A gene truncation strategy generating N- and C-terminal deletion variants of proteins for functional studies: mapping of the Sec1p binding domain in yeast Mso1p by a Mu in vitro transposition-based approach

Bacteriophage Mu in vitro transposition constitutes a versatile tool in molecular biology, with applications ranging from engineering of single genes or proteins to modification of genome segments or entire genomes. A new strategy was devised on the basis of Mu transposition that via a few manipulation steps simultaneously generates a nested set of gene constructions encoding deletion variants of proteins. C-terminal deletions are produced using a mini-Mu transposon that carries translation stop signals close to each transposon end. Similarly, N-terminal deletions are generated using a transposon with appropriate restriction sites, which allows deletion of the 5′-distal part of the gene. As a proof of principle, we produced a set of plasmid constructions encoding both C- and N-terminally truncated variants of yeast Mso1p and mapped its Sec1p-interacting region. The most important amino acids for the interaction in Mso1p are located between residues T46 and N78, with some weaker interactions possibly within the region E79–N105. This general-purpose gene truncation strategy is highly efficient and produces, in a single reaction series, a comprehensive repertoire of gene constructions encoding protein deletion variants, valuable in many types of functional studies. Importantly, the methodology is applicable to any protein-encoding gene cloned in an appropriate vector

Kokemuksia kiusaamistilanteiden selvittämisestä työyhteisössä ja viranomaistahojen kanssa. Diskurssianalyyttinen tutkimus verkkokeskusteluista

siirretty Doriast

High-precision mapping of protein–protein interfaces: an integrated genetic strategy combining en masse mutagenesis and DNA-level parallel analysis on a yeast two-hybrid platform

Understanding networks of protein–protein interactions constitutes an essential component on a path towards comprehensive description of cell function. Whereas efficient techniques are readily available for the initial identification of interacting protein partners, practical strategies are lacking for the subsequent high-resolution mapping of regions involved in protein–protein interfaces. We present here a genetic strategy to accurately map interacting protein regions at amino acid precision. The system is based on parallel construction, sampling and analysis of a comprehensive insertion mutant library. The methodology integrates Mu in vitro transposition-based random pentapeptide mutagenesis of proteins, yeast two-hybrid screening and high-resolution genetic footprinting. The strategy is general and applicable to any interacting protein pair. We demonstrate the feasibility of the methodology by mapping the region in human JFC1 that interacts with Rab8A, and we show that the association is mediated by the Slp homology domain 1

A transposase strategy for creating libraries of circularly permuted proteins

A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions

Bacteriophage Mu integration in yeast and mammalian genomes

Genomic parasites have evolved distinctive lifestyles to optimize replication in the context of the genomes they inhabit. Here, we introduced new DNA into eukaryotic cells using bacteriophage Mu DNA transposition complexes, termed ‘transpososomes’. Following electroporation of transpososomes and selection for marker gene expression, efficient integration was verified in yeast, mouse and human genomes. Although Mu has evolved in prokaryotes, strong biases were seen in the target site distributions in eukaryotic genomes, and these biases differed between yeast and mammals. In Saccharomyces cerevisiae transposons accumulated outside of genes, consistent with selection against gene disruption. In mouse and human cells, transposons accumulated within genes, which previous work suggests is a favorable location for efficient expression of selectable markers. Naturally occurring transposons and viruses in yeast and mammals show related, but more extreme, targeting biases, suggesting that they are responding to the same pressures. These data help clarify the constraints exerted by genome structure on genomic parasites, and illustrate the wide utility of the Mu transpososome technology for gene transfer in eukaryotic cells

Transposition-based method for the rapid generation of gene-targeting vectors to produce Cre/Flp-modifiable conditional knock-out mice

