The identity of interferon-β 1 mRNA transcripts in human fibroblasts and namalva cells

Abstract

1. Introduction Depending on the mode of induction and the cell- type, human cells are able to produce interferons, which are categorized in 3 different groups: inter- feron (IFN)-o, Q and 7 [ 11. Studies at the DNA and protein level provide evidence that the o-group is not a homogenous population of molecules, but consists of several species, differing in gene sequence and in the physicochemical properties of the protein [2-41. [r(I):r(C)] (50 pg/ml) and cycloheximide (50 pg/ml) for 4 h [ 111. The induced cells were supplied by Rentschler Inc. (Laupheim). Lymphoblastoid Namalva cells (5 X 10” cells/ml) were induced by 40 HA-units/ ml of Sendai virus for 5 h. Cells were washed and harvested. For 1FN-o there exist 212 different genes, and 8 different gene-products have been identified [4]. In the case of IFN_P only 1 gene-product has been char- acterized [5-71, but under some conditions human fibroblasts can synthesize >l species of IFNQ mRNA [8,9]. A Namalva (lymphoblastoid) cell line produces, upon induction with Sendai virus, both o- and P-types of interferon. Namalva IFN$ mRNA consists of 2 size-classes; 1.9 kilobase (kb) and 1 .l kb, both absent from fibroblasts [lo]. This raises the question as to whether or not other IFN_P genes are turned on in Namalva cells, or if transcription is initiated and/or terminated at novel sites. We have used a cloned genomic DNA fragment containing the human IFNQr gene, as a probe for S 1 -mapping of the IFNj3 mRNA species produced in Sendaiinduced Namalva cells. We find that this Namalva IFN$ mRNA is indistin- guishable from the main species found in poly [r(I): r(C)]-induced fibroblasts, with respect to molecular size, and the starting and termination points of trans- cription. The genomic clone pCosIFN& has been described [7]. DNA fragment EcoRI-F containing the whole IFN& gene has been subcloned in pBR325. This subclone has been used for S 1 -mapping. 3’-End label- ling was done with ‘Klenow’ polymerase by ‘filling in’ [ 121. 5’-End labelling was done with T4 DNA kinase [ 141. Hybridisation and S 1 -mapping were performed as in [13]. DNA-sequencing was performed according to [ 141. 3. Results Based on size determinations in a denaturing 2% agarose gel the size of IFNQr mRNA seemed identical (? 50 nucleotides) for poly [r(I):r(C)]-induced tibro- blasts and Sendai virus-induced Namalva cells (fig.1). To substantiate these findings we carried out S 1 -map- ping experiments. As a hybridisation probe we used the 1.9 kb EcoRI-F fragment of pCosIFNQr, described in [7]. This DNA fragment contains the entire IFNQr gene as well as the flanking sequences at the 3’- and the 5’-end. The strategy used to determine the size of IFNQr mRNA in poly [r(I):r(C)]-induced human fibroblasts (FS4) and Sendaiinduced lymphoblastoid Namalva cells is shown in fig.2. 2. Materials and methods Interferon mRNA synthesis in human fibroblasts (FS4 cells) was induced in the presence of poly The size of the protected fragments is identical in poly [r(I):r(C)] -induced FS4 and in Sendai-induced Namalva cells (fig.3). The size of IFNQr mRNA with- out the poly(A)-tail is 840 nucleotides. The 5’-end