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At least 199 records · Page 11Linked to original sources
PubMed2004

Achieving immortality in the C. elegans germline.

Germline immortality is a topic that has intrigued theoretical biologists interested in aging for over a century. The germ cell lineage can be passed from one generation to the next, indefinitely. In contrast, somatic cells are typically only needed for a single generation and are then discarded. Germ cells may, therefore, harbor rejuvenation mechanisms that enable them to proliferate for eons. Such processes are thought to be either absent from or down-regulated in somatic cells, although cell non-autonomous forms of rejuvenation are formally possible. A thorough description of mechanisms that foster eternal youth in germ cells is lacking. The mysteries of germline immortality are being addressed in the nematode Caenorhabditis elegans by studying mutants that reproduce normally for several generations but eventually become sterile. The mortal germline mutants probably become sterile as a consequence of accumulating various forms of heritable cellular damage. Such mutants are abundant, indicating that several different biochemical pathways are required to rejuvenate the germline. Thus, forward genetics should help to define mechanisms that enable the germline to achieve immortality.

Aging↗
PubMed2005

Molecular characterization of ENU mouse mutagenesis and archives.

The large-scale mouse mutagenesis with ENU has provided forward-genetic resources for functional genomics. The frozen sperm archive of ENU-mutagenized generation-1 (G1) mice could also provide a "mutant mouse library" that allows us to conduct reverse genetics in any particular target genes. We have archived frozen sperm as well as genomic DNA from 9224 G1 mice. By genome-wide screening of 63 target loci covering a sum of 197 Mbp of the mouse genome, a total of 148 ENU-induced mutations have been directly identified. The sites of mutations were primarily identified by temperature gradient capillary electrophoresis method followed by direct sequencing. The molecular characterization revealed that all the identified mutations were point mutations and mostly independent events except a few cases of redundant mutations. The base-substitution spectra in this study were different from those of the phenotype-based mutagenesis. The ENU-based gene-driven mutagenesis in the mouse now becomes feasible and practical.

Animals↗
PubMed2006

Point mutations within the LdNT2 nucleoside transporter gene from Leishmania donovani confer drug resistance and transport deficiency.

Leishmania donovani, a protozoan parasite, expresses an unusual inosine/guanosine-specific transporter, LdNT2, the gene for which was cloned by functional rescue of a drug-resistant, LdNT2-deficient (FBD5) strain. In this investigation, we have uncovered and characterized the mutations within the LdNT2 open reading frame that are the basis for the drug-resistance and transport-incompetent phenotype of the FBD5 line. The FBD5 cells were shown to be compound heterozygotes in which both mutant ldnt2 alleles harbor discrete point mutations, each of which impaired transport function and conferred resistance to formycin B, the drug to which the clonal FBD5 line was selected. One of the mutant ldnt2 alleles encoded an S189L alteration in predicted transmembrane domain 5, while the second allele accommodated a null mutation at codon 376, which truncated the transporter just prior to transmembrane domain 8. In addition to the null transport phenotype, very little S189L ldnt2 mutant transporter targeted to the surface of the parasite. The bulk of the truncated ldnt2 appeared to be sequestered internally, possibly within the endoplasmic reticulum, but some of the truncated transporter seemed to be cell surface exposed. The ability to dissect mutations within a viable parasite offers LdNT2 as an attractive model for implementing a thorough forward genetic dissection of transporter function in a eukaryotic cell.

Animals↗
PubMed2006

Neuroendocrinology of protochordates: insights from Ciona genomics.

The genome for two species of Ciona is available making these tunicates excellent models for studies on the evolution of the chordates. In this review most of the data is from Ciona intestinalis, as the annotation of the C. savignyi genome is not yet available. The phylogenetic position of tunicates at the origin of the chordates and the nature of the genome before expansion in vertebrates allows tunicates to be used as a touchstone for understanding genes that either preceded or arose in vertebrates. A comparison of Ciona, a sea squirt, to other model organisms such as a nematode, fruit fly, zebrafish, frog, chicken and mouse shows that Ciona has many useful traits including accessibility for embryological, lineage tracing, forward genetics, and loss- or gain-of-function experiments. For neuroendocrine studies, these traits are important for determining gene function, whereas the availability of the genome is critical for identification of ligands, receptors, transcription factors and signaling pathways. Four major neurohormones and their receptors have been identified by cloning and to some extent by function in Ciona: gonadotropin-releasing hormone, insulin, insulin-like growth factor, and cionin, a member of the CCK/gastrin family. The simplicity of tunicates should be an advantage in searching for novel functions for these hormones. Other neuroendocrine components that have been annotated in the genome are a multitude of receptors, which are available for cloning, expression and functional studies.

