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W Doerfler

Publications and source records attributed to W Doerfler.

At least 91 records · Page 5Linked to original sources
PubMed1994

Late transcripts of adenovirus type 12 DNA are not translated in hamster cells expressing the E1 region of adenovirus type 5.

Hamster cells are completely nonpermissive for the replication of human adenovirus type 12 (Ad12), whereas types 2 and 5 can replicate in hamster cells. The Ad5-transformed hamster cell line BHK297-C131, which carries the left terminal 18.7% of the Ad5 genome and expresses at least the viral E1A region, can somehow complement Ad12 DNA replication and the transcription of the late Ad12 genes. Since the interaction of Ad12 with hamster cells must constitute a significant factor in the induction of Ad12 tumors in neonatal hamsters, we have continued to examine details of this abortive virus infection. The late Ad12 mRNAs in BHK297-C131 cells are polyadenylated but are synthesized in reduced amounts compared with the Ad12 products in Ad12-infected human cells, which are permissive for viral replication. The late mRNA derived from the Ad12 fiber gene has been assessed for its structural properties. By cloning cDNA transcripts from this region and determining their nucleotide sequences, the authenticity of the complete Ad12 fiber sequence and the completeness of the Ad12-typical tripartite leader have been confirmed. Moreover, in Ad12-infected BHK297-C131 cells the Ad12 virus-associated RNA, a virus-encoded translational activator with the correct nucleotide sequence, is synthesized. Nevertheless, the synthesis of detectable amounts of Ad12 virion-specific proteins, and in particular that of the main viral antigens, hexons and fibers, cannot be documented. Cellular factors needed to promote late mRNA translation might be missing, or inhibitory factors might exist in Ad12-infected BHK297-C131 cells.

Adenovirus E1 Proteins↗
PubMed1993

Differences in the accessibility of methylated and unmethylated DNA to DNase I.

DNase I binds in the minor groove of DNA and is used as an enzymatic tool to investigate the interaction of proteins with DNA. Here we show that the major groove located 5-methyldeoxycytidine can enhance or inhibit the cleavage rates of DNA by DNase I. This effect may be caused in part by changes in DNA structure affecting the accessibility of the minor groove of DNA to DNase I.

Base Sequence↗
PubMed1993

Triplet repeat sequences in human DNA can be detected by hybridization to a synthetic (5'-CGG-3')17 oligodeoxyribonucleotide.

The seemingly autonomous amplification of naturally occurring triplet repeat sequences in the human genome has been implicated in the causation of human genetic disease, such as the fragile X (Martin-Bell) syndrome, myotonic dystrophy (Curshmann-Steinert), spinal and bulbar muscular atrophy (Kennedy's disease) and Huntington's disease. The molecular mechanisms underlying these triplet amplifications are still unknown. We demonstrate here that a synthetic (CGG)17 oligodeoxyribonucleotide can be utilized as hybridization probe to visualize some of the triplet repeats in the human genome. This technique may help in studies aimed at the elucidation of the amplification mechanism.

Autoradiography↗
PubMed1993

Fractionated nuclear extracts from hamster cells catalyze cell-free recombination at selective sequences between adenovirus DNA and a hamster preinsertion site.

We have explored the mechanism of adenovirus type 12 (Ad12) DNA integration because of its importance for viral oncogenesis and as an example of insertional recombination. We have used a fractionated cell-free system from nuclear extracts of hamster cells and have partly purified nuclear proteins that could catalyze in vitro recombination. As recombination partners, the 20,880- to 24,049-nucleotide Pst I D fragment of Ad12 DNA and the hamster preinsertion sequence p7 from the Ad12-induced tumor CLAC1 have proven to recombine at higher frequencies than randomly selected adenoviral or cellular DNA sequences. A preinsertion sequence might carry elements essential in eliciting recombination. Patch homologies between the recombination partners seem to play a role in the selection of sites for recombination in vivo and in the cell-free system. Nuclear extracts from BHK21 cells were prepared by incubating the nuclei in 0.42 M (NH4)2SO4 and fractionated by Sephacryl S-300 gel filtration, followed by chromatography on Mono S and Mono Q columns. The purified products active in recombination contained a limited number of different protein bands, as determined by polyacrylamide gel electrophoresis and silver staining. The most highly purified fraction IV had helicase and topoisomerase I activities. We used two different methods to assess the in vitro generation of hamster DNA-Ad12 DNA recombinants upon incubation with the purified protein fractions: (i) transfection of the recombination products into recA- strains of Escherichia coli and (ii) the polymerase chain reaction by using amplification primers unique for each of the two recombination partners. In p7 hamster DNA, the nucleotide sequence 5'-CCTCTCCG-3' or similar sequences served repeatedly as a preferred recombination target for Ad12 DNA in the tumor CLAC1 and in five independent cell-free recombination experiments.

