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W Doerfler

Publications and source records attributed to W Doerfler.

At least 127 records · Page 7Linked to original sources
PubMed1990

The expression of the Autographa californica nuclear polyhedrosis virus genome in insect cells.

This report presents a synopsis of recently published work in our laboratory on the molecular biology of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The following studies have been summarized. (1) On the mode of transcription of the AcNPV genome in insect cells. (2) Translation of proteins encoded in the 81.2 to 85.0 map unit segment of AcNPV. (3) Inserts of insect cell DNA in the AcNPV genome. (4) Expression of influenza (fowl plague) virus haemagglutinin in Spodoptera frugiperda insect cells, and successful immunization of chickens. (5) Synthesis of the influenza virus haemagglutinin in insect larvae by recombinant AcNPV. This insect virus system will continue to serve as a model for research on the molecular biology of insects. Moreover, the baculovirus system has been recognized as a very efficient and safe eukaryotic expression vector.

Animals↗
PubMed1990

Interindividual concordance of methylation profiles in human genes for tumor necrosis factors alpha and beta.

The DNA in mammalian genomes is characterized by complex patterns of DNA methylation that reflect the states of all genetic activities of that genome. The modified nucleotide 5-methyldeoxycytidine (5mdC) can affect the interactions of specific proteins with DNA sequence motifs. The most extensively studied effect of sequence-specific methylations is that of the long-term silencing of eukaryotic (mammalian) promoters. We have initiated studies on the methylation status of parts of the human genome to view patterns of DNA methylation as indicators for genetic activities. In this report, analyses using both restriction enzyme--Southern blotting and the very precise genomic sequencing technique have been done. The genes for tumor necrosis factors (TNF) alpha and beta--in particular, their 5'-upstream and promoter regions--have been investigated in DNA isolated from human lymphocytes, granulocytes, and sperm. The results are characterized by a remarkable interindividual concordance of DNA methylation in specific human cell types. The patterns are identical in the DNA from one cell type for different individuals even of different genetic origins but different in the DNA from different cell types. As an example, in the DNA from human granulocytes of 15 different individuals (ages 20-48 yr, both sexes), 5mdC residues have been localized by the genomic sequencing technique in three identical sequence positions in the 5'-upstream region and in one downstream position of the gene encoding TNF-alpha. The promoter of this gene is free of 5mdC, and TNF-alpha is expressed in human granulocytes. The TNF-beta promoter is methylated in granulocytes from 9 different individuals, and TNF-beta is not expressed. In human lymphocytes, the main source of TNF-beta, the TNF-beta promoter is free of 5mdC residues. All 5'-CG-3' sites studied in the TNF-alpha and -beta genes are methylated in DNA from human sperm. In human cell lines HL-60, Jurkat, and RPMI 1788, the extent of DNA methylation in TNF-alpha and -beta genes has also been studied.

5-Methylcytosine↗
PubMed1990

An insertion of insect cell DNA in the 81-map-unit segment of Autographa californica nuclear polyhedrosis virus DNA.

In this report, a transposonlike insertion of Spodoptera frugiperda insect cell DNA was analyzed in single-plaque isolate E of the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV). The 634-base-pair insertion is characterized by an 18-base-pair terminal inverted repeat and carries an EcoRI site. This additional EcoRI site in the 81-map-unit segment of the DNA of plaque isolate E of AcNPV explains the difference between the EcoRI restriction map of the DNA from this isolate and those of the virus stocks used in other laboratories. Except for this insertion, the nucleotide sequence at the site of insertion in the DNA of plaque isolate E is identical to that of AcNPV E2 (G. E. Smith and M. D. Summers, Virology 89:517-527, 1978). The cellular DNA insertion in the AcNPV genome is represented many times in the S. frugiperda cell genome but has no detectable homology with DNAs from species other than lepidopteran insects. In S. frugiperda cells, the transposonlike insertion sequences are transcribed into cytoplasmic RNA. The transcription of these sequences is initiated within the cellular insertion element. As reported previously (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 61:3048-3057, 1987), in S. frugiperda cells infected with plaque isolate E of AcNPV, at least nine different size classes of AcNPV-specific RNAs are synthesized; in AcNPV E2-infected cells, similar size classes have been detected. The cellular insertion of plaque isolate E provides the initiation site for the synthesis of an additional RNA size class which is transcribed off viral DNA.

