She picked one of the tracts up and read it over. Her mother would have called him rough looking. A faded brown suede vest worn too tightly to be fashionable. Tousled black hair, salted with a little age and comfortably uncombed.
A scar on his right cheek that made him look dangerous. Jimmy smiled whenever he asked her for money. Come in. Something I heard in an old Dracula movie. He shrugged.
For half an instant he looked like her dead husband, Frank. He sat at a card table. A deck of cards was tabled in front of him. Tarot cards, she presumed. I make it a point never to terrify anyone on their first date. Feeling flustered, she sat down. I forget my manners. You meet so few people who shake your hand these days.
An energy source. The body has them all over it. Here, he touched his belly. And here and here. He touched his head and he almost touched his heart. Doris would have sworn that he flinched just before his knuckles touched his chest. Another smile fluttered upon his lips. He looked a little nervous like he had just broke wind. He smiled at that.
She could see the laughter hiding behind his eyes. It was a good laugh, not at her but with her. The laughter and something else moved behind his eyes like a dancing shadow. You can call me Val if Carnival had too many syllables to chew over. He looked her in the eye. He had dark eyes like mirrors in shadow. If she was younger, she might have wanted to meet him over coffee.
He laid out the first card. She saw the picture, a woman sitting on some sort of a chair. Was that her? The chair in the picture looked like a lawn chair to Doris. This is you, Carnival said. You have a problem. Someone expects something from you. She nodded, just slightly, trying too late to check herself.
He laid another card, a dark haired figure sitting atop a large black horse, staring hard at a star in a circle in his hand. The card was upside down. The Knight of Pentacles, reversed. Your son? He laid a third card down. Three long swords piercing a heart.
The sky behind the heart appeared to be raining. Three of hearts. A hard decision. Tears falling upon the ground. You have to cut some one away. Carnival looked in her eyes. She felt his eyes, analyzing. Reading her like a hand running over a well-thumbed book. It took Doris a moment to realize he was talking about her age. She nearly blushed. Stupid, that a woman of her age should worry but she did. Some things never changed. I look here. He touched the corner of his eye.
Where the crows dance. They never lie. He looked at her again like he could see through her eyes. Like Superman in the comic books. Open navigation menu. Close suggestions Search Search. User Settings. Skip carousel. Carousel Previous.
Carousel Next. What is Scribd? Cancel anytime. Start your free 30 days Read preview. Publisher: Steve Vernon. Released: May 12, ISBN: Format: Book.
This is a fantasy for those folks who HATE fantasy! Horror Fiction. About the author SV. Related Books. Reborn: Demon's Legacy by D. Snigger by G. Related categories Skip carousel. Hurry up, boy. Time has never learned how to crawl. This has got to be one of the stupidest stunts I have ever tried. Are you made of broken clocks? Hurry up. Poppa, I looked. There was nobody on the roof. I counted three painters. Open your eyes. Ask Shemp.
Who is Shemp? Ticking clocks usually go boom. Carnival ignored Poppa. Nuisances went away if you ignored them long enough.
And where would I go? This cage is stronger than a sour garlic milkshake. Have you been drinking molasses with your tea? You could steal them. A real Gypsy would have. Oh, wait, what am I saying? Stop lying to yourself. Carnival stared at the crack. He concentrated on it. Come on now baby. The ladder began to tremble. I see you, Carnival said. In the shadows, in the back. Was the church empty? Had he imagined her evil presence? No way. You see nothing, Val my boy. There is nobody here but echoes.
She is not female. She is just painted herself that way. You are done here, Carnival whispered. I am here to end you once and for all. I am here to finish you. Tell her a joke. Women love to laugh. A giggle and a wiggle go hand in hand. Tell her how much you earned last year.
She will laugh her head off. Poppa was funny when he wanted to. The grin took the edge off of his fears. And you could thank me for that. Thank you Poppa. He smiled and whispered her true name, stepping closer into the shadows. Shut up Poppa. You are trying to get yourself killed. Why piss on a dragon in her lair? You are wasting time, boy. Stop thinking so long and move. Come on sexy. Come on you wet dreaming wonder-box Carnival kept his eye focused on the crack in the ladder.
