Recombination between adenovirus type 12 DNA and a hamster preinsertion sequence in a cell-free system. Patch homologies and fractionation of nuclear extracts.
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Adenoviridae↗
Biomedical subjects
W Doerfler
Publications and source records attributed to W Doerfler.
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↗
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↗
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↗
DNA methylation: eukaryotic defense against the transcription of foreign genes?
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Adenoviridae↗
Differential factor binding at the promoter of early baculovirus gene PE38 during viral infection: GATA motif is recognized by an insect protein.
Regulatory elements interacting with DNA-binding proteins have been investigated in the promoter sequence of the early PE38 gene in the Autographa californica nuclear polyhedrosis virus (AcNPV). A GATA motif located 50 nucleotides upstream of the PE38 transcriptional start site is recognized differentially in the course of infection. As demonstrated by footprint and gel mobility shift assays, the GATA sequences TTATCT are protected by nuclear extracts from uninfected Spodoptera frugiperda cells and from S. frugiperda cells early postinfection (p.i.) but not by S. frugiperda cell extracts isolated 40 h p.i. We have compared the binding capacity of the insect GATA-like protein with that of the vertebrate GATA-1 factor identified as erythroid-specific factor. Our results indicate that a factor present in mouse erythroleukemia cells, presumably GATA-1, can bind to the insect GATA motif and vice versa. Evidence from transient expression studies suggests that the mutated GATA sequences do not influence PE38 promoter activity in cell culture.
Animals↗
Interference with protein binding at AP2 sites by sequence-specific methylation in the late E2A promoter of adenovirus type 2 DNA.
The in vitro methylation of the +6, +24, and -215 located 5'-CCGG-3' sequences in the late E2A promoter of adenovirus type 2 (Ad2) DNA abrogates promoter function. 5-Methyldeoxycytidine (5-mC) at positions +6 and +24 in both or either of the two DNA complements in the late E2A promoter abolishes the formation of a high-molecular-mass DNA-protein complex that is essential for promoter function. The formation of this complex can be competed for by an oligodeoxyribonucleotide with a consensus AP2 sequence, but not by AP1, AP3, or CREB sequences. The AP2 sites comprise the +6 and +24 located 5'-CCGG-3' sequences in the late E2A promoter; the AP1, AP3 and CREB sequences are in their immediate vicinity. Methylation of either the +6 or the +24 5' -CCGG-3' sequence also compromises formation of the DNA-protein complex. A 40 nucleotide pair oligodeoxyribonucleotide encompassing the -215 5' -CCGG-3' site in the late E2A promoter can also form DNA-protein complexes which is not affected by the introduction of a 5-mC residue in the -215 position. The data suggest that the AP2 protein together with other proteins is involved in the generation of a transcription-activating complex with the late E2A promoter of Ad2 DNA, and the formation of this complex is completely abolished when both the +6 and +24 5'-CCGG-3' sequences are methylated.
Adenovirus Early Proteins↗
Heterologous recombination between Autographa californica nuclear polyhedrosis virus DNA and foreign DNA in non-polyhedrin segments of the viral genome.
