Rtf2 is important for replication fork barrier activity of via splicing of .

Arrested replication forks, when restarted by homologous recombination, result in error-prone DNA syntheses and non-allelic homologous recombination. Fission yeast is a model fork barrier used to probe mechanisms of recombination-dependent restart. barrier activity is entirely dependent on the DNA binding protein Rtf1 and partially dependent on a second protein, Rtf2.

Prospective Evaluation of Low-Fat Diet Monotherapy in Dogs with Presumptive Protein-Losing Enteropathy.

For dogs with protein-losing enteropathy (PLE) and evidence of lymphangiectasia, the efficacy of low-fat diet as monotherapy or combined with prednisone remains poorly characterized. In this prospective, observational cohort study of 14 dogs with presumptive PLE and ultrasonographic evidence of lymphangiectasia, subjects were placed on various low-fat diets as monotherapy and prednisone was added if response was deemed inadequate. Dogs were assessed and scored at four recheck examinations across a 6 mo study period, including a final recheck ultrasound.

Rice () TIR1 and 5'adamantyl-IAA Significantly Improve the Auxin-Inducible Degron System in .

The auxin-inducible degron (AID) system is a powerful tool to induce targeted degradation of proteins in eukaryotic model organisms. The efficiency of the existing AID system is limited due to the fusion of the F-box protein TIR1 protein to the SCF component, Skp1 (Skp1-TIR1). Here, we report an improved AID system for that uses the TIR1 from (OsTIR1) not fused to Skp1.

Inhibition of MRN activity by a telomere protein motif.

The MRN complex (MRX in Saccharomyces cerevisiae, made of Mre11, Rad50 and Nbs1/Xrs2) initiates double-stranded DNA break repair and activates the Tel1/ATM kinase in the DNA damage response. Telomeres counter both outcomes at chromosome ends, partly by keeping MRN-ATM in check. We show that MRX is disabled by telomeric protein Rif2 through an N-terminal motif (MIN, MRN/X-inhibitory motif).

Live-cell single-molecule tracking highlights requirements for stable Smc5/6 chromatin association in vivo.

The essential Smc5/6 complex is required in response to replication stress and is best known for ensuring the fidelity of homologous recombination. Using single-molecule tracking in live fission yeast to investigate Smc5/6 chromatin association, we show that Smc5/6 is chromatin associated in unchallenged cells and this depends on the non-SMC protein Nse6. We define a minimum of two Nse6-dependent sub-pathways, one of which requires the BRCT-domain protein Brc1.

Replication dynamics of recombination-dependent replication forks.

Replication forks restarted by homologous recombination are error prone and replicate both strands semi-conservatively using Pol δ. Here, we use polymerase usage sequencing to visualize in vivo replication dynamics of HR-restarted forks at an S. pombe replication barrier, RTS1, and model replication by Monte Carlo simulation.

Long-term outcome following surgical and radiation treatment of vertebral angiomatosis in a cat.

CASE DESCRIPTION A 2-year-old 5.2-kg (11.4-lb) neutered male domestic shorthair cat was referred because of a 6-week history of progressive paraparesis.

Short-Term Prospective Clinical Evaluation of a Polyglycolic Acid Tibial Tuberosity Advancement Cage Implant.

This study investigated the short-term radiographic healing of the osteotomy following tibial tuberosity advancement (TTA), maintenance of patellar tendon angle (PTA), and complications in dogs receiving a polyglycolic acid (PGA) TTA cage. Patients diagnosed with unilateral cranial cruciate ligament disease requiring a 9- or 12-mm TTA cage were included. Twenty-six consecutive client-owned dogs were prospectively selected for this clinical study.

PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast.

PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage.

Prospective Randomized Clinical and Radiographic Evaluation of a Novel Bioabsorbable Biocomposite Tibial Tuberosity Advancement Cage Implant.

Objective: To evaluate the suitability of a novel bioabsorbable biocomposite cage (BC) implant for use in tibial tuberosity advancement (TTA) surgery in dogs with cranial cruciate ligament (CrCL) disease and to compare radiographic osteotomy healing scores and complications between groups that received either a BC or stainless steel cage (SSC).

Study Design: Prospective randomized clinical study.

Animals: Dogs with unilateral CrCL rupture (n=56).

Molecular Genetic Tools and Techniques in Fission Yeast.

The molecular genetic tools used in fission yeast have generally been adapted from methods and approaches developed for use in the budding yeast, Saccharomyces cerevisiae Initially, the molecular genetics of Schizosaccharomyces pombe was developed to aid gene identification, but it is now applied extensively to the analysis of gene function and the manipulation of noncoding sequences that affect chromosome dynamics. Much current research using fission yeast thus relies on the basic processes of introducing DNA into the organism and the extraction of DNA for subsequent analysis. Targeted integration into specific genomic loci is often used to create site-specific mutants or changes to noncoding regulatory elements for subsequent phenotypic analysis.

