DETECTION OF MESSENGER-RNAS ENCODING PEROXISOMAL PROTEINS BY NONRADIOACTIVE IN-SITU HYBRIDIZATION WITH DIGOXIGENIN-LABELED CRNAS

We have used a non-radioactive in situ hybridization (ISH) protocol fo r the detection of mRNAs encoding proteins localized in peroxisomes. I n this presentation the literature on detection of ''peroxisomal mRNAs '' is reviewed and the results obtained by application of the non-radi oactive method are compared with those obtained by hybridization with radioactive probes. Moreover, the special processing conditions and th e application of the method for the specific visualization of mRNAs co ding for several peroxisomal proteins with different abundance levels and distinct tissue distributions are presented. The combination of th e following technical details in the ISH procedure were found to be es sential for obtaining optimal sensitivity and good histological qualit y of the preparations: (a) perfusion-fixation with a fixative containi ng 4% depolymerized paraformaldehyde/0.05% glutaraldehyde, (b) the use of par affin embedding instead of frozen sections, (c) specific prote inase K-digestion time for each tissue, and (d) the use of digoxigenin -labelled cRNA probes (hydrolyzed to a length of about 200 bases) for detection. By using this technique, we were able to localize several p eroxisome-specific mRNAs with different degrees of abundance: (I) high -level (catalase and urate oxidase) and (2) low-level (all beta-oxidat ion enzymes and the 70-kDa peroxisomal membrane protein) in rat liver and kidney. The specificity of the method was confirmed by the negativ e results obtained with corresponding sense controls and the distinct positive staining patterns obtained for albumin and glyceraldehyde-3-p hosphate dehydrogenase (GAPDH) mRNAs. All transcripts for mRNAs encodi ng peroxisomal proteins were localized to the cytoplasm of hepatocytes , with all nuclei as well as epithelial cells of bile ducts and sinuso idal cells remaining negative. In rat kidney, the catalase transcripts were confined to proximal tubular epithelial cells, which is consiste nt with the high abundance of peroxisomes in this part of the nephron. In contrast, no transcripts for urate oxidase were present in the kid ney, corresponding to the absence of that protein in this organ. The t ranscripts for GAPDH on the other hand were localized in proximal and distal tubular epithelial cells as well as in collecting ducts. The ap plication of this technique to the rat adrenal gland and testis in rec ent unpublished studies have revealed exclusive localization of catala se transcripts to the adrenal cortex and to interstitial cells of Leyd ig, which are known to be rich in microperoxisomes. These observations demonstrate the suitability of this technique for accurate localizati on of mRNAs encoding peroxisomal proteins and for the analysis of alte rations in the expression of the corresponding genes under differ ent experimental conditions.