Joe DiMaggio would know what to do right now. Come on now, darling, he called. Come on cinder-britches. Sweet talk her, boy. Come on, you mouth breathing bimbo psycho queen. Her face stretched and flexed like a reflection in a funhouse mirror. Open up, baby.
Damn it. He wanted her. Not real ones, anyway. Tied up in a pretty blue bag. Carnival raised his voice, yelling as much at the succubus as at Poppa. Open wide! She opened like a door, a coffin, a canyon, like the mouth of a crescent moon. He rushed towards her holding the ladder out like a rickety shield.
Open wide and say aaaaaaaaaahhhhhhhhhhhh!!! Now what? Let go, boy. Let go, but look down first. Let go boy. They want you to let go. They want your company. Hum Biol ; 71 : 99— CAS Google Scholar.
Study of a strongly endogamous ethnic group. Ann Pediatr Paris ; 18 : — Nouv Rev Fr Hematol ; 16 : — Thromb Haemost ; 83 : — Br J Haematol ; : — Blood ; 86 : — Thromb Haemost ; 74 : — Platelets ; 9 : — Platelets ; 12 : — J Thromb Haemost ; 1 : — J Thromb Haemost ; 1 : J Clin Invest ; 67 : — Thromb Haemost ; : — Austerlitz F, Kalaydjieva L, Heyer E : Detecting population growth, selection and inherited fertility from haplotypic data in humans.
Genetics ; : — Nature ; : — French Senate report. Thromb Haemost ; 98 : — J Thromb Haemost ; 3 : — Fraser A : The Gypsies. Blackwell Publishers: Oxford, Google Scholar. Am J Hum Genet ; 69 : — BMC Med Genet ; 2 : 5. Council of Europe Press: Strasbourg, Am J Hum Genet ; 75 : — Reyniers A : Manouches. Tzigane studies ; 26 : 1— Download references.
You can also search for this author in PubMed Google Scholar. Correspondence to Mathieu Fiore. Reprints and Permissions. Fiore, M. Founder effect and estimation of the age of the French Gypsy mutation associated with Glanzmann thrombasthenia in Manouche families.
Eur J Hum Genet 19, — Download citation. Received : 27 January Revised : 14 March Accepted : 15 March Published : 13 April Issue Date : September Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article.
Provided by the Springer Nature SharedIt content-sharing initiative. Endocrine European Journal of Human Genetics Advanced search. Skip to main content Thank you for visiting nature. Download PDF. Subjects Haematological diseases Mutation Population genetics. The process of evolutionary transition between LTR-RTE and retroviruses is thought to involve multiple steps by which the element loses or gains the ability to transmit copies between cells versus the ability to replicate intracellularly.
But, typically, these two modes of transmission are incompatible because they require assembly in different sub-cellular compartments. Indeed, there is some evidence that gypsy may exhibit retroviral properties. Given that gypsy elements have been found to actively mobilize in neurons and glial cells during normal aging and in models of neurodegeneration, this raises the question of whether gypsy replication in somatic cells occurs via intracellular retrotransposition, intercellular viral spread, or some combination of the two.
These modes of replication in somatic tissues would have quite different biological implications. Here, we demonstrate that Drosophila gypsy is capable of both cell-associated and cell-free viral transmission between cultured S2 cells of somatic origin.
Further, we demonstrate that the ability of gypsy to move between cells is dependent upon a functional copy of its viral envelope protein. This argues that the gypsy element has transitioned from an RTE into a functional endogenous retrovirus with the acquisition of its envelope gene.
On the other hand, we also find that intracellular retrotransposition of the same genomic copy of gypsy can occur in the absence of the Env protein. Thus, gypsy exhibits both intracellular retrotransposition and intercellular viral transmission as modes of replicating its genome. Many of them are actually evolutionary relatives of retroviruses, and are able to replicate themselves and insert new copies into the host genome.
But unlike retroviruses, which are infectious and replicate by moving from one cell to another, retrotransposons replicate within one cell and re-insert their new copies back into the chromosomes of the cell in which they originated.
