Competitive Effects of Non-Controlling Minority Acquisitions – Sufficient to merit further EU merger regulation?

Partial ownership interests are a widespread phenomenon in modern corporate environment. Unless minority shareholding affords the target to exercise control over the target, they do currently not have to be notified to the European Commission under EU merger regime. However, economic research has long suggested that when linking competing or non-horizontally positioned undertakings particularly in industries with few competitors, minority shareholdings even far below the majority of shares or voting rights could lead to higher prices or lower output volumes to the detriment of consumers. The Commission has recognized this issue and proceeded to suggest an extension of the merger regime to catch also certain non-controlling minority acquisitions. Horizontal non-controlling minority shareholdings create a positive correlation between the sales revenues of the partial acquirer and target. Through the equity interest the acquirer will internalise a fraction, proportional to the financial rights attached to the shareholding, of the profit of the target. This will incentivise the acquirer to contribute to increasing the target’s business profits by increasing its own sales price (horizontal unilateral effects). When a minority stake is held in a vertically related or a conglomerate company, the minority acquirer could be allowed to hamper or eliminate the target’s rivals’ access either to inputs (input foreclosure) or customers (customer foreclosure), depending on which level of the supply chain the parties are (vertical unilateral effects). Under certain circumstances minority share acquisitions could also lessen competition because they facilitate collusion between companies active in the market (coordinated effects). Economic theory confirms that non-controlling minority shareholdings may under certain circumstances create anti-competitive effects that are unlikely to be remedies by pro-competitive effects. However, they are likely to be of less significant nature than anticompetitive effects created by full mergers. This derives fore mostly from the fact that a minority share acquirer carries all the costs associated with its unilateral action but will internalise only a fraction of the lost profits. This is likely to limit the acquirer’s incentive to raise price and the profitability of such behavior. Having in mind that the number of potentially problematic cases is expected to be next to negligible, the limited potential competitive effects of non-controlling minority share acquisitions cannot be seen to clearly merit extension of the scope of the EUMR. The system suggested by the Commission is particularly ill-fitted for such purpose given the clear lack of legal certainty and considerable administrative burden associated with it.

Partial characterization of genes from the embryonic axis of Melanoxylon brauna Schott. (Leguminosae-Caesalpinioideae) seeds

The objective of this study was to partially characterize some genes involved in the desiccation tolerance of the embryonic axis of Melanoxylon brauna seeds subjected, or not, to oven fast-drying. Seeds were initially dried rapidly in an oven at 40 ºC, 50 ºC, 60 ºC, 70 ºC, and 80 °C, for 24, 48 and 72 h and then subjected to germination tests and moisture content determination. Degenerate primers were designed for 19 genes. The CDNA was used as a template for PCR amplifications using the degenerate primers, and the PCR products obtained were purified, cloned and sequenced. The seeds showed a gradual reduction in percent germination with increasing temperature and drying time. Nucleotide sequences of the cloned fragments related to genes CAT1, SPS1, Abi5, Transk and PM25 were obtained. The similarity analysis with the sequences deposited in databases revealed similarities with genes CAT1, SPS1, Transk and PM25 from other plant species. The nucleotide sequences obtained from the respective genes will be used for designing specific primers for gene expression analyses during seed germination in order to understand the causes for loss of physiological quality of Melanoxylon brauna seeds.

Individual Crimes or a Sociological Phenomenon: A Critical discourse analysis of the 2014 Royal Canadian Mounted Police National Operational Overview on missing and murdered Indigenous women in Canada

