Bridge-induced chromosome translocation in yeast relies upon a Rad54/Rdh54-dependent, Pol32-independent pathway.

While in mammalian cells the genetic determinism of chromosomal translocation remains unclear, the yeast Saccharomyces cerevisiae has become an ideal model system to generate ad hoc translocations and analyze their cellular and molecular outcome. A linear DNA cassette carrying a selectable marker flanked by perfect homologies to two chromosomes triggers a bridge-induced translocation (BIT) in budding yeast, with variable efficiency. A postulated two-step process to produce BIT translocants is based on the cooperation between the Homologous Recombination System (HRS) and Break-Induced Replication (BIR); however, a clear indication of the molecular factors underlying the genetic mechanism is still missing. In this work we provide evidence that BIT translocation is elicited by the Rad54 helicase and completed by a Pol32-independent replication pathway. Our results demonstrate also that Rdh54 is involved in the stability of the translocants, suggesting a mitotic role in chromosome pairing and segregation. Moreover, when RAD54 is over-expressed, an ensemble of secondary rearrangements between repeated DNA tracts arise after the initial translocation event, leading to severe aneuploidy with loss of genetic material, which prompts the identification of fragile sites within the yeast genome

Characterization of nine independent <i>pol32</i>Δ/<i>pol32</i>Δ translocants.

<p>A) Southern hybridization of genomic DNA from all the <i>pol32Δ</i>/<i>pol32Δ</i> translocants with probes against kanamycin, <i>rim4</i> (chromosome VIII) and <i>ndj1</i> (chromosome XV) <i>loci</i> are shown. Lane 1:wild type San1, 2: <i>pol32Δ</i>/<i>pol32Δ</i>cl3, 3: <i>pol32Δ</i>/<i>pol32Δ</i>cl22, 4: <i>pol32Δ</i>/<i>pol32Δ</i>cl23, 5: <i>pol32Δ</i>/<i>pol32Δ</i>cl26, 6 <i>pol32Δ</i>/<i>pol32Δ</i>cl31, 7: <i>pol32Δ</i>/<i>pol32Δ</i>cl35, 8 <i>pol32Δ</i>/<i>pol32Δ</i>cl43, 9 <i>pol32Δ</i>/<i>pol32Δ</i>cl44, 10: <i>pol32Δ</i>/<i>pol32Δ</i>cl46. T indicates the band of the translocated chromosome. The question mark indicates unknown bands coming from a rearrangement of the left part of chromosome XV on the membrane hybridized with <i>ndj1</i> B) Scheme of the location of the probes used with respect to the breakpoints (underlined) C) The <i>RIM4</i> copy number is reported with its own legend on the right. The results are referred to the wild type (here represented by a line with a value of two); each bar is the result of nine independent readings (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060926#s4" target="_blank">Materials & Methods</a> for details).</p

CHEF and Southern hybridization analysis of translocants unbalanced for Rdh54 and Rad54 amount.

<p>A) A scheme showing the bridge between chromosome VIII and XV and the new hybrid chromosome (Translocant = T). The chromosomal positions of the probes and of the translocation breakpoints (bold and underlined) are reported. B) CHEF (left) and Southern hybridization (right), with a probe against kanamycin, of the translocants in <i>RDH54</i> and <i>RAD54</i> deletants. Lanes from left to right: 1, San1ΔMSH2::Kan (it indicates chromosome XV); in <i>rdh54Δ/rdh54Δ</i> background: 2, cl4; 3, cl7; 4, cl16; 5, cl23n; 6, cl23*; 7, cl30; 8, cl47; in CRAD54 background: 9, cl1; 10, cl4; in OeRAD54 background: 11, clw; 12, cl37; 13, cl38; 14, San1ΔDUR3::Kan (chromosome VIII). n means phenotypically normal and * indicates sectored colony (see text for details). C) CHEF and Southern bots of the following samples (from left to right): 1, San1; in <i>rdh54Δ/rdh54Δ</i> background: 2, cl4; 3, cl7; 4, cl16; 5, cl23; 6, cl30 and 7, cl47. In CRAD54 background: 8, cl1 and 9, cl4; in OeRAD54 background: 10, clw; 11, cl37 and 12, cl38; 13, <i>top1Δ/top1Δ</i>cl18; 14, <i>xrs2Δ/xrs2Δ</i>cl6; 15, San1, hybridized with probes for <i>brx1, hal9</i> (upper line) and <i>cdc33</i> (not shown because identical to <i>hal9</i>) for chromosome XV and with <i>crp1</i>, <i>rim4</i> and <i>ste20</i> for chromosome VIII (bottom line). Two clones, obtained in <i>TOP1</i> and <i>XRS2</i> deletants, were also included in the CHEF (lanes 13 and 14); <i>brx</i> and <i>hal9</i> hybridization disclosed the complete loss of the native chromosome XV in <i>xrs2Δ/xrs2Δ</i>cl6 (lane 14).</p

Characterization of the translocants between homologs and proposed model explaining the role of Rdh54.

