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W Doerfler

Publications and source records attributed to W Doerfler.

At least 109 records · Page 6Linked to original sources
PubMed1992

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↗
PubMed1991

Patterns of DNA methylation in selected human genes in different Hodgkin's lymphoma and leukemia cell lines and in normal human lymphocytes.

The human genome, like many other genomes, harbors highly specific patterns of DNA methylation which have not yet been systematically studied. In a limited investigation on the genes for tumor necrosis factors-alpha and -beta, a surprising interindividual concordance in the patterns of DNA methylation at the nucleotide level has been demonstrated earlier by using the genomic sequencing method on DNA from individuals of very different ethnic origins. Patterns of DNA methylation could perhaps serve as indicators for genetic activities. These activities would not have to be restricted to gene transcription but could relate to other genetic activities in the cell. DNA methylation patterns are known to be cell type-specific. We have now initiated a study of these DNA patterns in human lymphocytes and in human cell lines of different malignant origins. Several of the proto-oncogenes, parts of the genes for tumor necrosis factor-alpha and -beta, the insulin receptor and lamin C have been used as hybridization probes. We have relied to some extent on the documented observation that the methylation patterns at 5'-CCGG-3' (HpaII/MspI) sequences yield a reflection of patterns at all 5'-CG-3' sequences. Three main types of patterns have been observed. Some of the probed segments are completely unmethylated; others are fully methylated, most of the areas are partly methylated exhibiting complex patterns at the 5'-CCGG-3' sites. In different tumor cell lines, different DNA methylation patterns are apparent for the same DNA probes. Comparisons of the methylation patterns in a given DNA segment between DNA from primary normal human lymphocytes and DNA from different tumor cell lines reveal changes in these patterns in several instances.

Base Sequence↗
PubMed1991

DNA methylation profiles in the human genes for tumor necrosis factors alpha and beta in subpopulations of leukocytes and in leukemias.

The genomic sequencing technique has been applied to assess the state of methylation in the DNA from human leukocyte subpopulations from healthy individuals and in the DNA from several individuals with myeloid or lymphatic leukemias or non-Hodgkin lymphomas. Leukocyte populations were purified by the high-gradient magnetic cell sorting technique. In the human tumor necrosis factor alpha (TNF-alpha) gene segment between nucleotides 300 and 1150, the specific methylation profile in the DNA from human granulocytes and monocytes is maintained in three cases of myeloid leukemia. In one such case, all 5-methyl-2'-deoxycytidine residues have been replaced by cytidine. In a chronic lymphatic T-cell leukemia, all 5-methyl-2'-deoxycytidine residues have been substituted by cytidine. In normal B lymphocytes, in two cases of chronic lymphatic B-cell leukemias and two cases of non-Hodgkin lymphomas, all 5'-CG-3' sequences in this gene segment are devoid of methylation. In the TNF-beta gene, DNA methylation is decreased in several examples of acute or chronic myeloid leukemias in comparison to normal human granulocytes or monocytes, whose DNA is almost completely methylated between nucleotides 700 and 900. In human T and B lymphocytes, the main producers of TNF-beta, in three instances of chronic lymphatic leukemias and two cases of non-Hodgkin lymphomas, all 5'-CG-3' sequences are unmethylated in this region. The DNA from the human HeLa cell line is highly methylated at all 5'-CG-3' sequences in the TNF-alpha and -beta genes. The TNF-alpha gene is transcribed in the cells of one case of acute myeloid leukemia in which the analyzed region of the TNF-alpha gene is completely unmethylated. The TNF-beta gene is not transcribed in any of the malignant cells tested.

Adult↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1991

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↗
PubMed1990

Establishment of de novo DNA methylation patterns. Transcription factor binding and deoxycytidine methylation at CpG and non-CpG sequences in an integrated adenovirus promoter.