In this publication, we studied replication of gypsy, which is a well known retrotransposon in fruit flies. We found that gypsy has the ability to replicate within a cell, like a retrotransposon and also has the ability to replicate by moving to a new cell like a retrovirus. PLoS Genet 17 4 : e This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files. The funding agencies played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist. The genomes of plants and animals contain a substantial contribution of sequences derived from transposable elements TEs. In humans, for example, TE derived sequences represent nearly half of all genetic material [ 1 ]. TEs mainly act as selfish genetic elements that replicate within germline tissue, where their de novo inserted copies can be passed to offspring, allowing vertical spread within a population [ 2 , 3 ].
But in the case of the Type I TEs, known as retrotransposons RTEs , there is now compelling evidence that expression and even replication also occurs in somatic tissues and impacts both normal biology and a variety of age-related diseases [ 4 — 7 ]. DNA copies can be inserted into de novo chromosomal sites in the genome, thereby increasing copy number with each successive replication cycle [ 75 — 77 ]. Unlike exogenous retroviruses, both LINE and LTR-RTEs are primarily adapted to make use of an intracellular replication cycle, although there is some evidence for transfer via extracellular vesicles [ 78 — 80 ].
Functional LTR-RTEs encode gag and pol open reading frames, but unlike retroviruses they do not contain an envelope glycoprotein Env to mediate inter-cellular spread. Also, they generally target assembly of virus-like particles at the lumen of the ER to facilitate re-entry to the nucleus rather than at the extracellular membrane to facilitate release from the cell [ 81 ]. Such LTR-RTEs are believed to be the evolutionary ancestors of exogenous retroviruses, which emerged by a multi-step process that includes the gain of an Env gene [ 82 — 85 ] and re-targeting of assembly to the extracellular membrane.
Indeed, many genomes contain such ERVs, which straddle the evolutionary transition between LTR-retrotransposon and exogenous retrovirus. Gypsy elements in Drosophila , the murine IAP-E elements and the HERV-K elements in human genomes, for example, each retain the viral Env , and may therefore have the potential to act as either a virus or a retrotransposon.
Although the bulk of research into somatic retrotransposition has so far focused on LINE elements [ 5 ], the gypsy ERV also has been shown capable of replicating in somatic tissues in Drosophila , including glial cells, post-mitotic neurons, adipose tissues, and intestinal stem cells [ 10 , 11 , 16 , 33 , 58 , 67 , 86 , 87 ], and HERV-K expression has been detected in ALS patients and in several cancers [ 57 , 60 , 61 , 88 — 90 ].
The expression and replication within somatic tissues of ERVs, which encode functional Env proteins, highlights the importance of understanding their replication cycle. These elements sit on a spectrum between intracellular RTE and extracellular virus. It is not clear whether such elements replicate through intracellular transposition or whether their replication requires them to move genetic material between somatic cells via viral transmission [ 81 , 91 — 97 ].
We have addressed this question using cultured Drosophila S2 cells of macrophage lineage. We used a replication reporter system that we recently developed [ 11 ] as well as a series of novel reporters, to test whether or not gypsy replication occurs via intra-cellular transposition or intercellular viral transfer. We find that gypsy can transfer between separate populations of cells in cell culture using both cell-free and cell-associated modes of transmission.
We further demonstrate that both forms of transmission between cells requires an intact Env open reading frame ORF. Surprisingly, we also find that in the absence of Env , gypsy is able to efficiently complete intracellular retrotransposition. The gypsy-CLEVR reporter reliably marks cells in which replication of gypsy has occurred and in which a de novo cDNA copy has been reinserted into the genome.
This reporter system reliably reports replication of the exogenously supplied gypsy construct both in cell culture and in vivo [ 10 , 11 ]. The gypsy-CLEVR reporter expression requires the replication of gypsy to link the promoter to the reporter and requires the presence of Gal4 to drive the WM signal after replication [ 11 ]. To examine inter-cellular spread of gypsy, we also generated gypsy-mCherry, a more standard reporter of gypsy expression.
Gypsy-mCherry relies on the porcine teschovirus-1 2A P2A self-cleaving peptide [ 98 ] inserted between mCherry and Env Fig 1A so that the nuclear targeting of mCherry does not interfere with the localization of Env. As the translation of mCherry is linked directly to the env encoding spliced transcript of gypsy, this reporter is driven by the gypsy-endogenous promoter and does not require Gal4 to display fluorescent signal.
