A viral suppressor of gene silencing in plants

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

Tissue-specific knockout of the mouse Pig-a gene reveals important roles for GPI-anchored proteins in skin development

Glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed on plasma membranes of eukaryotes. More than 50 GPI-anchored proteins have been shown to be spatiotemporally expressed in mice with a deficiency of GPI-anchor biosynthesis that causes embryonic lethality. Here, we examine the functional roles of GPI-anchored proteins in mouse skin using the Cre-loxP recombination system. We disrupted the Pig-a gene, an X-linked gene essential for GPI-anchor biosynthesis, in skin. The Cre-mediated Pig-a disruption occurred in skin at almost 100% efficiency in male mice bearing two identically orientated loxP sites within the Pig-a gene. Expression of GPI-anchored proteins was completely absent in the skin of these mice. The skin of such mutants looked wrinkled and more scaly than that of wild-type mice. Furthermore, histological examination of mutant mice showed that the epidermal horny layer was tightly packed and thickened. Electron microscopy showed that the intercellular space was narrow and there were many small vesicles embedded in the intercellular space that were not observed in equivalent wild-type mouse skin preparations. Mutant mice died within a few days after birth, suggesting that Pig-a function is essential for proper skin differentiation and maintenance.

Identification and characterization of Saccharomyces cerevisiae EXO1, a gene encoding an exonuclease that interacts with MSH2

A two-hybrid screen was used to identify Saccharomyces cerevisiae genes encoding proteins that interact with MSH2. One gene was found to encode a homologue of Schizosaccharomyces pombe EXO1, a double-stranded DNA-specific 5′–3′ exonuclease. S. cerevisiae EXO1 interacted with both S. cerevisiae and human MSH2 in two-hybrid and coimmunoprecipitation experiments. exo1 mutants showed a mutator phenotype, and epistasis analysis was consistent with EXO1 functioning in the MSH2-dependent mismatch repair pathway. exo1 mutations were lethal in combination with rad27 mutations, and overexpression of EXO1 suppressed both the temperature sensitive and mutator phenotypes of rad27 mutants.

Horizontal gene transfer and mutation: Ngrol genes in the genome of Nicotiana glauca

Ngrol genes (NgrolB, NgrolC, NgORF13, and NgORF14) that are similar in sequence to genes in the left transferred DNA (TL-DNA) of Agrobacterium rhizogenes have been found in the genome of untransformed plants of Nicotiana glauca. It has been suggested that a bacterial infection resulted in transformation of Ngrol genes early in the evolution of the genus Nicotiana. Although the corresponding four rol genes in TL-DNA provoked hairy-root syndrome in plants, present-day N. glauca and plants transformed with Ngrol genes did not exhibit this phenotype. Sequenced complementation analysis revealed that the NgrolB gene did not induce adventitious roots because it contained two point mutations. Single-base site-directed mutagenesis at these two positions restored the capacity for root induction to the NgrolB gene. When the NgrolB, with these two base substitutions, was positioned under the control of the cauliflower mosaic virus 35S promoter (P35S), transgenic tobacco plants exhibited morphological abnormalities that were not observed in P35s-RirolB plants. In contrast, the activity of the NgrolC gene may have been conserved after an ancient infection by bacteria. Discussed is the effect of the horizontal gene transfer of the Ngrol genes and mutations in the NgrolB gene on the phenotype of ancient plants during the evolution of N. glauca.

Suppression of gene silencing: A general strategy used by diverse DNA and RNA viruses of plants

In transgenic and nontransgenic plants, viruses are both initiators and targets of a defense mechanism that is similar to posttranscriptional gene silencing (PTGS). Recently, it was found that potyviruses and cucumoviruses encode pathogenicity determinants that suppress this defense mechanism. Here, we test diverse virus types for the ability to suppress PTGS. Nicotiana benthamiana exhibiting PTGS of a green fluorescent protein transgene were infected with a range of unrelated viruses and various potato virus X vectors producing viral pathogenicity factors. Upon infection, suppression of PTGS was assessed in planta through reactivation of green fluorescence and confirmed by molecular analysis. These experiments led to the identification of three suppressors of PTGS and showed that suppression of PTGS is widely used as a counter-defense strategy by DNA and RNA viruses. However, the spatial pattern and degree of suppression varied extensively between viruses. At one extreme, there are viruses that suppress in all tissues of all infected leaves, whereas others are able to suppress only in the veins of new emerging leaves. This variation existed even between closely related members of the potexvirus group. Collectively, these results suggest that virus-encoded suppressors of gene silencing have distinct modes of action, are targeted against distinct components of the host gene-silencing machinery, and that there is dynamic evolution of the host and viral components associated with the gene-silencing mechanism.