We used the expression vector system of Autographa californica nuclear polyhedrosis virus (AcNPV) and Spodoptera frugiperda insect cells to study mechanisms of recombination in insect cells. We concentrated on the isolation and analysis of heterologous recombinants. The E1 region of human adenovirus type 2 (Ad2) was inserted into regions of the AcNPV genome which lacked apparent homologies to the polyhedrin region. Out of a total of 122 recombinant AcNPV plaques, which hybridized to Ad2 DNA in plaque annealing experiments, 13 recombinants proved heterologous, and 5 of these recombinants could be grown to titers that facilitated virus replication and further investigations of the recombinant DNA. Restriction and Southern blot analyses for all of the recombinants and nucleotide sequence determinations for one of them permitted the mapping of the sites of foreign DNA integration into the AcNPV genome for the heterologous recombinants. These sites were located in the EcoRI-C (map units 42.5-52.4), the EcoRI-L (map units 69.5-72.5), the EcoRI-O (map units 32.6-34.5), and the EcoRI-Q (map units 88.2-89.7) segments of the plaque isolate E AcNPV genome. Two of the heterologous recombinants carried the insert in the EcoRI-L fragment. The nucleotide sequence determinations across the sites of junction between the AcNPV DNA and the foreign (Ad2) DNA in one of the heterologous recombinants, AcNPV-Ad2E1-D, revealed no sequence similarities at or close to the sites of junctions. A short sequence of six nucleotides was deleted from the original EcoRI-O sequence of AcNPV at the site of insertion. The inserted Ad2E1 DNA fragment comprised nucleotides 183-2763; thus nucleotides at the termini had been deleted. In the usual polyhedrin gene-located recombinants, the foreign Ad2 DNA segment was fused to the polyhedrin promoter and recombined presumably via polyhedrin sequence segments in the vector into the polyhedrin gene of AcNPV. In one of the control recombinants, AcNPV-Ad2E1-192, the Ad2E1 DNA segment between nucleotides 1 and 3117 (out of 3322 original nucleotides) was inserted in an inverted orientation between nucleotides -115 and +735 of the polyhedrin gene of AcNPV. This particular polyhedrin sequence was deleted in the process. It was uncertain how this recombinant had been generated. The infectivities of the polyhedrin-located recombinant AcNPV-Ad2E1-192 and of the five heterologous recombinants were compared by single-cycle growth curves to the infectivity of non-recombinant AcNPV.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Patterns of DNA methylation are indistinguishable in different individuals over a wide range of human DNA sequences.
Patterns of DNA methylation at 5'-CCGG-3' and 5'-GCGC-3' sequences were determined in about 570 kb, equivalent to about 0.02% of the human genome, by using HpaII and HhaI restriction endonucleases, respectively, and randomly selected cosmid clones of human DNA as hybridization probes. Many of these human DNA sequences were of the repetitive type. The DNAs from human lymphocytes, from a mixture of all blood cells or from several established human cell lines (HeLa, KB, 293, or DEV) were included in these analyses. In the segments of the human genome investigated, the patterns of DNA methylation were characterized by often completely or partly methylated 5'-CCGG-3' or by partly methylated 5'-GCGC-3' sequences. Even among individuals of different genetic origins (East-Asian or Caucasian), these patterns of DNA methylation proved indistinguishable by the method applied. The cytokine-dependent stimulation of human lymphocytes to replicate in culture did not affect the stability of these patterns. In the same DNA sequences from several human cell lines, much lower levels of DNA methylation were observed. In human cell lines some of the investigated sequences were unmethylated. The results presented lend credence to the notion that the human genome exhibits highly cell type-specific patterns of DNA methylation which are often indistinguishable among different individuals even of different genetic backgrounds.
Cells, Cultured↗
Persistence or loss of preimposed methylation patterns and de novo methylation of foreign DNA integrated in transgenic mice.
In cultured mammalian cells, foreign DNA can be integrated into the host genome. Foreign DNA is frequently de novo methylated in specific patterns with successive cell generations. The sequence-specific methylation of promoter sequences in integrated foreign DNA is associated with the long-term inactivation of eukaryotic genes. We have now extended these experiments to studies on transgenic mice. As in previous work, a construct (pAd2E2AL-CAT) has been used which consists of the late E2A promoter of adenovirus type 2 (Ad2) DNA fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT). This construct has been integrated in the non-methylated in the 5'-CCGG-3' premethylated form in the genomes of transgenic mice. DNA from various organs was analyzed by HpaII/MspI cleavage to assess the state of methylation in 5'-CCGG-3' sequences. The results demonstrate that the transgenic construct is in general stable. Non-methylated constructs have remained partly non-methylated for four generations or can become de novo methylated at all or most 5'-CCGG-3' sequences in the founder animal. Preimposed patterns of 5'-CCGG-3' methylation have been preserved for up to four generations beyond the founder animal. In the testes of two different founder animals and two F1 males, the transgenic DNA has become demethylated by an unknown mechanism. In all other organs, the transgenic DNA preserves the preimposed 5'-CCGG-3' methylation pattern. In the experiments performed so far we have not observed differences in the transmission of methylation patterns depending on whether the transgene has been maternally or paternally inherited. The 5'-CCGG-3' premethylated transgene does not catalyze CAT activity in several organs, except in one example of the testes of an animal in which the transgenic construct has become demethylated. In contrast, when the nonmethylated construct has been integrated and remained largely non-methylated, CAT activity has been detected in extracts from some of the organs.