Animals↗
PubMed2006

Pumping RNA: nuclear bodybuilding along the RNP pipeline.

Cajal bodies (CBs) are nuclear subdomains involved in the biogenesis of several classes of small ribonucleoproteins (RNPs). A number of recent advances highlight progress in the understanding of the organization and dynamics of CB components. For example, a class of small Cajal body-specific (sca) RNPs has been discovered. Localization of scaRNPs to CBs was shown to depend on a conserved RNA motif. Intriguingly, this motif is also present in mammalian telomerase RNA and the evidence suggests that assembly of the active form of telomerase RNP occurs in and around CBs during S phase. Important steps in the assembly and modification of spliceosomal RNPs have also been shown to take place in CBs. Additional experiments have revealed the existence of kinetically distinct subclasses of CB components. Finally, the recent identification of novel markers for CBs in both Drosophila and Arabidopsis not only lays to rest questions about the evolutionary conservation of these nuclear suborganelles, but also should enable forward genetic screens for the identification of new components and pathways involved in their assembly, maintenance and function.

Animals↗
PubMed2005

A new view of an old pore.

Despite reports suggesting a potential role in membrane fusion, the V(0) subunit of the (+)H/ATPase has remained an unlikely candidate for the fusion pore. In this issue of Cell, Hiesinger and coworkers (Hiesinger et al., 2005) present a forward genetic screen that reveals it to be necessary for synaptic vesicle fusion, independent of its role in vesicle acidification.

Animals↗
PubMed2023

Anticancer benzoxaboroles block pre-mRNA processing by directly inhibiting CPSF3.

A novel class of benzoxaboroles was reported to induce cancer cell death but the mechanism was unknown. Using a forward genetics platform, we discovered mutations in cleavage and polyadenylation specific factor 3 (CPSF3) that reduce benzoxaborole binding and confer resistance. CPSF3 is the endonuclease responsible for pre-mRNA 3'-end processing, which is also important for RNA polymerase II transcription termination. Benzoxaboroles inhibit this endonuclease activity of CPSF3 in vitro and also curb transcriptional termination in cells, which results in the downregulation of numerous constitutively expressed genes. Furthermore, we used X-ray crystallography to demonstrate that benzoxaboroles bind to the active site of CPSF3 in a manner distinct from the other known inhibitors of CPSF3. The benzoxaborole compound impeded the growth of cancer cell lines derived from different lineages. Our results suggest benzoxaboroles may represent a promising lead as CPSF3 inhibitors for clinical development.

Cleavage And Polyadenylation Specificity Factor↗
PubMed2004

New models for the study of Mycobacterium-host interactions.

The outcome of Mycobacterium infection is determined by a series of complex interactions between the bacteria and host immunity. Traditionally, mammalian models and cultured cells have been used to study these interactions. Recently, ameba (Dictyostelium), fruit flies (Drosophila) and zebrafish, amenable to forward genetic screens, have been developed as models for mycobacterial pathogenesis. Infection of these hosts with mycobacteria has allowed the dissection of intracellular trafficking pathways (Dictyostelium) and the roles of phagocytic versus antimicrobial peptide responses (Drosophila). Real-time visualization of the optically transparent zebrafish embryo/larva has elucidated mechanisms by which Mycobacterium-infected leukocytes migrate and subsequently aggregate into granulomas, the hallmark pathological structures of tuberculosis.

Animals↗
PubMed2006

Learning from stargazin: the mouse, the phenotype and the unexpected.

The stargazin gene (also referred to as Cacng2) has been identified by forward genetics in a spontaneous mouse mutant with ataxic gait, upward head-elevating movements (hence the name stargazer for the mouse) and episodes of spike-wave discharges. Stargazin is related to the gamma-1 subunit of skeletal muscle voltage-dependent calcium channel (VDCC), and a deficit in its role as auxiliary VDCC subunit was proposed to underlie the epileptic phenotype of the mouse; yet, a conclusive demonstration of stargazin function in VDCC regulation is still lacking. In contrast, stargazin and its three closely related isoforms gamma-3, gamma-4 and gamma-8 were shown to function as auxiliary subunits for a very different ion channel - the AMPA-type glutamate receptor - prominently regulating early intracellular transport, synaptic targeting and anchoring, and ion channel functions of this major excitatory receptor in the brain.

Animals↗
PubMed2004

Identification of thermosensory and olfactory neuron-specific genes via expression profiling of single neuron types.

