Adenovirus (Ad) contains linear double-stranded DNA with a molecular weight of 20-30 X 10 6 daltons (8). Its DNA molecule covalently binds to the terminal proteins at the 5' termini (1,9,(11)(12)(13). The methods of purification of Ad DNA have been well established (2, 6, 10), but since these methods require virion purification, they are not so convenient for preparation of DNA from many different samples or small scale preparation of non-labeled DNA. Horwitz (1976) extracted Ad DNA from many different samples of infected cells by the modified Hirt method (3) and purified the DNA by sucrose gradient centrifugation.In this paper, we describe a rapid and simple method of purifying Ad DNA directly from infected cells without ultracentrifugation. This is based on the unique nature of Ad DNA that its 5' termini are linked covalently to the terminal proteins, so that the viral DNA-terminal protein complex extracted from infected cells by the Hirt method (3) is simply separated from RNA and fragmented cell DNA by phenol extraction, and the viral DNA is isolated from the interphase of the phenol extraction by ethanol precipitation and protease digestion followed by phenol extraction. We also applied this method for the preparation of poliovirus RNA, in which the 5' terminal is linked to a protein (16) from infected cells.Tront strain of canine Ad type 2(CAd2) obtained from Dr. S. Konishi, Tokyo University, was grown on a monolayer culture of canine kidney cell line MDCK maintained in Eagle's minimum essential medium supplemented with I % calf serum.The following procedure was designed to obtain 5 to 10 flg of viral DNA which were sufficient for a few trials concerning restriction endonuclease analysis. The cell cultures were prepared in 55 X 105 mm bottles with a culture area of 38 cm 2. The cells were infected with CAd2 at 5 to 10 TCID so per cell. When most cells showed cytopathic effects (usually 35 to 40 hr after infection), the culture medium was removed, and the cells were lysed with 2 ml of 1% SDS, 10 mM Tris-HCl and 10 mM EDTA, pH 7.5, at room temperature for 10 min. The viscous lysate was transferred to a plastic centrifuge tube and incubated at 37 C for 5 min during which the tube was gently inverted 20 times. Most of cellular DNA was removed 817

SUMMARYWe describe a method of sample preparation to detect scrapie-associated fibril (SAF) proteins in small amounts of scrapie-infected mouse tissues by Western blot analysis using an antiserum to a synthetic peptide that corresponds to the N-terminal region of hamster prion protein. SAF proteins were efficiently detected in brain tissue by this procedure. The proteins were also detected in preparations from spleen and lymph node. SAF proteins were detected in brain samples at 24 weeks after intraperitoneal infection. Using spleen samples, the proteins were detected from mice in the preclinical stage (from 4 weeks after infection), clinical symptoms of scrapie were observed in some mice from 22 weeks after infection.

Isolation of citrate-utilizing variants of Escherichia coli has been demonstrated (5), but no genetic study of citrate-utilizing ability (Cit+) has been reported. This paper deals with isolation of Cit+ E. coli strains from pigeons, pigs and cattle, and the instability and transferability of citrate-utilizing ability in the strains, indicating that the ability is controlled by a plasmid.Thirteen of 36 strains of E. coli isolated from cloacal swabs of 22 pigeons in a pigeonry in Sapporo, Hokkaido, grew on Simmons' citrate agar (Eiken) within 1 to 3 days. Twelve of 33 E. coli strains obtained from 33 composite fecal samples of 33 pens in a piggery in the same district utilized citrate. In addition, one of 24 E. coli strains isolated from feces of seven cattle on a farm in Hokkaido was citrate-positive. These three kinds of animals were kept in epidemiologically-unrelated places. In basic biochemical reactions except citrate utilization, the Cit+ strains could not be differentiated from the Cit-strains and a typical E. coli strain (ATCC11775). The Cit+ strains were resistant to three to six antimicrobial agents including chloramphenicol (CM), and carried thermosensitive R plasmids (3). A genetic study of the R plasmids of the representative strains indicated that the R plasmids showed no fertility inhibition and no restriction against phage lambda, and were classified into incompatibility group H (4).Three Cit+ E. coli strains [HT58 from pigeons, carrying R plasmid conferring resistance to tetracycline (TC), CM, streptomycin (SM) and sulfadimethoxine (SA); KE10 from pigs, carrying R plasmid conferring resistance to TC, CM, SM, SA and kanamycin (KM) ; and C53 from cows, carrying R plasmid conferring resistance to TC, CM, SM and SA] were used in this genetic study. Two methionine-requiring F-derivatives [ML1410 resistant to nalidixic acid (NA) and ML1410RFP resistant to rifampicin (RIF)] of E. coli K-12 strain were employed as recipients. In this paper, only the data of the HT58 strain are tabulated.The effect of passaging Cit+ strains in penassay broth (Difco) at 25 C, 37 C and 42 C was investigated. After each passage, the cultures were streaked onto desoxycholate-hydrogen sulfide-lactose agar plates (DHL, Eiken), and more than 100 colonies grown on the plates were impressed on Simmons' citrate agar plates contain-357

The nucleotide sequences of insertion sequences IS3411L (left) and IS3411R (right), present as direct terminal repeats in the citrate utilization of citrate utilization transposon Tn3411, and of IS3411 (generated by intramolecular recombination between IS3411L and IS3411R) were determined. The three IS3411 elements (IS3411R, IS3411L, and IS3411) were 1,309 base pairs long and identical in DNA sequence. IS3411 had 27-base-pair terminal inverted repeats with three bases mismatched and one long open reading frame (240 amino acids) that was proposed to be a transposase.