Selecting a promoter for driving transgene expression is one of the most important factors to consider in a transformation project. Information about the native regulation of the promoter activity is important, but it is also necessary to consider how that activity will be affected when integrated into the genome of the transformed plants. Study of a promoter performance in individually transformed lines provides useful information in this area. The maize ubiquitin 1 (Ubi-1) promoter has been widely used to drive constitutive transgene expression in monocotyledonous plants. However, lack of data on its activity in individual transformed wheat lines constitutes a gap in the understanding and predictability of this promoter's performance. In this paper, we began addressing this problem by examining the expression of the marker gene uidA, coding for P-glucuronidase (GUS), under the control of the maize Ubi-1 promoter in individual transgenic wheat (Eiticum aestivum L.) lines from different wheat varieties. The expression of uidA driven by this promoter depended to a great extent on the specific transformation event. Whilst expression was strong and constitutive in all tissues in some of the lines analysed, there were also transgenic lines in which GUS activity was restricted to only a few tissues. In general the maize Ubi-1 promoter had strong activity in young, metabolically active tissues and in pollen grains.
The promoterless maize ubiquitin first exon and intron fragment can drive gusA expression in immature tritordeum inflorescences and immature wheat scutella. In fluorescence assays, this fragment induces gusA expression in tritordeum inflorescences to 50 times higher than background. The activity of the complete promoter, exon and intron cassette was up to 20000-fold higher than background but the maize ubiquitin promoter in isolation had very low activity. A construct with the maize alcohol dehydrogenase first exon and intron had low activity, visible in histochemical assays. Both intron sequences have promoter-like features and in the ubiquitin intron there is a sequence homologous to the opaque-2-binding box. We suggest that the combination of these elements may explain the promoter activity detected in these introns.
Drosophila Schneider 2 (S2) cells have been available for approximately 40 years. Their use has intensified over the past 15 years: resolution of the whole Drosophila melanogaster genome and the amenability of S2 cells for siRNA-based studies are some of the reasons for their growing use. This review covers recent publications on use of S2 cells for research and manufacturing and points to some possible future developments in their use in the vaccine field. Relatively few groups have systematically developed the system to enable expression of challenging proteins. They demonstrated that these cells can constitute a robust, efficient protein expression system, with specific advantages such as homogeneous glycosylation profile; reproducibility between production runs; options for cultivation modes, including perfusion; and no cell lysis, leading to relatively low levels of contaminating host cell proteins. The platform has shown to be particularly well adapted for the production of challenging viral, malaria and immunotherapy antigens.
Pea enation mosaic virus "PEMV# is a bipartite virus\ transmitted mechanically and by aphids in the circulative manner[ Virus capsid proteins\ coded by RNA!0 are involved in aphid transmissibility[ Using an antiserum against a European "UK# aphid!transmissible isolate\ the capsid proteins of six PEMV isolates\ each aphid!trans! missible or non!transmissible\ were compared using Western blots[ All isolates have a major coat protein of approximately 11 kDa\ but also additional proteins[ RNA!0 complementary DNAs corresponding to ORFs 3 and 4 of isolate SP were cloned and sequenced[ This had 84) homology at the nucleotide level and a 85) homology at the amino acid level with the sequence of a North American isolate[ Most amino acid di}erences were in the region corresponding to ORF 4\ some of which could result in potential changes in the con! formation of the protein[ This is the _rst report on the sequence of an European PEMV isolate and the _rst direct comparison of viral capsid complexity in di}erent PEMV isolates[ Zusammenfassung Vergleich von Isolaten des Erbsenenationenmosaikvirus "PEMV#] Sequenz der Hu Ãllproteingene und der fu Ãr die Blattlausu Ãber! tragungsproteine codierenden Gene eines europa Ãischen Isolates Das Erbsenenationenmosaikvirus "PEMV# ist ein zweige! teiltes Virus\ das mechanisch und durch Blattla Ãuse zirku! lativ u à bertragen wird[ Von RNA!0 codierte Hu à llproteine des Virus spielen fu à r die Blattlausu à bertragbarkeit eine Rolle[ Mit einem Antiserum gegen ein blattlausu à bertrag! bares Isolate aus dem Vereinigten Ko à nigreich wurden die Hu à llproteine von sechs blattlausu à bertragbaren bzw[ nicht u à bertragbaren PEMV!Isolaten in Western blots vergli! U[ S[ Copyright Clearance Center Code Statement] 9820Ð0674:88:3697Ð9280 , 03[99:9 chen[ Alle Isolate besitzen ein gro)es Hu à llprotein von ca[ 11 kDa\ aber au)erdem weitere Proteine[ RNA!0 cDNAs\ die mit den ORFs 3 und 4 des Isolats SP kor! responierten\ wurden kloniert und sequenziert[ Auf Nukleotidebene wurde eine Homologie von 84 ) mit einem nordamerkanischen Isolat festgestellt\ auf Amino! sa Ãureebene eine Homologie von 85 )[ Die meisten Unterschiede bei den Aminosa Ãuren fanden sich in der ORF 4 entsprechenden Region\ einige von ihnen sind mo à glicherweise fu à r eine Konformationsa Ãnderung des Proteins verantwortlich[ Dies ist der erste Bericht u à ber die Sequenz eines europa Ãischen PEMV!Isolats und der erste direkte Vergleich der Komplexita Ãt von Virushu à llen verschiedener PEMV!Isolate[
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