Adenoviridae↗
PubMed1993

Probing DNA-protein interactions in vitro with the CpG DNA methyltransferase.

A sensitive method was devised to monitor the in vitro binding of nuclear proteins from HeLa cells presumably to the major groove of DNA. Upon the incubation of DNA with nuclear extracts, the complexed DNA was incubated with the CpG DNA methyltransferase from Spiroplasma species. Subsequently, the DNA was repurified, and the location of the methylated cytidine residues was determined by the hydrazine reaction of the DNA sequencing method. By using as DNA substrate the VAI (virus associated) region of human adenovirus type 2 (Ad2) DNA or specific Alu sequences associated with a number of human genes, it was documented that those segments of DNA that were protected by bound proteins against the reaction with DNasel also escaped in vitro methylation by the CpG DNA methyltransferase. This new footprinting method provides a sensitive indicator for in vitro DNA--protein interactions which are specific for the major groove of DNA.

Adenoviridae↗
PubMed1993

DNA methylation in the Alu sequences of diploid and haploid primary human cells.

We have investigated DNA methylation in human Alu sequences, both in general and in specific Alu sequences associated with the genes for alpha 1 globin, tissue plasminogen activator (tPA), adrenocorticotropic hormone (ACTH) and angiogenin. We studied DNAs from lymphocytes, granulocytes, brain, heart muscle and sperm, and from the human HeLa and KB cell lines by using cleavage with methylation-sensitive restriction enzymes combined with Southern blot hybridization and by using genomic sequencing. The results can be summarized as follows. (i) In differentiated primary human cells, Alu elements are often highly methylated even when they are in very 5'-CG-3'-rich regions. This finding is not consistent with the notion that hypermethylation would be a sufficient condition in itself for 5'-CG-3' sequences to undergo loss of 5-methyl-deoxycytidine (5-mC) due to deamination and subsequent mutation. (ii) There are distinct differences in the levels of methylation in the specific Alu sequences. (iii) Alu elements in the DNA of haploid spermatozoa are much less methylated than in diploid cells. Preliminary data indicate that spermatozoa contain Alu-specific RNAs. (iv) The results of cell-free transcription experiments with Alu elements suggest that the in vitro transcription of Alu elements can be inhibited by 5'-CG-3' methylation. High levels of 5'-CG-3' methylation in Alu elements could contribute to their general transcriptional inactivity. (v) The patterns of methylation observed in the Alu elements and in the surrounding sequences are characterized by cell type specific interindividual concordance.

Adrenocorticotropic Hormone↗
PubMed1993

Non-homologous recombination between adenovirus and AcNPV DNA fragments in cell-free extracts from insect Spodoptera frugiperda nuclei.

In previous work, we have developed a cell-free system from nuclear extracts of hamster cells to study the mechanism of integrative recombination between adenovirus type 12 (Ad12) DNA and hamster cell DNA (Jessberger et al., 1989; Tatzelt et al., 1992). We have also demonstrated that in insect cells the left terminal fragment of Ad2 DNA can insert by non-homologous recombination into the 32.6 to 34.5 map unit (EcoRI-O) fragment and into other segments of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA (Xiong et al., 1991). We have now imitated this recombination event in vitro by incubating the E1 fragment of Ad2 DNA and the EcoRI-O fragment of AcNPV DNA, both in the plasmid-cloned circular forms, with partly purified nuclear extracts from Spodoptera frugiperda insect cells. Proteins from these extracts have been fractionated by gel filtration. After the reextraction of DNA from the incubation mixture, recombinants generated in this cell-free system have been identified directly with the polymerase chain reaction (PCR) by using Taq polymerase and appropriate primers which are unique to either of the two reaction partners. The recombinants identified are all different. The results of control experiments argue against the possibility that unspecific reaction products might have been generated during PCR. Nucleotide sequence determinations in some of the recombinants localize the sites of genetic exchange between the two partners and assess the non-homologous nature of the reaction. The recombinants are characterized by the presence of short patch homologies at or close to the sites of linkage between the reaction partners, as described earlier in the hamster cell system (Tatzelt et al., 1992). The occurrence of recombinants in the cell-free system can also be demonstrated by a biological test in which potential recombinants are isolated by transfection into recA- strains of Escherichia coli.

Adenoviridae↗
PubMed1993

The state of DNA methylation in the promoter and exon 1 regions of the human gene for the interleukin-2 receptor alpha chain (IL-2R alpha) in various cell types.

