We have developed a subtractive cloning procedure based on the hybridization of single-stranded cDNA libraries constructed in ITH3M, a vector containing the phage M13 origin of replication. We have used this strategy to isolate three transcripts whose abundance is increased in scrapieinfected brain. DNA sequence analysis showed that they represent glial fibrillary acidic protein, metallothionein II, and the B chain of a-crystallin; the latter two may represent a response to stress.Scrapie is the best studied example of the subacute spongiform virus encephalopathies (1), a group of slow, transmissible, neurodegenerative diseases characterized by a long latent period, an insidious onset, and a progressive course that in scrapie results in ataxia, wasting, prostration, and death (for reviews, see refs. 1-3). Established human diseases in this class include Creutzfeld-Jakob disease and kuru (1). Scrapie, a disease of sheep and goats, has been passaged in hamsters (4), the source of tissue used in this study.In this work, a subtractive cloning procedure was developed and used to isolate several cDNAs whose transcripts are modulated in scrapie-infected hamster brain. Nine recombinants were sequenced and shown to represent three different genes, encoding glial fibrillary acidic protein (GFAP), metallothionein, and the B chain of a-crystallin.** GFAP (5, 6) and its mRNA (7,8) RNA Preparation. Brains were shattered on dry ice in a sealed plastic bag, suspended in 5 M guanidinium thiocyanate/8% 2-mercaptoethanol/50 mM Hepes/5 mM EDTA, pH 7.5 (5 ml per brain), at 20°C (10), and immediately homogenized by using an STD Tissuemizer with either a 182EN (two brains) or a 100EN (one brain) generator (Tekmar, Cincinnati, OH) at full speed for 10 sec. Four volumes of 6 M LiCl was added and the mixture was kept overnight at 0°C, after which it was either used directly or stored at -70°C before centrifugation at 40,000 x g for 1 hr at 4°C. The pellet was resuspended in 4 M LiCl at 0°C and centrifuged as above. The washed pellet was dissolved in 10 ml of0.5% NaDodSO4/20 mM Hepes/10 mM EDTA, pH 7.5, and incubated with proteinase K (Bethesda Research Laboratories) at 100 ,ug/ml for 1 hr at 37°C. The solution was then extracted twice with phenol/chloroform (1:1, wt/vol) and once with chloroform and the RNA was ethanol-precipitated. mRNA was purified from total RNA by benzoylcellulose chromatography as described by Roberts (11). Benzoylcellulose was the gift of N. Chaudhari and W. Hahn (University of Colorado School of Medicine, Denver).cDNA Synthesis and Library Construction. mRNA (2 ,ug) from two brains was dissolved in 5 ,u1 of HE (10 mM Hepes/1 mM EDTA, pH 7.5) and 15 ,u1 of water, heated to 65°C for 1 min, and cooled on ice. cDNA was synthesized by using Moloney murine leukemia virus reverse transcriptase (Bethesda Research Laboratories) as recommended by the vendor, with random hexanucleotide (Pharmacia) at 0.5 mg/ ml as primer. The second strand was synthesized as described by Okayama and Berg (12). Non-self-complementary...
We have isolated two recombinant cDNAs whose corresponding RNAs have an increased abundance in scrapie-infected hamster brain. DNA sequence analysis has shown that these two recombinants represent the genes for sulfated glycoprotein 2 and transferrin. The abundance of sulfated glycoprotein 2 RNA is increased in hippocampus from patients with Alzheimer disease and Pick disease, whereas transferrin RNA is not strongly modulated in these conditions. Expression of two previously identified scrapie-modulated genes, encoding glial fibrillary acidic protein and metallothionein, is also increased in both of these neurodegenerative diseases.
We have developed a system where double-stranded cDNA can be amplified using a synthetic oligonucleotide primer and the polymerase chain reaction, generating cDNA libraries in vitro. Using a library subtraction strategy (1), scrapie and control brain in vitro cDNA libraries were used to identify sequences whose expression is modulated in scrapie infection. One of these sequences represents beta-2 microglobulin, while the other two have not been previously described. The use of in vitro libraries offers increased speed and efficiency of construction, and their subtraction is more efficient and powerful, compared with the previous system (1).
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