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PubMed2004

Genetic, genomic, and bioinformatic tools for studying breast cancer progression.

Cancer susceptibility is a complex interaction of an individual's genetic composition and environmental exposures. Huge strides have been made in understanding cancer over the past 100 years, from the recognition of cancer as a genetic disease, to the identification of specific carcinogens, isolation of oncogenes and the recognition of tumor suppressors. Analysis of high-risk familial cancers has led to the discovery of new tumor suppressor genes and important cancer pathways. These families however represent only a small fraction of cancer in the general population. Most cancer instead probably results of an intricate interaction of polymorphic susceptibility genes with the sea of environmental exposures that humans experience. Although the central cadre of cancer genes is known, little is understood about the peripheral genes that likely comprise the polymorphic susceptibility loci. The challenge for cancer genetics is therefore to move forward from the Mendelian genetics of the rare familial cancer syndromes into the field of quantitative trait loci, susceptibility factors, and modifier genes. By identifying the genes that modulate an individual's susceptibility to cancer after an environmental exposure, researchers will be able to gain important insights into human biology, cancer prevention and treatment. The most efficient strategies to identify and characterize modifier loci will likely be those that are transdisciplinary, encompassing a variety of different "-omic" technologies. The application of a "trans-omic" techniques in our laboratory to the study of breast cancer dissemination is presented here as an illustration of the strategy.

Journal Article↗
PubMed2005

Making waves in cancer research: new models in the zebrafish.

The zebrafish (Danio rerio) has proven to be a powerful vertebrate model system for the genetic analysis of developmental pathways and is only beginning to be exploited as a model for human disease and clinical research. The attributes that have led to the emergence of the zebrafish as a preeminent embryological model, including its capacity for forward and reverse genetic analyses, provides a unique opportunity to uncover novel insights into the molecular genetics of cancer. Some of the advantages of the zebrafish animal model system include fecundity, with each female capable of laying 200-300 eggs per week, external fertilization that permits manipulation of embryos ex utero, and rapid development of optically clear embryos, which allows the direct observation of developing internal organs and tissues in vivo. The zebrafish is amenable to transgenic and both forward and reverse genetic strategies that can be used to identify or generate zebrafish models of different types of cancer and may also present significant advantages for the discovery of tumor suppressor genes that promote tumorigenesis when mutationally inactivated. Importantly, the transparency and accessibility of the zebrafish embryo allows the unprecedented direct analysis of pathologic processes in vivo, including neoplastic cell transformation and tumorigenic progression. Ultimately, high-throughput modifier screens based on zebrafish cancer models can lead to the identification of chemicals or genes involved in the suppression or prevention of the malignant phenotype. The identification of small molecules or gene products through such screens will serve as ideal entry points for novel drug development for the treatment of cancer. This review focuses on the current technology that takes advantage of the zebrafish model system to further our understanding of the genetic basis of cancer and its treatment.

Animals↗
PubMed1983

The genetic activity of anthramycin, tomaymycin and sibiromycin in bacterial forward- and reverse-mutation assays and in the mouse bone-marrow micronucleus test.

The genetic activity of the structurally similar antitumor antibiotics anthramycin, tomaymycin and sibiromycin was evaluated in the standard Ames Salmonella/microsome mutagenicity assay, a Salmonella typhimurium forward-mutation assay and the micronucleus test. None of the test drugs showed any significant genetic activity in forward or reverse Salmonella mutation assays. The ability of mouse-liver enzymes to produce mutagens from the drugs was examined in the Salmonella reverse-mutation assay and was generally negative. As the concentrations of sibiromycin increased, some activity was detected in the presence of liver S-9 fractions from Aroclor-induced mice. This observation could not be verified at higher concentrations in the reverse-mutation assay due to cytotoxicity, and in the forward-mutation assay due to interference with the selection process by S-9. Cytogenetic evaluation of anthramycin and tomaymycin in the micronucleus test also gave negative results. However, significant increases in the frequency of micronucleated polychromatic erythrocytes were observed in the bone marrow of sibiromycin-treated mice. The results suggest that, except for some possible activity of sibiromycin, these drugs are generally devoid of any marked genetic activity in the test systems employed.

Aminoglycosides↗
PubMed1978

Quantitative forward mutation assay in Salmonella typhimurium using 8-azaguanine resistance as a genetic marker.

We have developed a quantitative forward mutation assay using Salmonella typhimurium, in which resistance to the purine analog 8-azaguanine is used as a genetic marker. We present the assay protocol, the concentration-dependent toxicity and mutagenicity of five known mutagens (N-methyl-N'-nitro-N-nitrosoguanidine, ICR-191, 9-aminoacridine, dimethylnitrosamine, and benzo[a]pyrene), and reconstruction experiments testing the assay for possible bias. The relative merits of forward versus reverse mutation assays are discussed.

Azaguanine↗
PubMed1993

Estimation of nonadditive genetic variances for a total-merit model including maternal effects.

