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Biomedical subjects

W Doerfler

Publications and source records attributed to W Doerfler.

At least 163 records · Page 9Linked to original sources
PubMed1987

Insertion of adenovirus type 12 DNA in the vicinity of an intracisternal A particle genome in Syrian hamster tumor cells.

In the adenovirus type 12 (Ad12)-induced hamster tumor T1111(2) about 10 Ad12 genome equivalents were integrated at different sites. One of the integrated copies proved unstable and was lost from the cellular genome or rearranged upon passage of the cell line, H1111(2), established from this tumor. This unstable site of junction between the left terminus of Ad12 DNA and hamster DNA and the preinsertion site from BHK21 hamster cells was cloned, sequenced, and analyzed. The junction site showed several peculiarities. At the left terminus of Ad12 DNA, the first 64 nucleotides were deleted. At a distance of 127 nucleotides to the left from this junction site, an internal dispersed fragment of Ad12 DNA comprising nucleotides 1290 to 1361 of the authentic Ad12 DNA sequence was inserted into cellular DNA in an inverted orientation relative to the complete Ad12 genome that was located in its vicinity. The 127-nucleotide sequence between the intact Ad12 genome and the separate 72-base-pair (bp) Ad12 DNA fragment was cellular, but it was not identical to the preinsertion sequence at this location. The sequences flanking the termini of the dispersed 72-bp Ad12 DNA fragment were characterized by direct repeats of 9 or 10 nucleotides. To the left of Ad12 nucleotide 1361 in the separate 72-bp fragment, about 620 cellular nucleotides followed which were identical at the occupied and at the preinsertion sites. It was conceivable that the separate 72-bp Ad12 DNA fragment and the cellular sequence of 127 bp to its right had been transposed en bloc from another unknown location. Abutting the 620 nucleotides of cellular DNA to the left of this block, the 3'-terminal sequence of an endogenous, intracisternal A particle (IAP) genome of hamster cells was detected. The possible significance of the proximity of an IAP sequence to an inserted Ad12 genome with respect to the transformation event, to the instability at this site, or to the transcriptional activity of this region is not known. The 620 bp of cellular DNA between the 72-bp Ad12 DNA fragment and the end of the long terminal repeat of the hamster IAP sequence was apparently of a unique type. Transcriptional activity was not found in the approximate region between nucleotides -620 (to the left) and +350 (to the right) relative to the site of Ad12 DNA insertion, but was found outside these boundaries.

Adenoviridae↗
PubMed1986

Characterization of a member of the highly repeated long interspersed rat DNA family with long open reading frames.

A highly repetitive long interspersed sequence from rat DNA has been isolated and partly characterized. This sequence comprises at least a 1300 base-pair and a 2400 base-pair EcoRI fragment and probably additional elements. The 2400 base-pair segment has been analyzed in detail. It appears to be part of the chromosomal DNA in rat cells. The 2400 base-pair repeat is likely to be distributed over several regions in the rat genome. The 2400 base-pair segment has been cloned, mapped for restriction sites, and part of its nucleotide sequence has been determined. The 2400 base-pair sequence is a member of a typical highly repetitive long interspersed sequence with high copy number and restriction site polymorphism. There are sequence homologies to mouse and human DNA. A striking homology has been detected to the flanking sequences of a repetitive mouse DNA sequence that has been described to be located adjacent to one of the kappa-immunoglobulin variable genes. Elements in the 2400 base-pair rat repeat are transcribed in cells from most rat organs and from several continuous rat cell lines. This RNA from rat cell lines was found polyadenylated or not polyadenylated. The nucleotide sequence of parts of the 2400 base-pair DNA segment revealed open reading frames for polypeptide sequences. Such open reading frames have been detected in two different segments of the 2400 base-pair DNA repeat. Open reading frames exist in the two complementary strands in the same DNA segment. The hypothetical polypeptide whose sequence has been determined in toto has a length of 190 amino acid residues and is enriched in hydrophobic amino acids, reminiscent of the amino acid composition in membrane proteins. Hence, it is conceivable that the 2400 base-pair repeat sequence from rat DNA, at least in part, encodes messenger RNAs that might be translated into functional proteins.

Amino Acid Sequence↗
PubMed1986

Chromosomal localization of integrated adenovirus DNA in productively infected and in transformed mammalian cells.