Animals↗
PubMed1989

Sequence-specific methylation in a downstream region of the late E2A promoter of adenovirus type 2 DNA prevents protein binding.

In studies on the promoter-inhibitory effect of sequence-specific DNA methylations, the late E2A promoter of human adenovirus type 2 (Ad2) has been used as an experimental tool. Upon the in vitro methylation of 5'-CCGG-3' (HpaII) sequences at nucleotides +24, +6 and -215 relative to nucleotide + 1, the site of transcriptional initiation, promoter inhibition or inactivation has been demonstrated in transient expression tests in Xenopus laevis oocytes (Langner et al., 1984), in mammalian cells (Langner et al., 1986), after the genomic fixation of the promoter in conjunction with a reporter gene in mammalian cells (Müller & Doerfler, 1987), and in a cell-free transcription system employing nuclear extracts of human HeLa cells (Dobrzanski et al., 1988). Possible explanations for the inhibitory effect of three 5-methyldeoxycytidine (5-mC) residues in a promoter sequence are structural alterations in DNA or the positive or negative modulation of the sequence-specific binding of proteins. This modulation could be indirect at the level of protein-protein interactions. A synthetic oligodeoxyribonucleotide of 50 base-pairs (bp) or a restriction endonuclease fragment of 73 bp in length, which comprised the +24 and +6 5'-CCGG-3' sequences of the late E2A promoter, were methylated or hemimethylated at these two sites, or were left unmethylated and were subsequently incubated with a partly purified (heparin-Sepharose) nuclear extract of human HeLa cells. Protein binding was monitored by electrophoretic migration delay of the 32P-labeled 50 bp oligodeoxyribonucleotide or the 73 bp fragment on polyacrylamide gels. The formation of one of the DNA-protein complexes in this analysis was compromised when 5'-CCGG-3' methylated oligodeoxyribonucleotides were used in the binding assays. Similar results were obtained when the 50 bp oligodeoxyribonucleotide was hemimethylated in either complement.The formation of the same complex could also be obliterated by adding the same non-methylated oligodeoxyribonucleotide as competitor to the reaction mixture. The methylated oligodeoxyribonucleotide did not act as a competitor, nor did a randomly composed oligodeoxyribonucleotide of identical length. The results show that protein binding is abolished by methylation of those sequences in the late E2A promoter whose methylation inhibits promoter function. The abrogation of protein binding has been observed with a 50 bp or 73 bp fragment. With a 99 bp or a 377 bp fragment, binding differences between the unmethylated and the 5'-CCGG-3' methylated late E2A promoter are not apparent.

Adenovirus Early Proteins↗
PubMed1989

Chromatin structure and levels of expression and DNA methylation in the E3 region of chromosomally integrated adenovirus type 12 DNA.

Integrated adenovirus type 12 (Ad12) genes in Ad12-transformed cell lines were investigated for chromatin structure, expression levels and states of DNA methylation. The E3 region in the Ad12-transformed cell line HA12/7 is hypermethylated and not expressed. The same region in the Ad12-transformed hamster cell lines T637 and A2497-3 is transcribed and undermethylated (Kruczek, I. and Doerfler, W. (1982) EMBO J. 1, 409-414). There was no significant difference in the DNase I sensitivity of the E3 region when nuclei of the aforementioned cell lines were incubated with this nuclease. In contrast, incubation of these nuclei with the restriction endonuclease PstI and subsequent cleavage of the DNA with BamHI generated an additional 0.9 kbp fragment in T637 and A2497-3 DNA which was not observed after treating HA12/7 nuclei and DNA in the same way. This finding was interpreted as indicative of differences in the chromatin structure of the E3 region depending on its state of transcriptional activity and its level of methylation. The E1 and major late promoter regions, which were transcriptionally active and inactive, respectively, in all three cell lines investigated, did not exhibit differences in sensitivity towards DNase I or PstI treatment of nuclei. More refined technology will be required to compare the chromatin structure of active versus inactive genes.

Adenovirus Early Proteins↗
PubMed1989

Recombination in hamster cell nuclear extracts between adenovirus type 12 DNA and two hamster preinsertion sequences.