Adenoviridae↗
Proteins encoded in the 81.2- to 85.0-map-unit fragment of Autographa californica nuclear polyhedrosis virus DNA can be translated in vitro and in Spodoptera frugiperda cells.
We have previously demonstrated that five open reading frames exist in the nucleotide sequence of the 81.2- to 85.0-map-unit (m.u.) segment of plaque isolate E of Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The corresponding polypeptides are 9.8, 12.1, 36.6, 25.0, and 48.2 kDa in size (C. Oellig, B. Happ, T. Müller, and W. Doerfler, J. Virol. 63:1494, 1989), and we have investigated whether these proteins can be translated in infected cells. On subfragments of this viral DNA segment, mRNAs were selected from AcNPV-infected Spodoptera frugiperda insect cells at different times postinfection (p.i.). The in vitro translation of these RNAs in a rabbit reticulocyte-derived cell-free translation system yielded polypeptides of approximately 10 to 11, 12 to 14, 28, 36 to 38, and 48 to 50-kDa which were commensurate in size with the theoretically expected values. mRNAs for the 28- and 48- to 50-kDa proteins were identified by their translation products at 6 h p.i., and mRNAs for the 10- to 11-, 12- to 14-, and 36- to 38-kDa proteins were identified by their translation products at 12 h p.i. We constructed an AcNPV recombinant which carried in its polyhedrin gene the 3.9-kbp EcoRI-HindIII (81.8 to 84.8 m.u.) subfragment of the EcoRI J segment. Nucleotide sequence determinations revealed that the intact polyhedrin promoter lay adjacent to the additional 81.8- to 84.8-m.u. fragment in this recombinant. In S. frugiperda cells, which were infected with the recombinant AcNPV, a protein of 36 to 38 kDa was detected at 44 h p.i. in larger amounts than after infection with the nonrecombinant virus. However, there was no evidence for larger amounts of RNA derived from the 81.8- to 84.8-m.u. fragment in recombinant-infected cells. Recombinant-infected cells lacked the polyhedrin polypeptide. The synthesis of the 36- to 38-kDa polypeptide in recombinant- or AcNPV-E-infected S. frugiperda cells could be demonstrated by immunoprecipitation experiments. Peculiarly, this polypeptide was present in the cytoplasm as a 64-kDa glycoprotein. These data corroborate the notion that at least some of the open reading frames encoded in the 81.2- to 85.0-m.u. segment of AcNPV can be expressed in S. frugiperda cells.
Amino Acid Sequence↗
Adenovirus type 2 VAI RNA transcription by polymerase III is blocked by sequence-specific methylation.