Henderson described a method to reduce the number of mixed-model equations when estimating additive and nonadditive genetic variances or predicting additive and nonadditive genetic merits. The extension to a maternal effects model is straight-forward. When maternal genetic effects are strictly additive, an algebraic identity was found that reduces by a factor of two the order of a matrix that must be inverted each round to account for the genetic covariances among direct and maternal genetic effects. An algorithm for derivative-free restricted maximum likelihood was developed based on Henderson's total-merit model that is the basis for a reduced number of equations. The same values for the logarithm of the likelihood can be calculated from components of the equations for the total-merit model and from components of the equations for the individual effects model. The computational properties of the equations for the total-merit model, however, do not lend themselves to sparse-matrix methods. Both memory and time requirements were much greater for the total-merit model than for the individual-effects model for a data set of 871 animals and a model with additive, dominance, and additive x additive direct and additive maternal genetic effects. Approximately 14 times more memory was required, although the number of equations decreased from 3,773 to 2,031. Computing time per round increased by a factor of 50.

Algorithms↗
PubMed2004

Molecular genetic approaches to studying fertilization in model systems.

In a wide range of experimental systems, a variety of both forward and reverse genetic approaches are becoming available for the study of the molecules involved in fertilization. An integration of these methods with the antibody-based and biochemical studies traditionally used in fertilization research is enabling rapid advancements in our understanding of this process. We highlight some of the recent advances resulting from these genetic methods and their applications in these systems.

Animals↗
PubMed1999

Yeast forward and reverse 'n'-hybrid systems.

Since its original description almost 10 years ago, the yeast two-hybrid system has been used extensively to identify protein-protein interactions from many different organisms. Simultaneously, a number of 'variations on a theme' based on the original concept have been described. In one set of variations, systems were developed to detect other macromolecular interactions: DNA-protein (one-hybrid), RNA-protein (RNA-based three-hybrid) and small molecule-protein interactions (ligand-based three-hybrid). These different versions are collectively referred to here as 'n-hybrid systems'. In another set of variations, the original configuration of the two-hybrid fusion proteins was modified to expand the range of possible protein-protein interactions that could be analyzed. For example, systems were developed to detect trimeric interactions, ligand-receptor interactions or interactions that require particular post-translational modifications. Finally, the original concept was turned upside down and 'reverse n-hybrid systems' were developed to identify mutations, peptides or small molecules that dissociate macromolecular interactions. These reagents can be used to validate, in the relevant biological systems, the potential interactions identified with the 'forward n-hybrid systems'. The powerful genetic selections of the forward and reverse n-hybrid systems are proving useful in proteomic projects aimed at generating macromolecular interaction maps.

Animals↗
PubMed2004

Chemical genetics for therapeutic target mining.

Chemical genetics is an emerging research field that utilises biologically active small molecules to study biological functions of genes and their products. The direct regulation of the protein function by the biologically active small molecules can alternate the gene mutagenesis studies utilised in conventional genetics. Like conventional genetics, chemical genetics can be divided into two concepts - 'forward' and 'reverse' chemical genetics. These approaches of chemical genetics have a tremendous impact on both functional genomics and drug development. This review focuses on the two ways in which chemical genetics can be used for therapeutic target mining and their practical application in drug development, particularly, in angiogenesis-related diseases.

Angiogenesis Inhibitors↗
PubMed2003

Understanding the molecular responses to hypoxia using Drosophila as a genetic model.

We have previously discovered that Drosophila melanogaster could recover from extended periods of anoxia (0% oxygen) with no apparent consequential injuries. We have since employed forward and reverse genetic approaches to decipher the molecular basis for anoxia tolerance. In so doing, we have identified several independent mutant lines that demonstrated increased sensitivity to anoxia. Characterization of one of these mutants resulted in the identification of a dADAR gene that plays a role in the sensitivity to low levels of oxygen. We have also used microarrays to study all known Drosophila genes, the expression of which may be altered by hyoxia. Microarrays experiments have generated a large body of information that is being currently analyzed. We believe that these undertakings will provide insight into the genetic mechanisms of hypoxia tolerance and ischemic injuries.

Adenosine Deaminase↗
PubMed2001

Rethinking genetic strategies to study complex diseases.

Understanding the genetic basis of complex diseases is turning out to be difficult, prompting a widespread (re-)evaluation of the relevant issues. 'Forward' and 'reverse' genetics strategies have been applied arguably in a manner only suitable for much simpler diseases. It would now be beneficial to pay detailed attention to experimental design, and to increase study scales dramatically. Ultimately, this would lead to completely hypothesis-free, truly comprehensive, multi-platform investigations. Such studies would maximize the chances of finding data patterns indicative of real etiology, although many aspects of complex disease causation might simply be too intricate and inconsistent to ever be deciphered. Therefore, considerable technology development is an immediate priority, along with parallel advances in bioinformatics and biostatistics systems aimed at discriminating between marginal signals and background noise within extremely large, diverse and complex data sets. Community standards and open data sharing will be essential ingredients for success in this exciting 21st-century challenge.

Genetic Diseases, Inborn↗
PubMed2005

Cyclic peptides, a chemical genetics tool for biologists.