Pro gradu -tutkielma käsittelee Kanadan poliisivoimien, Royal Canadian Mounted Police (RCMP), vuoden 2014 operationaalista raporttia Kanadan kadonneista ja murhatuista alkuperäiskansojen naisista. Heitä katoaa ja murhataan Kanadassa suhteessa huomattavasti enemmän kuin muita naisia. Aihe on hyvin ajankohtainen sekä kriittinen ja siihen on paikallisten organisaatioiden lisäksi kiinnittänyt huomiota mm. Amnesty International ja Yhdistyneet Kansakunnat. Tilannetta pidetään jo ihmisoikeuskriisinä ja sen puolesta puhuminen voidaan nähdä osana laajempaa kansannousua alkuperäiskansojen oikeuksien puolesta. Kanadan hallitusta sekä poliisivoimia on syytetty välinpitämättömyydestä ja rasismista, poliiseja jopa väkivallasta alkuperäiskansojen naisia kohtaan. Kanadan hallitus ei myönnä ongelman olevan sosiologinen ilmiö, vaan pääministerin mukaan naisten katoamiset ja murhat ovat yksittäisiä rikoksia. Tilanteen taustalla on laajempi ongelma, joka juontaa juurensa kolonialismista, alkuperäiskansojen pakkokoulutuksesta ja heidän kulttuurinsa sekä identiteettinsä tuhoamisesta. Ennakkoluulot ja stereotypiat elävät yhä vahvana luoden heikon aseman alkuperäiskansojen edustajille ja tässä tapauksessa erityisesti naisille, joita myös usein syytetään omista ongelmistaan. Tutkielma selvittää, onko tämä operationaalinen raportti eriarvoisuutta, stereotypioita sekä ennakkoluuloja ylläpitävä ja tukeeko se näkemystä sosiologisesta ilmiöstä. Poliisivoimat ovat yhteiskunnassa vaikutusvaltaisessa asemassa ja voivat edesauttaa, jatkaa ja voimistaa syrjivää ajattelua, käytöstä ja viestintää. Vaikka he myös ajavat alkuperäiskansojen etuja, tulee heidän toimintaansa ja täten tätä kyseistä raporttia tarkastella kriittisesti. Tutkielma keskittyy kriittiseen diskurssianalyysiin (Fairclough 1995, Wodak & Meyer 2009). Raportin teksti ei ole avoimesti diskriminoivaa ja keskittyy tilastojen ja lukujen raportointiin sekä vertailuun, luoden samalla kuvan asianomaisista sekä tilanteesta yleisesti. Makroanalyysi (van Dijk 1977, 1980) mahdollistaa korkeamman tason ajatusten ja ideoiden esille tuonnin paljastaen ns. rivien välistä löytyvän viestinnän. Näistä makroanalyysin tuloksista etsitään erilaisia diskurssistrategioita (Reisigl & Wodak 2001), joiden avulla luodaan mm. kuva positiivisesta itsestä ja negatiivisesta toisesta. Näitä tuloksia tuetaan lyhyellä multimodaalisella analyysillä, joka keskittyy lähinnä raportin kahteen kuvaan (Kress & van Leeuwen 2006). Lopuksi tuloksista keskustellaan suhteessa yhteiskunnan valtasuhteisiin (Foucault 1976), rasismiin ja seksismiin. Analyysin perusteella raportti on selkeästi diskriminoiva. Se tuo esiin vanhoja stereotypioita ja ennakkoluuloja, esittää tilanteen alkuperäiskansojen sisäisenä ongelmana, jättää mainitsematta tilanteen taustat ja luo hyvin positiivisen kuvan poliisivoimista (RCMP). Tällä tavoin se ylläpitää yhteiskunnan epätasapainoisia valtasuhteita ja alkuperäiskansojen naisten heikompaa ja marginalisoitua asemaa. Raportti todistaa kuinka syvällä Kanadan yhteiskunnassa jopa institutionaalisella tasolla nämä ajatukset piilevät, sillä diskriminaatio ulottuu tapausten syistä niiden käsittelyyn ja raportointiin. Tilanne on huolestuttava ja voidaan nähdä sosiologisena ilmiönä. Poliisivoimien tulisi lisätä sisäistä koulutusta asian suhteen sekä hallituksen tutkia tätä ilmiönä ja paneutua sen selvittämiseen laaja-alaisesti ja läpinäkyvästi.