The gene for the human interleukin-2 receptor alpha chain (IL-2R alpha) is expressed only in stimulated, not in resting, human T lymphocytes. This gene, in conjunction with others, plays a pivotal role in eliciting the T cell-mediated immune response. We have investigated the promoter and exon 1 region, the nucleotide -300 to +300 region of this gene relative to the position of transcriptional initiation at nucleotide +1, particularly with respect to the extent of DNA methylation at the 5'-CG-3' sequences and its changes upon induction. By using RNA transfer analyses and the in vivo footprinting technique, we have confirmed the previously reported finding that, upon stimulation of lymphocytes by phytohemagglutinin (PHA) plus interleukin-2 (IL-2), the IL-2R alpha gene can be induced to be transcribed. The region of the IL-2R alpha gene analyzed for 5'-CG-3' methylation by the genomic sequencing method or a polymerase chain reaction-based method subsequent to HpaII or HhaI cleavage of the DNA does not seem to be significantly methylated in most cell types tested, except for the cytidine residue in position +198 which is partly methylated. In the DNA of cells from a chronic B cell lymphatic leukemia 5'-CCGG-3' sequences in the exon 1 region are almost completely unmethylated. These results suggest that the promoter of a gene that is crucial in promoting the immune response and may have to be activated momentarily, will not be silenced by a long-term mechanism like DNA methylation. It is striking that the absence of DNA methylation in this promoter and exon 1 segment also extends to cell types not directly associated with the immune response and even to continuous cell lines.

Base Sequence↗
PubMed1993

Patterns of frog virus 3 DNA methylation and DNA methyltransferase activity in nuclei of infected cells.

The iridovirus frog virus 3 (FV3) can replicate in culture in fat head minnow (FHM) fish cells or in BHK-21 hamster cells. Viral DNA replication commences about 3 h after infection of FHM cells with FV3. Between 3 and 6 h postinfection (p.i.), a portion of the intranuclear FV3 DNA is partly unmethylated. At later times, p.i., all of the viral DNA in the nuclear and cytoplasmic compartments is methylated at the 5'-CCGG-3' sequences. Cytoplasmic FV3 DNA has not been found unmethylated. We have cloned viral DNA fragments from methylated virion DNA. By using the genomic sequencing technique, it has been demonstrated for segments of the FV3 DNA replicated both in FHM fish and BHK21 hamster cells that in a stretch encompassing a total of 350 bp, all of the analyzed 5'-CG-3' dinucleotides are methylated. The modified nucleotide 5-methyldeoxycytidine is present exclusively in the 5'-CG-3' dinucleotide combination. In the cloned FV3 DNA fragment p21A, an open reading frame has been located. The 5' region of this presumptive viral gene is also methylated in all 5'-CG-3' positions. DNA methyltransferase activity has been detected in the nuclei of FV3-infected FHM cells at 4, 11, and 20 h p.i. In the cytoplasmic fraction, comparable activity has not been observed. These data are consistent with the interpretation that FV3 DNA is newly synthesized and de novo methylated in the nuclei of infected FHM cells and subsequently exported into the cytoplasm for viral assembly.

Amino Acid Sequence↗
PubMed1993

A unique mitigator sequence determines the species specificity of the major late promoter in adenovirus type 12 DNA.

Human adenovirus type 12 (Ad12) cannot replicate in hamster cells, whereas human cells are permissive for Ad12. Ad12 DNA replication and late-gene and virus-associated RNA expression are blocked in hamster cells. Early Ad12 genes are transcribed, and the viral DNA can be integrated into the host genome. Ad12 DNA replication and late-gene transcription can be complemented in hamster cells by E1 functions of Ad2 or Ad5, for which hamster cells are fully permissive (for a review, see W. Doerfler, Adv. Virus Res. 39:89-128, 1991). We have previously demonstrated that a 33-nucleotide mitigator sequence, which is located in the downstream region of the major late promoter (MLP) of Ad12 DNA, is responsible for the inactivity of the Ad12 MLP in hamster cells (C. Zock and W. Doerfler, EMBO J. 9:1615-1623, 1990). A similar negative regulator has not been found in the MLP of Ad2 DNA. We have now studied the mechanism of action of this mitigator element. The results of nuclear run-on experiments document the absence of MLP transcripts in the nuclei of Ad12-infected BHK21 hamster cells. Surprisingly, the mitigator element cannot elicit its function in in vitro transcription experiments with nuclear extracts from both hamster BHK21 and human HeLa cells. Intact nuclear topology and/or tightly bound nuclear elements that cannot be eluted in nuclear extracts are somehow required for recognition of the Ad12 mitigator. Electrophoretic mobility shift assays have not revealed significant differences in the binding of proteins from human HeLa or hamster BHK21 cells to the mitigator sequence in the MLP of Ad12 DNA or to the corresponding sequence in Ad2 DNA. We have converted the sequence of the mitigator in the MLP of Ad12 DNA to the equivalent sequence in the MLP of Ad2 DNA by site-directed mutagenesis. This construct was not active in hamster cells. When the Ad12 mitigator, on the other hand, was inserted into the Ad2 MLP, the latter's function in hamster cells was not compromised. Deletions in the 5' upstream region of the Ad12 MLP have provided evidence for the existence of additional sequences that codetermine the deficiency of the Ad12 MLP in hamster cells. The amphifunctional YY1 protein from HeLa cells can bind specifically to the mitigator and to upstream elements of the MLP of Ad12 DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenoviruses, Human↗
PubMed1992

Adenovirus DNA replication facilitates binding of the MLTF/USF transcription factor to the viral major late promoter within infected cells.

