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MicroRNAs (miRNAs) attenuate gene expression by means of translational inhibition and mRNA degradation. They are abundant, highly conserved, and predicted to regulate a large number of transcripts. Several hundred miRNA classes are known, and many are associated with cell proliferation and differentiation. Many exhibit tissue-specific expression, which aids in evaluating their functions, and it has been assumed that their high level of sequence conservation implies a high level of expression conservation. A limited amount of data supports this, although discrepancies do exist. By comparing the expression of [approximately equal to] 100 miRNAs in medaka and chicken with existing data for zebrafish and mouse, we conclude that the timing and location of miRNA expression is not strictly conserved. In some instances, differences in expression are associated with changes in miRNA copy number, genomic context, or both between species. Variation in miRNA expression is more pronounced the greater the differences in physiology, and it is enticing to speculate that changes in miRNA expression may play a role in shaping the physiological differences produced during animal development. chick | evolution | medaka | miRNA | zebrafish

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.02.030 Byline: Ronald H.A. Plasterk (1) Abstract: Micro RNAs (miRNAs) are [approximately equal to]22 nucleotide single-stranded noncoding RNA molecules that bind to target messenger RNAs (mRNAs) and silence their expression. This Essay explores the importance of miRNAs in animal development and their possible roles in disease and evolution. Author Affiliation: (1) Hubrecht Laboratory (KNAW), Utrecht University, 3584CT Utrecht, the Netherlands

The rat is the most extensively studied model organism and is broadly used in biomedical research. Current rat disease models are selected from existing strains and their number is thereby limited by the degree of naturally occurring variation or spontaneous mutations. We have used ENU mutagenesis to increase genetic variation in laboratory rats and identified a recessive mutant, named tornado, showing aberrant circling behavior, hyperactivity, and stereotypic head shaking. More detailed analysis revealed profound deafness due to disorganization and degeneration of the organ of Corti that already manifests at the onset of hearing. We set up a single nucleotide polymorphism (SNP)-based mapping strategy to identify the affected gene, revealing strong linkage to the central region of chromosome 1. Candidate gene resequencing identified a point mutation that introduces a premature stopcodon in Myo7a. Mutations in human MYO7A result in Usher syndrome type 1B, a severe autosomal inherited recessive disease that involves deafness and vestibular dysfunction. Here, we present the first characterized rat model for this disease. In addition, we demonstrate proof of principle for the generation and cloning of human disease models in rat using ENU mutagenesis, providing good perspectives for systematic phenotypic screens in the rat.

Here, we describe a systematic search for synthetic gene interactions in a multicellular organism, the nematode Caenorhabditis elegans. We established a high-throughput method to determine synthetic gene interactions by genome-wide RNA interference and identified genes that are required to protect the germ line against DNA double-strand breaks. Besides known DNA-repair proteins such as the C. elegans orthologs of TopBP1, RPA2, and RAD51, eight genes previously unassociated with a double-strand-break response were identified. Knockdown of these genes increased sensitivity to ionizing radiation and camptothecin and resulted in increased chromosomal nondisjunction. All genes have human orthologs that may play a role in human carcinogenesis. DNA double-strand break | synthetic gene interactions | Caenorhabditis elegans

Protein misfolding and the formation of aggregates are increasingly recognized components of the pathology of human genetic disease and hallmarks of many neurodegenerative disorders. exemplified by polyglutamine diseases, the propensity for protein misfolding is associated with the length of polyglutamine expansions and age-dependent changes in protein-folding homeostastasis suggesting a critical role for a protein homeostatic buffer. To identify the complement of protein factors that protects cells against the formation of protein aggregates, we tested transgenic Caenorhabditis elegans strains expressing polyglutamine expansion yellow fluorescent protein fusion proteins at the threshold length associated with the age-dependent appearance of protein aggregation. We used genome-wide RNA interference to identify genes that, when suppressed, resulted in the premature appearance of protein aggregates, Our screen identified 186 genes corresponding to five principal classes of polyglutamine regulators; genes involved in RNA metabolism, protein synthesis, protein folding, and protein degradation; and those involved in protein trafficking. We propose that each of these classes represents a molecular machine collectively comprising the protein homeostatic buffer that responds to the expression of damaged proteins prevent their misfolding and aggregation. protein misfolding | neurodegenerative diseases