The Tabby phenotype is caused by mutation in a mouse homologue of the EDA gene that reveals novel mouse and human exons and encodes a protein (ectodysplasin-A) with collagenous domains

Mouse Tabby (Ta) and X chromosome-linked human EDA share the features of hypoplastic hair, teeth, and eccrine sweat glands. We have cloned the Ta gene and find it to be homologous to the EDA gene. The gene is altered in two Ta alleles with a point mutation or a deletion. The gene is expressed in developing teeth and epidermis; no expression is seen in corresponding tissues from Ta mice. Ta and EDA genes both encode alternatively spliced forms; novel exons now extend the 3′ end of the EDA gene. All transcripts recovered have the same 5′ exon. The longest Ta cDNA encodes a 391-residue transmembrane protein, ectodysplasin-A, containing 19 Gly-Xaa-Yaa repeats. The isoforms of ectodysplasin-A may correlate with differential roles during embryonic development.

Further examination of the Xist promoter-switch hypothesis in X inactivation: Evidence against the existence and function of a P0 promoter

The onset of X inactivation coincides with accumulation of Xist RNA along the future inactive X chromosome. A recent hypothesis proposed that accumulation is initiated by a promoter switch within Xist. In this hypothesis, an upstream promoter (P0) produces an unstable transcript, while the known downstream promoter (P1) produces a stable RNA. To test this hypothesis, we examined expression and half-life of Xist RNA produced from an Xist transgene lacking P0 but retaining P1. We confirm the previous finding that P0 is dispensable for Xist expression in undifferentiated cells and that P1 can be used in both undifferentiated and differentiated cells. Herein, we show that Xist RNA initiated at P1 is unstable and does not accumulate. Further analysis indicates that the transcriptional boundary at P0 does not represent the 5′ end of a distinct Xist isoform. Instead, P0 is an artifact of cross-amplification caused by a pseudogene of the highly expressed ribosomal protein S12 gene Rps12. Using strand-specific techniques, we find that transcription upstream of P1 originates from the DNA strand opposite Xist and represents the 3′ end of the antisense Tsix RNA. Thus, these data do not support the existence of a P0 promoter and suggest that mechanisms other than switching of functionally distinct promoters control the up-regulation of Xist.

Abnormal dendritic spines in fragile X knockout mice: Maturation and pruning deficits

Fragile X syndrome arises from blocked expression of the fragile X mental retardation protein (FMRP). Golgi-impregnated mature cerebral cortex from fragile X patients exhibits long, thin, tortuous postsynaptic spines resembling spines observed during normal early neocortical development. Here we describe dendritic spines in Golgi-impregnated cerebral cortex of transgenic fragile X gene (Fmr1) knockout mice that lack expression of the protein. Dendritic spines on apical dendrites of layer V pyramidal cells in occipital cortex of fragile X knockout mice were longer than those in wild-type mice and were often thin and tortuous, paralleling the human syndrome and suggesting that FMRP expression is required for normal spine morphological development. Moreover, spine density along the apical dendrite was greater in the knockout mice, which may reflect impaired developmental organizational processes of synapse stabilization and elimination or pruning.