A cell-free system of nuclear extracts from BHK21 cells has been developed to catalyse recombination in vitro between the DNA of adenovirus type 12 (Ad12) and two different hamster preinsertion sequences. The pBR322 cloned 1768 bp fragment p7 and the 3.1 kbp fragment p16 from BHK21 hamster DNA had previously been identified as the preinsertion sites corresponding to the junctions between Ad12 DNA and hamster DNA in cell line CLAC1 and in the Ad12-induced tumour T1111(2), respectively. Preinsertion sequences, which had recombined previously with foreign (Ad12) DNA, might again be recognized by the recombination system even in a cell-free system. PstI cleaved Ad12 DNA and the circular or the EcoRI linearized p7 or p16 preinsertion sequences were incubated with nuclear extracts. Recombinants were isolated by transfecting the DNA into recA- Escherichia coli strains and by screening for Ad12 DNA-positive colonies. Without a selectable eukaryotic marker, all Ad12 DNA positive recombinants were registered. Out of a total of greater than 90 p7-Ad12 DNA recombinants, 21 were studied by restriction-hybridization, and four by partial nucleotide sequence analyses. Among the p16-Ad12 DNA recombinants, four were analysed. The sites of linkage between Ad12 DNA and p7 or p16 hamster DNA were all different and distinct from the original CLAC1 or T1111(2) junction site between Ad12 and hamster DNA. The in vitro recombinants were not generated by simple end-to-end joining of the DNA fragments used in the reaction but by genetic exchange. Thirteen of the 25 recombinants were derived from the 61-71 map unit fragment of Ad12 DNA. Recombination experiments between Ad12 DNA and four randomly selected unique or repetitive hamster DNA sequences of 1.5-6.2 kbp in length did not yield recombinants. Apparently, the p7 and p16 hamster preinsertion sequences recombined with Ad12 DNA with a certain preference.

Adenoviridae↗
PubMed1989

DNA-DNA dot hybridization technique used as DNA determination method in the alkaline elution analysis of DNA damage.

A DNA-DNA ('Southern') dot hybridization technique was adapted for use as a quantitative DNA detection method during alkaline elution analysis of irradiated rat cell material. In comparison to standard microfluorometric methods, similar gamma-ray dose-response relationships were obtained with less than 1% of the cell material when the dot hybridization assay was used. When a highly repetitive, long interspersed DNA element of the rat genome is used as a hybridization probe, as few as 10(4) cells of rat tissue or rat cell culture cells per sample with approx. 50 ng of DNA were sufficient to detect single-strand breaks and protein cross-links in the DNA of rat hepatocytes and cells of the nasal epithelium after in vitro gamma-irradiation. Since highly repetitive DNA elements are available from nearly all higher eukaryotes, this alternative approach of detecting DNA in alkaline elution analysis is generally proposed for tissues which yield only low amounts of cell material and/or which are difficult to label by radioactive DNA precursors.

Animals↗
PubMed1989

Arrangement and expression of integrated adenovirus type 12 DNA in the transformed hamster cell line HA12/7: amplification of Ad12 and c-myc DNAs and evidence for hybrid viral-cellular transcripts.

In the genome of the adenovirus type 12 (Ad12)-transformed hamster cell line HA12/7 about three copies of the viral DNA are fixed by integration. The results of blot-hybridization, molecular cloning, and nucleotide sequencing experiments suggest a model for the arrangement of Ad12 DNA molecules in which the left hand terminus of one of the Ad12 DNA copies is linked to unique hamster DNA. The right hand end of this DNA molecule is fused to an inverted copy of a left terminal approximately 4.3 kb fragment of Ad12 DNA. This ensemble is followed by the second Ad12 DNA copy whose right terminus is again joined to an inverted, supernumerary left terminal approximately 4.3 kb Ad12 DNA fragment. There is a third Ad12 DNA copy whose right terminus is linked to cellular DNA. In this sequence arrangement, the left terminus of Ad12 DNA is overrepresented, as had been shown earlier (S. Stabel, W. Doerfler and R.R. Friis (1980) J. Virol. 36, 22-40). In the presented model, cellular DNA sequences are interspersed in between the three copies of Ad12 DNA. In the left terminus of the integrated Ad12 DNA, transcription of RNA is initiated which extends out into cellular DNA. The interviral DNA junctions are also transcribed. The c-myc gene in cell line HA12/7 is amplified about 10-fold and considerably more c-myc RNA has been identified in the Ad12-transformed cells than in BHK21 or in LSH hamster cells. It has been shown previously that the E1 region of Ad12 DNA is transcribed into mRNA in HA12/7 cells (Ortin et al. (1976) J. Virol. 20, 355-372). It remains to be investigated whether c-myc amplification and expression are related to the transformed phenotype of HA12/7 cells.