The in situ hybridization technique has been used to localize adenovirus type 12 (Ad12) genomes on specific chromosomes of Ad12-transformed hamster cells as well as on specific chromosomes of human cells productively infected with Ad12. Hamster cell lines T637 and A2497-2 contain 20 to 22 and 17 copies of Ad12 DNA, respectively, in an integrated form. The results of in situ hybridization experiments demonstrate that the Ad12 DNA molecules are predominantly located on chromosomes 2, 7, 11 to 13, and 15 in cell line T637, and on chromosomes 7, 20, and perhaps 16 to 19 in cell line A2497-2. These data further document that viral DNA is chromosomally integrated and does not persist in a huge extrachromosomal, episomal structure. There is evidence from previous work that adenovirus DNA can also be covalently linked to human cellular DNA after productive adenovirus infection of human cells. In keeping with these earlier findings, we have now been able to show by in situ hybridization experiments that early in productive infection, i.e., 2 or 6 hr after infection, Ad12 genomes are predominantly associated with a limited number of human chromosomes. It appears biologically significant that these chromosomes, which belong to groups A and CII, have been the same ones in several independent infection experiments. Other chromosomes may also carry smaller amounts of viral DNA. In situ hybridization of Ad12 DNA to chromosomes of uninfected human cells yields no significant chromosomal association of viral DNA. The signal to noise ratio of grain counts over chromosomes from Ad12-infected cells to areas devoid of chromosomes is 5.6. The results presented raise the question of whether Ad12 DNA can integrate at selective sites in human chromosomes and whether this insertion event plays an essential role in early steps of viral transcription or replication. There is no direct evidence to suggest a biologic function for viral DNA integration in the productive infection cycle.

Adenoviruses, Human↗
PubMed1986

Computer analyses on the structure of junction sites between adenovirus DNA and cellular DNA.

In previous work we have cloned and determined the nucleotide sequence of sites of linkage between mammalian cell DNA and foreign (viral) DNA. These investigations have been performed to study details of the mechanism of recombination in mammalian cells. Cloned lines of adenovirus-transformed cells have been used in the analyses because they constituted cell populations in which the foreign DNA had been fixed at certain sites in the cellular genomes. In the present investigation, these nucleotide sequences at sites of linkage have been subjected to computer-aided analyses. A number of sequence motifs have been determined; sequence features common to all junction sites have not been discernible. In some instances, patch homologies have been detected. At several sites of junction, the cellular DNA sequences seem to be transcriptionally active, even in cells that do not carry foreign DNA. Transcriptional activity may be a necessary but perhaps not sufficient precondition for recombination of mammalian DNA sequences with foreign DNA.

Adenoviridae↗
PubMed1986

N6-methyldeoxyadenosine residues at specific sites decrease the activity of the E1A promoter of adenovirus type 12 DNA.

The activity of eukaryotic promoters is highly sensitive to site-specific modifications by DNA methylations. We have used the E1A promoter of adenovirus type 12 (Ad12) DNA to investigate the effects of methylations at different promoter sites on its activity. The chloramphenicol acetyltransferase gene has served as an activity indicator. Activity of the E1A promoter is lost or markedly decreased by deoxycytidine methylation of two HpaII (5'-C-C-G-G-3') or seven HhaI (5'-G-C-G-C-3') sites upstream from the 3' located T-A-T-A signal. There are two T-A-T-A signals in the E1A promoter of adenovirus type 12 DNA, one T-A-T-T-A-T sequence starting at nucleotide 276 (5' located), a second T-A-T-T-T-A-A sequence starting at nucleotide 414 (3' located). Deoxycytidine methylations at two AluI (5'-A-G-C-T-3') sites downstream from the 5' located T-A-T-A signal have no effect on promoter activity. When one EcoRI (5'-G-A-A-T-T-C-3') or one TaqI (5'-T-C-G-A-3') sequence at 281 base-pairs upstream or 61 base-pairs downstream from the 5' located E1A T-A-T-A signal, respectively, is deoxyadenosine methylated, the promoter becomes inactive. Deoxyadenosine methylation at one MboI (5'-G-A-T-C-3') site, which is located 127 nucleotides downstream from the 5' located T-A-T-A signal, fails to decrease E1A promoter activity. There is no conspicuous anatomical relation of any of these sites to the two presumptive enhancer sequences in the E1A promoter. We conclude that 5-deoxymethylcytidine or N6-methyldeoxyadenosine residues have to be introduced at highly specific promoter sites to inactivate the promoter. These sites are probably different for different promoters.