Most C. elegans sensory neuron types consist of a single bilateral pair of neurons, and respond to a unique set of sensory stimuli. Although genes required for the development and function of individual sensory neuron types have been identified in forward genetic screens, these approaches are unlikely to identify genes that when mutated result in subtle or pleiotropic phenotypes. Here, we describe a complementary approach to identify sensory neuron type-specific genes via microarray analysis using RNA from sorted AWB olfactory and AFD thermosensory neurons. The expression patterns of subsets of these genes were further verified in vivo. Genes identified by this analysis encode 7-transmembrane receptors, kinases, and nuclear factors including dac-1, which encodes a homolog of the highly conserved Dachshund protein. dac-1 is expressed in a subset of sensory neurons including the AFD neurons and is regulated by the TTX-1 OTX homeodomain protein. On thermal gradients, dac-1 mutants fail to suppress a cryophilic drive but continue to track isotherms at the cultivation temperature, representing the first genetic separation of these AFD-mediated behaviors. Expression profiling of single neuron types provides a rapid, powerful, and unbiased method for identifying neuron-specific genes whose functions can then be investigated in vivo.

Amino Acid Sequence↗
PubMed2005

Defective skeletogenesis with kidney stone formation in dwarf zebrafish mutant for trpm7.

Development of the adult form requires coordinated growth and patterning of multiple traits in response to local gene activity as well as to global endocrine and physiological effectors. An excellent example of such coordination is the skeleton. Skeletal development depends on the differentiation and morphogenesis of multiple cell types to generate elements with distinct forms and functions throughout the body. We show that zebrafish touchtone/nutria mutants exhibit severe growth retardation and gross alterations in skeletal development in addition to embryonic melanophore and touch-response defects. These alterations include accelerated endochondral ossification but delayed intramembranous ossification, as well as skeletal deformities. We show that the touchtone/nutria phenotype results from mutations in trpm7, which encodes a transient receptor potential (TRP) family member that functions as both a cation channel and kinase. We find trpm7 expression in the mesonephric kidney and show that mutants develop kidney stones, indicating renal dysfunction. These results identify a requirement for trpm7 in growth and skeletogenesis and highlight the potential of forward genetic approaches to uncover physiological mechanisms contributing to the development of adult form.

Abnormalities, Multiple↗
PubMed2006

nsf is essential for organization of myelinated axons in zebrafish.

BACKGROUND: Myelinated axons are essential for rapid conduction of action potentials in the vertebrate nervous system. Of particular importance are the nodes of Ranvier, sites of voltage-gated sodium channel clustering that allow action potentials to be propagated along myelinated axons by saltatory conduction. Despite their critical role in the function of myelinated axons, little is known about the mechanisms that organize the nodes of Ranvier. RESULTS: Starting with a forward genetic screen in zebrafish, we have identified an essential requirement for nsf (N-ethylmaleimide sensitive factor) in the organization of myelinated axons. Previous work has shown that NSF is essential for membrane fusion in eukaryotes and has a critical role in vesicle fusion at chemical synapses. Zebrafish nsf mutants are paralyzed and have impaired response to light, reflecting disrupted nsf function in synaptic transmission and neural activity. In addition, nsf mutants exhibit defects in Myelin basic protein expression and in localization of sodium channel proteins at nodes of Ranvier. Analysis of chimeric larvae indicates that nsf functions autonomously in neurons, such that sodium channel clusters are evident in wild-type neurons transplanted into the nsf mutant hosts. Through pharmacological analyses, we show that neural activity and function of chemical synapses are not required for sodium channel clustering and myelination in the larval nervous system. CONCLUSIONS: Zebrafish nsf mutants provide a novel vertebrate system to investigate Nsf function in vivo. Our results reveal a previously unknown role for nsf, independent of its function in synaptic vesicle fusion, in the formation of the nodes of Ranvier in the vertebrate nervous system.

Action Potentials↗
PubMed2006

Atmnd1-delta1 is sensitive to gamma-irradiation and defective in meiotic DNA repair.

The efficient repair of double-strand breaks (DSBs) in genomic DNA is crucial for the survival of all organisms. Mnd1 is suggested to promote the strand invasion step during meiotic recombination. We used a forward genetics approach, through the search for mutants, to characterize the Arabidopsis homologue of Mnd1. Atmnd1 null mutants exhibit normal vegetative and flower development. In contrast, during prophase I, chromosomes become fragmented resulting in random distribution of the fragments between polyads. Male and female meiosis are defective and strong accumulation of AtRAD51 was observed in atmnd1-delta1 nuclei. These results suggest that similarly to its yeast and animal homologues, AtMnd1 plays a role in DSB repair during meiosis. In addition, like its human homologue AtMnd1 is expressed in somatic cells. AtMnd1 expression is strongly induced by gamma-rays and null mutants show increased sensibility to gamma-rays. Taken together, these results suggest that AtMnd1 is also required for DSB repair in somatic cells.