Adenoviridae↗
PubMed1989

Genomic sequencing reveals a 5-methylcytosine-free domain in active promoters and the spreading of preimposed methylation patterns.

Previous work demonstrated an inverse correlation between methylation at the three 5'-CCGG-3' sequences in positions +24, +6, and -215 relative to the cap site of the late E2A promoter of adenovirus type 2 (Ad2) DNA and its activity. In the study presented here, we used the genomic sequencing method to detect 5-methyl-2'-deoxycytidine (m5dC) residues in 5'-CG-3' sequences other than the 5'-CCGG-3' (Hpa II) sites. The patterns of methylation in all 5'-CG-3' sequences over a region of about 180 base pairs required for gene activity in the late E2A promoter of integrated Ad2 DNA were determined in cell lines that carry this promoter in an active or inactive state. In cell lines HE1 and uc2, the late E2A promoter is active and all thirteen 5'-CG-3' sequences between positions +24 and -160 are unmethylated. In cell line HE2, the same promoter is permanently shut off and all 5'-CG-3' sequences are methylated in both strands. Thus, the inverse correlation is perfect in these cell lines over a region of about 180 base pairs in the late E2A promoter. The same promoter segment was analyzed in cell lines mc23 and mc40, in which a late E2A promoter-chloramphenicol acetyltransferase (CAT) gene construct had been genomically fixed after in vitro 5'-CCGG-3' methylation and subsequent transfection. In cell line mc23, the preimposed methylation pattern was stable and the CAT gene was inactive. Genomic sequencing confirmed the presence of m5dC in the 5'-CCGG-3' sequences and revealed the spreading of methylation to neighboring 5'-CG-3' sequences along the entire promoter. Some of these sites were hemimethylated. In cell line mc40, several of the 120 integrated copies became demethylated in positions +24 and +6, but the promoter was methylated in some of the copies upstream of position -50. Cell line mc40 expressed the CAT gene.

5-Methylcytosine↗
PubMed1989

Induction of transcription factor AP-1 by adenovirus E1A protein and cAMP.

Treatment of adenovirus-infected mouse S49 cells with cAMP analogs leads to the transcriptional induction of early viral genes. E1A proteins and cAMP work in synergy to activate several of these genes. We now demonstrate that the transcription factor AP-1 is modestly induced by cAMP in S49 cells and induced to significantly higher levels by cAMP in the presence of E1A proteins. Cytoplasmic levels of c-fos and junB mRNAs are rapidly increased by cAMP, and the induction is substantially stronger in the presence of E1A protein. The AP-1 activity binds efficiently to both AP-1 and activating transcription factor (ATF)/cAMP response element binding protein (CREB)-binding sites present in E1A-inducible promoters and presumably plays a role in the transcriptional activation of adenovirus genes by E1A proteins and cAMP.

Adenovirus Early Proteins↗
PubMed1989

Synthesis of biologically active influenza virus hemagglutinin in insect larvae.

The hemagglutinin of influenza (fowl plague) virus was expressed in larvae of Heliothis virescens by using recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) as a vector. Animals were infected with the recombinant virus either by parenteral injection or by feeding. For oral uptake, recombinant virus occluded in polyhedra obtained from cultured Spodoptera frugiperda cells after coinfection with authentic AcNPV was used. Immunohistological analyses of infected animals revealed that the hemagglutinin was expressed only in those tissues that are also permissive for the replication of authentic AcNPV. These tissues included hypodermis, fat body, and tracheal matrix. After oral infection, hemagglutinin was also detected in individual gut cells. The amount of hemagglutinin synthesized in larvae after parenteral infection was 0.3% of the total protein, compared with 5% obtained in cultured insect cells. The hemagglutinin was transported to the cell surface and expressed in polarized cells only at the apical plasma membrane. It was processed by posttranslational proteolysis into the cleavage products HA1 and HA2. Oligosaccharides were attached by N-glycosidic linkages and were smaller than those found on hemagglutinin obtained from vertebrate cells. Hemagglutinin from larvae expressed receptor binding and cell fusion activities, but quantitation of the hemolytic capacity revealed that it was only about half as active as hemagglutinin from vertebrate or insect cell cultures. Chickens immunized with larval tissues containing hemagglutinin were protected from infection with fowl plague virus. These observations demonstrate that live insects are able to produce a recombinant membrane protein of vertebrate origin in biologically active form.

