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The utility of siRNA transcripts produced by RNA polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA viruses.

Short interfering double-stranded RNAs (siRNAs) expressed under the control of an RNA polymerase I promoter system were used to target gene expression of influenza A and West Nile virus. Decreased RNA and protein expression was induced in a sequence-specific manner-reducing sequence complementarity from 21 to 17 nucleotides abrogated the siRNA effect. Reduced M(2) expression resulted in a decrease in total and infectious influenza A virus production. WNV protein expression, genomic RNA, and infectious virus production were all dramatically reduced by siRNAs targeting two distinct viral sequences. The data demonstrate the utility of plasmid-driven siRNAs in regulating the expression of single viral genes, global viral gene expression, as a potential antiviral treatment, and as a genetic tool for viruses whose genomes are difficult to manipulate.

Animals↗

A new opaque variant of maize by a single dominant RNA-interference-inducing transgene.

In maize, alpha-zeins, the main protein components of seed stores, are major determinants of nutritional imbalance when maize is used as the sole food source. Mutations like opaque-2 (o2) are used in breeding varieties with improved nutritional quality. However, o2 works in a recessive fashion by affecting the expression of a subset of 22-kD alpha-zeins, as well as additional endosperm gene functions. Thus, we sought a dominant mutation that could suppress the storage protein genes without interrupting O2 synthesis. We found that maize transformed with RNA interference (RNAi) constructs derived from a 22-kD zein gene could produce a dominant opaque phenotype. This phenotype segregates in a normal Mendelian fashion and eliminates 22-kD zeins without affecting the accumulation of other zein proteins. A system for regulated transgene expression generating antisense RNA also reduced the expression of 22-kD zein genes, but failed to give an opaque phenotype. Therefore, it appears that small interfering RNAs not only may play an important regulatory role during plant development, but also are effective genetic tools for dissecting the function of gene families. Since the dominant phenotype is also correlated with increased lysine content, the new mutant illustrates an approach for creating more nutritious crop plants.

Genes, Dominant↗

Construction of a BALB/c congenic mouse, C.C3H-Lpsd, that expresses the Lpsd allele: analysis of chromosome 4 markers surrounding the Lps gene.

Development of a congenic BALB/c mouse strain that contains a segment of chromosome 4 including the Lpsd allele of the lipopolysaccharide (LPS)-hyporesponsive C3H/HeJ strain is presented. On the basis of LPS-induced spleen cell mitogenesis, macrophage tumor necrosis factor secretion, and tyrosine phosphorylation in vitro and lethality in galactosamine-sensitized mice in vivo, the C.C3H-Lpsd strain provides a model of LPS hyporesponsiveness that is comparable to that of the parental C3H/HeJ strain. Analysis of markers in this region indicates that length of the donor fragment is approximately 5.5 centimorgans. Thus, the C.C3H-Lpsd strain provides an important genetic tool for analysis of markers in this region and for examining functional effects of Lpsd expression on the BALB/c background.

Alleles↗

Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases.

The intriguing biology of stem cells and their vast clinical potential is emerging rapidly for gene therapy. Bone marrow stem cells, including the pluripotent haematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and possibly the multipotent adherent progenitor cells (MAPCs), are being considered as potential targets for cell and gene therapy-based approaches against a variety of different diseases. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. The apparently high self-renewal potential makes them strong candidates for delivering genes and restoring organ systems function. However, the high proliferative potential of MSCs, now presumed to be self-renewal, may be more apparent than real. Although expanded MSCs have great proliferation and differentiation potential in vitro, there are limitations with the biology of these cells in vivo. So far, expanded MSCs have failed to induce durable therapeutic effects expected from a true self-renewing stem cell population. The loss of in vivo self-renewal may be due to the extensive expansion of MSCs in existing in vitro expansion systems, suggesting that the original stem cell population and/or properties may no longer exist. Rather, the expanded population may indeed be heterogeneous and represents several generations of different types of mesenchymal cell progeny that have retained a limited proliferation potential and responsiveness for terminal differentiation and maturation along mesenchymal and non-mesenchymal lineages. Novel technology that allows MSCs to maintain their stem cell function in vivo is critical for distinguishing the elusive stem cell from its progenitor cell populations. The ultimate dream is to use MSCs in various forms of cellular therapies, as well as genetic tools that can be used to better understand the mechanisms leading to repair and regeneration of damaged or diseased tissues and organs.