<p>A) Characterization through colony-PCR of cl44 and cl94. The presence of spacer2 (lane 1 = primers sp2/sp2Fw), spacer3 (lane 2 = primers sp3/Dur3’int) and spacer1 (lane3 = primers Sp1/Dur3R) are shown from left to right in the wild type strain YF123, in the translocant 44 (<i>rdh54Δ</i>/<i>rdh54Δ</i> background) and in the translocant 94 (wt YF123) respectively. In the panel on the right the amplification size of the region surrounding spacer3 in clone 44 and 94 is compared (DurEXT/Dur3’int). The partial sequence of these fragments is shown in details on panel C. B) Two hypothetical models to explain the loss or the retaining of spacer sp3 in the wild type cl 94 (I, II) and in the <i>rdh54Δ</i>/<i>rdh54Δ</i>cl 44 respectively (III, IV). C) DNA consensus from the alignment of a partial sequence of cl44 and cl94 around spacer3 confirming that only in the first one the spacer is present (here indicated as green, small letters). The capital letters indicate the nucleotides present in both clones.</p

Chromosome stability in <i>rdh54Δ/rdh54Δ</i> translocants.

<p>A) Kanamycin gene copy number ( = <i>N</i>) obtained by quantitative PCR in the six <i>rdh54Δ/rdh54Δ</i> translocants compared with the control (K). The control K is a strain where one copy of kanamycin stably replaces the <i>DUR3</i> gene. B) Gene copy number ( = <i>N</i>) of <i>BRX1</i> in <i>rdh54Δ/rdh54Δ</i> translocants compared with the wild type (that has two copies of this gene). <i>BRX1</i> is located within chromosome XV (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060926#pone-0060926-g002" target="_blank">Figure 2A</a>) and on the translocated chromosome XV–VIII. Each histogram is the result of nine independent readings. C) Loss (%) of the chromosome carrying kanamycin, indicated by lack of growth on G418 medium (in red), versus viability (in blue) derived by replica-plating. The strains were grown overnight and plated without selection and then replicated on G418. The results are expressed in percentage. 1: wild type strain, 2: K, 3: <i>rdh54Δ/rdh54Δ</i>cl4, 4: <i>rdh54Δ/rdh54Δ</i>cl7, 5: <i>rdh54Δ/rdh54Δ</i>cl16, 6: <i>rdh54Δ/rdh54Δ</i>cl23, 7: <i>rdh54Δ/rdh54Δ</i>cl30, 8 <i>rdh54Δ/rdh54Δ</i>cl47.</p

KO and translocation (T) events with short and long homologies between two homologous chromosomes VIII.

<p>The two homologs are labeled here as chrVIII <b>a</b> and <b>b</b>, and are bridged by a DNA cassette carrying kanamycin resistance (KAN). On the top, the molecular event that can give rise to KO (left) and to T (right) as previously reported <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060926#pone.0060926-Tosato2" target="_blank">[9]</a> using a cassette with short homologies (40 nt). F and Y are the two possible chromosome configurations for the KO as reported in the text. The only translocant found was cl77 while the opposite configuration was never detected. At the bottom (framed area), the different result obtained with a homology of 400 nt (reported in this work). On the left the transformants with the KO in the homologous chrVIII <b>a</b> (F); it is shown that spacer sp2 is lost from both chromosomes VIII; on the contrary, in the transformants where the KO happened in the homolog chrVIII <b>b</b> (Y) sp2 is present on both chromosomes VIII. This LOH, with the absence of sp2 on both chromosomes, is maintained also in the two translocants cl94 and cl44 (frame, right) while cl44 only, which is in <i>rdh54Δ</i>/<i>rdh54Δ</i> background, has retained also sp3 as it is shown experimentally in details in Fig. 6C.</p

The representation of the onset of the Covid-19 pandemic and the consequent lockdown in Italy: A psychosocial research by SPS, Studio di Psicosociologia of Rome