Administration, Oral↗
PubMed1989

Reactivation of a methylation-silenced gene in adenovirus-transformed cells by 5-azacytidine or by E1A trans activation.

In the adenovirus type 2 (Ad2)-transformed hamster cell line HE3, the integrated late E2A promoter of Ad2 DNA is inactive, is methylated at all three 5'-CCGG-3' sequences, and can be reactivated by growing the cells in the presence of 50 microM 5-azacytidine (5-azaC). The three 5'-CCGG-3' sequences then become demethylated. Demethylation and reactivation are stable over 30 passages even after the removal of 5-azaC. The dormant late E2A promoter in cell line HE3 can also be reactivated by transfecting the cells with recombinant plasmids that carry the left terminal E1A and part of the E1B region of Ad2 DNA or the E1A 13S cDNA, but not with plasmids containing the E1A 12S cDNA. The E1A 13S cDNA encodes the 289-amino-acid trans-activating protein of Ad2. The E1A-mediated reactivation of the late E2A promoter is not accompanied by its demethylation in both DNA complements. Cell line HE3 produces constitutively E1A-encoded mRNAs and reactivates the methylated late E2A promoter-chloramphenicol acetyltransferase gene construct after transfection into HE3 cells. Constitutive levels of the endogenous E1A gene products in HE3 cells are detectable but, paradoxically, appear insufficient to reactivate the endogenous, chromosomally integrated E2A gene.

Adenovirus Early Proteins↗
PubMed1989

Defect of adenovirus type 12 replication in hamster cells: absence of transcription of viral virus-associated and L1 RNAs.

The nonpermissive interaction of hamster cells with human adenovirus type 12 (Ad12) is characterized by a total block of Ad12 DNA replication and late transcription, whereas most of the early functions of Ad12 DNA can be transcribed. Ad2 can replicate in hamster cells. The replication and late transcription defects of Ad12 DNA can be complemented to a certain extent by the E1B functions of Ad2 DNA. This complementation fails, however, to lead to the synthesis of the late Ad12 proteins and to the assembly of infectious virions. It will now be demonstrated that the Ad12 L1 (late genes of group 1) and virus-associated (VA) RNAs are not transcribed in hamster cells. Synthesis of these RNAs in productively infected human cells or Ad2-infected hamster cells is readily detectable by S1 nuclease protection experiments and Northern (RNA) blotting. Similarly, the Ad2-transformed hamster cell line BHK-Ad2E1 fails to complement L1 and VA RNA syntheses after superinfection with Ad12. However, Ad12 infection of the Ad5-transformed hamster cell line BHK297-C131 leads to the transcription of the Ad12 L1 and VA segments. This difference in complementation by the two transformed hamster cell lines might be accounted for by functions in the segment of Ad5 DNA extending between map units 30 and 40 and persisting in the Ad5-transformed hamster cells or by hamster host cell functions which might be operative in cell line BHK297-C131 but not in BHK-Ad2E1 or BHK-21 hamster cells.

Adenoviruses, Human↗
PubMed1989

Adenovirus L1 52- and 55-kilodalton proteins are required for assembly of virions.

A variant of adenovirus type 5 that contained a mutation within the L1 52- and 55-kilodalton (52/55K) protein-coding region was isolated. The mutant, termed ts369, produced L1 52/55K proteins with a two-amino-acid substitution and was temperature sensitive. Temperature-shift experiments indicated that the ts369 defect was late in the viral growth cycle. DNA replication and synthesis of late proteins occurred normally in ts369-infected cells at the nonpermissive temperature, but mature virions were not produced. Rather, capsidlike particles associated with the left-terminal region of the viral chromosome accumulated. These incomplete particles could not be chased into mature virions when the infected cells were shifted to the permissive temperature. However, previously synthesized proteins could be assembled into virions in the presence of a protein synthesis inhibitor upon shiftdown from the nonpermissive temperature, suggesting that the inactivation of the L1 52/55K proteins was reversible. These results indicate that the adenovirus L1 52/55K proteins play a role in the assembly of infectious virus particles.

Adenoviridae↗