Animals↗

[New concepts for the study of anticancer drug resistance].

In the past decade, numerous mechanisms of resistance have been described, involving the availability of the drug at the target or the availability of the target itself, but resistance to cell death induction remains far from being understood. The involvement of p53, of Bcl2 and related proteins, of the Fas/Fas-L system and other membrane death receptor pathways, have especially been studied. However, conflicting results have been published concerning the impairment of apoptosis in resistance to cytotoxic drugs. This has shed important doubts on the currently accepted view, which presents apoptosis as a universal determinant of drug activity. These discrepancies are likely to be related to the cell-type specificity of apoptotic pathways and further research is warranted to get a complete picture of the role of cell death inhibition as a drug resistance mechanism. New genetic tools have been recently made available for the study of anticancer drug resistance. Differential or subtractive analyses of gene expression in drug-sensitive and drug-resistant cell lines or tumors have allowed the identification of genes which are potentially responsible for drug resistance, and which had not been recognized previously by the usual analytic approaches. The generation of genetic suppressor elements represents a more functional approach since they can be selected upon the actual resistance properties of the cell lines. Global transcriptome analysis can be performed through the use of cDNA microarrays, either for the comparison of drug-sensitive and resistant cell lines, or for the study of drug effects on gene expression. This may allow the identification of drug-response genes (whose expression is altered by the drug) and of signaling and metabolic pathways involved in drug activity.

Apoptosis↗

RNAi: ancient mechanism with a promising future.

RNA interference (RNAi) is a gene silencing mechanism that has been conserved in evolution from yeast to man. Double stranded RNA, which is either expressed by cellular genes for small non-coding RNAs, by parasitic nucleic acids, such as viruses or transposons, or is expressed as an experimental tool, becomes processed into small RNAs, which induce gene silencing by a variety of different means. RNAi-induced gene silencing controls gene expression at all levels, including transcription, mRNA stability and translation. We are only beginning to understand the physiological roles of the RNAi pathway and the function of the many small non-coding RNA species, which are found in eukaryotic genomes. Here we review the known functions of genes in RNAi in various species, the experimental use and design of small RNAs as a genetic tool to dissect the function of mammalian genes and their potential as therapeutic agents to modulate gene expression in patients.

Animals↗

Utilization of Drosophila eye to probe the functions of two mammalian serine/threonine kinases, Snk and HsHPK.

Here we report a quick functional analysis of two mammalian serine/threonine kinases, a serum inducible kinase (Snk) and Homo sapiens hepatoma protein kinase (HsHPK), using Drosophila eye as a model system. We generated transgenic fly lines carrying constructs of both kinases under control of the GAL upstream activating sequence (UAS). Each UAS line was then crossed to a line in which GAL4 expression was driven by one of the following promoters, eyeless (ey), glass or decapentaplegic. Thus, different kinase mutants can be ectopically expressed in a promoter-dependent manner. We observed that the ectopic expression of either the wild-type or active form of Snk driven by the glass promoter resulted in a rough-eye phenotype. Nevertheless, the ectopic expression of HsHPK under the control of the ey promoter resulted in a small-eye phenotype. The results of this study demonstrated that ectopic expression of these two mammalian genes could be achieved by the regulation of Drosophila promoters. In addition, the effects of these ectopically expressed genes on eye development could be an implication of their functions with respect to cell proliferation and differentiation. Thus, Drosophila eye, with the powerful genetic tools and vast information on eye development available, can be a useful system to probe the functions of mammalian genes in the postgenome era.