The activity of the adenovirus major late promoter is substantially increased as the infection proceeds from the early to late phase. To gain insight into the regulation of this promoter, we analyzed protein-DNA interactions by in vivo DMS and DNasel footprinting during the course of adenovirus infection. Little or no protein interaction at promoter sequences was detected early (5 hr) after infection but strong interactions at the major late transcription factor (MLTF/USF) binding site and at the TATA box were evident late (12 hr) after infection. Comparison of in vivo and in vitro footprints revealed that the in vivo interaction late after infection results from binding of the cellular transcription factor MLTF/USF. Nuclear extracts prepared from uninfected cells as well as cells harvested at 5 and 12 hr after infection contained similar levels of MLTF/USF footprint activity, therefore the lack of a detectable interaction early after infection is not due to reduced levels of the factor early in the viral growth cycle. Viral DNA replication was required for MLTF/USF binding at the major late promoter. These results indicate that DNA replication participates in the regulation of adenovirus late gene expression by facilitating the binding of a transcription factor to the major late promoter.

Adenoviruses, Human↗
PubMed1992

Recombination between adenovirus type 12 DNA and a hamster preinsertion sequence in a cell-free system. Patch homologies and fractionation of nuclear extracts.

We have previously described a cell-free recombination system derived from hamster cell nuclear extracts in which the in vitro recombination between a hamster preinsertion sequence, the cloned 1768 base-pair p7 fragment, and adenovirus type 12 (Ad12) DNA has been demonstrated. The nuclear extracts have now been subfractionated by gel filtration on a Sephacryl S-300 column. The activity promoting cell-free recombination elutes from the Sephacryl S-300 matrix with the shoulder and not the peak fractions of the absorbancy profile. By using these protein subfractions, in vitro recombinants have been generated between the p7 preinsertion sequence and the 60 to 70 map unit fragment of Ad12 DNA, which has previously shown high recombination frequency. In all of the analyzed recombinants thus produced in vitro, striking patchy homologies have been observed between the p7 and Ad12 junction sequences, and between Ad12 DNA or p7 DNA and pBR322 DNA. The patchy homologies are similar to those found earlier during the analyses of some of the junction sequences in integrated Ad12 genomes in Ad12-induced hamster tumor cell lines. Proteins in the shoulder fractions of the gel-filtration experiment can form specific complexes with double-stranded synthetic oligodeoxyribonucleotides corresponding to several p7 and Ad12 DNA sequences. These sequences participate in the recombination reactions catalyzed by the same column fractions in the shoulder of the absorbancy profile. Such proteins have not been found in the peak fractions. Further work will be required to ascertain that the cell-free recombination system mimics certain elements of the mechanisms of integrative recombination and to purify the cellular components essential for recombination.

Adenoviridae↗
PubMed1992

Alterations in the levels of expression of specific cellular genes in adenovirus-infected and -transformed cells.

There is increasing evidence that changes in the transcriptional program of cellular genes in virus-transformed cells can contribute to virus transformation. It is, therefore, important to study altered expression patterns of cellular genes in adenovirus-infected and -transformed cells. We have used 40 different cellular genes or gene segments as hybridization probes to analyze the cytoplasmic RNA from adenovirus type 2 (Ad2)-infected KB cells, from Ad5-transformed human cells (293) or from several Ad2- or adenovirus type 12 (Ad12)-transformed hamster cell lines. Many of the genes probed were not expressed in human or hamster cells. Transcription of the ADPRT and the heat shock protein 70 genes was increased in Ad2-infected KB cells and in 293 cells. In Ad2-infected KB cells, c-myc gene transcription was decreased. In 293 cells and in three adenovirus-transformed hamster cell lines (T637, BHK21-Ad2E1A-E1B, and BHK21-Ad2 HindIII-G), the transcription of the c-jun gene was increased, whereas c-myc transcription was decreased in the latter two cell lines. The data presented here demonstrate that, among 40 different mammalian gene probes, alterations in steady state levels of RNA were detected for five of these genes. These results suggest major alterations in transcription patterns in adenovirus-infected and -transformed cells.

Actins↗