Retinoic acid X receptor in the diploblast, Tripedalia cystophora

Nuclear hormone receptors comprise a characteristic family of transcription factors found in vertebrates, insects and nematodes. Here we show by cDNA and gene cloning that a Cnidarian, Tripedalia cystophora, possesses a retinoid receptor (jRXR) with remarkable homology to vertebrate retinoic acid X receptors (RXRs). Like vertebrate RXRs, jRXR binds 9-cis retinoic acid (Kd = 4 × 10−10 M) and binds to the DNA sequence, PuGGTCA as a monomer in vitro. jRXR also heterodimerizes with Xenopus TR beta on a thyroid responsive element of a direct repeat separated by 4 bp. A jRXR binding half-site capable of interacting with (His6)jRXR fusion protein was identified in the promoters of three T. cystophora crystallin genes that are expressed highly in the eye lens of this jellyfish. Because crystallin gene expression is regulated by retionoid signaling in vertebrates, the jellyfish crystallin genes are candidate in vivo targets for jRXR. Finally, an antibody prepared against (His6)jRXR showed that full-length jRXR is expressed at all developmental stages of T. cystophora except the ephydra, where a smaller form replaces is. These data show that Cnidaria, a diploblastic phylum ancestral to the triploblastic invertebrate and subsequent vertebrate lineages, already have an RXR suggesting that RXR is an early component of the regulatory mechanisms of metazoa.

Origins and antiquity of X-linked triallelic color vision systems in New World monkeys

It is known that the squirrel monkey, marmoset, and other related New World (NW) monkeys possess three high-frequency alleles at the single X-linked photopigment locus, and that the spectral sensitivity peaks of these alleles are within those delimited by the human red and green pigment genes. The three alleles in the squirrel monkey and marmoset have been sequenced previously. In this study, the three alleles were found and sequenced in the saki monkey, capuchin, and tamarin. Although the capuchin and tamarin belong to the same family as the squirrel monkey and marmoset, the saki monkey belongs to a different family and is one of the species that is most divergent from the squirrel monkey and marmoset, suggesting the presence of the triallelic system in many NW monkeys. The nucleotide sequences of these alleles from the five species studied indicate that gene conversion occurs frequently and has partially or completely homogenized intronic and exonic regions of the alleles in each species, making it appear that a triallelic system arose independently in each of the five species studied. Nevertheless, a detailed analysis suggests that the triallelic system arose only once in the NW monkey lineage, from a middle wavelength (green) opsin gene, and that the amino acid differences at functionally critical sites among alleles have been maintained by natural selection in NW monkeys for >20 million years. Moreover, the two X-linked opsin genes of howler monkeys (a NW monkey genus) were evidently derived from the incorporation of a middle (green) and a long wavelength (red) allele into one chromosome; these two genes together with the (autosomal) blue opsin gene would immediately enable even a male monkey to have trichromatic vision.

Regulation of dopaminergic pathways by retinoids: Activation of the D2 receptor promoter by members of the retinoic acid receptor–retinoid X receptor family

Dopamine is a neuromodulator involved in the control of key physiological functions. Dopamine-dependent signal transduction is activated through the interaction with membrane receptors of the seven-transmembrane domain G protein-coupled family. Among them, dopamine D2 receptor is highly expressed in the striatum and the pituitary gland as well as by mesencephalic dopaminergic neurons. Lack of D2 receptors in mice leads to a locomotor parkinsonian-like phenotype and to pituitary tumors. The D2 receptor promoter has characteristics of a housekeeping gene. However, the restricted expression of this gene to particular neurons and cells points to a strict regulation of its expression by cell-specific transcription factors. We demonstrate here that the D2 receptor promoter contains a functional retinoic acid response element. Furthermore, analysis of retinoic acid receptor-null mice supports our finding and shows that in these animals D2 receptor expression is reduced. This finding assigns to retinoids an important role in the control of gene expression in the central nervous system.

Virus-sized self-assembling lamellar complexes between plasmid DNA and cationic micelles promote gene transfer

Gene therapy is based on the vectorization of genes to target cells and their subsequent expression. Cationic amphiphile-mediated delivery of plasmid DNA is the nonviral gene transfer method most often used. We examined the supramolecular structure of lipopolyamine/plasmid DNA complexes under various condensing conditions. Plasmid DNA complexation with lipopolyamine micelles whose mean diameter was 5 nm revealed three domains, depending on the lipopolyamine/plasmid DNA ratio. These domains respectively corresponded to negatively, neutrally, and positively charged complexes. Transmission electron microscopy and x-ray scattering experiments on complexes originating from these three domains showed that although their morphology depends on the lipopolyamine/plasmid DNA ratio, their particle structure consists of ordered domains characterized by even spacing of 80 Å, irrespective of the lipid/DNA ratio. The most active lipopolyamine/DNA complexes for gene transfer were positively charged. They were characterized by fully condensed DNA inside spherical particles (diameter: 50 nm) sandwiched between lipid bilayers. These results show that supercoiled plasmid DNA is able to transform lipopolyamine micelles into a supramolecular organization characterized by ordered lamellar domains.