Animals↗

A biometrical genome search in rats reveals the multigenic basis of blood pressure variation.

A genome-wide search for multiple loci influencing salt-loaded systolic blood pressure (NaSBP) variation among 188 F2 progeny from a cross between the Brown-Norway and spontaneously hypertensive rat strains was pursued in an effort to gain insight into the polygenic basis of blood pressure regulation. The results suggest that loci within five to six genomic regions collectively explain approximately 43% of the total NaSBP variation exhibited among the 188 F2 progeny. Many of these loci are in regions that previous studies have not implicated in blood pressure regulation. Ultimately, however, this study not only sheds light on the multigenic basis of blood pressure but provides further evidence that the identification of the genetic determinants of polygenic traits in mammals is possible with modern biometrical and molecular genetic tools in controlled settings (i.e., breeding paradigm and model organism).

Animals↗

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo.

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.

Animals↗

Genetic elements of Thermococcales.

This minireview summarizes our current knowledge about archaeal genetic elements in the hyperthermophilic order Thermococcales in the phylum Euryarchaeota. This includes recent work on the first virus of Pyrococcus, PAV1, the discovery of novel unique virus morphotypes in hot deep-sea environments, and preliminary observations on novel cryptic plasmids. We also review the work accomplished over the last 5 years in the development of genetic tools for members of the Pyrococcus and Thermococcus genera, mainly in our laboratories.

Antigens, Archaeal↗

The genetic revolution in artificial reproduction: a view of the future.

With the completion of the human genome project, micro-array technology offers the potential to open up a whole new vista in assisted reproduction. In the next 10-20 years we will be able to screen each human embryo for all numerical chromosomal abnormalities as well as many genetic diseases. Micro-array analysis may permit the screening of multiple alleles for monogenetic diseases and polygenic diseases, including diabetes, hypertension and schizophrenia. In the near future, it may be possible to assess an individual's genetic predisposition for cardiovascular disease, all types of cancer and infectious diseases. In the distant future, it may even be possible to screen for any genetic trait, e.g. stature, baldness, obesity, hair colour, skin colour or even IQ. Although it is still uncertain what molecular genetic tools may be available, we can be sure that some of these trends will have major consequences on the future of assisted reproduction and society at large.

Chromosome Aberrations↗

Novel cassette-based shuttle vector system for gram-positive bacteria.

Our understanding of staphylococcal pathogenesis depends on reliable genetic tools for gene expression analysis and tracing of bacteria. Here, we have developed and evaluated a series of novel versatile Escherichia coli-staphylococcal shuttle vectors based on PCR-generated interchangeable cassettes. Advantages of our module system include the use of (i) staphylococcal low-copy-number, high-copy-number, thermosensitive and theta replicons and selectable markers (choice of erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin); (ii) an E. coli replicon and selectable marker (ampicillin); and (iii) a staphylococcal phage fragment that allows high-frequency transduction and an SaPI fragment that allows site-specific integration into the Staphylococcus aureus chromosome. The staphylococcal cadmium-inducible P(cad)-cadC and constitutive P(blaZ) promoters were designed and analyzed in transcriptional fusions to the staphylococcal beta-lactamase blaZ, the Vibrio fischeri luxAB, and the Aequorea victoria green fluorescent protein reporter genes. The modular design of the vector system provides great flexibility and variety. Questions about gene dosage, complementation, and cis-trans effects can now be conveniently addressed, so that this system constitutes an effective tool for studying gene regulation of staphylococci in various ecosystems.

Artificial Gene Fusion↗

RNA silencing as a tool for exploring gene function in ascomycete fungi.