In a genome-wide analysis of the active transposons in Caenorhabditis elegans we determined the localization and sequence of all copies of each of the six active transposon families. Most copies of the most active transposops, Tc1 and Tc3, are intact but individually have a unique sequence, because of unique patterns of single-nucleotide polymorphisms. The sequence of each of the 32 Tc1 elements is invariant in the C. elegans strain N2, which has no germline transposition. However, at the same 32 Tc1 loci in strains with germline transposition, Tc1 elements can acquire the sequence of Tc1 elements elsewhere in the N2 genome or a chimeric sequence derived from two dispersed Tc1 elements. We hypothesize that during double-strand-break repair after Tc1 excision, the template for repair can switch from the Tc1 element on the sister chromatid or homologous chromosome to a Tc1 copy elsewhere in the genome. Thus, the population of active transposable elements in C. elegans is highly dynamic because of a continuous exchange of sequence information between individual copies, potentially allowing a higher evolution rate than that found in endogenous genes.

The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 [F.sub.1] fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 [F.sub.1] fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated., Forward genetic screens in the zebrafish have identified thousands of mutants defective in many kinds of biological processes (1, 2). Cloning of the affected genes is accelerated by both an [...]

Mismatch-repair-deficient mutants were initially recognized as mutation-prone derivatives of bacteria, and later mismatch repair deficiency was found to predispose humans to colon cancers (HNPCC). We generated mismatch-repair-deficient Caenorhabditis elegans by deleting the msh-6 gene and analyzed the fidelity of transmission of genetic information to subsequent generations, msh-6-defective animals show an elevated level of spontaneous mutants in both the male and female germline; also repeated DNA tracts are unstable. To monitor DNA repeat instability in somatic tissue, we developed a sensitive system, making use of heat-shock promoter-driven lacZ transgenes, but with a repeat that puts this reporter gene out of frame. In genetic msh-6-deficient animals lac[Z.sup.+] patches are observed as a result of somatic repeat instability. RNA interference by feeding wild-type animals dsRNA homologous to msh-2 or msh-6 also resulted in somatic DNA instability, as well as in germline mutagenesis, indicating that one can use C. elegans as a model system to discover genes involved in maintaining DNA stability by large-scale RNAi screens.

The sgs-1 (suppressor of activated G[[alpha].sub.s]) gene encodes one of the four adenylyl cyclases in the nematode C. elegans and is most similar to mammalian adenylyl cyclase type IX. We isolated a complete loss-of-function mutation in sgs-1 and found it to result in animals with retarded development that arrest in variable larval stages. sgs-1 mutant animals exhibit lethargic movement and pharyngeal pumping and (while not reaching adulthood) have a mean life span that is >50% extended compared to wild type. An extensive set of reduction-of-function mutations in sgs-1 was isolated in a screen for suppressors of a neuronal degeneration phenotype induced by the expression of a constitutively active version of the heterotrimeric G[[alpha].sub.s] subunit of C. elegans. Although most of these mutations change conserved residues within the catalytic domains of sgs-1, mutations in the less-conserved transmembrane domains are also found. The sgs-1 reduction-of-function mutants are viable and have reduced locomotion rates, but do not show defects in pharyngeal pumping or life span.

Posttranscriptional gene silencing in Caenorhabditis elegans results from exposure to double-stranded RNA (dsRNA), a phenomenon designated as RNA interference (RNAi), or from co-suppression, in which transgenic DNA leads to silencing of both the transgene and the endogenous gene. Here we show that single-stranded RNA oligomers of antisense polarity can also be potent inducers of gene silencing. As is the case for co-suppression, antisense RNAs act independently of the RNAi genes rde-1 and rde-4 but require the mutator/RNAi gene mut-7 and a putative DEAD box RNA helicase, mut-14. Our data favor the hypothesis that gene silencing is accomplished by RNA primer extension using the mRNA as template, leading to dsRNA that is subsequently degraded., RNA interference (RNAi) in animals, co-suppression in plants, and quelling in fungi appear to be manifestations of cellular defense mechanisms against the invasion of foreign nucleic acids such as viruses [...]

