Search PubMed⌕ Search

Biomedical subjects

W Doerfler

Publications and source records attributed to W Doerfler.

At least 235 records · Page 13Linked to original sources
PubMed1979

High molecular weight virus DNA in KB cells infected with ts mutants of adenovirus type 2 under permissive and non-permissive conditions.

Temperature-sensitive (ts) mutants of adenovirus type 2 (Ad2), which are deficient in virus DNA synthesis at the non-permissive temperature, have been used to investigate whether virus DNA replication is required for the occurrence of high mol. wt. Ad2 DNA (greater than 100S, 50 to 90S) in human cells productively infected with Ad2. The high mol. wt. virus DNA has been previously shown to consist of virus and cellular DNA molecules covalently linked. The present data indicate that after infection with DNA-ts mutants, the production of high mol.wt. virus DNA is much less sensitive to restrictive conditions than the synthesis of unit length (34S) Ad2 DNA. This finding lends further support to the idea that the occurrence of high mol. wt. virus DNA is independent of the synthesis of unit length virus DNA.

Adenoviruses, Human↗
PubMed1979

Transcription of the genome of adenovirus type 12. V. Kinetic and size-class analyses of nuclear and messenger RNA in productive infection.

In human KB cells productively infected with adenovirus type 12 (Ad12) the kinetics of synthesis of Ad12-specific RNA were investigated. Using 2 h labelling periods, striking differences were observed in the time course and the rate of synthesis of virus RNA comparing the nuclear, cytoplasmic and polysomal RNA fractions. A protracted onset of virus RNA synthesis was followed by an exponential increase between 15 and 40 h p.i.; reaching a maximum at 40 to 45 h p.i., the fraction of the newly synthesized Ad12-specific RNA remained constant up to 66 h in the nucleus, but decreased in the cytoplasm. Characteristic patterns were found in the size distribution of Ad12-specific messenger RNA synthesized early and late in the infection cycle. By using 10 min labelling periods, the size distribution and the degree of polyadenylation of the primary transcripts were determined. Early in infection, the size distribution of the virus-specific nuclear RNA resembled that of the mRNA, whereas late after infection most of the label was found in high mol. wt. RNA sedimenting between 30 and 55S. Eighty percent of the nuclear RNA was polyadenylated and belonged to the high mol. wt. RNA class. During the early phase of infection, approx. 20% of the virus genome was found to be transcribed symmetrically, and later after infection the entire Ad12 genome. Symmetrical (self-complementary) transcripts were preferentially drived from the terminal parts of Ad12 DNA.

Adenoviruses, Human↗
PubMed1979

In vitro translation of adenovirus type 12-specific mRNA isolated from infected and transformed cells.

The early and late gene products of human adenovirus type 12 (Ad12), as well as the viral proteins synthesized in an Ad12-transformed cell line, were identified by translation of viral mRNA in an in vitro protein-synthesizing system. Cytoplasmic RNA was isolated from permissive KB or nonpermissive BHK cells infected with Ad12 and from Ad12-transformed HA12/7 cells. Virus-specific RNA was selected by hybridization to Ad12 DNA covalently bound to cellulose. Viral RNA was then translated in a fractionated rabbit reticulocyte cell-free system or in wheat germ S-30 extracts. The proteins synthesized were characterized by immunoprecipitation and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. RNA prepared from KB cells late after infection with Ad12 elicited the synthesis of most of the structural polypeptides of the virion and at least two presumably nonstructural Ad12 proteins. When viral RNA isolated early after infection of KB cells with Ad12 was translated in vitro, 10 polypeptides were observed: E-68K, E-50K, E-42K, E-39K, E-34K, E-21K, E-19K, E-13K, E-12K, and E-10K. Ad12-specific RNA was also isolated from the Ad12-transformed hamster cell line HA12/7, which contains several copies of the Ad12 genome integrated in the host genome. The RNA codes for at least seven polypeptides with molecular weights very similar to those of the early viral proteins.

Adenoviruses, Human↗
PubMed1978

[Mutagenic and teratogenic effects of cigarette smoke. A summary of experimental and clinical observations (author's transl)].

