Genetic engineering of baker’s and wine yeasts using formaldehyde hyperresistance-mediating plasmids

Yeast multi-copy vectors carrying the for maldehyde-resistance marker gene SFA have proved to be a valuable tool for research on industrially used strains of Saccharomyces cerevisiae. The genetics of these strains is often poorly understood, and for various reasons it is not possible to simply subject these strains to protocols of genetic engineering that have been established for laboratory strains of S. cerevisiae. We tested our vectors and protocols using 10 randomly picked baker’s and wine yeasts all of which could be transformed by a simple protocol with vectors conferring hyperresistance to formaldehyde. The application of formaldehyde as a selecting agent also offers the advantage of its biodegradation to CO2 during fermentation, i.e., the selecting agent will be consumed and therefore its removal during down-stream processing is not necessary. Thus, this vector provides an expression system which is simple to apply and inexpensive to use. Key words: · Yeast · Transformation · Hyperresistance to formaldehyd

Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene’s influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT–PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2{Delta}, rlf2{Delta} and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2{Delta} pso4-1 and rlf2{Delta} pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae

The impact of earnings per share targets in executive remuneration contracts on company accounting choices

This study concerns itself with executive share option plans that have earnings per share targets and examines whether the existence of such vesting criteria results in opportunistic behaviour by managers or represents efficient contracting. Accounting choices by management are studied to see whether earnings per share targets in various executive remuneration components are associated with (1) the disclosure of alternative earnings per share, (2) earnings management defined as abnormal working capital accruals and (3) earnings management defined as meeting or beating analysts' forecasts. To begin with, the current study tests for an association between the disclosure of alternative earnings per share figures and earnings per share performance criteria in executive share options. Following Healy (1985) it is argued that situations might exist where executives are aware they will not meet the target or will overshoot the target giving rise to incentives to manage earnings downwards. There are also situations where executives expect to miss the target but have incentives (and scope) to manage earnings upwards. The study then proceeds to measure earnings management using a modified Jones (1991) model. A proxy for target growth in earnings per share is developed. The third and final section of the current study considers meeting or beating analysts' forecasts as the earnings management metric. Prior research provides evidence that meeting or beating analysts' forecasts is rewarded by the stock market and as the payout from executive share options is linked to share price, executives have incentives to meet or beat analysts' forecasts.Regression analysis, in the form of either logit or ordinary least squares is employed in all three sections of this study. The results suggest that earnings management is associated with earnings per share vesting targets in executive share option plans. Moreover, the findings as a whole suggest that the introduction of earnings per share targets as a vesting criterion in executive share options resulted in opportunistic behaviour by management.This thesis adopts an agency theory framework and contributes to the literature on corporate governance and executive remuneration by identifying a specific contractual setting where management is especially sensitive to reported earnings numbers. This particular setting is novel. Additionally, the research design facilitated the testing of whether or not executive share options with an earnings per share growth target result in opportunistic behaviour on the part of managers or represent efficient contracting.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Blue dot taxi system in the Western Cape : systems and technology overview

Papers presented virtually at the 41st International Southern African Transport Conference on 10-13 July 2040The Western Cape Department of Transport and Public Works (DTPW) initiated the Blue Dot Taxi pilot project to encourage, monitor, and reward behaviour change in the minibus taxi (MBT) industry through an incentive programme for compliant participants. The incentive is determined by the participant’s performance against specified standards designed to improve road safety and service quality for passengers and the broader public. To enable this, each participating taxi is fitted with hardware, including a telematics device and a driver tag. This hardware links the taxi and driver to the incentive system, which routinely checks that compliance requirements have been met and measures performance of the participants against the standards. Participants are enrolled via a registration portal, and their vehicles are inspected using a purpose-built depot application. Compliance checks include daily eNatis and operating licence validity checks, amongst others. All this information feeds into the Intelligent Transport Solution, which monitors and measures performance through a Business Intelligence platform, and calculates incentive payments. Passengers can also rate taxis via WhatsApp and USSD and report incidents, which automatically impacts the incentive calculation and improves accountability of participating MBT services. The government-owned technology is easily scalable and has been successfully used to reduce instances of speeding and harsh driving, and improve passenger perception

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function. A human–yeast chimera was indeed capable of restoring the wild-type phenotype of the yeast mutant strain. In addition, immunodepletion of SNEV from human nuclear extracts resulted in a decrease of in vitro pre-mRNA splicing efficiency. Furthermore, as part of our analysis of protein–protein interactions within the CDC5L complex, we found that SNEV interacts with itself. The self-interaction domain was mapped to amino acids 56–74 in the protein's sequence and synthetic peptides derived from this region inhibit in vitro splicing by surprisingly interfering with spliceosome formation and stability. These results indicate that SNEV is the human orthologue of yeast PRP19, functions in splicing and that homo-oligomerization of SNEV in HeLa nuclear extract is essential for spliceosome assembly and that it might also be important for spliceosome stability

A genome-wide CRISPR/Cas9 screen reveals the requirement of host sphingomyelin synthase 1 for infection with Pseudorabies virus mutant gD–Pass

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked