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An aggregate present in cell-free extracts of Drosophila melanogaster tissue culture cells, sedimenting at 20 to 30S, contains hsps 23, 26 and 27. Hsp 23 was purified from this aggregate and a monospecific antibody was raised against it. Immunofluorescence microscopy showed the presence of hsp 23 preferentially in nuclei after heat shock, while on return to 25 degrees C, hsp 23 was reduced in nuclei and increased in the cytoplasm. Thus the immunofluorescence observations reported here unambiguously confirm for hsp 23 earlier reports that heat shock proteins are mainly found in nuclei after heat shock and that upon return to 25 degrees C, they move to the cytoplasm.

Simian-virus40-specific T-antigen was isolated by immunoprecipitation. From other studies we have proof that the T-antigen described in this work is coded by the viral DNA. The molecular weight estimated from electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels of T-antigen (1, 4,1) of a part of SV40 DNA ("early region") that comprises about 45% of total length (5, 6). Evidence discussed elsewhere suggested that early 19S mRNA directs synthesis of a single, rather large protein, the SV40-specific T-antigen, which is required to induce lytic and abortive infection, to initiate and maintain cell transformation, and probably also to induce primary tumors in animals (details in refs. 4 and 7).In the present work we isolated T-antigen by immunoprecipitation. The T-antigen isolated in this way is shown to be coded by the viral DNA. We compared the molecular properties of T-antigen in abortive and lytic infection. Our The results were the same whether crude viral lysates or highly purified viral preparations were used for infection. Virus (0.3 ml per dish) was adsorbed at 370 for 90 min; the cultures were then covered with 10 ml of medium containing 10% bovine serum; in most experiments primary mouse kidney cultures were covered with serum-free medium (4).Parallel cultures were mock-infected with 0.3 ml of medium and then treated as SV40-infected cultures.Cultures were labeled with [asS]methionine Ci/ mmol; The Radiochemical Centre, Amersham, England) by addition of 3 ml per dish of prewarmed (370) medium (E serum) which contained only 2 glg/ml of unlabeled methionine (instead of 30 .ug) and 50 ,Ci/ml of [a5S]methionine. At the end of the labeling period the cultures were washed twice with 5 ml of cold (20) isotonic phosphate buffer; then the cells were gently scraped off in 1 ml of the same phosphate buffer with a silicone policeman and centrifuged for 5 min at 500 X g.Preparation of "Soluble Extracts." The pellets were suspended at concentrations varying between 107 and 108 cells in 5 ml of extraction buffer (2.5 mM Tris-HCl, pH 7.4, 0.5 M LiCl, 1 mM EDTA), sonicated (MSE 100 W Ultrasonic Disintegrator) four times for 15 sec in an ice-water bath, and then immediately centrifuged for 30 min at 30,000 X g at 40. The supernatant was removed and is referred to as "soluble extract." In early experiments phenylmethylsulfonyl fluoride (0.1-0.3 mg/ml) and dithiothreitol (1 mM) were present in the extraction buffer.Immunoprecipitation. Most experiments were performed with sera (anti-T) obtained from Syrian hamsters bearing tumors induced by inoculation of an SV40-transformed hamster cell line (9). One preparation was a gift of Drs. E. and P. May, others were made in our laboratory. Hamster anti-T sera (batches 3 X 1888 and 4 X 21) were received from the National Cancer Institute (Bethesda, Md.). The anti-T sera used had specific complement fixing titers of 160-640. Control serum was a pool of 15 normal adult Syrian hamster

An indirect immunoferritin technique for the localizationThe idea of coupling antibody to ferritin, a marker easily recognized in electron microscopy, was first presented by Singer (1959). Since then a number of authors, in particular Morgan et al., (1961 a, b,), and Iater Dales et al., (1965), Kalnins et al., (1966), and others, have applied ferritin-labeled antibodies to the study of virusinfected cells, using the direct immunoferritin technique, localizing antigens with purified, specific, ferritin-coupled antibody. The indirect or sandwich technique, in which whole serum containing specific antibody is added to cells followed by purified, ferritin-coupled, specific antiglobulin has been used only in the study of cell surface antigens (Baxendall et al., 1962) where fixation is unnecessary and the problems of non-specific localization are minimized. The present communication deals with the detection and localization of intracellular viral antigens by the sandwich technique with the use of double labeled (Hsu et al., 1963) fluorescein and ferritincoupled antiglobulins.Human adenovirus type 12 (A-12) antigens were chosen for study because of previous experience with their localization by immunofluorescence (Levinthal et al., 1966) and because of the existence of an antigen produced early in the virus cycle (neoantigen, T antigen) of characteristic delicate, fibrous-filamentous shape, detected initially by immunofluorescence (Pope and Rowe, 1964) and demonstrated subsequently by direct immunoferritin (Kalnins et al., 1966) Furthermore, the complex adenovirus cycle is associated with the intranuclear accumulation of structural viral antigens in the form of dots, rings and large central and peripheral nuclear masses which are readily detected by immunofluorescence and standard electron microscopic methods. For

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