Individual Crimes or a Sociological Phenomenon: A Critical discourse analysis of the 2014 Royal Canadian Mounted Police National Operational Overview on missing and murdered Indigenous women in Canada

Pro gradu -tutkielma käsittelee Kanadan poliisivoimien, Royal Canadian Mounted Police (RCMP), vuoden 2014 operationaalista raporttia Kanadan kadonneista ja murhatuista alkuperäiskansojen naisista. Heitä katoaa ja murhataan Kanadassa suhteessa huomattavasti enemmän kuin muita naisia. Aihe on hyvin ajankohtainen sekä kriittinen ja siihen on paikallisten organisaatioiden lisäksi kiinnittänyt huomiota mm. Amnesty International ja Yhdistyneet Kansakunnat. Tilannetta pidetään jo ihmisoikeuskriisinä ja sen puolesta puhuminen voidaan nähdä osana laajempaa kansannousua alkuperäiskansojen oikeuksien puolesta. Kanadan hallitusta sekä poliisivoimia on syytetty välinpitämättömyydestä ja rasismista, poliiseja jopa väkivallasta alkuperäiskansojen naisia kohtaan. Kanadan hallitus ei myönnä ongelman olevan sosiologinen ilmiö, vaan pääministerin mukaan naisten katoamiset ja murhat ovat yksittäisiä rikoksia. Tilanteen taustalla on laajempi ongelma, joka juontaa juurensa kolonialismista, alkuperäiskansojen pakkokoulutuksesta ja heidän kulttuurinsa sekä identiteettinsä tuhoamisesta. Ennakkoluulot ja stereotypiat elävät yhä vahvana luoden heikon aseman alkuperäiskansojen edustajille ja tässä tapauksessa erityisesti naisille, joita myös usein syytetään omista ongelmistaan. Tutkielma selvittää, onko tämä operationaalinen raportti eriarvoisuutta, stereotypioita sekä ennakkoluuloja ylläpitävä ja tukeeko se näkemystä sosiologisesta ilmiöstä. Poliisivoimat ovat yhteiskunnassa vaikutusvaltaisessa asemassa ja voivat edesauttaa, jatkaa ja voimistaa syrjivää ajattelua, käytöstä ja viestintää. Vaikka he myös ajavat alkuperäiskansojen etuja, tulee heidän toimintaansa ja täten tätä kyseistä raporttia tarkastella kriittisesti. Tutkielma keskittyy kriittiseen diskurssianalyysiin (Fairclough 1995, Wodak & Meyer 2009). Raportin teksti ei ole avoimesti diskriminoivaa ja keskittyy tilastojen ja lukujen raportointiin sekä vertailuun, luoden samalla kuvan asianomaisista sekä tilanteesta yleisesti. Makroanalyysi (van Dijk 1977, 1980) mahdollistaa korkeamman tason ajatusten ja ideoiden esille tuonnin paljastaen ns. rivien välistä löytyvän viestinnän. Näistä makroanalyysin tuloksista etsitään erilaisia diskurssistrategioita (Reisigl & Wodak 2001), joiden avulla luodaan mm. kuva positiivisesta itsestä ja negatiivisesta toisesta. Näitä tuloksia tuetaan lyhyellä multimodaalisella analyysillä, joka keskittyy lähinnä raportin kahteen kuvaan (Kress & van Leeuwen 2006). Lopuksi tuloksista keskustellaan suhteessa yhteiskunnan valtasuhteisiin (Foucault 1976), rasismiin ja seksismiin. Analyysin perusteella raportti on selkeästi diskriminoiva. Se tuo esiin vanhoja stereotypioita ja ennakkoluuloja, esittää tilanteen alkuperäiskansojen sisäisenä ongelmana, jättää mainitsematta tilanteen taustat ja luo hyvin positiivisen kuvan poliisivoimista (RCMP). Tällä tavoin se ylläpitää yhteiskunnan epätasapainoisia valtasuhteita ja alkuperäiskansojen naisten heikompaa ja marginalisoitua asemaa. Raportti todistaa kuinka syvällä Kanadan yhteiskunnassa jopa institutionaalisella tasolla nämä ajatukset piilevät, sillä diskriminaatio ulottuu tapausten syistä niiden käsittelyyn ja raportointiin. Tilanne on huolestuttava ja voidaan nähdä sosiologisena ilmiönä. Poliisivoimien tulisi lisätä sisäistä koulutusta asian suhteen sekä hallituksen tutkia tätä ilmiönä ja paneutua sen selvittämiseen laaja-alaisesti ja läpinäkyvästi.

