A toolbox of stable integration vectors in the fission yeast Schizosaccharomyces pombe

Schizosaccharomyces pombe is a widely used model organism to study many aspects of eukaryotic cell physiology. Its popularity as an experimental system partially stems from the ease of genetic manipulations, where the innate homology-targeted repair is exploited to precisely edit the genome. While vectors to incorporate exogenous sequences into the chromosomes are available, most are poorly characterized. Here we show that commonly used fission yeast vectors, which upon integration produce repetitive genomic regions, yield unstable genomic loci. We overcome this problem by designing a new series of Stable Integration Vectors (SIV) that target four different prototrophy genes. SIV produce non-repetitive, stable genomic loci and integrate predominantly as single copy. Additionally, we develop a set of complementary auxotrophic alleles that preclude false-positive integration events. We expand the vector series to include antibiotic resistance markers, promoters, fluorescent tags and terminators, and build a highly modular toolbox to introduce heterologous sequences. Finally, as proof of concept, we generate a large set of ready-to-use, fluorescent probes to mark organelles and cellular processes with a wide range of applications in fission yeast research

Up-regulation of ectonucleotidase activity after cortical stab injury in rats

The objective of this study was to examine the changes in the activity and expression of ectonucleotidase enzymes in the model of unilateral cortical stab injury (CSI) in rat. The activities of ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1) and ecto 50-nucleotidase were assessed by measuring the levels of ATP, ADP and AMP hydrolysis in the crude membrane preparations obtained from injured left cortex, right cortex, left and right caudate nucleus, whole hippocampus and cerebellum. Significant increase in NTPDase and ecto 50-nucleotidase activities was observed in the injured cortex following CSI, whereas in other brain areas only an increase in ecto 50-nucleotidase activity was seen. Immunohistochemical analysis performed using antibodies specific to NTPDase 1 and ecto 50-nucleotidase demonstrated that CSI induced sig-nificant changes in enzyme expression around the injury site. Immunoreactivity patterns obtained for NTPDase 1 and ecto 50-nucleotidase were compared with those obtained for glial fibrillary acidic protein, as a marker of astrocytes and complement receptor type 3 (OX42), as a marker of microglia. Results suggest that up-regulation of ectonucleotidase after CSI is catalyzed by cells that activate in response to injury, i.e. cells immunopositive for NTPDase 1 were predominantly microglial cells, whereas cells immunopositive for ecto 50-nucleotidase were predominantly astrocytes

Povećana izraženost receptora vaskularnog endotelijalnog faktora rasta u recidivu karcinoma dojke

Uvod. Histološki gradus (HG), koji označava zrelost tumora, i nuklearni gradus(NG), koji predstavlja nuklearnu morfometriju, važni su prognostički faktorikarcinoma dojke. Sa progresijom tumora povezan je i vaskularni endotelijalnifaktor rasta (VEGF). Cilj rada je da se korelira stepen diferenciranosti tumorasa izraženošću receptora za VEGF u recidivu karcinoma dojke.Metode. Ukupno je analizirano 40 uzoraka recidiva karcinoma dojke i 45uzoraka primarnog tumora. Određeni su histološki i nuklearni gradusi i uprimarnom tumoru i u recidivu, te izraženost receptora za VEGF.Rezultati. U ispitivanom uzorku najučestaliji je duktalno invazivni karcinom(75,3%). Histološki gradus I je češći od gradusa III i bilo ih je 27,4% naspram13,7%. Bilo je 24,7% slučajeva nuklearnog gradusa (NG) I, 46,6% slučajevaNG II i 28,8% slučajeva NG III. Od ukupno 85 testiranih uzoraka na receptoreza VEGF samo je u jednom uzorku primarnog karcinoma dojki bio pozitivanreceptor za VEGF, ali je u 15,1% uzoraka recidiva receptor za VEGF biopozitivan. Ispitivanjem odnosa histoloških gradusa i receptora za VEGF nijenađena statistička značajna razlika u broju uzoraka sa pozitivnim receptoromza VEGF između različitih histoloških gradusa (Pearson Hi2=4,79; p=0,571).Međutim, nađena je značajna razlika u pozitivnosti receptora za VEGF uuzoricima različitih nuklearnih gradusa. Receptor za VEGF je bio srazmjernomanje zastupljen u NG II u odnosu na druga dva gradusa (Mann WhitheyU= 272,0; p=0,05 za NG I i NG II i Mann Whithey U=306.000 p=0,02 za NGII i NG III).Zaključak. Receptor za VEGF je bio pozitivan u uzorcima recidiva karcinomadojke i ekspresija receptora VEGF bila je češća u slabije diferenciranimkarcinomima

Gamete fusion triggers bipartite transcription factor assembly to block re-fertilization.

The ploidy cycle, which is integral to sexual reproduction, requires meiosis to halve chromosome numbers as well as mechanisms that ensure zygotes are formed by exactly two partners <sup>1-4</sup> . During sexual reproduction of the fungal model organism Schizosaccharomyces pombe, haploid P and M cells fuse to form a diploid zygote that immediately enters meiosis <sup>5</sup> . Here we reveal that rapid post-fusion reconstitution of a bipartite transcription factor blocks re-fertilization. We first identify mutants that undergo transient cell fusion involving cytosol exchange but not karyogamy, and show that this drives distinct cell fates in the two gametes. The P partner undergoes lethal haploid meiosis, whereas the M cell persists in mating. The zygotic transcription that drives meiosis is rapidly initiated first from the P parental genome, even in wild-type cells. This asymmetric gene expression depends on a bipartite complex formed post-fusion between the cytosolic M-cell-specific peptide Mi and the nuclear P-cell-specific homeobox protein Pi <sup>6,7</sup> , which captures Mi in the P nucleus. Zygotic transcription is thus poised to initiate in the P nucleus as fast as Mi reaches it after fusion, a design that we reconstruct using two synthetic interactors localized to the nucleus and the cytosol of two respective partner cells. Notably, delaying zygotic transcription-by postponing Mi expression or deleting its transcriptional target in the P genome-leads to zygotes fusing with additional gametes, thus forming polyploids and eventually aneuploid progeny. The signalling cascade to block re-fertilization shares components with, but bifurcates from, meiotic induction <sup>8-10</sup> . Thus, a cytoplasmic connection upon gamete fusion leads to asymmetric reconstitution of a bipartite transcription factor to rapidly block re-fertilization and induce meiosis, ensuring genome maintenance during sexual reproduction