We have developed a system for the enzymatic in vitro synthesis of non-radioactively labeled RNA which is derivatized with the hapten digoxigenin (DIG). The labeling reaction as well as the conditions for hybridization and detection of hybrids by an antibody-conjugate and a coupled colour reaction were analyzed and adapted for high sensitivity and low background. In addition, data on the performance and sensitivity of digoxigenin-labeled RNA probes in Southern and Northern blots are presented.

FAS1, the structural gene of the pentafunctional fatty acid synthetase subunit beta in Saccharomyces cerevisiae has been sequenced. Its reading frame represents an intron-free nucleotide sequence of 5,535 base pairs, corresponding to a protein of 1,845 amino acids with a molecular weight of 205,130 daltons. In addition to the coding sequence, 1,468 base pairs of its 5'-flanking region were determined. S1 nuclease mapping revealed two transcriptional initiation sites; 5 and 36 base pairs upstream of the translational start codon. Within the flanking sequences two TATATAAA boxes, several A-rich and T-rich blocks and a TAG...TATGTT...TATGTT...TTT sequence were found and are discussed as transcriptional initiation and termination signals, respectively. The order of catalytic domains in the cluster gene was established by complementation of defined fas1 mutants with overlapping FAS1 subclones. Acetyl transferase (amino acids 1-468) is located proximal to the N-terminus of subunit beta, followed by the enoyl reductase (amino acids 480-858), the dehydratase (amino acids 1,134-1,615) and the malonyl/palmityl transferase (amino acids 1,616-1,845) domains. One major inter-domain region of about 276 amino acids with so far unknown function was found between the enoyl reductase and dehydratase domains. The substrate-binding serine residues of acetyl, malonyl and palmityl transferases were identified within the corresponding domains. Significant sequence homologies exist between the acyl transferase active sites of yeast and animal fatty acid synthetases. Similarly, a putative sequence of the enoyl reductase active site was identified.

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A novel highly sensitive non-radioactive DNA labeling and detection system based on the ELISA principle has been developed. DNA is modified with the cardenolide-hapten digoxigenin by enzymatic incorporation of digoxigenin-labeled deoxyuridine-triphosphate with Klenow enzyme. Digoxigenin is linked to dUTP via an 11-atom linear spacer (Dig-[ll]-dUTP). Following hybridization of membrane-bound target-DN A with a digoxigenin-labeled probe, the hybrids are detected by an ELISA reaction using digoxigenin-specific antibodies covalently coupled to the marker enzyme alkaline phosphatase [(Dig):CLAP].This binding of antibody: marker enzyme-conjugate is followed by an enzyme-catalysed coupled redox reaction with the colour substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium salt (NBT) giving rise to a deepblue coloured, water-insoluble precipitate directly adhering to the membrane.The digoxigenin system allows the detection of 0.1 pg homologous DNA within 16 h in dot-and Southernblots on nitrocellulose or nylon membranes avoiding any significant background even after a prolonged period of color development. Due to its high sensitivity and specificity, the new system is appropriate for detection of single-copy genes in genomic blots as well as for Northern, slot, colony, plaque and in situ hybridizations. intestine alkaline phosphatase; Dig, digoxigenin; Dig-[ll]-dUTP, digoxigenin-(9-succinyl-e-aminocaproyl[5-(3-aminoallyl)-2'-deoxyuridine-5'-triphosphate], Na4 (see Fig. 2); (Dig), digoxigenin-specific sheep polyclonal antibody (Fab-fragments); EDTA, ethylenediamine tetraacetic acid, disodium salt; ELISA, enzyme-linked immuno-sorbent assay; ß-Gal, ß-galactosidase from E. coli; Klenow enzyme, large fragment of E. coli DNA polymerase I; NBT, nitroblue tetrazolium salt (2,2'-di-p-nitrophenyl-5,5'-diphenyI-3,3'-dimethoxy-4,4'-diphenylene]ditetrazolium chloride); POD, peroxidase from horse-radish; SDS, sodium dodecyl sulphate (lauryl sulphate, sodium salt); SSC, standard saline citrate (0.15M NaCl, 0.015M Na citrate, pH 7.0);Tris, tris[hydroxymethyl]aminomethane. Zusammenfassung: Ein neues hochsensitives, nichtradioaktives DNA Markierungs-und Detektionssystem, das auf dem ELISA-Prinzip basiert, wurde entwickelt. DNA wird mit dem Cardenolid-Hapten Digoxigenin über enzymatischen Einbau von Digoxigenin-markierten Desoxyuridintriphosphaten mit Hilfe von Klenow-Enzym modifiziert. Digoxigenin ist mit dUTP über einen 11 Atome langen linearen Spacer verbunden (Dig-[ll]-dUTP). Im Anschluß an die Hybridisierung zwischen Membran-gebundener nachzuweisender DNA und einer Digoxigenin-markierten Sonde werden die Hybride über eine ELISA-Reaktion mit Hilfe von solchen Digoxigenin-spezifischen Antikörpern nachgewiesen, an die das Markierungsenzym Alkalische Phosphatase kovalent gebunden ist [