Adenoviridae↗
PubMed1986

Expression of the influenza virus haemagglutinin in insect cells by a baculovirus vector.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) has played a major role in studies on the molecular biology of insect DNA viruses. Recently, this system has been effectively adapted as a highly efficient vector in insect cells for the expression of several mammalian genes. A cDNA sequence of the influenza (fowl plague) virus haemagglutinin gene has been inserted into the BamHI site of the pAc373 polyhedrin vector. Spodoptera frugiperda cells were co-transfected with this construct, pAc-HA651, and authentic AcNPV DNA. Recombinant virus was selected by adsorption of transfected cells to erythrocytes followed by serial plaque passages on S. frugiperda cells. We have determined the site of insertion of the haemagglutinin gene into the AcNPV genome by restriction enzyme cleavage and Southern blot hybridization analyses using haemagglutinin cDNA as a probe. The influenza haemagglutinin gene is located in the polyhedrin gene of AcNPV DNA. Immunofluorescent labelling, immunoprecipitation and immunoblot analyses with specific antisera revealed that S. frugiperda cells produce immune reactive haemagglutinin after infection with the recombinant virus. The haemagglutinin is expressed at the cell surface and has haemolytic capacity that has been activated by post-translational proteolytic cleavage. When chickens were immunized with S. frugiperda cells expressing haemagglutinin, they developed haemagglutinin-inhibiting and neutralizing antibodies and were protected from infection with fowl plague virus. These observations demonstrate that the haemagglutinin is processed in insect cells in a similar fashion as in fowl plaque virus-infected vertebrate cells and that it has full biological activity.

Amino Acid Sequence↗
PubMed1986

Trans effect of the E1 region of adenoviruses on the expression of a prokaryotic gene in mammalian cells: resistance to 5' -CCGG- 3' methylation.

The plasmid construct pSVO-CAT has been used to test adenovirus promoter activities in the unmethylated or methylated state. We have now observed that the E2A late promoter of adenovirus type 2 (Ad2) DNA also activated the chloramphenicol acetyltransferase (CAT) gene upon transfection of the pAd2E2A-CAT construct into mammalian cells, and it was inactivated by specific methylations of three 5' -CCGG- 3' sites. Similar results had been reported previously after microinjecting promoter-methylated constructs into oocytes of Xenopus laevis. Surprisingly, it was found that the pSVO-CAT construct, which lacked eukaryotic promoter sequences, was able to express the CAT gene upon transfection into human or hamster cells that harbored and constitutively expressed the E1 region of Ad2 or Ad5 DNA. In these cells, the expression of the pAd2E2A-CAT construct was enhanced, but it was only partly sensitive to DNA methylation, possibly because DNA methylation was counteracted directly or indirectly by E1 functions. The pSVO-CAT construct was also expressed in HeLa or BHK21 cells upon cotransfection with a plasmid carrying the HindIII-G fragment of Ad2 DNA that contained the E1A region and part of the E1B region. By mapping pSVO-CAT-specific RNAs, we could demonstrate that pSVO-CAT activity in Ad2- or Ad5-transformed cells was mediated by prokaryotic promoter-like sequences in the pBR322 section of the construct, and it presumably functioned via trans-activation mediated by the E1 region. This trans-activation of pSVO-CAT in adenovirus-transformed cells was partly insensitive to DNA methylation.

Acetyltransferases↗
PubMed1985

DNA methyltransferases: activity minigel analysis and determination with DNA covalently bound to a solid matrix.

We describe two methods that facilitate detection and characterization of DNA methyltransferases: activity gel analysis and the use of DNA-cellulose or DNA-Sepharose in DNA methylation reactions. The first permits identification of catalytic subunits, determination of the influence of proteolysis, and evolutionary or developmental studies. The second allows accurate and fast determination of DNA methyltransferase activities in crude extracts and during purification.

Catalysis↗
PubMed1985

The promoter of the late p10 gene in the insect nuclear polyhedrosis virus Autographa californica: activation by viral gene products and sensitivity to DNA methylation.

