Germ-line expression of the I factor, a functional LINE from the fruit fly Drosophila melanogaster, is positively regulated by reactivity, a peculiar cellular state

I factor is a functional LINE (long interspersed nucleotidic element) which is mobilized in the germ-line of dysgenic SF females during I-R hybrid dysgenesis. Such females are obtained when an oocyte from a reactive stock, devoid of I factors but characterized by a level of reactivity, i.e. its potential for hybrid dysgenesis, is fertilized by a spermatozoon from an I factor-containing inducer stock. In a previous paper we described the expression of an I factor-lacZ fusion. Expression was detected in the ovaries of reactive and dysgenic flies only. In this paper we show that this transgenic activity can be quantified and depends upon the maternally inherited reactivity. Reactivity is not just a permissive state and modifiers of the reactivity level such as heat treatment and ageing change the level of expression of our transgenic fusion accordingly. Moreover, ageing through generations has the same cumulative and reversible effect on both reactivity and I factor expression. Using our fusion as a test for reactivity we show that the silencing of I factor after its introduction into a reactive genome may not be established in a single generation.     

Germ-line expression of the I factor,a functional LINE from the fruit fly Drosophila melanogaster,is positively regulated by reactivity,a peculiar cellular state

I factor is a functional LINE (long interspersed nucleotidic element) which is mobilized in the germ-line of dysgenic SF females during I-R hybrid dysgenesis. Such females are obtained when an oocyte from a reactive stock, devoid of I factors but characterized by a level of reactivity, i.e. its potential for hybrid dysgenesis, is fertilized by a spermatozoon from an I factor-containing inducer stock. In a previous paper we described the expression of an I factor-lacZ fusion. Expression was detected in the ovaries of reactive and dysgenic flies only. In this paper we show that this transgenic activity can be quantified and depends upon the maternally inherited reactivity. Reactivity is not just a permissive state and modifiers of the reactivity level such as heat treatment and ageing change the level of expression of our transgenic fusion accordingly. Moreover, ageing through generations has the same cumulative and reversible effect on both reactivity and I factor expression. Using our fusion as a test for reactivity we show that the silencing of I factor after its introduction into a reactive genome may not be established in a single generation.     

Transport of galectin-3 between the nucleus and cytoplasm. I. Conditions and signals for nuclear import

Galectin-3, a factor involved in the splicing of pre-mRNA, shuttles between the nucleus and the cytoplasm. We have engineered a vector that expresses the fusion protein containing the following: (a) green fluorescent protein as a reporter of localization, (b) bacterial maltose-binding protein to increase the size of the reporter polypeptide, and (c) galectin-3, whose sequence we wished to dissect in search of amino acid residues vital for nuclear localization. In mouse 3T3 fibroblasts transfected with this expression construct, the full-length galectin-3 (residues 1-263) fusion protein was localized predominantly in the nucleus. Mutants of this construct, containing truncations of the galectin-3 polypeptide from the amino terminus, retained nuclear localization through residue 128; thus, the amino-terminal half was dispensable for nuclear import. Mutants of the same construct, containing truncations from the carboxyl terminus, showed loss of nuclear localization. This effect was observed beginning with truncation at residue 259, and the full effect was seen with truncation at residue 253. Site-directed mutagenesis of the sequence ITLT (residues 253-256) suggested that nuclear import was dependent on the IXLT type of nuclear localization sequence, first discovered in the Drosophila protein Dsh (dishevelled). In the galectin-3 polypeptide, the activity of this nuclear localization sequence is modulated by a neighboring leucine-rich nuclear export signal.     

The cellular localization pattern of Varicella-Zoster virus ORF29p is influenced by proteasome-mediated degradation

Varicella-zoster virus (VZV) open reading frame 29 (ORF29) encodes a single-stranded DNA binding protein. During lytic infection, ORF29p is localized primarily to infected-cell nuclei, whereas during latency it appears in the cytoplasm of infected neurons. Following reactivation, ORF29p accumulates in the nucleus. In this report, we analyze the cellular localization patterns of ORF29p during VZV infection and during autonomous expression. Our results demonstrate that ORF29p is excluded from the nucleus in a cell-type-specific manner and that its cellular localization pattern may be altered by subsequent expression of VZV ORF61p or herpes simplex virus type 1 ICP0. In these cases, ORF61p and ICP0 induce nuclear accumulation of ORF29p in cell lines where it normally remains cytoplasmic. One cellular system utilized by ICP0 to influence protein abundance is the proteasome degradation pathway. Inhibition of the 26S proteasome, but not heat shock treatment, resulted in accumulation of ORF29p in the nucleus, similar to the effect of ICP0 expression. Immunofluorescence microscopy and pulse-chase experiments reveal that stabilization of ORF29p correlates with its nuclear accumulation and is dependent on a functional nuclear localization signal. ORF29p nuclear translocation in cultured enteric neurons and cells derived from an astrocytoma is reversible, as the protein's distribution and stability revert to the previous states when the proteasomal activity is restored. Thus, stabilization of ORF29p leads to its nuclear accumulation. Although proteasome inhibition induces ORF29p nuclear accumulation, this is not sufficient to reactivate latent VZV or target the immediate-early protein ORF62p to the nucleus in cultured guinea pig enteric neurons.     