Sequence-specific methylation of the promoter and adjacent regions in mammalian genes transcribed by RNA polymerase II leads to the inhibition of these genes. So far, RNA polymerase III-transcribed genes have not been investigated in depth. We therefore studied methylation effects on the RNA polymerase III-transcribed VAI gene of adenovirus type 2 DNA. The VAI gene contains 20 5'-CG-3' dinucleotides, of which 4 (20%) can be methylated by HpaII (5'-CCGG-3') and HhaI (5'-GCGC-3'). Three of these 5'-CG-3' sequences are located close to the internal regulatory region of the VAI segment. An unmethylated, a 5'-CCGG-3'- and 5'-GCGC-3'-methylated, and a 5'-CG-3'-methylated pUC18 construct containing the VAI and VAII regions were transfected into mammalian cells. In many experiments, an inactivating effect of 5'-CCGG-3' and 5'-GCGC-3' DNA methylation on the VAI region was not observed. In contrast, methylation of all 20 5'-CG-3' sequences in the VAI region by a CpG-specific DNA methyltransferase from Spiroplasma species did interfere with VAI transcription. Transcription of the VAI- and VAII- and of the VAI-containing constructs was also shown to be inhibited in an in vitro cell-free transcription system after the constructs had been methylated at the 5'-CCGG-3' and 5'-GCGC-3' sequences or at all 5'-CG-3' sequences. When an oligodeoxyribonucleotide which carried the internal control block A of the VAI region was methylated at three 5'-CG-3' sequences, the formation of a complex with HeLa nuclear proteins was abrogated. The results presented support the notion that the VAI gene transcribed by the DNA-dependent RNA polymerase III is also inactivated by methylation of the decisive 5'-CG-3' sequences.
Adenoviruses, Human↗
Spreading of DNA methylation across integrated foreign (adenovirus type 12) genomes in mammalian cells.
The establishment of de novo-generated patterns of DNA methylation is characterized by the gradual spreading of DNA methylation (I. Kuhlmann and W. Doerfler, J. Virol. 47:631-636, 1983; M. Toth, U. Lichtenberg, and W. Doerfler, Proc. Natl. Acad. Sci. USA 86:3728-3732, 1989; M. Toth, U. Müller, and W. Doerfler J. Mol. Biol. 214:673-683, 1990). We have used integrated adenovirus type 12 (Ad12) genomes in hamster tumor cells as a model system to study the mechanism of de novo DNA methylation. Ad12 induces tumors in neonate hamsters, and the viral DNA is integrated into the hamster genome, usually nearly intact and in an orientation that is colinear with that of the virion genome. The integrated Ad12 DNA in the tumor cells is weakly methylated at the 5'-CCGG-3' sequences. These sequences appear to be a reliable indicator for the state of methylation in mammalian DNA. Upon explantation of the tumor cells into culture medium, DNA methylation at 5'-CCGG-3' sequences gradually spreads across the integrated viral genomes with increasing passage numbers of cells in culture. Methylation is reproducibly initiated in the region between 30 and 75 map units on the integrated viral genome and progresses from there in either direction on the genome. Eventually, the genome is strongly methylated, except for the terminal 2 to 5% on either end, which remains hypomethylated. Similar observations have been made with tumor cell lines with different sites of Ad12 DNA integration. In contrast, the levels of DNA methylation do not seem to change after tumor cell explanation in several segments of hamster cell DNA of the unique or repetitive type. Restriction (HpaII) and Southern blot experiments were performed with selected cloned hamster cellular DNA probes. The data suggest that in the integrated foreign DNA, there exist nucleotide sequences or structures or chromatin arrangements that can be preferentially recognized by the system responsible for de novo DNA methylation in mammalian cells.
Adenoviridae↗
Processing of influenza virus hemagglutinin in insect cells.
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Animals↗
Patterns of DNA methylation--evolutionary vestiges of foreign DNA inactivation as a host defense mechanism. A proposal.