Arabidopsis↗
PubMed2006

fester, A candidate allorecognition receptor from a primitive chordate.

Histocompatibility in the primitive chordate, Botryllus schlosseri, is controlled by a single, highly polymorphic locus, the FuHC. By taking a forward genetic approach, we have identified a locus encoded near the FuHC, called fester, which is polymorphic, polygenic, and inherited in distinct haplotypes. Somatic diversification occurs through extensive alternative splicing, with each individual expressing a unique repertoire of splice forms, both membrane bound and potentially secreted, all expressed in tissues intimately associated with histocompatibility. Functional studies, via both siRNA-mediated knockdown and direct blocking by monoclonal antibodies raised against fester, were able to disrupt predicted histocompatibility outcomes. The genetic and somatic diversity, coupled to the expression and functional data, suggests that fester is a receptor involved in histocompatibility.

Alleles↗
PubMed2004

RNA interference for analysis of gene function in trypanosomatids.

Gene-specific silencing by RNA interference is a valuable tool for analysis of gene function in the protozoan parasite Trypanosoma brucei. The development of tetracycline-regulated vectors for production of double-stranded RNA has facilitated its widespread use. RNA interference provides a fast and efficient method for determining whether a gene is essential for growth and viability, reveals mechanistic information on gene function, and has greatly enhanced our understanding of complex biological processes. Finally, the creation of an RNA interference-based library has allowed, for the first time, an approach for conducting forward genetic experiments in this organism.

Animals↗
PubMed2006

Components of the protein quality control system are expressed in a strain-dependent manner in the mouse hippocampus.

Inbred mouse strains are used in forward-genetic experiments, designed to uncover genes contributing to their highly distinct neurophenotypes and multiple reports of variations in mutant phenotypes due to genetic background differences in reverse-genetic approaches have been published. Information on strain-specific protein expression-phenotypes however, is limited and a comprehensive screen of an effect of strain on brain protein levels has not yet been carried out. Herein a proteomic approach, based upon two-dimensional gel electrophoresis (2-DE) coupled to mass spectrometry (MALDI-TOF/TOF) was used to show significant genetic variation in hippocampal protein levels between five mouse strains. Considering recent evidence for the importance of the intracellular protein quality control system for synaptic plasticity-related mechanism we decided to focus on the analysis of molecular chaperones and components of the ubiquitin-proteasome system. Sixty-six spots, depicting 36 proteins have been unambiguously identified by mass spectrometry. Quantification revealed strain-dependent levels of 18 spots, representing 12 individual gene products. We thus present proteome analysis of hippocampal tissues of several mouse strains as suitable tool to address fundamental questions about genetic control of protein levels and to demonstrate molecular networks of protein metabolism and chaperoning. The findings are useful for designing future studies on these cascades and interpretation of results show that data on brain protein levels cannot be simply extrapolated among different mouse strains.

Animals↗
PubMed2006

In vivo cell biology: following the zebrafish trend.

A deeper understanding of the mechanisms of cell behavior is essential if we want to comprehend how an organism develops and functions. Changes in cellular processes, including the orientation of cell divisions, cell shape, polarity, differentiation and migration, account for tissue rearrangements during development and homeostasis. The in vivo relevance of in vitro findings is being constantly debated and the need for in vivo systems becoming more pressing. The zebrafish (Danio rerio) might become the vertebrate system of choice for a wide spectrum of biological questions that need to be investigated in vivo at cellular and subcellular resolutions. Here, we discuss some recent studies in which the zebrafish was used to gain insight into cell-biological mechanisms. Although this model system has been predominantly appreciated for its amenability to forward genetics, current advances in imaging technology and an increasing number of transgenic lines are bringing it closer to its full potential.

Animals↗
PubMed2006

Mutagenesis strategies in zebrafish for identifying genes involved in development and disease.

It has been nearly a decade since the completion of two large-scale chemical mutagenesis screens in zebrafish, and two years since the completion of a large-scale insertional mutagenesis. In this article, we use the accumulated data from these screens to compare the efficiency of each mutagen to isolate mutants and to identify mutated genes, and argue that the two mutagens target the same set of genes. We then review how both forward genetic screens and reverse genetic techniques, such as morpholinos and TILLING, and transgenics are being used to develop models of human disease.

Animals↗