Expression of cloned newcastle disease virus genes in E. coli and mammalian cells

31 EXPRESSION OF TEE P GENE OF RS VIRUS IN HAMKALIAN CELLS. H.N. Baybutt, D. Flint, J. Coveney and C.R. Prinale. Department of Biological Sciences, University of Warwick, Coventry, CV4 i'AL,UK. The fusion (F) protein gene of the RSS-2 strain (subgroup A) of human respiratory syncytial virus was transferred into the pMSG inducible expression vector and amplified using the shuttle vector properties of pMSG. The xanthine-guaninephosphoribosyltransferasegene of E.coli present in this vector was used to obtain stably transformed Wish cells by selection for growth in HAT medium. Insertion of the F gene adjacent to the Mouse Mammary Tumour Virus LTR present in this construct was obtained in both orientations. Dexamethasone induced expression of the F protein, monitored by immunodot blot assay, was obtained only from clones with inserts in the correct orientation. The kinetics of dexamethasone regulated expression of the F protein gene in Wish cells has been investigatedby Western Blotting and FACS analysis. This expression system in conjunction with in vitro mutagenesis is being used to analyse F protein function.

32 EXPRESSION OF CLONED NEWCASTLE DISEASE VIRUS GENES IN E_ CQJ~ AN5 MAMMALIANCELLS NEIL S. MILLAR, EMMERSON

I.

BARRY VIPOND,

MICHAEL STEWARD AND PETER T.

Department of Biochemistry, University Newcastle upon Tyne, NE2 4HH, UK

of Newcastle

upon

Tyne,

We have previously constructed cDNA libraries to the Beaudette C and Ulster 2C strains of Newcastle disease virus (NDV), and determined the nucleotide sequence of the HN and F genes of both strains and the M and L genes of Beaudette C. Cl ones spanning the entire coding regions have been assembled by ligation of fragments from smaller, overlapping cDNA clones. After removal of 5’ non-coding regions by either Ba131 or site directed deletion, such full length genes have been inserted downstream of strong promoters in a range of plasmid expression vectors and their expression in prokaryotic and eukaryotic cells is presently being investigated. The effects of site directed deletion and site specific mutagenesis on expression and processing of the gene products will also be investigated.

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