Colony Polymerase Chain Reaction with Schizosaccharomyces pombe.

When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S.

Extraction of Chromosomal DNA from Schizosaccharomyces pombe.

Extraction of DNA from Schizosaccharomyces pombe cells is required for various uses, including templating polymerase chain reactions (PCRs), Southern blotting, library construction, and high-throughput sequencing. To purify high-quality DNA, the cell wall is removed by digestion with Zymolyase or Lyticase and the resulting spheroplasts lysed using sodium dodecyl sulfate (SDS). Cell debris, SDS, and SDS-protein complexes are subsequently precipitated by the addition of potassium acetate and removed by centrifugation.

Identifying Products of Recombinase-Mediated Cassette Exchange (RMCE) in Schizosaccharomyces pombe.

Homologous recombination is highly efficient when mediated between two identical target sequences by recombination enzymes such as Cre. Exploiting this, recombinase-mediated cassette exchange (RMCE) was developed for the genetic manipulation of eukaryotic cells, including those of Schizosaccharomyces pombe RMCE can be summarized in three stages: (1) A loxP-ura4(+)-loxM3 cassette is introduced into the genome using standard homologous recombination techniques to create a "base strain." (2) A Cre-expression plasmid carrying a protein tag or replacement gene flanked by loxP and loxM3 is introduced into the cell.

Transformation of Schizosaccharomyces pombe: Protoplast Procedure.

Transformation of Schizosaccharomyces pombe with DNA requires the conditioning of cells to promote DNA uptake followed by cell growth under conditions that select and maintain the plasmid or integration event. The three main methodologies are electroporation, treatment with lithium cations, and transformation of protoplasts. The protocol for protoplast transformation, which is described here, is more complicated than those for electroporation or lithium acetate and thus less often used.

Transformation of Schizosaccharomyces pombe: Lithium Acetate/ Dimethyl Sulfoxide Procedure.

Transformation ofSchizosaccharomyces pombewith DNA requires the conditioning of cells to promote DNA uptake followed by cell growth under conditions that select and maintain the plasmid or integration event. The three main methodologies are electroporation, treatment with lithium cations, and transformation of protoplasts. The lithium acetate method described here is widely used because it is simple and reliable.

Transformation of Schizosaccharomyces pombe: Electroporation Procedure.

Transformation ofSchizosaccharomyces pombewith DNA requires the conditioning of cells to promote DNA uptake followed by cell growth under conditions that select and maintain the plasmid or integration event. The three main methodologies are electroporation, treatment with lithium cations, and transformation of protoplasts. This protocol describes transformation by electroporation.

Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet.

Crystal structure of a Fanconi anemia-associated nuclease homolog bound to 5' flap DNA: basis of interstrand cross-link repair by FAN1.

Fanconi anemia (FA) is an autosomal recessive genetic disorder caused by defects in any of 15 FA genes responsible for processing DNA interstrand cross-links (ICLs). The ultimate outcome of the FA pathway is resolution of cross-links, which requires structure-selective nucleases. FA-associated nuclease 1 (FAN1) is believed to be recruited to lesions by a monoubiquitinated FANCI-FANCD2 (ID) complex and participates in ICL repair.

Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.

Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells.

Optimisation of the Schizosaccharomyces pombe urg1 expression system.

The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition.

Imaging diagnosis-MRI characteristics of a fourth ventricular cholesterol granuloma in a dog.

A 2-year-old male American Bulldog experienced paroxysmal staggering, altered consciousness, and hyperesthesia. Magnetic resonance (MR) imaging enabled recognition of a fourth ventricular mass causing compression of the cerebellum and brainstem and obstructive hydrocephalus. The mass was uniformly T2-hyperintense and predominantly T1-hypointense.

Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus.

The lack of a rapid and efficient system to regulate transcriptional induction in the fission yeast Schizosaccharomyces pombe is currently a limitation of this model eukaryote. The commonly used nmt1 promoter has excellent dynamic range and a low "off-state" transcription, but takes 14-16 hours to induce upon thiamine withdrawal. Conversely, other induction systems have rapid response times, but suffer from a limited dynamic range and/or relatively high levels of off-state transcription.

Safety and correlation of test results of combined ultrasound-guided fine-needle aspiration and needle core biopsy of the canine spleen.

The safety and diagnostic value of combined splenic fine-needle aspiration (FNA) and needle core biopsy (NCB) is unknown. Forty-one dogs with splenic lesions were studied prospectively. Safety was assessed in 38 dogs and no complications were encountered.