Two interferon mRNAs in human fibroblasts: in vitro translation and Escherichia coli cloning studies.

Abstract

Two mRNA species that produce biologically active interferon were isolated from human fibroblasts and studied by size fractionation and cloning in Escherichia coil plasmid pBR322. The major fibroblast interferon (Hu IFN-B1) is coded for by the smaller of the two mRNAs, an 11S species, 900 nucleotides long, which in cell-free systems yields a 20,000 M, protein. The second interferon mRNA species (Hu is 14S, about 1300 nucleotides long, and codes for another protein of 23,000-26,000 Mr. The two interferon mRNAs do not cross-hybridize. Both are induced by poly(rIrC), but IFN-,2 mRNA is induced to about 10% in cells by cycloheximide treatment alone whereas under these conditions IFN-P1 is not induced.The major interferon produced by human fibroblasts was recently purified and its amino-terminal amino acid sequence determined by Knight et al. (1). An interferon mRNA 12S fraction from human fibroblasts was cloned in Escherichia coli, and the recombinant DNA nucleotide sequence of the resulting clones was shown to contain the codons corresponding to the structure determined by protein sequencing (2, 3). During the course of similar studies, we observed that human fibroblasts actually contain two interferon mRNAs. As shown here, these two RNAs differ in their size and in their translation products; studies of cDNA recombinant clones in E. coli show that the two mRNAs do not readily cross-hybridize. Sehgal and Sagar (4) have also recently succeeded in separating the two interferon mRNAs from human fibroblasts by mercury-agarose gel electrophoresis.MATERIALS AND METHODS mRNA Preparation and Translation. Poly(rI-rC) superinduction of human FS11 fibroblasts, preparation of poly(A)+ mRNA, sucrose gradient purification, and mRNA translation in micrococcal nuclease-treated rabbit reticulocyte lysates were described in detail (5). Antibodies to human fibroblast interferon were produced and used for immunoprecipitation of the [35S]methionine-labeled translation products with staphylococcal protein A-Sepharose, as before (5). Interferon mRNA injection into Xenopus laevis oocytes was carried out according to Raj and Pitha (6) Assay of Interferon by (2'-5')Oligo(A) Synthetase E. Translation products from reticulocyte lysates or from the medium of mRNA-injected oocytes were diluted 1:10 to 1:15 and 0.1 ml was added to a 96-well microtiter plate. After 18 hr at 37°C, cells were lysed with Nonidet P40, and the lysates were adsorbed on poly(rI-rC)-agarose beads, which were then incubated with a-[32P]ATP (0.3 Ci/mmol; 2.5 mM; 1 Ci = 3.7 X 1010 becquerels) as detailed (7). After 20 hr at 30°C, the supernatant was digested by bacterial alkaline phosphatase and the amount of (2'-5')ApA formed was analyzed by paper electrophoresis at pH 3.5.Cloning of cDNAs. Procedures established by Rougeon (8) were used. cDNA was prepared from sucrose gradient RNA fractions (4 ,ug) with reverse transcriptase (RNA-dependent DNA polymerase) from avian myeloblastosis virus (J. Beard), (dT)12_18, and 4 mM pyrophosphate (9). Double-stranded cDNA was ...

Mechanisms of differential regulation of interleukin‐6 mRNA accumulation by tumor necrosis factor alpha and lymphotoxin during monocytic differentiation

“…Signal transduction pathways controlling IL-6 gene expression involve activation of protein kinase C as well as cAMP [1]. A tissue specific preferential utilization of distinct initiation sites suggests distinct regulatory mechanisms controlling IL-6 gene expression in different cell species [4,10,11]. Mechanisms that account for differential regulation of the IL-6 gene during ontogeny of a distinct cell type are, however, still unknown.…”

Induction and regulation of the 26-kDa protein in the absence of synthesis of beta-interferon mRNA in human cells

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“…poly(C)-induced and also cycloheximidetreated [6] human fibroblasts an mRNA that is translated in a reticulocyte cell-free system into a 23 -26-kDa protein. This protein can be precipitated with a partially purified antiserum directed against partially purified HuIFN-P. After cloning the corresponding cDNA [6] they could purify this mRNA on its cloned cDNA and translate it into a 23 -26-kDa-protein with antiviral and (2'-5')(A),, synthetase-inducing activities. This protein was named IFN-P, and is probably distinct [7] from the IFN-P, described by Sehgal et al [8,9].…”

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Tumor necrosis factor inhibits collagen and fibronectin synthesis in human dermal fibroblasts

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