Production of digoxigenin-labelled RNA probes and the detection of cytokine mRNA in rat spleen and brain by in situ hybridization.

Non-radioactive in situ hybridization is a sensitive method for determining the site of production for secretory molecules such as cytokines. We report here on the central and peripheral induction of proinflammatory cytokines by endotoxin, and outline procedures for the generation and application of rat-specific digoxigenin (Dig)-labelled RNA probes for the localization of mRNA by in situ hybridization. Rats were injected either intravenously (i.v.) or intracerebroventricularly (i.c.v.) with vehicle or lipopolysaccharide (LPS) and sacrificed at various time intervals post-injection. Rats were then perfused with 4% paraformaldehyde and the spleens and brains were removed and cryoprotected in 30% sucrose. Dig-labelled, rat-specific, antisense and sense RNA probes were generated by in vitro transcription from PCR-derived templates. Positive staining with all the antisense probes was cytoplasmic, whereas the sense probes showed no staining. Numerous tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) mRNA positive cells were observed in the marginal zone and in the red pulp of the spleen after iv LPS injections, whereas sections from saline-treated animals showed minimal cytokine mRNA expression. Cells positive for TNF-alpha and IL-1beta mRNA were detectable in the brain after i.c.v. injections of LPS, but not after icv injection of vehicle. An antisense probe for c-fos was utilized in these studies as a positive control for our procedure due to its anatomically specific expression in the rat brain after LPS. In conclusion we have demonstrated that in situ hybridization with Dig-labelled RNA probes is an efficient, sensitive and reliable tool to localize cytokine mRNA production in rat tissue.

Molecular characterization of an NAD-specific glutamate dehydrogenase gene inducible by L-glutamine. Antisense gene pair arrangement with L-glutamine-inducible heat shock 70-like protein gene.

The gene for an NAD-specific glutamate dehydrogenase (NAD-GDH) that is allosterically activated by NADP+ (non-substrate) was cloned, and its physical structure and nucleotide sequence was determined. The gene consists of 9 introns and 10 exons; the 10th and largest exon, which is 1863 nucleotides long, is at the 3'-end of the gene. The shortest exon of 33 base pairs is the first and is located at the 5'-end of the gene. The large exon is in perfect register along the complementary strand with a heat shock 70 (HSP)-like protein gene. The NAD-GDH gene is inducible with L-glutamine, just as the HSP 70-like protein gene (LéJohn, H.B., Cameron, L.E., Yang, B., MacBeath, G., Barker, D.S., and Williams, S.A. (1994) J. Biol. Chem. 269, 4513-4522). The phenomenon of anti-parallel coupling of two genes is named antisense gene pair. By Northern and Western blotting techniques, we obtained indirect evidence that the gene is expressed in vivo. The gene encodes a protein of M(r) 118,740 which consists of 1063 amino acid residues. The 5' and 3' borders of the gene display typical but unproven promoter motifs of CCAAT, TATAAT, and AAATAAAA polyadenylation signal bounded by a pyrimidine-rich transcription termination-type format. Restriction endonuclease site mapping of all the genomic clones isolated that carry most or all of the gene, and of the genome itself, gave hybridization patterns that are consistent with the interpretation that the organism, Achlya klebsiana, has only one form of the gene. 3'-End-labeling of a 5.2-kb XbaI DNA fragment (carrying the antisense gene pair) that was then asymmetrically cleaved to produce two single 3'-end-labeled pieces that were used as probes on L-glutamine-induced cell poly(A)+ RNA, showed that the end-labeled DNA equivalent to the HSP 70-like protein mRNA hybridized to a 3.4-kb transcript and the end-labeled DNA equivalent to the NAD-GDH mRNA hybridized to a 2.4-kb transcript.