The "hot money" phenomenon in Brazil

Because of its high interest rates, Brazil attracts more and more speculative capital flows, called "hot money", under the form of foreign loans, direct or portfolio investments. Actually, the country is directly involved in a carry-trade strategy that tends to appreciate the real, what penalizes the Brazilian exportations of manufactured products. Moreover, capital inflows are extremely volatile, and their departure, causing a fall in loans granted to the Brazilian private banks, could provoke a dangerous burst of the speculative bubble they have contributed to form in the Brazilian real estate sector.

Understanding round-tripping phenomenon in Russian context – case Cyprus

Russian FDI has a few peculiarities. One of them is round-tripping. Round-tripping is defined as transfer of funds abroad, usually to offshore financial centers (OFCs), and then bringing all or some of the investment back as foreign investment. Russian context was chosen for this study because the share of round-tripping investments from country’s total FDI is extensive. However, it needs to be addressed that this is not just a Russian phenomenon. Round-tripping is used by many developed and developing countries, and most of the countries have their own designated destinations for this kind of capital, much like Cyprus is the main destination for Russian capital. It is important to study this phenomenon further, since it falsifies FDI statistics and can lead to poor decisions on state level. Theoretical part of the study tries to determine weather traditional FDI and internationalization theories fit to explain the Russian round-tripping phenomenon. Traditional FDI and internationalization theories are first introduced in general terms, and then further examined in Russian context. In traditional endogenic FDI theories, when the capital is formed in one country it goes abroad to find better profits. At a first glance, this seemed not to be the case in round-tripping. However, during the study it became rather clear that with few adjustments and changes in perspective, traditional theories could be used to explain round-tripping phenomenon. For example, OLI paradigm can be further developed into OLIH paradigm with ‘H’ representing the important home country institutions. Transaction based view and resource seeking theories were also seen well equipped to explain round-tripping with a change in perspective. The latter part of the study focused on holistic understanding of Russian –Cyprian investment relationship. Study aims to shed light into the determinants and consequences of this phenomenon for both countries involved. The two share historical, cultural and political ties, but most importantly common financial interests. Russian companies seek security and financial knowledge to maneuver their assets and Cyprian economy largely is dependent on their disproportionally large financial sector. Consequences for Cyprian economy were seen in current economic crisis, when the need for their financial services diminished. Russian government on the other hand is losing vast amounts of tax money due to this phenomenon. A rather extreme view was also introduced in this study. Round-tripping phenomenon and OFCs are an important reason why corruption exists, since if one does not have a way to make ill-gained money legitimate why try to ill-gain the money at the first place. The most important finding of the study is that round-tripping companies are in a better competitive position than genuine and purely domestic investor due to their institutional knowledge.

The Literary relations between La Fontaine and the "Astrée" of Honoré d'Urfé : a dissertation presented to the faculty of the department of philosophy of the university of Pennsylvania in partial fulfilment of the requirements for the degree of doctor of philosophy / by Walther P. Fischer

Collection : Publications of the University of Pennsylvania ; n° 6

The Midnight express phenomenon : a historical materialist approach to the reception of the film Midnight express /

This thesis examines the Midnight Express phenomenon focusing on the film's reception by audiences in Europe, North America, and Turkey between 1978-2003. Using and enhancing the "historical materialist approach" to film reception developed by Janet Staiger, the thesis considers the historical determinants of the film's nationally and culturally differential readings in different periods and of the transformations in those readings. The thesis argues that while Midnight Express was most likely read in the late 1970s as an attempt to reaffirm American social identity by projecting Turks as an instance of the negative Other, there has been an important shift in the reception of the film in the West during the 1990s due to the changes in the discursive contexts in which the film has been circulating. One does not observe any specific reference to Turkish prisons as a part of the issue of human rights violations in Turkey in the initial reception of the film by European and American critics, whereas these issues appear to be important constituents of a particular reception of the film in the West in the present. The thesis explains this shift by pointing to the constitution of a particular discourse on human rights violations in Turkey after 1980, and especially throughout the 1990s, which has become a part of the discursive repertoires of the Western audience. Therefore, the thesis argues that today, Midnight Express functions as a more legitimate political statement about Turkey in the eyes of some Western audiences than it had been in the 1970s. On the other hand, parallel to the increasing desire of Turkey to connect itself to the West, particularly to become a member of the European Union, one observes an immense increase in the belief in and defense against the negative effects of Midnight Express on Turkey's international representation since the 1990s. The historical and current discourses that audiences, both in Turkey and abroad, bring into play suggest that these audiences engage with Midnight Express by assuming or denying not only the subject positions constructed by the film text but also certain history-specific extra-filmic subject positions produced by other social and discursive formations.