The THO complex is required for nucleolar integrity in Drosophila spermatocytes

The THO complex is a conserved multisubunit protein complex that functions in the formation of export-competent messenger ribonucleoprotein (mRNP). Although the complex has been studied extensively at the single-cell level, its exact role at the multicellular organism level has been poorly understood. Here, we isolated a novel Drosophila male sterile mutant, garmcho (garm). Positional cloning indicated that garm encodes a subunit of the Drosophila THO complex, THOC5. Flies lacking THOC5 showed a meiotic arrest phenotype with severe nucleolar disruption in primary spermatocytes. A functional GFP-tagged fusion protein, THOC5-GFP, revealed a unique pattern of THOC5 localization near the nucleolus. The nucleolar distribution of a testis-specific TATA binding protein (TBP)-associated factor (tTAF), SA, which is required for the expression of genes responsible for sperm differentiation, was severely disrupted in mutant testes lacking THOC5. But THOC5 appeared to be largely dispensable for the expression and nuclear export of either tTAF target mRNAs or tTAF-independent mRNAs. Taken together, our study suggests that the Drosophila THO complex is necessary for proper spermatogenesis by contribution to the establishment or maintenance of nucleolar integrity rather than by nuclear mRNA export in spermatocytes.     

Utilization of the IR hybrid dysgenesis system in Drosophila to test in vivo mobilization of synthetic SINEs sharing 3' homology with the I factor

The current model of short interspersed nuclear element (SINE) mobility suggests that these non-coding retroposons are able to recruit for their own benefits the enzymatic machinery encoded by autonomous long interspersed nuclear elements (LINEs). The recent characterization of potential SINE-LINE partner pairs that share common 3' end sequences concurs with this model and has led to a potent picture of tRNA-derived SINEs consisting of a tripartite functional structure (Mol. Cell. Biol. 16 (1996) 3756; Mol. Biol. Evol. 16 (1999) 1238; Proc. Natl. Acad. Sci. USA 96 (1999) 2869). This structure consist of a 5' polIII tRNA-related promoter region, a central conserved domain and a variable 3' region with homology to the 3' end of LINEs, believed to be essential to direct recognition by the LINE proteins. To test this model in vivo, we have designed synthetic SINEs possessing this 'canonical' structure, including 3' homology to the 3' UTR of the LINE I factor from Drosophila. These synthetic elements were introduced in a Drosophila reactive strain, and SINE retroposition was assessed following dysgenic crosses that are known to induce high levels of I factor germinal transposition. In the progeny from the dysgenic crosses 3400-4000 flies were analyzed but no retroposed copy of the chimeric SINEs was detected, indicating that what is assumed to be a typical SINE structure is not sufficient per se to allow efficient trans-mobilization of our synthetic SINEs by an actively amplifying partner LINE. Alternatively, the apparent absence of natural fly SINEs may underline intrinsic properties of fly biology that are incompatible with the genesis and/or propagation of SINE-like elements.     

Functions of Varicella-zoster virus ORF23 capsid protein in viral replication and the pathogenesis of skin infection

The assembly of herpesvirus capsids is a complex process involving interactions of multiple proteins in the cytoplasm and in the nucleus. Based on comparative genome analyses, varicella-zoster virus (VZV) open reading frame 23 (ORF23) encodes a conserved capsid protein, referred to as VP26 (UL35) in other alphaherpesviruses. Mutagenesis using a VZV bacterial artificial chromosome system showed that ORF23 was dispensable for replication in vitro. However, the absence of ORF23 disrupted capsid assembly in a melanoma cell line. Expression of ORF23 as a red fluorescent protein (RFP) fusion protein appeared to have a dominant negative effect on replication that was rescued by ORF23 expression from a nonnative site in the VZV genome. In contrast to its VP26 homolog, ORF23 has an intrinsic nuclear localization capacity that was mapped to an SRSRVV motif at residues 229 to 234 in the extreme C terminus of ORF23. In addition, coexpression with ORF23 resulted in nuclear import of the major capsid protein, ORF40. VZV ORF33.5 also translocated ORF40, which may provide a redundant mechanism in vitro but appears insufficient to overcome the dominant negative effect of the monomeric RFP-ORF23 (mRFP23) fusion protein. ORF23 was required for VZV infection of human skin xenografts, indicating that ORF33.5 does not compensate for lack of ORF23 in vivo. These observations suggest a model of VZV capsid assembly in which nuclear transport of the major capsid protein and associated proteins requires ORF23 during VZV replication in the human host. If so, ORF23 expression could be a target for a novel antiviral drug against VZV.     

