here the first common (allele frequency in a Swedish population, 28%) genetic variant in a fundamental oxidative stress protection gene with a defined phenotype.

Alleles↗
PubMed2002

Designed transcription factors as structural, functional and therapeutic probes of chromatin in vivo. Fourth in review series on chromatin dynamics.

Despite its central importance in gene regulation, chromatin in mammalian cells remains relatively poorly understood-a predicament due to the paucity of robust genetic tools in mammals, the complexity of the chromatin remodeling machinery, and the dynamic properties of chromatin in vivo. Here we review recent developments in understanding endogenous mammalian gene regulation via the use of designed transcription factors (TFs). These include mutated forms of naturally occurring TFs that exhibit dominant-negative activity, and designed proteins with novel, predetermined DNA-binding specificities. Systematic targeting of designed TFs to particular promoters is helping to illuminate the complex rules that chromatin imposes on TF access and action in vivo. We evaluate the potential applications of these proteins as probes of mammalian chromatin-based regulatory pathways and their potential for the therapy of human disease, highlighting leukemia in particular.

Animals↗
PubMed2004

Differential oocyte-specific expression of Cre recombinase activity in GDF-9-iCre, Zp3cre, and Msx2Cre transgenic mice.

Oocyte-specific deletion of ovarian genes using Cre/loxP technology provides an excellent tool to understand their physiological roles during folliculogenesis, oogenesis, and preimplantation embryonic development. We have generated a transgenic mouse line expressing improved Cre recombinase (iCre) driven by the mouse growth differentiation factor-9 (GDF-9) promoter. The resulting transgenic mouse line was named GDF-9-iCre mice. Using the floxed ROSA reporter mice, we found that Cre recombinase was expressed in postnatal ovaries, but not in heart, liver, spleen, kidney, and brain. Within the ovary, the Cre recombinase was exclusively expressed in the oocytes of primordial follicles and follicles at later developmental stages. The expression of iCre of GDF-9-iCre mice was shown to be earlier than the Cre expression of Zp3Cre and Msx2Cre mice, in which the Cre gene is driven by zona pellucida protein 3 (Zp3) promoter and a homeobox gene Msx2 promoter, respectively, in the postnatal ovary. Breeding wild-type males with heterozygous floxed germ cell nuclear factor (GCNF) females carrying the GDF-9-iCre transgene did not produce any progeny having the floxed GCNF allele, indicating that complete deletion of the floxed GCNF allele can be achieved in the female germline by GDF-9-iCre mice. These results suggest that GDF-9-iCre mouse line provides an excellent genetic tool for understanding functions of oocyte-expressing genes involved in folliculogenesis, oogenesis, and early embryonic development. Comparison of the ontogeny of the Cre activities of GDF-9-iCre, Zp3Cre, and Msx2Cre transgenic mice shows there is sequential Cre activity of the three transgenes that will allow inactivation of a target gene at different points in folliculogenesis.

Animals↗
PubMed2004

Fatty acid-binding proteins--insights from genetic manipulations.

Fatty acid-binding proteins (FABPs) belong to the conserved multigene family of the intracellular lipid-binding proteins (iLBPs). These proteins are ubiquitously expressed in vertebrate tissues, with distinct expression patterns for the individual FABPs. Various functions have been proposed for these proteins, including the promotion of cellular uptake and transport of fatty acids, the targeting of fatty acids to specific metabolic pathways, and the participation in the regulation of gene expression and cell growth. Novel genetic tools that have become available in recent years, such as transgenic cell lines, animals, and knock-out mice, have provided the opportunity to test these concepts in physiological settings. Such studies have helped to define essential cellular functions of individual FABP-types or of combinations of several different FABPs. The deletion of particular FABP genes, however, has not led to gross phenotypical changes, most likely because of compensatory overexpression of other members of the iLBP gene family, or even of unrelated fatty acid transport proteins. This review summarizes the properties of the various FABPs expressed in mammalian tissues, and discusses the transgenic and ablation studies carried out to date in a functional context.

Animals↗
PubMed2002

A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.

Double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful reverse genetic tool to silence gene expression in multiple organisms including plants, Caenorhabditis elegans, and Drosophila. The discovery that synthetic double-stranded, 21-nt small interfering RNA triggers gene-specific silencing in mammalian cells has further expanded the utility of RNAi into mammalian systems. Here we report a technology that allows synthesis of small interfering RNAs from DNA templates in vivo to efficiently inhibit endogenous gene expression. Significantly, we were able to use this approach to demonstrate, in multiple cell lines, robust inhibition of several endogenous genes of diverse functions. These findings highlight the general utility of this DNA vector-based RNAi technology in suppressing gene expression in mammalian cells.

Animals↗
PubMed1992

The recombinant congenic strains for analysis of multigenic traits: genetic composition.