The mutagenic effect of compounds in cigarette smoke condensate has been demonstrated by the Ames test. A small percentage of cells is transformed into malignant cells. In animal experiments, evidence was found for the teratogenic effects of the contents of cigarette smoke. Among the published epidemiological statistical observations on the effects on humans are a reduction of the birth weight due to maternal smoking, an increase in perinatal mortality if the father smokes and twice the incidence of deformities in children of fathers who smoke heavily. The knowledge of these connections should be of significance for family planning consultation.

Birth Weight↗
PubMed1978

Patterns of integration of viral DNA sequences in the genomes of adenovirus type 12-transformed hamster cells.

The patterns of integration of the viral genome have been analyzed in four hamster cell lines transformed by adenovirus type 12 (Ad12). It has previously been shown that in each of the cell lines HA12/7, T637, A2497-2 and A2497-3, the viral genome persists in multiple copies, and that different parts of the viral DNA are represented non-stoichiometrically (Fanning and Doerfler, 1976). All four cell lines are oncogenic when injected into hamsters. The DNA from each of the cell lines was extracted and cleaved in different experiments with restriction endonucleases Bam HI, Bgl II, Eco RI, Hind III, Hpa II or Sma I. The DNA fragments were separated on 1% agarose slab gels and transferred to nitrocellulose filters by the Southern technique. Ad12 DNA sequences were detected by hybridization to Ad12 DNA, which was 32 P-labeled by nick translation, and by subsequent autoradiography. In some experiments, the 32P-labeled Eco RI restriction endonuclease fragments of Ad12 DNA were used to investigate the distribution of specific segments of the viral genome in the cellular DNA. For each cell line, a distinct and specific pattern of integrated viral DNA sequences is observed for each of the restriction endonucleases used. Moreover, viral sequences complementary to the isolated Eco RI restriction endonuclease fragments are also distributed in patterns specific for each cell line. There are striking differences in integration patterns among the four different lines; there are also similarities. Because the organization of cellular genes in virus-transformed as compared to normal cells has not yet been determined, conclusions about the existence or absence of specific integration sites for adenovirus DNA appear premature. Analysis of the integration patterns of Ad12 DNA in the four hamster lines investigated reveals that some of the viral DNA molecules are fragmented prior to or during integration. Analysis with specific restriction endonuclease fragments demonstrates that the Eco RI B, D and E fragments, comprising a contiguous segment from 0.17-0.62 fractional length units of the viral DNA, remain intact during integration in a portion of the viral DNA molecules. Although each cell line carries multiple copies of Ad12 DNA, the viral DNA sequences are concentrated in a small number of distinct size classes of fragments. This finding is compatible with, but does not prove, the notion that at least a portion of the viral DNA sequences, is integrated into repetitive sequences, or else that the integrated viral sequences have been amplified after integration. In the three cell lines which were tested, the integration pattern is stable over many generations, with continuous passage-twice weekly-of cells for 6-7 months. In the three cell lines which were examined, the integration pattern is identical in a number of randomly isolated clones. Hence it can be concluded that the patterns of integration are identical among all cells in a population of a given line of transformed cells.

Adenoviruses, Human↗
PubMed1978

Integration of adenovirus type 2 DNA in productively infected cells: results of sequential hybridization experiments.

In human KB cells permissively infected with adenovirus type 2, the high mol. wt. forms of virus DNA have been characterized. These size classes of virus DNA sediment at greater than 100S and 40 to 90S in alkaline sucrose density gradients. Considerable evidence from a series of earlier communications supports the notion that the high mol. wt. forms of virus DNA represent virus DNA sequences covalently linked to cellular DNA. 3H-labelled high mol. wt. adenovirus type 2 DNA from productively infected cells can be shown to hybridize to virus DNA fixed to filters. In the present paper we demonstrate that on alkali elution of the DNA from the filters used in the first step of the hybridization experiment, the labelled DNA re-hybridizes to cellular DNA in the second step of a sequential hybridization experiment. The order of performing the two successive hybridization experiments can be reversed and very similar results are obtained. These data provide conclusive evidence for the covalent linkage of virus and cellular DNA sequences in KB cells productively infected with adenovirus type 2.

Adenoviruses, Human↗
PubMed1978

Morphological revertants of adenovirus type 12-transformed hamster cells.