We also generated a version of this construct in which the Env protein coding sequence was deleted Fig 1A. Star denotes Gal4 dependence for fluorescence but not replication. Quantification is presented as totals cells counted from 3 near equivalent sets of biological replicates. Together, these findings confirm our previous report [ 11 ] that gypsy-CLEVR labels S2 cells in which gypsy replication has occurred. Both of these constructs produce a nuclear localized mCherry signal when expressed Fig 1B.
For this set of experiments, we used an actin5c-driven mCherry pAc-H2B-mCherry as a transfection control. Therefore, the gypsy-CLEVR and gypsy-mCherry groups of reporter constructs reliably label cells where gypsy has replicated or is expressed respectively, but these experiments do not discriminate between intercellular and intra-cellular replication cycles.
We next used the gypsy-CLEVR reporter to test whether gypsy is capable of transmitting between cells grown in contact. Cells were then mounted and imaged after 48 hours in co-culture. On the other hand, intercellular transmission of the gypsy-CLEVR followed by integration into the Gal4 expressing recipient cell genome would yield reporter expression. We also used a co-culture control in which one population of cells had been transfected with a Gal4 dependent UAS-WM and the other with the Gal4 itself.
The expectation is that there should be no intercellular transmission of the WM transcript when it is not associated with the gypsy-CLEVR construct. A Cartoon schematic showing the experimental design of the co-culture assay. Separate populations of S2 cells are transfected with gypsy-CLEVR or tubulin-Gal4 constructs for 48 hours, washed, and then mixed together in equal proportions for further incubation of 48 hours before imaging.
We see clear evidence that gypsy is able to transmit between cells in this cell-associated co-culture assay. In contrast, we observed no WM positive cells when the UAS-WM transfected cells were co-cultured with Gal4 transfected cells, indicating that the reporter cannot move between cells when it is not associated with gypsy.
Thus, gypsy constructs that are unable to generate cDNAs for reinsertion, due to deletion or mutation of the PBS also are unable to report expression in recipient cells grown in contact.
We next tested whether gypsy is capable of cell-free transmission between S2 cells that are not grown in direct cell contact. This assay is conceptually similar to that of the gypsy-CLEVR reporter in co-culture described above. However, in this case, we used a transwell system that utilizes a semi-permeable barrier 0. This construct is capable of replicating independently of Gal4, but cannot express the reporter from the integrated pro-virus unless Gal4 is present.
We again separately transfected either the gypsy-CLEVR reporter or Gal4, and we grew these in a transwell cell culture plate to separate the two populations of cells. The culture plates used possess a membrane permeabilized by 0. A separate population of cells was transfected with Gal4 alone.
Cells transfected with either the CLEVR constructs or the Gal4 were allowed to incubate on their own for 48 hours, after which the cells were washed by centrifugation and seeded on opposite sides of the membrane in the transwell plate cell-culture dish Fig 3A.
After an additional incubation of 48 hours in the transwell cell culture plate, both donor and recipient populations were separately mounted and imaged to detect both transfer and directionality of transfer. Expression of the WM reporter was indicative of transfer, as none of the plasmids transfected can produce the WM signal on their own. A Cartoon schematic showing the experimental design of the transwell assay.
Separate populations of S2 cells are transfected with gypsy-CLEVR or tubulin-Gal4 constructs for 48 hours, washed, and then re-seeded on separate sides of a 0.
B Fluorescent images showing WM labeled cells in the tubulin-Gal4 cell recipient population. No WM labeled cells were detected in the donor populations and are not shown. To accomplish this, we used several independent PCR strategies. With this approach, we detected a product in cells from either side of the transwell assay.
As an independent confirmation that does not rely on nested PCR, we used primers that detect the presence of either the GFP or mCherry reporters. Because the recipient cells were not transfected with the plasmid, such DNA should only be present if the virus was transferred through the transwell system and was then used to produce a viral DNA. Here too, we detected both GFP and mCherry in the recipient cells from the same 4 out of 6 transwell experiments in which we used the intact gypsy-CLEVR construct and in 0 of 6 wells that used the primer binding site mutant construct S2C Fig.
