The pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2,5-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and ␤ 2 -microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2,5-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.

Prion-induced chronic neurodegeneration has a substantial inflammatory component, and the activation of glia cells may play an important role in disease development and progression. However, the functional contribution of cytokines to the development of the gliosis in vivo was never systematically studied.

An efficient purification protocol for infectivity causing a transmissible spongiform encephalopathy (TSE) is described. From fractions purified by this protocol about 3 × 108 LD50 but only 3 ng of nucleic acids per gram of brain material can be isolated from all TSE-affected brains (hamster, human, sheep, cattle). By PAGE such fractions from brains of infected and control hamsters contained only one distinct nucleic acid band of 1.5 kb together with some broader smear of nucleic acid material. Although distilled water was used for such purifications, quite often a similar nucleic acid band was isolated from blanks containing no brain material. In all instances this material proved to be DNA. The result challenges the potentially important claim that purified infectious preparations of TSE-specific amyloid are free of nucleic acids of viral size. Nucleic acids isolated by other groups from diseased brain were not detected in preparations isolated by the new protocol. The application of this purification protocol in future studies will be helpful to decide whether TSEs are caused by agents containing nucleic acid or by protein only.

A method for the partial purification of scrapie infectivity from hamster brain is described. About a 100–1000‐fold, 20‐fold, and 200‐fold enrichment in scrapie infectivity with respect to protein, RNA, and DNA content has been achieved using differential centrifugation, enzyme and detergent treatment. The inbred CLAC strain of hamsters used in our experiments contained about 10 times less infectivity in brain than has been found in randomly bred animals or other inbred strains.

DNA methylase has been purified 660-fold from nuclei from regenerating rat liver. The enzyme is able to methylate single stranded (ss) and double stranded (ds) DNA, the only reaction product being 5-methylcytosine. Previously unmethylated double stranded DNA from prokaryotes (M.luteus) as well as from eukaryotes (Ascaris suis) can serve as substrates. The synthetic copolymers (dG-dC)n . (dC-dG)n and (dG,dC)n are also methylated. While SV40 DNA is almost not methylated, PM2 DNA is a good substrate even in the supercoiled form. The enzyme methylates 1 in 17 bases in heterologous M.luteus DNA, but only 1 in 590 in homologous rat liver DNA. The high methylation level of M.luteus DNA, an analysis of the methylated pyrimidine isostichs and a preliminary dinucleotide analysis suggest that all the CpGs in a DNA can be methylated.

Methylation of cytosine in the DNA inhibits the transcription by RNA polymerase II in higher eukaryotes, but has no infhtence on RNA polymerase I transcription. The effect on RNA polymerase III was unknown, so far. Two polymerase III genes: a type 1 5s rRNA gene and a type 2 tRNA gene were methylated in vitro with a purified eukaryotic DNA methyltransferase (EC2.1.1.37) and their transcription was analyzed in Xenopus oocytes. The 5s rRNA gene, an oocyte 5s rRNA gene from X. luevis which is subject to developmental inactivation, was not alfected by methylation. Conversely, transcription of the tRNA gene was 8m inhibited by methylation with the eukaryotic methyltransferase. H/au1 and HpaII methylation left its transcription unaffected.

We identi¢ed cDNAs coding for homologues to tetrapod prion proteins (PrPs) in Atlantic salmon (Salmo salar) and Japanese pu¡er¢sh (Fugu rubripes), which were termed 'similar to PrPs' (stPrPs). Besides signi¢cant sequence homologies the ¢sh stPrPs display characteristic structural features in common with tetrapod PrPs. In addition, two stPrPs were shown to be highly expressed in brain tissue. None of the so far identi¢ed PrP-homologues of ¢sh resembles doppel. Hence, the duplication of the PrP gene, which generated doppel, may have occurred not in ¢sh but later in the tetrapod lineage. The identi¢cation of ¢sh PrPs provides a basis to address concerns about a possible susceptibility of ¢sh to prion infections.

The biological importance of DNA methylation for gene expression in eukaryotes is becoming increasingly evident, and a direct role of methylation in gene expression has been suggested by an analysis of the infectivity of integrated retroviral genomes in a transfection assay. These studies, however, did not address whether specific methylatable residues are involved in gene regulation. Methylation by sequence-specific bacterial DNA methylases has been shown to suppress the expression of some genes, but not others. To investigate the effect of methylation on gene expression without having to rely on sequence-specific methylases, a rat liver enzyme was used to methylate in vitro all C-G dinucleotides of a proviral genomic clone. This treatment reduced the biological activity of Moloney murine leukaemia virus (M-MuLV) proviral DNA by more than three orders of magnitude, whereas complete methylation of 35 HpaII sites in the same DNA had only a marginal effect. The rat methylase-induced inactivation was reversible, as treatment of recipient cells with 5-azacytidine rendered the non-infectious viral genomes biologically active. This suggests that methylation in other C-G dinucleotides than those detectable with restriction enzymes can be crucial for gene expression.