Morphological revertants have been isolated from one line of adenovirus type 12-transformed hamster cells. This line, T637, is oncogenic in hamsters and contains multiple copies of the virus genome per cell. Different parts of the virus genome are represented in non-stoichiometric amounts and the virus DNA persists in the cells in an integrated form. The pattern of integrated virus genomes has been determined by the blotting technique. In the T637 line, morphological revertants arise spontaneously at relatively high frequency. Two of these revertants have been cloned. In contrast to the T637 line, the revertants F10 and G12 exhibit fibroblastic morphology. The patterns of integrated virus genomes in the revertants differs markedly from that of the T637 line; one of the revertant cell lines, F10, appears to have lost all virus DNA sequences. The morphological revertants continue to express the oncogenic phenotype, although the time required to produce tumours in animals appears to be prolonged compared to the parental BHK21 and the T637 cell lines. A number of biological parameters of the revertant lines have also been investigated.

Adenoviridae↗
PubMed1977

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.

Adenoviruses, Human↗
PubMed1977

Characterization of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells.

The properties of the pH 4.0 endonuclease from adenovirus-type-2-infected KB cells were determined. The enzyme has a molecular weight of approximately 40000. Its pH optimum is at pH 4.0, it is not inhibited by ethylenediaminetetraacetate (EDTA), and it is active at temperatures up to 60 degree C. The enzyme cleaves adenovirus DNA in a stepwise manner. The limit digestion product has a molecular weight of 120000-200000. There is evidence that the cleavage reaction proceeds via an initial single-strand nick. Under the conditions tested the endonuclease did not seem to reveal a high degree of specificity as to the recognition of cleavage sites, or else the sites recognized occurred very frequently.

Adenoviruses, Human↗
PubMed1977

The fate of type 7 adenovirions in lysosomes of HeLa cells.

Properties of type 7 adenovirions in lysosomes of HeLa cells were studied 12 hr postinfection. Viral particles were transferred to the lysosomes very quickly after initiation of penetration, i.e., after 10 min of incubation at 37 degrees. No morphological modification of the virion was detected for 6 hr postinfection. However, by 12 hr postinfection, the virion was no longer recognizable. Most of the virus remained infectious for 2 hr, whereas after 12 hr the infectivity was abolished. Soon after the adsorption of the virus on the cell membrane at 4 degrees, the viral DNA in the virion became sensitive to pancreatic DNase, and this sensitivity increased during the first 2 hr of incubation at 37 degrees. This result suggests that some modification in the architecture of the virion occurred before transfer to the lysosomes. The adenovirus 7 (Ad 7) DNA extracted from the lysosomes appeared intact for 6 hr postinfection and was found to cosediment at 34 S with the Ad 2 DNA marker. Comparable activities of free acid phosphatase were found in lysosomes isolated from uninfected control cells and from infected cells. In in vitro experiments, lysosomal acid DNase and pancreatic DNase were shown to degrade Ad 7 DNA at similar rates; however, in vivo, intralysosomal Ad 7 DNA was only partially sensitive to lysosomal DNase.

Acid Phosphatase↗
PubMed1977

Integrated viral sequences in adenovirus type 12-transformed hamster cells.

The physical state of the viral genome in four lines of hamster cells transformed by adenovirus type 12 (Ad12) has been investigated. The four lines of transformed cells originated from hamster cells after infection with Ad12 at multiplicities ranging from 5-350 plaque-forming units per cell. The DNA from transformed cells has been restricted with the Sal I endonuclease from Streptomyces albus which cleaves adenovirus DNA more frequently than DNA from adenovirus-transformed hamster cells. Thus after cleavage by the Sal I enzyme, it is possible to separate free adenovirus DNA sequences from these which are covalently linked to cellular DNA in transformed hamster cells. The results of sequential hybridization experiments in which the Sal I-treated DNA from transformed cells is first annealed to Ad12 DNA on filters, then eluted, and finally hybridized to hamster cell DNA, support the model of Ad12 DNA integrated in multiple fragments into the host genome. Further experiments will be required to characterize the host sequences adjacent to adenovirus DNA and to compare these sequences in different lines of Ad12 transformed cells.

Adenoviridae↗