The Drosophila mobile element jockey is a LINE element and contains coding sequences homologous to retroviral proteins

A detailed investigation of Drosophila melanogaster mobile dispersed repetitive element jockey is performed. Its structural features resemble those of LINE elements. Sequencing of the complete jockey 5020 bp in length revealed two long open reading frames ORF1 and ORF2 overlapping with a frameshift-1. Judging by amino acid homologies, ORF1 encodes a nucleic acid binding protein, characteristic of replication competent retroviruses; the 3' part of ORF2 encodes an RNA-dependent DNA polymerase which has an amino acid sequence, similar to recently published sequences of LINE elements of Drosophila, Trypanosoma and mammals. This fact demonstrates their evolutionary relationship. Sequencing of several deleted copies of jockey revealed the absence of the major part of ORF2, though the rest of the element, including the ends, is highly conservative.     

The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein.

The erect wing (ewg) locus of Drosophila melanogaster encodes a vital function important for the development of the nervous system and the indirect flight muscles. In order to understand the ewg function at a molecular level, cDNA clones were isolated. Sequence analysis of cDNAs revealed a single open reading frame (ORF) encoding a protein of 733 residues. The translational start for this ORF is a CTG codon. A 225-amino-acid region of this protein is 71% identical to the DNA binding region of the Strongylocentrotus purpuratus P3A2 DNA binding protein. Additionally, the ORF contains large acidic and basic domains characteristic of those in proteins involved in nuclear regulatory functions. Immunoblot analysis using polyclonal anti-EWG antisera generated against a bacterial fusion protein reveals a single, 116-kDa protein present throughout development, beginning at approximately stage 12 of embryogenesis, which is enriched in adult heads and absent from embryos carrying certain ewg alleles. Additionally, we show that EWG is localized specifically to the nuclei of virtually all embryonic neurons. Finally, a minigene consisting of an ewg cDNA under control of the hsp70 promoter can provide the ewg function in transgenic ewg mutant flies.     

Artificial and epigenetic regulation of the I factor, a nonviral retrotransposon of Drosophila melanogaster

The I factor (IF) is a LINE-like transposable element from Drosophila melanogaster. IF is silenced in most strains, but under special circumstances its transposition can be induced and correlates with the appearance of a syndrome of female sterility called hybrid dysgenesis. To elucidate the relationship between IF expression and female sterility, different transgenic antisense and/or sense RNAs homologous to the IF ORF1 have been expressed. Increasing the transgene copy number decreases both the expression of an IF-lacZ fusion and the intensity of the female sterile phenotype, demonstrating that IF expression is correlated with sterility. Some transgenes, however, exert their repressive abilities not only through a copy number-dependent zygotic effect, but also through additional maternal and paternal effects that may be induced at the DNA and/or RNA level. Properties of the maternal effect have been detailed: (1) it represses hybrid dysgenesis more efficiently than does the paternal effect; (2) its efficacy increases with both the transgene copy number and the aging of sterile females; (3) it accumulates slowly over generations after the transgene has been established; and (4) it is maintained for at least two generations after transgene removal. Conversely, the paternal effect increases only with female aging. The last two properties of the maternal effect and the genuine existence of a paternal effect argue for the occurrence, in the IF regulation pathway, of a cellular memory transmitted through mitosis, as well as through male and female meiosis, and akin to epigenetic phenomena.     

In vivo RNA localization of I factor, a non-LTR retrotransposon, requires a cis-acting signal in ORF2 and ORF1 protein

According to the current model of non-LTR retrotransposon (NLR) mobilization, co-expression of the RNA transposition intermediate, and the proteins it encodes (ORF1p and ORF2p), is a requisite for the formation of cytoplasmic ribonucleoprotein complexes which contain necessary elements to complete a retrotransposition cycle later in the nucleus. To understand these early processes of NLR mobilization, here we analyzed in vivo the protein and RNA expression patterns of the I factor, a model NLR in Drosophila. We show that ORF1p and I factor RNA, specifically produced during transposition, are co-expressed and tightly co-localize with a specific pattern (Loc+) exclusively in the cytoplasm of germ cells permissive for retrotransposition. Using an ORF2 mutated I factor, we show that ORF2p plays no role in the Loc+ patterning. With deletion derivatives of an I factor we define an RNA localization signal required to display the Loc+ pattern. Finally, by complementation experiments we show that ORF1p is necessary for the efficient localization of I factor RNA. Our data suggest that ORF1p is involved in proper folding and stabilization of I factor RNA for efficient targeting, through Loc+ patterning, to the nuclear neighborhood where downstream steps of the retrotransposition process occur.     

The Drosophila single-minded gene encodes a nuclear protein with sequence similarity to the per gene product

Mutations in the single-minded (sim) gene of Drosophila result in the loss of the precursor cells giving rise to the midline cells of the embryonic central nervous system. We have examined the structure of the sim product by sequencing a sim cDNA clone, and have also determined the subcellular localization of the protein and its developmental expression by staining embryos with an antiserum against a sim fusion protein. The results indicate that sim is a nuclear protein specifically expressed along the midline of the neuroepithelium, the same subset of cells that are missing in the mutant. No similarity is observed between sim and any known nuclear protein, but, surprisingly, it is similar to the Drosophila period (per) locus gene product, which controls the periodicity of biological rhythms.