The genomics era has presented scientists with an overwhelming amount of sequence information that encodes for a continuously expanding array of metabolic pathways and regulatory networks. In order to unravel this elaborate maze, effective approaches need to be pioneered, and cyclic peptides represent an exciting new tool that can aid this challenging task. There are considerable benefits to cyclic peptides including enhanced protein binding affinity and metabolic stability, but perhaps most important is the ability to biologically synthesize them using intein chemistry. This capacity for genetic encoding allows biologists access to diverse, intracellular small-molecule libraries that can be tailored for numerous chemical genetics approaches. An increasing number of molecular strategies are being developed to aid in the implementation of cyclic peptides in forward and reverse genetics experiments, which will provide researchers with a powerful toolbox to complement existing genomic methodologies.

Animals↗
PubMed2000

Forward with BACs: new tools for herpesvirus genomics.

The large, complex genomes of herpesviruses document the high degree of adaptation of these viruses to their hosts. Not surprisingly, the methods developed over the past 30 years to analyse herpesvirus genomes have paralleled those used to investigate the genetics of eukaryotic cells. The recent use of bacterial artificial chromosome (BAC) technology in herpesvirus genetics has made their genomes accessible to the tools of bacterial genetics. This has opened up new avenues for reverse and forward genetics of this virus family in basic research, and also for vector and vaccine development.

Alleles↗
PubMed2003

Genetic underpinnings of autoimmunity--lessons from studies in arthritis, diabetes, lupus and multiple sclerosis.

Autoimmunity has been studied for more than four decades, but its genetic origins have remained a mystery. The recent past has witnessed an exponential growth in our understanding of autoimmunity resulting from both forward and reverse genetic approaches. More than 40 genes have been shown to precipitate systemic autoimmunity when genetically manipulated. In addition, reverse genetic studies in various autoimmune diseases have successfully guided researchers to specific locations on the genome that are associated with disease susceptibility. Buried within these genomic intervals lies a further treasure chest of autoimmunity genes. Efforts to unmask these culprit genes have yielded the very first clues about how an elaborate cast of players may be at work to orchestrate autoimmunity.

Arthritis↗
PubMed2002

The mouse: genetics meets behaviour.

Genetic studies in the mouse are important in the elucidation of molecular pathways that underlie behaviour. The advantages of the mouse for behavioural studies include an extensive array of genetic technologies and an elaborate behavioural repertoire that can be used to create models of human disease. This review discusses the relative advantages of forward and reverse genetic approaches to studying the genetic basis of behaviour in the mouse, and the complexities that behavioural studies need to address, such as phenotypic variability, genetic background effects and pleiotropy.

Animals↗
PubMed2004

A GFP-based assay reveals a role for RHD3 in transport between the endoplasmic reticulum and Golgi apparatus.

We describe the use of a secreted form of Aequoria victoria green fluorescent protein (secGFP) in a non-invasive live cell assay of membrane traffic in Arabidopsis thaliana. We show that in comparison to GFP-HDEL, which accumulates in the endoplasmic reticulum (ER), secGFP generates a weak fluorescence signal when transported to the apoplast. The fluorescence of secGFP in the apoplast can be increased by growth of seedlings on culture medium buffered at pH 8.1, suggesting that apoplastic pH is responsible, at least in part, for the low fluorescence intensity of seedlings expressing secGFP. Inhibition of secGFP transport between the ER and plasma membrane (PM), either by Brefeldin A (BFA) treatment or by genetic intervention results in increased intracellular secGFP accumulation accompanied by an increase in the secGFP fluorescence intensity. secGFP thus provides a valuable tool for forward and reverse genetic analysis of membrane traffic and endomembrane organisation in Arabidopsis. Using this assay for quantitative sublethal perturbation of secGFP transport, we identify a role for root hair defective 3 (RHD3) in transport of secreted and Golgi markers between the ER and the Golgi apparatus.

Arabidopsis↗
PubMed2002

Science review: key inflammatory and stress pathways in critical illness - the central role of the Toll-like receptors.

A pure reductionist approach can sometimes be used to solve an exceptionally complicated biologic problem, and sepsis is nothing if not complicated. A serious infection promptly leads to changes in many aspects of host physiology, including alterations in circulation, metabolism, renal, hepatic, and neuroendocrine function; all of these changes happen at once, and each influences one another. It is difficult to tease apart a problem of this sort, if only because the systems affected are so profoundly interactive. The key to understanding sepsis, insofar as we do understand it at present, was found in the use of genetic tools to study the very earliest events that take place at the interface of the pathogen and the host. The continued application of both forward and reverse genetic methods, in both mammals and insects, is steadily revealing the central biochemical events that occur during infection.

Animals↗
PubMed1997

Turn to the worm!

Caenorhabditis elegans will be the first multicellular animal to have its entire genome sequenced. This is not just good news for those currently working in the field, but also for those trying to understand the biology of more complex animals, including humans. C elegans is a relatively simple animal that is amenable to studies of genetics and developmental processes that are common to all animals, making this an attractive model in which to study basic processes that are altered in human disease. Powerful forward and reverse genetics mean that virtually any gene of interest can be studied at the functional level.

Animals↗