Identification of semaphorin E as a non-MDR drug resistance gene of human cancers

To improve cancer chemotherapy, a better understanding of the molecular mechanisms of drug resistance is essential. To identify the molecules responsible for drug resistance that is unrelated to MDR1 or MRP gene products, a eukaryotic expression cDNA library of cis-diamminedichloroplatinum(II) (CDDP)-resistant ovarian cancer TYKnuR cells was introduced into Cos-7 cells. After repeated CDDP selection, cDNA homologous to murine semaphorin E was isolated from surviving cells. Human semaphorin E (H-sema E) was overexpressed in CDDP-resistant cell lines and was readily induced not only by diverse chemotherapeutic drugs but also by x-ray and UV irradiation. Transfection of H-sema E conferred a drug-resistant phenotype to CDDP-sensitive cells. In addition, the aberrant expression of H-sema E protein was detected immunohistochemically in 14 of 42 (33.3%) recurrent squamous cell carcinomas removed at autopsy after extensive radiochemotherapy. Recently, another member of the semaphorin family, CD100, was shown to significantly improve the viability of B lymphocytes. These results suggest the involvement of semaphorins in diverse cell survival mechanisms.

CXR, a chicken xenobiotic-sensing orphan nuclear receptor, is related to both mammalian pregnane X receptor (PXR) and constitutive androstane receptor (CAR)

Nuclear receptors constitute a large family of ligand-modulated transcription factors that mediate cellular responses to small lipophilic molecules, including steroids, retinoids, fatty acids, and exogenous ligands. Orphan nuclear receptors with no known endogenous ligands have been discovered to regulate drug-mediated induction of cytochromes P450 (CYP), the major drug-metabolizing enzymes. Here, we report the cloning of an orphan nuclear receptor from chicken, termed chicken xenobiotic receptor (CXR), that is closely related to two mammalian xenobiotic-activated receptors, the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR). Expression of CXR is restricted to tissues where drug induction of CYPs predominantly occurs, namely liver, kidney, small intestine, and colon. Furthermore, CXR binds to a previously identified phenobarbital-responsive enhancer unit (PBRU) in the 5′-flanking region of the chicken CYP2H1 gene. A variety of drugs, steroids, and chemicals activate CXR in CV-1 monkey cell transactivation assays. The same agents induce PBRU-dependent reporter gene expression and CYP2H1 transcription in a chicken hepatoma cell line. These results provide convincing evidence for a major role of CXR in the regulation of CYP2H1 and add a member to the family of xenobiotic-activated orphan nuclear receptors.

The human sex-reversing ATRX gene has a homologue on the marsupial Y chromosome, ATRY: Implications for the evolution of mammalian sex determination

Mutations in the ATRX gene on the human X chromosome cause X-linked α-thalassemia and mental retardation. XY patients with deletions or mutations in this gene display varying degrees of sex reversal, implicating ATRX in the development of the human testis. To explore further the role of ATRX in mammalian sex differentiation, the homologous gene was cloned and characterized in a marsupial. Surprisingly, active homologues of ATRX were detected on the marsupial Y as well as the X chromosome. The Y-borne copy (ATRY) displays testis-specific expression. This, as well as the sex reversal of ATRX patients, suggests that ATRY is involved in testis development in marsupials and may represent an ancestral testis-determining mechanism that predated the evolution of SRY as the primary mammalian male sex-determining gene. There is no evidence for a Y-borne ATRX homologue in mouse or human, implying that this gene has been lost in eutherians and its role supplanted by the evolution of SRY from SOX3 as the dominant determiner of male differentiation.