Effect of diurnal cycling temperatures on cardiovascular- ventilatory function in statically-acclimated rainbow trout, Salmo gairdneri

Four groups of rainbow trout, Salmo gairdneri, were acclimated to 2°, 10°, and 18°e, and to a diurnal temperature cycle (100 ± 4°C). To evaluate the influence of cycling temperatures in terms of an immediate as opposed to acclimatory response various ventilatory-cardiovascular rate functions were observed for trout, either acclimated to cycling temperatures or acclimated to constant temperatures and exposed to a diurnal temperature cycle for the first time (10° ± 4°C for trout acclimated to 10°C; 18°+ 4°C for trout acclimated to l8°e). Gill resistance and the cardiac to ventilatory rate ratio were then calculated. Following a post preparatory recovery period of 36 hr, measurements were made over a 48 hour period with the first 24 hours being at constant temperature in the case of statically-acclimated fish followed by 24 hours under cyclic temperature conditions. Trout exhibited marked changes in oxygen consumption (Vo ) with temp- 2 erature both between acclimation groups, and in response to the diurnal temperature cycle. This increase in oxygen uptake appears to have been achieved by adjustment of ventilatory and, to some extent, cardiovascular activity. Trout exhibited significant changes in ventilatory rate (VR), stroke volume (Vsv), and flow (VG) in response to temperature. Marked changes in cardiac rate were also observed. These findings are discussed in relation to their importance in convective oxygen transport via water and blood at the gills and tissues. Trout also exhibited marked changes in pressure waveforms associated with the action of the resp; ratory pumps with temperature. Mean differenti a 1 pressure increased with temperature as did gill resistance and utilization. This data is discussed in relation to its importance in diffusive oxygen transport and the conditions for gas exchange at the gills. With one exception, rainbow trout were able to respond to changes in oxygen demand and availability associated with changes in temperature by means of adjustments in ventilation, and possibly pafusion, and the conditions for gas exchange at the gills. Trout acclimated to 18°C, however, and exposed to high cyclic temperatures, showed signs of the ventilatory and cardiovascular distress problems commonly associated with low circulating levels of oxygen in the blood. It appears these trout were unable to fully meet the oxygen requirements associated with c~ling temperatures above 18°C. These findings were discussed in relation to possible limitations in the cardiovascular-ventilatory response at high temperatures. The response of trout acclimated to cycling temperatures was generally similar to that for trout acclimated to constant temperatures and exposed to cycling temperatures for the first time. This result suggested that both groups of fish may have been acclimated to a similar thermal range, regardless of the acclimation regime employed. Such a phenomenon would allow trout of either acclimation group to respond equally well to the imposed temperature cycle. Rainbow trout showed no evidence of significant diurnal rhythm in any parameters observed at constant temperatures (2°, 10°, and 18° C), and under a 12/12 light-dark photoperiod regime. This was not taken to indicate an absence of circadian rhythms in these trout, but rather a deficiency in the recording methods used in the study.

Isolation and partial characterization of host cell wall surface glycoproteins: their involvement in agglutination, attachment and appressorium formation by piptocephalis virginiana