We have developed a pHANNIBAL-like silencing vector, pSilent-1, for ascomycete fungi, which carries a hygromycin resistance cassette and a transcriptional unit for hairpin RNA expression with a spacer of a cutinase gene intron from the rice blast fungus Magnaporthe oryzae. In M. oryzae, a silencing vector with the cutinase intron spacer (147 bp) showed a higher efficiency in silencing of the eGFP gene than did those with a spacer of a GUS gene fragment or a longer intron (850 bp) of a chitin binding protein gene. Application of pSilent-1 to two M. oryzae endogenous genes, MPG1 and polyketide synthase-like gene, resulted in various degrees of silencing of the genes in 70-90% of the resulting transformants. RNA silencing was also induced by a pSilent-1-based vector in Colletotrichum lagenarium at a slightly lower efficiency than in M. oryzae, indicating that this silencing vector should provide a useful reverse genetic tool in ascomycete fungi.

Base Sequence↗

Development gone awry: congenital heart disease.

Significant advances in the understanding of the molecular and genetic basis of congenital heart disease have emerged from gene inactivation studies in mice and from human genetic investigations. However, the ability to utilize information gleaned from animal models to inform clinical care of patients depends on an accurate anatomic analysis and presentation in terms that are meaningful to the clinical pediatric cardiologist. Likewise, the enormous depth and breadth of accumulated clinical experience can inform the developmental biologist and can highlight the importance and interrelationships of particular phenotypes. The explosion of potentially informative genetic tools demands that basic scientists and clinicians concerned with congenital cardiac disease enhance the ongoing bidirectional dialogue. In some cases, categories of congenital disease familiar to clinicians are not recognized by developmental biologists, and mechanisms accepted by the biologist seem inconsistent with clinical experience. In this review, we summarize some of the more clinically significant forms of congenital heart disease, and we highlight relevant genetic and developmental pathways.

Animals↗

Studying phenotypic evolution using multivariate quantitative genetics.

Quantitative genetics provides a powerful framework for studying phenotypic evolution and the evolution of adaptive genetic variation. Central to the approach is G, the matrix of additive genetic variances and covariances. G summarizes the genetic basis of the traits and can be used to predict the phenotypic response to multivariate selection or to drift. Recent analytical and computational advances have improved both the power and the accessibility of the necessary multivariate statistics. It is now possible to study the relationships between G and other evolutionary parameters, such as those describing the mutational input, the shape and orientation of the adaptive landscape, and the phenotypic divergence among populations. At the same time, we are moving towards a greater understanding of how the genetic variation summarized by G evolves. Computer simulations of the evolution of G, innovations in matrix comparison methods, and rapid development of powerful molecular genetic tools have all opened the way for dissecting the interaction between allelic variation and evolutionary process. Here I discuss some current uses of G, problems with the application of these approaches, and identify avenues for future research.

Biological Evolution↗

Familial Mediterranean fever at the millennium. Clinical spectrum, ancient mutations, and a survey of 100 American referrals to the National Institutes of Health.

