Baculovirus superinfection: a probable restriction factor on the surface display of proteins for library screening

In addition to the expression of recombinant proteins, baculoviruses have been developed as a platform for the display of complex eukaryotic proteins on the surface of virus particles or infected insect cells. Surface display has been used extensively for antigen presentation and targeted gene delivery but is also a candidate for the display of protein libraries for molecular screening. However, although baculovirus gene libraries can be efficiently expressed and displayed on the surface of insect cells, target gene selection is inefficient probably due to super-infection which gives rise to cells expressing more than one protein. In this report baculovirus superinfection of Sf9 cells has been investigated by the use of two recombinant multiple nucleopolyhedrovirus carrying green or red fluorescent proteins under the control of both early and late promoters (vAcBacGFP and vAcBacDsRed). The reporter gene expression was detected 8 hours after the infection of vAcBacGFP and cells in early and late phases of infection could be distinguished by the fluorescence intensity of the expressed protein. Simultaneous infection with vAcBacGFP and vAcBacDsRed viruses each at 0.5 MOI resulted in 80% of infected cells coexpressing the two fluorescent proteins at 48 hours post infection (hpi), and subsequent infection with the two viruses resulted in similar co-infection rate. Most Sf9 cells were re-infectable within the first several hours post infection, but the reinfection rate then decreased to a very low level by 16 hpi. Our data demonstrate that Sf9 cells were easily super-infectable during baculovirus infection, and super-infection could occur simultaneously at the time of the primary infection or subsequently during secondary infection by progeny viruses. The efficiency of super-infection may explain the difficulties of baculovirus display library screening but would benefit the production of complex proteins requiring co-expression of multiple polypeptides.

Boots Book-lovers' Library and the novel: The impact of a circulating library market on twentieth-century fiction

In a Report for the Society of Bookmen in 1928, British publishers estimated that between a quarter to two thirds of all the books they published went to four circulating libraries: Boots, Smith’s, Mudie’s, and The Times bookclub. This essay examines the literary impact of one of the largest of these, Boots Book-lovers’ Library (1899-66), which by 1935 had around 400 libraries attached to their high-street pharmacies catering for the tastes of over one million subscribers a year. Compared to the wealth of studies examining the influence of the library market in the Victorian period, the significance of the subscription libraries as key distributors of fiction in the twentieth century is not well known. But private libraries expanded rapidly in the early twentieth century to cater for what Sidney Dark termed a ‘new reading public’, and records in publishers’ archives indicate that authors routinely adapted their unpublished manuscripts in order to meet the perceived demands of this library reader. This article examines the impact of the Boots Book-lovers’ Library market on authors’ practices of writing and revision, and on literary marketing and censorship. It focuses in particular on the author James Hanley (1897-1985), using unpublished correspondence in the Chatto & Windus archive at the University of Reading to demonstrate how the publisher’s sense of the tastes and expectations of the Boots library reader influenced the editorial process.

Construction of a Festuca pratensis BAC library for map-based cloning in Festulolium substitution lines

Introgression in Festulolium is a potentially powerful tool to isolate genes for a large number of traits which differ between Festuca pratensis Huds. and Lolium perenne L. Not only are hybrids between the two species fertile, but the two genomes can be distinguished by genomic in situ hybridisation and a high frequency of recombination occurs between homoeologous chromosomes and chromosome segments. By a programme of introgression and a series of backcrosses, L. perenne lines have been produced which contain small F. pratensis substitutions. This material is a rich source of polymorphic markers targeted towards any trait carried on the F. pratensis substitution not observed in the L. perenne background. We describe here the construction of an F. pratensis BAC library, which establishes the basis of a map-based cloning strategy in L. perenne. The library contains 49,152 clones, with an average insert size of 112 kbp, providing coverage of 2.5 haploid genome equivalents. We have screened the library for eight amplified fragment length polymorphism (AFLP) derived markers known to be linked to an F. pratensis gene introgressed into L. perenne and conferring a staygreen phenotype as a consequence of a mutation in primary chlorophyll catabolism. While for four of the markers it was possible to identify bacterial artificial chromosome (BAC) clones, the other four AFLPs were too repetitive to enable reliable identification of locus-specific BACs. Moreover, when the four BACs were partially sequenced, no obvious coding regions could be identified. This contrasted to BACs identified using cDNA sequences, when multiple genes were identified on the same BAC.

A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2

We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST) markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus.

A genealogy of the kings of England in papal Avignon: British Library, Egerton MS. 1500

This is one of three articles in eBLJ (2013) dedicated to the "Abreujamen de las estorias", British Library Egerton MS 1500. The other two articles are: Federico Botana,' The Making of "L'Abreujamen de las estorias": British Library Egerton MS 1500', eblj (2013), article 16. Alexander Ibarz, 'The Provenance of the "Abreujamens de las estorias" (London, British Library, Egerton MS. 1500) and the Identification of Scribal Hands (c. 1323)' eblj (2013), article 17. Leglu's article, eblj (2013), article 18, examines the various political agendas that emerge in the depiction of the kings of Britain and England, from Brutus to Edward II.