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, to the mycoparasite, Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 a.u. mg- t ) as compared with the nonhost extract (21 a.li. mg- t ). SDS-polyacrylamide gel electrophoresis of the cell wall proteins revealed four protein bands, a, b, c, and d (Mr 117, 100, 85 and 64 kd, respectively) at the host surface, but not at the nonhost surface, except for the faint band c. Deletion of proteins b or c from the host cell wall protein extract significantly reduced its agglutinating activity. Proteins band c, obtained as purified preparations by a series of procedures, were shown to be two glycoproteins. Carbohydrate analysis by gas chromatography demonstrated that glucose and Nacetylglucosamine were the major carbohydrate components of the glycoproteins. It was further shown that the agglutinating activity of the pure preparation containing both band c was 500-850 times that of the single glycoproteins, suggesting the involvement of both glycoproteins in agglutination. The results suggest that the glycoproteins band c are the two subunits of agglutinin present at the host cell surface. The two glycoproteins band c purified from the host cell wall protein extract were further examined after various treatments for their possible role in agglutination, attachment and appressorium formation by the mycoparasite. Results obtained by agglutination and attachment tests showed: (1) the two glycoprotein-s are not only an agglutinin responsible for the mycoparasite spore agglutination, but may also serve as a receptor for the specific recognition, attachment and appressorium formation by the mycoparasite; (2) treatment of the rnycoparasite spores with various sugars revealed that arabinose, glucose and N-acetylglucosamine inhibited the agglutination and attachment activity of the glycoproteins, however, the relative percentage of appressorium formation was not affected by the above sugars; (3) the two glycoproteins are relatively stable with respect to their agglutinin and receptor functions. The present results suggest that the agglutination and attachment may be mediated directly by certain sugars present at the host and mycoparasite cell surfaces while the appressorlum formation may be the response of complementary combinations of both sugar and protein, the two parts of the glycoproteins at the interacting surfaces of two fungi.

Isolation and partial characterization of a complementary protein from the mycoparasite, Piptocephalis virginiana, which specifically binds to two glycoproteins b and c of the host cell surface

Presence of surface glycoprotein in Piptocephalis virginiana that recognizes the host glycoproteins band c, reported earlier from our laboratory, was detected by immunofluorescence microscopy. Germinated spores of P. virginiana treated with Mortierella pusilla cell wall protein extract, primary antibodies prepared against glycoproteins band c and FITC-goat anti-rabbit IgG conjugate showed fluorescence. This indicated that on the surfaces of the biotrophic mycoparasite P. virginiana , there might be a complementary molecule which recognizes the glycoproteins band c from M. pusilla. Immunobinding analysis identified a glycoprotein of Mr 100 kDa from the mycoparasite which binds with the host glycoproteins band c, separately as well as collectively. Purification of this glycoprotein was achieved by (i) 60% ammonium sulfate precipitation, (ii) followed by heat treatment, and (iii) Sephadex G-IOO gel filtration. The glycoprotein was isolated by preparative polyacrylamide gel electrophoresis by cutting and elution. The purity of the protein ·was ascertained by SDS-PAGE and Western blot analysis. Positive reaction to periodic acid-Schiff reagent revealed the glycoprotein nature of this 100 kDa protein. Mannose was identified as a major sugar component of this glycoprotein by using a BoehringerMannheim Glycan Differentiation Kit. Electrophoretically purified glycoprotein was used to raIse polyclonal antibody in rabbit. The specificity of the antibody was determined by dot-immunobinding test and western-blot analysis. Immunofluorescence mIcroscopy revealed surface localization of the protein on the germ tube of Piptocephalis virginiana. Fluorescence was also observed at the surfaceJ of the germinated spores and hyphae of the host, M. pusilla after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein and FITC-goat anti-rabbit IgG conjugate.

Partial characterization of the cloned dihydrofolate reductase gene of Saccharomyces cerevisiae