A fine febbraio 2020, in SPS4ci siamo chiesti quali fossero i vissuti evocati dalla pandemia Covid-19 in esordio, e quali fatti “derivassero” da tali vissuti. A tal fine abbiamo interpellato 419 persone, tra l’1 marzo e il 5 maggio 2020. Il corpus raccolto è stato analizzato con l’Analisi Emozionale del Testo (AET). Si ipotizzava che la pandemia avesse destrutturato le modalità abituali di rapporto, e pensavamo stessero emergendo dimensioni relazionali inedite. I nostri dati dicono che l’individualismo abituale, di avida competitività, è in crisi. In risposta alla destrutturazione dello schema relazionale amico/nemico, alla base della socialità, è emerso un nuovo individualismo. La rappresentazione del pericolo insito nel contagio pandemico ci ha reso, tutti, potenzialmente nemici gli uni degli altri. Tutti siamo vissuti come potenzialmente nemici di tutti, a meno di non essere dichiaratamente malati. I malati, di contro, non sono vissuti come nemici: sono un’alterità scissa, relegata in un altrove lontano da chi è “sano”. Le cure, nel lockdown, erano confinate nell’ospedale, caratterizzate dall’isolamento, dall’emergenza, dalla morte esperita nel peggiore dei modi. L’altrove è stato reificato in un ospedale diventato sintomatico del fallimento del sistema sanitario. Si è costituito un “noi” qui insieme, sani e maniacalmente felici, e un “loro”contagiati, dannati, isolati e “altrove”. Internet, consentendo vicinanza senza contatto, è diventata un nuovo contesto di socialità. Ha permesso di ridiventare umani, ovvero amici, a meno che non si dimostri il contrario. Ma la nuova amicalità è fondata sulla scissione dall’altro dannato: la coppia malato/curante, e tutti gli esclusi, per diverse motivazioni, dalla protezione del lockdown. Dalla nuova socialità è escluso anche il vissuto dello stare chiusi in casa con gli abituali conviventi, dove emerge la violenza delle relazioni familiari obbligate. Si evidenziano altri esclusi dal noi maniacalmente amicale: gli anziani che non usano internet e che più di tutti rischiano di morire. C’è poi una cultura che, entro il fallimento delle relazioni sociali abituali, sottolinea l’impotenza delle istituzioni (politiche, sanitarie, mediatiche etc.) nella contingenza pandemica. Infine, c’è una cultura pre-lockdown, fatta della paura che porterà a scegliere l’isolamento. Manca, nei dati, il mondo produttivo, che non ha ritrovato, per gli interpellati dalla ricerca –nel periodo di tempo da noi considerato –un codice emozionale condiviso che potesse raccogliersi in un cluster. La ricerca aveva anche un obiettivo di intervento: quello di creare un contesto in cui l’evento pandemia potesse essere interpretato, entro un setting di partecipazione. Oltre a effettuare una pubblicazione rapida dei dati, intendiamo promuovere gruppi di discussione su internet con i partecipanti. La creazione di un contesto di condivisione è anche un motivo dell’alto numero di Autori.At the end of February 2020, in SPS2we asked ourselves what were the experiences evoked by the Covid-19 pandemic in its debut, and what facts “derived”from these experiences. To this end, we interviewed 419 people, between 1 March and 5 May 2020. The collected corpus was analyzed through the Emotional Text Analysis (AET). It was assumed that the pandemic had deconstructed the usual ways of relating, and we thought that new relational dimensions were emerging. Our data show that habitual individualism, of greedy competitiveness, is in crisis. A new individualism has emerged in response to the deconstruction of the friend/foerelational schema, at the basis of sociality. The representation of the danger inherent in the pandemic contagion has made us all potentially enemies of each other. We have all lived as potentially enemies of all, unless we are admittedly sick. The sick, on the other hand, are not experienced as enemies: they are a split otherness, relegated to an elsewhere far from those who are “healthy”. Duringthe lockdown, treatments were confined to the hospital, characterized by isolation, emergency, death experienced in the worst way. The othernesswas reified in a hospital that became symptomatic of the failure of the health system. A “we”has formed here together, healthy and maniacally happy, and a “them”infected, damned, isolated and “elsewhere”. The Internet, by allowing contactless proximity, has become a new context of sociality. It has allowed us to become human again, or friends, unless proven otherwise. But the new friendship is based on the split from the damned other: the sick/caring couple, and all those excluded, for various reasons, from the protection of the lockdown. The experience of being closed at home with the usual cohabitants is also excluded from the new sociality, where the violence of forced family relationships emerges. There are others excluded from a maniacally friendlyus: the elderly who do not use the internet and who most of all risk dying. There is also a culture that, within the failure of habitual social relations, underlines the powerlessness of institutions (political, health, media, etc.) in the pandemic contingency. Finally, there is a pre-lockdown culture, made up of fear that will lead to chooseisolation. In the data, the productive world is missing, which for those interviewed by the research did not find -in the period of time we considered -a shared emotional code that could be gathered in a cluster. The research also had an intervention objective: to create a context in which the pandemicevent could be interpreted, within a setting of participation. In addition to publishing the data quickly, we intend to promote discussion groups onthe internet with participants. The creation of a sharing context is also areason for the high number of Author