Peer reviewe

Imaging and imagination: understanding the endo-lysosomal system

Lysosomes are specialized compartments for the degradation of endocytosed and intracellular material and essential regulators of cellular homeostasis. The importance of lysosomes is illustrated by the rapidly growing number of human disorders related to a defect in lysosomal functioning. Here, we review current insights in the mechanisms of lysosome biogenesis and protein sorting within the endo-lysosomal system. We present increasing evidence for the existence of parallel pathways for the delivery of newly synthesized lysosomal proteins directly from the trans-Golgi network (TGN) to the endo-lysosomal system. These pathways are either dependent or independent of mannose 6-phosphate receptors and likely involve multiple exits for lysosomal proteins from the TGN. In addition, we discuss the different endosomal intermediates and subdomains that are involved in sorting of endocytosed cargo. Throughout our review, we highlight some examples in the literature showing how imaging, especially electron microscopy, has made major contributions to our understanding of the endo-lysosomal system today

Vesivoimatuotannon optimointi-ohjelma

Opinnäytetyön tarkoituksena oli kehittää vesivoimalaitosten matemaattisesta mallinnuksesta toimiva ja käyttäjäystävällinen ohjelma, jolla suunnitellaan PVO-Pool Oy:n käyttökeskuksesta ohjattavien vesivoimalaitosten tuotannot. Ohjelma sisältää simulointi- ominaisuuden, jolla simuloidaan vesivoimalaitosten yläaltaan pinnankorkeuksien muutosta. Ohjelman perustana on vesivoimalaitoksen käytettävissä oleva vesimäärä. Juoksutetun veden määrästä riippuvat vesivoimalaitosten antamat tehot ja pinnankorkeudet. Juoksutuksen määrä optimoidaan tuntikohtaisen sähkön hintaennusteen mukaan. Kalleimmilla tunneilla juoksutetaan enemmän kuin halvimmilla tunneilla. Käyttäjällä on mahdollisuus muokata laskennallista suunnitelmaa omien kokemusten ja näkemysten mukaan. Ohjelman tavoitteena oli saavuttaa pienellä suunnittelun tarkennuksella vuositasolla merkittävä tuoton lisäys. Ohjelman tekemisessä pyrin ottamaan huomioon käyttäjän näkökulman ja tekemään siitä mahdollisimman helppokäyttöisen käyttäjilleen.The purpose of this final year project was to develop workable and user-friendly software of mathematic modelling of hydropower plants. The software will be used for controlling the output of the hydropower plants operated from the PVO-Pool control room. The software includes a simulation feature. The change of the headpond water level will be simulated by this software. The basis of this software is the available amount of water of a hydropower plant. The amount of water run determines the power and the head-pond level. The amount of water run will be optimised using the forecasts of electricity prices. On highpriced hours more water will be run than on low-priced hours. The user has the possibility to edit the calculated plan using his own experience and opinion. One aim for this software was to reach a significant increase of income annually through minor correction of planning. When making this software I tried to take into account the viewpoint of the user and to make it as user-friendly as possible

CARING FOR LGBT-PATIENTS IN FINLAND AND GERMANY: A COMPARISON OF PREPARATION, ATTITUDE, AND RESOURCES OF NURSES

The purpose of this thesis was to find out if the health needs of LGBT (lesbian, gay, bisexual, transgender) patient’s play a role in the curriculum of nursing education, if personal influence affects the professional attitude when caring for these patients, and if and what kind of resources are available to further educate oneself for nursing of LGBT patients. The research was focused to find answers to these questions by gaining and analysing answers from Finnish and German nurses, and compare their answers in the end. The aim was to collect enough data for comparison and to be able to highlight problems within the fields of variables. The long-term aim was to gain answers that provide important information to health care educators and providers to improve the curriculum and resources, in order to impact the treatment and visibility of LGBT patients. The theoretical framework dealt with different topics of health care needs of LGBT patient’s, showing percentages obtained due to published researches in relation to heterosexual patients, and highlighting how and why the different health needs must be taken into account by health care professionals. The results were obtained via quantitative research, done with two online surveys – one for Finnish nurses and one for German nurses. The results showed that the majority of Finnish and German nurses did not receive education about LGBT patient’s health care during the curriculum of their nursing education. Furthermore, personal attitude is not an influencing factor in how non- heterosexual patients are treated professionally. Resource availability differs between both countries, with Finnish work places providing more resources for their workers than Germany. Additionally, more Finnish nurses than German ones use available resources. The nurses of both countries would educate themselves further on the topic in form a schooling offered by their workplace, and would also recommend such a schooling to colleagues