Together, the imaging and PCR findings demonstrate that gypsy is able to transmit between cells that are not in contact. The fact that such transfer only occurs when the reporter is tethered to an intact gypsy that is able to replicate demonstrates the specificity of this assay. The unidirectional nature of transfer from gypsy-CLEVR expressing cells to tubulin-Gal4 expressing recipient cells, also supports the conclusion that gypsy acts as an infectious retrovirus in cell-culture, capable of cell-free transmission.
Enveloped viruses encode a surface glycoprotein that mediates recognition of cellular receptors and fusion with the cell membrane. Retroviral Env genes thus are required both for cell-free and cell-associated transmission. As a further test of the requirement for Env, we also tested whether co-transfection of a pActin-Env pAc-Env was able to rescue the env-deficient virus in trans.
A pAc-H2B-mCherry plasmid was used as a transfection control. Following this incubation period, the cells were washed via centrifugation and transferred into the transwell cell culture plate above recipient S2 cells Fig 4A. Because the gypsy-mCherry constructs do not require presence of Gal4 to visualize reporter expression, the recipient cells used here were untransfected. Following a hour incubation in the transwell cell culture plate, both donor and recipient populations of cells were mounted and imaged for expression of nuclear mCherry.
One population is transfected with the gypsy-mCherry constructs for 48 hours, washed, and placed opposite untransfected S2 cells separated by a 0. B Fluorescent images showing mCherry labeled cells in the S2 cell recipient population. Donor populations are not shown.
C Quantification showing the percentage of cells expressing mCherry for the pAc-H2B-mCherry control and gypsy-mCherry constructs for both the donor and recipient populations in the transwell assay. Here, all of the transfected donor population are expected to express nuclear mCherry, and the recipient population of cells would express mCherry if gypsy had transferred across the membrane.
In the recipient population grown opposite to the control pAc-H2B-mCherry, no cells were found to express the mCherry label, as expected. But this number dropped to near zero 0. This strongly supports the conclusion that the gypsy Env gene is required for transmission.
This deficiency in intercellular transmission with the Env deleted construct also could be rescued when Env was expressed in trans. Together, these results confirm that gypsy is capable of cell-free transmission, but also show that this transmission is reliant upon the presence of functional Env.
The above findings indicate that gypsy retains the ability to transmit between cells under both cell-associated and cell-free conditions, and such transmission is Env dependent. Unlike retroviruses, LTR-retrotransposons typically utilize an intracellular replication cycle that is not env-dependent, but intracellular replication also requires significant differences in targeting within the cell. The Env dependent inter-cellular transmission of gypsy would necessitate assembly at the extracellular membrane.
We wondered therefore if gypsy, which classically has been thought of as a retrotransposon, is even capable of replicating intracellularly. Because the gypsy-CLEVR reporter labels cells only after reverse transcription and template switching [ 10 , 11 ], this reporter provides a means to distinguish replication events from mere expression.
Each of the above two constructs were tested both with and without pAc-Env to provide Env expression in trans Fig 5A. After transfection, the populations of cells were incubated for 48 hours, mounted and imaged to detect the presence of the WM reporter. This is consistent with the robust levels of gypsy replication in S2 cells that we have observed previously [ 11 ]. When this gypsy-CLEVR construct was co transfected with both tubulin-Gal4 and an additional source of pAc-Env expressed in trans, the fraction of labeled cells remained at 3.
Taken together, these findings demonstrate that gypsy is capable of intracellular retrotransposition that is independent of Env. From an evolutionary perspective, it is thought that LTR-RTEs are the likely ancestors of retroviruses, and all vertebrate retroviruses come from a single lineage [ 85 , 99 ].
The emergence of retroviruses is thought to have involved a multi-step process that includes targeting to the cell membrane and the incorporation of a surface glycoprotein Env. This process also has likely occurred in reverse, as some ERVs have lost their Env and developed the ability to re-target internally, and in some cases, these have even been called RTEs, despite the different evolutionary history.
0コメント