A model in which MUT-7, guided by transposon-derived dsRNA, represses transposition by degrading transposon-specific messengers and prevents thereby transposase production and transposition, is proposed. It appears that NRAi may work by dsRNA-directed enzymatic RNA degradation. One Tc1 silenced strain of C. elegans has been has been mutagenized. Mutants in which Tc1 was activated in the germline were isolated.

Caenorhabditis elegans uses chemosensation to determine its course of development. Young larvae can arrest as dauer larvae in response to increasing population density, which they measure by a nematode-excreted pheromone, and decreasing food supply. Dauer larvae can resume development in response to a decrease in pheromone and increase in food concentration. We show here that two novel G protein alpha subunits (GPA-2 and GPA-3) show promoter activity in subsets of chemosensory neurons and are involved in the decision to form dauer larvae primarily through the response to dauer pheromone. Dominant activating mutations in these G proteins result in constitutive, pheromone-independent dauer formation, whereas inactivation results in reduced sensitivity to pheromone, and, under certain conditions, an alteration in the response to food. Interactions between gpa-2, gpa-3 and other genes controlling dauer formation suggest that these G proteins may act in parallel to regulate the neuronal decision making that precedes dauer formation.

We present an approach to map large numbers of Tc1 transposon insertions in the genome of Caenorhabditis elegans. Strains have been described that contain up to 500 polymorphic Tc1 insertions. From these we have cloned and shotgun sequenced over 2000 Tc1 flanks, resulting in an estimated set of 400 or more distinct Tc1 insertion alleles. Alignment of these sequences revealed a weak Tc1 insertion site consensus sequence that was symmetric around the invariant TA target site and reads CAYATATRTG. The Tc1 flanking sequences were compared with 40 Mbp of a C. elegans genome sequence. We found 151 insertions within the sequenced area, a density of [approximately equal to]1 Tc1 insertion in every 265 kb. As the rest of the C. elegans genome sequence is obtained, remaining Tc1 alleles will fall into place. These mapped Tc1 insertions can serve two functions: (i) insertions in or near genes can be used to isolate deletion derivatives that have that gene mutated; and (ii) they represent a dense collection of polymorphic sequence-tagged sites. We demonstrate a strategy to use these Tc1 sequence-tagged sites in fine-mapping mutations.

Maternal G proteins were found to play a major role in the orientation of early cell division axes possibly by coupling a membrane signal to centrosome position. The G proteins in the nematode Caenorhabditis elegans were observed to learn about the function of G proteins in vivo and to identify other active elements in the signaling cascade. G beta, which is needed in many developmental stages, is involved in the regulation of behaviors such as locomotion and egg-laying.

Integration of human immunodeficiency virus (HIV) DNA into the human genome requires the virus-encoded integrase (IN) protein, and therefore the IN protein is a suitable target for antiviral strategies. To find a potent HIV IN inhibitor, we screened a 'synthetic peptide combinatorial library.' We identified a hexapeptide with the sequence HCKFWW that inhibits IN-mediated 3'-processing and integration with an [IC.sub.50] of 2 [mu]M. The peptide is active on IN proteins from other retroviruses such as HIV-2, feline immunodeficiency virus, and Moloney murine leukemia virus, supporting the notion that a conserved region of IN is targeted. The hexapeptide was also tested in the disintegration reaction. This phosphoryl-transfer reaction can be carried out by the catalytic core of IN alone, ahd the peptide HCKFWW was found to inhibit this reaction, suggesting that the hexapeptide acts at or near the catalytic site of IN. Identification of an IN hexapeptide inhibitor provides proof of concept for the approach, and, moreover, this peptide may be useful for structure-function analysis of IN.

Over the past two decades, the small soil nematode Caenorhabditis elegans has become established as a major model system for the study of a great variety of problems in biology and medicine. One of its most significant advantages is its simplicity, both in anatomy and in genomic organization. The entire haploid genetic content amounts to 100 million base pairs of DNA, about 1/30 the size of the human value. As a result, C. elegans has also provided a pilot system for the construction of physical maps of larger animal and plant genomes, and subsequently for the complete sequencing of those genomes. By mid-1995, approximately one-fifth of the complete DNA sequence of this animal had been determined. Caenorhabditis elegans provides a test bed not only for the development and application of mapping and sequencing technologies, but also for the interpretation and use of complete sequence information. This article reviews the progress so far toward a realizable goal--the total description of the genome of a simple animal., Caenorhabditis elegans has many attractive features as an experimental system[1]. The life cycle is simple and rapid, with a 3-day generation time, and populations can be grown with ease on [...]