The proposals in this review are based on experimental work on the integration of foreign DNA in mammalian cells, on the establishment of specific de novo patterns of DNA methylation, and on the inhibition of transcription by the sequence-specific methylation of promoter sequences. It is suggested that eukaryotic cells have developed several mechanisms of defense against the uptake, integration, and continued expression of foreign DNA. In the course of evolution and continuing at present, cells have been exposed to foreign DNA, entire genomes or fragments of them. A particularly problematic organ system in that respect must be digestive tract in higher organisms. The defense mechanisms are thought to be the following: (i) degradation and/or excretion of foreign DNA; (ii) excision and loss of previously integrated DNA from the host genome; (iii) targeted inactivation of foreign genes by sequence-specific methylation. Genes whose products could be advantageous to the transformed cells can somehow be selectively excluded from this silencing mechanism. In part, the specificity of de novo methylation must reside in the DNA methyltransferase systems of the host cell. However, nucleotide sequence, structure, and chromatin arrangement in the foreign DNA could also play an important role. Since defense processes must have been activated many times in evolution, patterns of DNA methylation as they can be observed today, may represent vestiges of evolution, i.e. the sum total of selective de novo methylations, possibly demethylations, and mutations.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
The significance of DNA methylation patterns: promoter inhibition by sequence-specific methylation is one functional consequence.
This paper presents a review of previously published results from my laboratory on the inactivating or inhibiting function of sequence-specific methylation on promoter activity. In this study, viral promoters, mostly those from adenovirus type 2 (Ad2) or type 12 (Ad12), have been used. It has also been shown that the transcriptional block of these methylated viral promoters can, at least partly, be reversed by a transactivating protein or by the presence of a strong enhancer. We have also adduced evidence that the methylation of the late E2A promoter of Ad2 DNA at positions +6 and +24 from the cap site of this promoter interferes with the binding of one or several proteins at these particular sites, at least when 50 or 73 base-pair long fragments of this promoter have been used for studies on protein binding. With a 377 base-pair fragment, binding differences between the unmethylated and the 5'-CCGG-3' methylated late E2A promoter are not obvious. By applying the genomic sequencing technique developed by Church & Gilbert (1984), the patterns of methylation in all 5'-CG-3' dinucleotides in the late E2A promoter in the active or inactive state in different Ad2-transformed cell lines have been determined. It has been found that 5-methyldeoxycytidine residues introduced into foreign DNA, which is then integrated into the mammalian cell genome, can lead to the spreading of methylation starting from the point of initial methylation. We have begun to investigate whether certain patterns of methylation in mammalian DNA can also influence biological processes other than promoter activity. We have developed a cell-free system using nuclear extracts from hamster cells to study recombination between Ad12 DNA and hamster pre-insertion sites into which Ad12 DNA had previously integrated. The DNAs used in recombination experiments are in the unmethylated or the methylated form. Some speculative aspects have also been discussed in this review. Could existing patterns of methylation in mammalian (human) DNA represent composites of several interdigitating patterns each one of which might have a different signal value? Can a 5-methyldeoxycytidine group in DNA modulate DNA-protein interactions in a positive or negative way, for proteins which could have positively or negatively regulating functions? Patterns of methylation appear to be relatively stable over many years for cell lines propagated in culture. Are patterns of methylation stable also in different parts of the human chromosome? To what extent are these patterns inheritable?
Adenovirus Early Proteins↗
Revertants of adenovirus type-12-transformed hamster cells have lost part of the viral genomes.
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Adenoviridae↗
Specificity and mode of cleavage of the pH 4.0 endonuclease from adenovirus type 2 - infected KB cells.
Adenovirus type 2 or lambda DNA was digested with the pH 4.0 endonuclease, purified from adenovirus 2-infected KB cells. The enzyme produces a limit digest of approximate size in the range of 140-210 base pairs long. The termini of the DNA fragments generated by the endonuclease digestion had 3'-P and 5'-OH groups. The 3' and 5' end groups of the products were analyzed. Our data indicate that 3' end group was a purine (68-76%), dA occuring about twice the frequency of dG. The 5' end group was either dG or dC with equal frequency. Data obtained by treatment of the 5' labeled endonuclease product of lambda DNA with single-strand specific S1 nuclease from Asperigillus oryzae or exonuclease VII from Escherichia coli indicated that the majority of the products had a short 5' protruding ends. The mode of cleavage of this endonuclease seems to be through initial formation of several single-strand breaks with some base specificity. If these breaks are at close proximity on opposite strands, double-stranded fragments with protruding ends are generated.
Adenoviruses, Human↗