Abstract
Rat peritoneal exudate cells produce two interleukin 6 (IL6) messenger RNA species, a major 1200 nucleotide and a 5-fold less abundant, 2400-nucleotide species. A cDNA clone representing the major species was isolated, and sequenced. The 1055-base pair insert covered the 3'-nontranslated region, the 211 triplet coding region and most of the 5'-nontranslated region. The derived rat IL6 amino acid sequence was 93 and 58% identical, respectively, with mature murine and human IL6. Rat IL6 lacks N-glycosylation sites but contains a fifth cysteinyl residue in addition to the 4 residues shared in conserved positions with murine and human IL6. Stable murine L cell and human HeLa-derived cell lines were established by cotransfection with rat IL6 cDNA and a selectable neomycin resistance marker. These lines secrete 9-fold increased amounts of functional IL6 over their respective parental cells. A stable rat macrophage-derived cell line, RM-SV1, was established by transformation with simian virus 40. IL6 and Il1 mRNA levels are inducible 20- and 3.5-fold, respectively, in this line by treatment with lipopolysaccharide with kinetics characteristic of macrophages. A set of three overlapping genomic DNA clones was isolated and a 10-kilobase DNA segment was sequenced containing the rat IL6 gene plus 2.9 kilobases of 5'-flanking and 1.3 kilobases of 3'-flanking sequences. The two transcription start sites used in RM-SV1 cells were mapped within 5 base pairs of each other. The exon/intron boundaries are conserved with the murine and human IL6 genes. The two IL6 mRNA species are generated by alternative polyadenylation at sites separated by a distance of 1.2 kilobases. The intervening region contains a repetitive element 72-80% identical with the rat and murine consensus L1 family sequences.
Highlights
20- and 3.5-fold, respectivelyin, this line by treatmentIL6 is produced by manydifferent cell types including with lipopolysaccharidewith kinetics characteristicof fibroblasts, macrophages/monocytes, T and B lymphocytes, macrophages
We describe the establishment of a rat macrophage-derived cell line that was used as a convenientsource of mRNA and as a tool for studies of the expression of this gene in macrophage-like cells
The exon/intron block structure of the gene has been det.ermined, and we report the presence of two IL6 mRNA species derived from the single copy rat IL6 gene by alternative lpolyadenylation
Summary
Animals and Materials-Male Fisher 344 rats (Simonsen Laboratories, Gilroy, CA) were used for the preparationof primary peritoneal exudate cell cultures and RNA preparation.Laboratory chemicals and standard enzymes for molecular biology were from J. The final T7 polymerase product was dried and redissolved in 50 pl of H20.For the annealing reaction, 12 pg of total RNA (extracted from the LPS-stimulated macrophage-like cell line RM-SV1, Results) were combined with 1 p1 of the T7 polymerase product and the RNA was denatured in a 30-p1 volume containing 80%formamide for 10 min at 75 “C. The reaction mixture was incubated for 25 min a t 37 “C,and the RNase-resistant RNA products were extracted once with phenol, precipitated and washed with ethanol, and lyophilized to dryness. They were resuspended in 6 pl of formamide sample buffer Southern Blot Analysis of Genomic DNA-The analysis was performed as described (39)
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