A study has been conducted to describe a new typing method developed for Actinobacillus actinomycetemcomitans. Genomic fingerprinting produced common bands for all strains and classified the bacterium into 29 distinct types. Use the method on 10 other clinically relevant bacterial species produced the same species-specific and strain-specific bands. Computer analysis of the band patterns allowed determination of the similarity of coefficients. These results suggest that genomic fingerprinting by restriction fragment end labeling is an efficient typing method for bacterial species.

The Caenorhabditis elegans odc-1 gene and its cDNA was cloned and characterized to determine the Ornithine decarboxylase (ODC) enzyme's developmental function. An odc-1 null mutant was also isolated to offer supplementary characterization. Assay analysis of ODC activity found no substantial activity level, indicating the factor's dispensability for laboratory nematode growth.

The goa-1 gene encoding the alpha subunit of the heterotrimeric guanosine triphosphate-binding protein (G protein) [G.sub.o] from Caenorhabditis elegans is expressed in most neurons, and in the muscles involved in egg laying and male mating. Reduction-of-function mutations in goa-1 caused a variety of behavioral defects including hyperactive movement, premature egg laying, and male impotence. Expression of the activated [G.sub.o] alpha subunit [Gα.sub.o]) in transgenic nematodes resulted in lethargic movement, delayed egg laying, and [reduced mating efficiency. Induced expression of activated [Gα.sub.o] in adults was sufficient to cause these phenotypes, indicating that [Gα.sub.o] mediates behavior through its role in neuronal function and the functioning of specialized muscles., The heterotrimeric G protein [G.sub.o] is abundantly expressed in mammalian and insect neurons and growth cones, suggesting that it is important for both neuronal development and function (1). Although G. [...]

A study of Tc3 transposase expression from heat-shock promoters in transgenic nematodes helps understand the Tc3 transposition pathway. The linear extrachromosomal Tc3 DNA that appears during transposition is cut incompletely, with two nucleotides being absent from the 5' ends. The nucleotides at the donor site lead to a distinct footprint. A model that depicts the excision and integration of the Tc3 element and target replication and traces the roots of the somatic footprints is suggested, based on the structure of the extrachromosomal DNA element.

Transposons are used in the tagging of genes, in the generation of transgenic flies and to perform reverse genetics. The P elements of the Drosophila, when excised, cause the breakage in the DNA, which is repaired by ectopic templates. This repair introduces 8000 binding proteins of foreign DNA. This indicates that the shuttling of long sequences into the chromosomes is caused by the transposon allele. The introduction of the required fusion as a transgene into a strain results in the formation of the precise gene fusions outside a living cell.

The genotype that determines the phenotype of the Caenorhabditis elegans was sought to be understood by the establishment of a mutant library by developing a scheme to screen the library for the insertion of mutants of the C. elegans. These insertions are used in the isolation of genuine gene knockouts. The Tc1 transposon was found to induce deletions of flanking DNA regularly and was used to inactivate genes by establishing a frozen library of 5000 nematode lines mutagenized by Tc1 insertions.

A study was done on the integrase protein of HIV-2 using mutational analysis. It was shown that donor cut, strand transfer and disintegration are carried out in the same way by immobilized and soluble integrase protein which were observed after prolonged incubation. These findings suggest that only one active site can accomplish both hydrolysis of the specific phosphodiester bond 3' of the conserved CA-3' and the transesterification reactions that results in the actual strand transfer.

Genomes are databases sensitive to invasion by viruses. In recent years, a defense mechanism has been discovered, which turns out to be conserved among eukaryotes. The system can be compared to the immune system in several ways: It has specificity against foreign elements and the ability to amplify and raise a massive response against an invading nucleic acid. The latter property is beginning to be understood at the molecular level., All genomes of complex organisms are potential targets of invasion by viruses and transposable elements. Forty-five percent of the human genome consists of remnants of previous transposon/virus invasions and elements [...]