The cloned dihydrofolate reductase gene of Saccharomyces cerevisiae (DFR 1) is expressed in Escherichia coli. Bacterial strain JF1754 transformed with plasmids containing DFR 1 is at least 5X more resistant to inhibition by the folate antagonist trimethoprim. Expression of yeast DFR 1 in E. coli suggests it is likely that the gene lacks intervening sequences. The 1.8 kbp DNA fragment encoding yeast dhfr activity probably has its own promotor, as the gene is expressed in both orientations in E. coli. Expression of the yeast dhfr gene cloned into M13 viral vectors allowed positive selection of DFR 1 - M13 bacterial transfectants in medium supplemented with trimethoprim. A series of nested deletions generated by nuclease Bal 31 digestion and by restriction endonuclease cleavage of plasmids containing DFR 1 physically mapped the gene to a 930 bp region between the Pst 1 and Sal 1 cut sites. This is consistent with the 21,000 molecular weight attributed to yeast dhfr in previous reports. From preliminary DNA sequence analysis of the dhfr DNA fragment the 3' terminus of DFR 1 was assigned to a position 27 nucleotides from the Eco Rl cut site on the Bam Hi - Eco Rl DNA segment. Several putative yeast transcription termination consensus sequences were identified 3' to the opal stop codon. DFR 1 is expressed in yeast and it confers resistance to the antifolate methotrexate when the gene is present in 2 - 10 copies per cell. Plasmid-dependent resistance to methotrexate is also observed in a rad 6 background although the effect is somewhat less than that conferred to wild-type or rad 18 cells. Integration of DFR 1 into the yeast genome showed an intermediate sensitivity to folate antagonists. This may suggest a gene dosage effect. No change in petite induction in these yeast strains was observed in transformed cells containing yeast dhfr plasmids. The sensitivity of rad 6 , rad 18 and wild-type cell populations to trimethoprim were unaffected by the presence of DFR 1 in transformants. Moreover, trimethoprim did not induce petites in any strain tested, which normally results if dhfr is inhibited by other antifolates such as methotrexate. This may suggest that the dhfr enzyme is not the only possible target of trimethoprim in yeast. rad 6 mutants showed a very low level of spontaneous petite formation. Methotrexate failed to induce respiratory deficient mutants in this strain which suggested that rad 6 might be an obligate grande. However, ethidium bromide induced petites to a level approximately 50% of that exhibited by wild-type and rad 18 strains.

The isolation, partial purification and preliminary characterization of a growth factor from chick embryo brains

Chicl( brain growth factor (CBGF) is a mitogen isolated from embryonic chick brains thought to have a potential role as a trophic factor involved in nerve dependent amphibian limb regeneration. In addition, CBGF stimulates 3H-thymidine incorporation in chick embryo brain astrocytes in vitro. In this study, cultured chick embryo brain non-neuronal cells were employed in a bioassay to monitor CBGF activity throughout various stages of its pllrification. Cell culture and assay conditions were optimized. Nonneuronal cells grew best on collagen-coated culture dishes in complete medium, were most responsive to a growth stimulus [10% fetal bovine serum (FBS)] at the second and third subcultures, and were healthiest when rendered "quiescent" in medium supplemented with 1% FBS. The most effective bioassay conditions consisted of a minimum 14.5 hour "quiescence" time (24 hours was used), a 6 hour "prestimulation" time, and a 24 hour 3H-thymidine labeling time. Four-day subconfluent primary non-neuronal cells consisted of 6.63% GFAP positive cells; as a result cultures were thought to be mainly composed of astroblasts. CBGF was purified from 18-day chick embryo brains by ultrafiltration through Amicon PM-30 and YM-2 membranes, size exclusion chromatography through a Biogel P6 column, and analytical reverse-phase high-performance liquid chromatography (rp-HPLC). The greatest activity resided in rp-HPLC fraction #7 (10 ng/ml) which was as effective as 10% FBS at stimulating 3H-thymidine incorporation in chick embryo brain nonneuronal cells. Although other researchers report the isolation of a mitogenic fraction consisting of 5'-GMP from the embryonic chick brain, UV absorbance spectra, rp-HPLC elution profiles, and fast atom bombardment (FAB) mass spectra indicated that CBGF is neither 5'-GMP nor 51-AMP. 2 Moreover, commercially available 5t-GMP was inhibitory to 3H-thymidine incorporation in the chick non-neuronal cells, while Sf-AMP had no effect. Upon treatment with pronase, the biological activity of fraction P6-3 increased; this increase was nearly 30% greater than what would be expected from a simple additive effect of any mitogenic activity of pronase alone together with P6-3 alone. This may suggest the presence of an inhibitor protein. The bioactive component may be a protein protected by a nucleoside/nucleotide or simply a nucleoside/nucleotide acting alone. While the FAB mass spectrum of rp-HPLC fraction #7 did not reveal molecular weight or sequence information, the ion of highest molecular weight was observed at m/z 1610; this is consistent with previous estimations of CBGF's size. 3