Regarded as the most common and best understood of the hereditary periodic fever syndromes, familial Mediterranean fever (FMF) is a recessively inherited disease of episodic fever with some combination of severe abdominal pain, pleurisy, arthritis, and a characteristic ankle rash. The flares typically last for up to 3 days at a time, and most patients are completely asymptomatic between attacks; if untreated with prophylactic colchicine, some patients later develop amyloidosis and renal failure. The recent cloning of the FMF gene on the short arm of chromosome 16p, and the subsequent finding that its tissue expression is limited to granulocytes, has helped to explain the dramatic accumulation of neutrophils at the symptomatic serosal sites; the wild-type gene likely acts as an upregulator of an anti-inflammatory molecule or as a downregulator of a pro-inflammatory molecule. For nearly half a century, FMF was thought to cluster primarily in non-Ashkenazi Jews, Arabs, Armenians, and Turks, although the screening of the 8 known mutations in an American cohort has identified substantial numbers of people from the Ashkenazi Jewish and Italian populations in the United States who also have this disease. Nevertheless, the symptoms often go unrecognized and patients remain undiagnosed for years, not receiving the highly efficacious colchicine therapy; their histories often include multiple laparotomies, laparoscopies, and psychiatric evaluations. The combinations of clinical manifestations among FMF patients are quite heterogeneous, but our American cohort did not establish any connections between individual mutations and specific clinical pictures--as is seen in other diseases like cystic fibrosis, in which distinct genotypes target certain organ systems. Specifically, the data from our American series are insufficient to evaluate the hypothesis that the M694V/M694V genotype confers a more severe phenotype, or increases the risk of amyloidosis; but both our data and the recent literature (160) indicate that amyloidosis can occur in FMF patients with only 1 copy, or no copies, of the M694V mutation. It appears that specific MEFV mutations are probably not the sole determinants of phenotype, and that unknown environmental factors or modifying genes act as accomplices in this disease. Although we hope the discovery of the FMF gene will allow the diagnosis of FMF to become genetically accurate, the reality is that both clinical and genetic tools must still be used together unless mutations are identified on both of a patient's chromosomes. Physicians should be careful not to rule out the diagnosis in patients of high-risk ethnic backgrounds just because of atypical clinical features, as our data indicate that MEFV mutations are sometimes demonstrable in such patients. At the same time, physicians cannot yet rely solely on a genetic diagnosis because we have not yet identified a sufficient spectrum of mutations, and it is not currently feasible to examine every patient's full DNA sequence for the entire gene; screening an ethnically consistent and clinically positive patient for the 8 known mutations frequently identifies a mutation on only 1 chromosome, and genetic analysis of other classic cases will often reveal none of the 8 mutations. Still, our data suggest that ethnic background is an important predictor of finding 1 of the presently known mutations, and the knowledge of ancestries atypical for FMF can suggest the diagnosis of other hereditary periodic fever syndromes. As the list of FMF-associated MEFV mutations is expanded, and/or new sequencing technologies permit more rapid screening, the value and interpretation of genetic testing for FMF will become more straightforward. Moreover, as the pathophysiology of this disorder becomes less of a hypothesis and more of an understood entity, it is likely that treatment options will broaden beyond the use of daily prophylactic colchicine. (ABSTRACT TRUNCATED)

Adult↗

The Pseudozyma flocculosa actin promoter allows the strong expression of a recombinant protein in the Pseudozyma species.

Fungi belonging to the recently classified genus Pseudozyma possess some unique properties such as biocontrol activity, production of rare antimicrobial glycolipids and production of recombinant proteins. In this work, we report the first cloning of a promoter endogenous to the multi-faceted yeast-like Pseudozyma flocculosa, that of the actin gene. The promoter region lacked typical TATA or CAAT box but displayed three putative GC box and two CT-rich regions. As in other related basidiomycetes, only one copy of the actin gene was present in the genome of P. flocculosa. The activity of the actin promoter was compared to that of the HSP70 promoter from Ustilago maydis in two Pseudozyma species. In P. flocculosa, the actin promoter allowed the expression of a very high amount of GFP protein (27.8 mg g(-1) total protein) compared to those obtained with the HSP70 promoter in liquid culture. By contrast, the levels of GFP expression obtained in liquid culture were similar with the actin or the HSP70 promoter in Pseudozyma antarctica. A similar pattern of GFP expression was observed in solid culture. The cloning of this new promoter offers a unique genetic tool to further exploit and study the unusual properties of fungi from the Pseudozyma genus.

Actins↗

Genetic manipulation of antibiotic-producing Streptomyces.

The genetic manipulation of Streptomyces species has been facilitated by the development of versatile cloning vectors, robust gene transfer systems and transposable elements. These molecular genetic tools have been used to construct antibiotic-producing strains with improved properties and recombinants for the production of hybrid glycopeptide and macrolide antibiotics, novel anthelminthic agents and novel cytotoxic anthracyclines.

Anthelmintics↗