The genetic control of susceptibility to many common diseases, including cancer, is multigenic both in humans and in animals. This genetic complexity has presented a major obstacle in mapping the relevant genes. As a consequence, most geneticists and molecular biologists presently focus on "single gene" diseases. To make the multigenic diseases accessible to genetic and molecular analysis, we developed a novel genetic tool, the recombinant congenic strains (RCS) in the mouse (4). The RC strains are produced by inbreeding of mice of the second backcross generation between two inbred strains, one of which serves as the "donor" and the other as the "background" strain. A series of RCS consists of approximately 20 strains, each carrying a different set of genes: approximately 12.5% genes from the common donor inbred strain, the remaining 87.5% from the common background inbred strain. As the set of donor strain genes in each RC strain is different, the nonlinked genes of the donor strain involved in the control of a multigenic trait, e.g., cancer susceptibility, become distributed into different RC strains where they can be analyzed one by one. Hence, the RCS system transforms a multigenic trait into a series of single gene traits, where each gene contributing to the multigenic control can be mapped and studied separately. Recently we demonstrated that the RCS system is indeed capable of resolving multigenic traits, which are hardly analyzable otherwise, by mapping four new colon tumor susceptibility loci (8; P. C. Groot, C. J. A. Moen, W. Dietrich, L. F. M. van Zutphen, E. S. Lander, and P. Demant, unpublished results). For successful application of the RCS system, extensive genetic characterization of the individual recombinant congenic strains is essential. In this paper we present detailed information about the genetic composition of three series of RC strains on the basis of typing of 120-180 markers distributed along all autosomes. The data indicate that the relative representation of the donor strain genes in the RC strains does not deviate from the theoretical expectation, and that the RC strains achieved a very high degree of genetic homogeneity and for all practical purposes can be considered inbred strains. The density and distribution of markers reported here permits an effective mapping of unknown genes of donor strain origin at almost all autosomal locations. Much of this information has been obtained using the new class of genetic markers, the simple sequence repeat polymorphisms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1999

A single-step transconjugation system for the introduction of unmarked deletions into Actinobacillus pleuropneumoniae serotype 7 using a sucrose sensitivity marker.

Research on the porcine respiratory tract pathogen Actinobacillus pleuropneumoniae requires the availability of improved genetic tools. Therefore, using the sacB gene of Bacillus subtilis, we developed a sucrose-based counterselection system that allows rapid curing of an Escherichia coli-A. pleuropneumoniae shuttle vector as well as the introduction of unmarked mutations into the A. pleuropneumoniae chromosome. A cassette containing the Tn903 kanamycin resistance determinant (km(r)) and the sacB gene expressed from the A. pleuropneumoniae omlA promoter was introduced by homologous recombination into the ureC gene of A. pleuropneumoniae. The resultant stable plasmid cointegrates were kanamycin-resistant, sucrose-sensitive, and urease-positive. A simple counterselection on sucrose-containing agar plates without an additional transconjugation step allowed the efficient isolation of urease-negative A. pleuropneumoniae mutants that had lost the km(r)-sacB cassette.

Actinobacillus pleuropneumoniae↗
PubMed2002

Genome shuffling of Lactobacillus for improved acid tolerance.

Fermentation-based bioprocesses rely extensively on strain improvement for commercialization. Whole-cell biocatalysts are commonly limited by low tolerance of extreme process conditions such as temperature, pH, and solute concentration. Rational approaches to improving such complex phenotypes lack good models and are especially difficult to implement without genetic tools. Here we describe the use of genome shuffling to improve the acid tolerance of a poorly characterized industrial strain of Lactobacillus. We used classical strain-improvement methods to generate populations with subtle improvements in pH tolerance, and then shuffled these populations by recursive pool-wise protoplast fusion. We identified new shuffled lactobacilli that grow at substantially lower pH than does the wild-type strain on both liquid and solid media. In addition, we identified shuffled strains that produced threefold more lactic acid than the wild type at pH 4.0. Genome shuffling seems broadly useful for the rapid evolution of tolerance and other complex phenotypes in industrial microorganisms.

Catalysis↗
PubMed1993

Chromosome mapping in Alcaligenes eutrophus CH34.

Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34. In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated. Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37 degrees C and is characteristic of many metallotolerant strains of A. eutrophus. Plasmid pULB113 (RP4::miniMu) was used to map the available mutations. Twenty-five loci were ordered in a circular map. pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37 degrees C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A. eutrophus CH34 chromosome with a small number of crosses.

Alcaligenes↗
PubMed2026

Development of a new recombineering system for Edwardsiella species.

Edwardsiella species are important aquaculture pathogens that also cause opportunistic infections in humans, necessitating efficient genome editing tools to study their pathogenesis and develop control strategies. In this study, we identified and characterized six endogenous recombinases pairs from Edwardsiella and its phages. Among these, the BAS_MS17 system exhibited the highest recombination efficiency in E. piscicida EIB202&#x394;p. Extending homology arms from 150 bp to 200 bp improved editing efficiency by 2-fold, while the addition of Redg or Plug further enhanced recombination by 3-fold and 2.5-fold, respectively, without compromising accuracy (100%). More importantly, when applied to E. piscicida sdu12S, Redg or Plug improved the editing efficiency by 8-fold and 7-fold, respectively. Deletion of the phage-derived single-strand binding protein (SSB) reduced efficiency to 25% of the BAS_MS17 level, whereas expression of the endogenous RecA-family SSB (rSSB) increased recombinant yield by 5-fold, highlighting functional conservation. Furthermore, SSB proteins from heterologous hosts failed to enhance recombination efficiency. Using the optimized system, we successfully knocked out ten distinct genes, including virulence-associated loci, with editing accuracy exceeding 85%. Phenotypic analysis revealed that luxR, but not the other tested genes, contributes to biofilm formation. Virulence evaluation results showed that aroA, fur, and hfq are critical virulence-associated factors. Collectively, this streamlined recombineering system provides a simple, rapid, and efficient genetic tool for Edwardsiella, supporting mechanistic studies of virulence and the development of live attenuated vaccine candidates.

Edwardsiella piscicida↗