The establishment of de novo patterns of DNA methylation in mammalian genomes is characterized by the gradual spreading of methylation, which has been documented to occur across an entire integrated adenovirus genome as well as at the nucleotide level in the integrated late E2A promoter of adenovirus type 2. By applying the techniques of genomic sequencing and dimethylsulfate or DNase I genomic footprinting in vivo, we have now demonstrated that the spreading of methylation in cell lines that carry the late E2A promoter with three in vitro pre-methylated 5'-CCGG-3' sequences initially involves a DNA domain of this promoter that is devoid of bound proteins. Subsequently, methylation further spreads to neighboring regions, and the patterns of complexed transcription factors are altered. Evidence has been adduced that DNA methylation at sequences homologous to the AP-1 and octamer binding factor sites interferes with protein binding. In contrast, the methylation of sequences in the vicinity of but not involving sequences homologous to an AP-2 site still permits the binding of proteins to these sites. It is significant that during the spreading of methylation a few 5'-CG-3' sequences can remain hemimethylated for several cell generations, before they also become methylated in both complements. Moreover, in cell line HE2, the integrated, heavily methylated late E2A promoter has been shown by the genomic sequencing technique to contain 5-methyldeoxycytidine residues, not only in all 5'-CG-3' dinucleotides but also in a 5'CA-3' and a 5'-CT-3' dinucleotide sequence. Hence, 5-methyldeoxycytidine occurs in a silenced mammalian DNA sequence also in dinucleotides other than 5'-CG-3'. This finding raises the question of whether 5-methyldeoxycytidine in non-5'-CG-3' dinucleotides can be maintained in the methylated state during continuous cell propagation.

Adenoviridae↗
PubMed1990

Eukaryotic DNA methylation: facts and problems.

Patterns of DNA methylation in complex genomes like those of mammalian cells have been viewed as indicators of different levels of genetic activities. It is as yet unknown how these complicated patterns are generated and maintained during cell replication. There is evidence from many different biological systems that the sequence-specific methylation of promoters in higher eukaryotes is one of the important factors in controlling gene activity at a long-term level. In general, the fifth nucleotide 5-methyldeoxycytidine can be considered as a modulator of protein-DNA interactions. The degree and direction of this modulation has to be assessed experimentally in each individual instance. The establishment of de novo patterns of DNA methylation is characterized by the gradual non-random spreading of DNA methylation by an essentially unknown mechanism. In this review, some of the general concepts of DNA methylation in mammalian systems are presented, and research currently performed in the authors' laboratory has been summarized.

Animals↗
PubMed1990

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↗
PubMed1990

A mitigator sequence in the downstream region of the major late promoter of adenovirus type 12 DNA.

Human adenovirus type 12 (Ad12) replicates in permissive human host cells, but undergoes an abortive infection cycle in non-permissive hamster cells. Ad12 DNA cannot replicate and late viral genes are not expressed in hamster cells, whereas most of the early viral mRNAs are synthesized. We have shown previously that the major late promoter of Ad12 DNA (Ad12 MLP; nucleotides -228 to +435 relative to nucleotide +1 as the site of transcriptional initiation) does not function in uninfected or in Ad12-infected hamster BHK21 cells. The transcriptional defect of Ad12 DNA in hamster cells has thus been, at least partly, localized to the viral MLP. As expected, this construct is active in permissive human cells. Here, we show that the sequence between nucleotides +249 and +435 in the Ad12 MLP is in some way responsible for the late transcriptional block of this promoter in hamster cells. An Ad12 MLP--CAT construct comprising nucleotides -228 to +248 shows striking activity in hamster cells, and its activity is very markedly enhanced in Ad2- or Ad12-infected hamster or human cells compared with the nucleotide -228 to +435 construct. By using exonuclease Bal31, a series of Ad12 MLP--CAT gene assemblies were constructed which carry deletions of increasing lengths in the downstream part of the Ad12 MLP. Activity measurements of these constructs in BHK21 and in HeLa cells have located the presumptive mitigator element to the Ad12 sequence between nucleotides +320 and +352 of the MLP. It is also demonstrated that in the nucleotide -228 to +248 MLP construct, transcription is initiated at the authentic Ad12 MLP cap site after the transfection of both hamster and human cells. The localization of this cap site in the nucleotide sequence of the Ad12 MLP indicates the similarity to the comparable start site in the MLP of Ad2 DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1990

The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector.

The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.

Animals↗