In lepidopteran insect cells infected with the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV), two major late viral gene products are expressed: the polyhedrin, a 28 000 mol. wt. protein which makes up the mass of the nuclear inclusion bodies, and a 10 000 mol. wt. protein (p10) whose function is unknown. The nucleotide sequences of these strong promoters conform to those of other eukaryotic promoters and are rich in AT base pairs. We used the pSVO-CAT construct containing the prokaryotic gene chloramphenicol acetyl transferase (CAT) to study the function of the p10 gene promoter in insect and mammalian cells. Upon transfection of the pAcp10-CAT construct, which contained 402 bp of the p10 gene of AcNPV DNA in the HindIII site of pSVO-CAT, CAT activity was determined. The p10 gene promoter was inactive in human HeLa cells and in uninfected Spodoptera frugiperda insect cells. The same promoter was active, however, in AcNPV-infected S. frugiperda cells and exhibited optimal activity when cells were transfected 18 h after infection with the insect virus. This finding demonstrated directly that the p10 gene promoter required other viral gene products for its activity in insect cells. The nature of these products was unknown. The p10 gene promoter sequence contained one 5'-CCGG-3' site 40 bp upstream from the cap site of the gene and two such sites 178 and 192 bp downstream from the ATG initiation codon of the gene. Since Drosophila DNA or S. frugiperda DNA contained no 5-methylcytosine or extremely small amounts of it, we were interested in determining the effect of site-specific methylations on the p10 gene insect virus promoter. Methylation at the 5'-CCGG-3' sites led to a block of this promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed1985

Species dependence of the major late promoter in adenovirus type 12 DNA.

In hamster cells human adenovirus type 12 (Ad12) is deficient in DNA replication and late gene expression whereas adenovirus type 2 (Ad2) can replicate. Functions located in the E1 region of the Ad2 or adenovirus type 5 (Ad5) genome can complement the deficiencies of the Ad12 genome in hamster cells, but, infectious viral particles are not produced. We have now investigated the activity of the major late promoter of Ad2 and of Ad12 DNA in human and hamster cells. This promoter governs the expression of most of the late viral functions. We have inserted the major late promoter (MLP) of Ad2 or of Ad12 DNA in front of the chloramphenicol acetyl transferase gene in the pSVO-CAT construct. Upon transfection into uninfected human and hamster cells, the pAd12MLP-CAT construct shows no significant activity; the pAd2MLP-CAT construct exhibits low activity. In Ad12-infected human cells, both constructs are active. These findings support the notion that other viral factors are required for MLP activity of Ad2 or Ad12 DNA in permissive human cells. In Ad2-infected hamster cells, both the pAd2MLP-CAT and the pAd12MLP-CAT constructs are active. Apparently, the Ad12 MLP can be activated by Ad2 functions, as already demonstrated for the entire Ad12 genome in double-infected cells or in Ad2- or Ad5-transformed cells superinfected with Ad12. In Ad12-infected hamster cells, however, the MLP of Ad12 DNA is inactive but that of Ad2 DNA shows activity. Thus the MLP of Ad12 DNA somehow differentiates between cellular auxiliary functions of different species.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenoviruses, Human↗
PubMed1985

DNA homologies between the genomes of Choristoneura fumiferana and Autographa californica nuclear polyhedrosis viruses.

The DNA sequence homology between the genomes of Choristoneura fumiferana and Autographa californica multicapsid nuclear polyhedrosis viruses (CfMNPV and AcMNPV) were compared by hybridization of nick-translated [32P]CfMNP[V DNA to restricted AcMNPV genome. In the presence of 5 x SSC and 50% formamide the CfMNPV DNA exhibited extensive homology to the AcMNPV genome. When the stringency conditions of hybridization were lowered, we observed hybridization to almost all the EcoRI fragments of AcMNPV. We then utilized the cloned EcoRI fragments from both genomes to obtain more detailed information, and to localize the hybridizing fragments on the EcoRI physical map of AcMNPV. It was clear that some CfMNPV clones hybridized to more than one fragment of the AcMNPV genome indicating that there has been some DNA sequence rearrangement in the AcMNPV genome.

Base Sequence↗
PubMed1985

Increased infectivity of extracellular adenovirus type 12.

Human adenovirus type 12 was propagated on human embryonic kidney cells, and the specific infectivities of intra- and extracellular virus particles were compared between 48 and 104 h after infection. Released virions exhibited a specific infectivity of up to 10 times higher than that of intracellular particles. The increased infectivity was apparently not due to enhanced rates of adsorption or penetration of extracellular virus. There may be a delay in the onset of viral DNA replication in intracellular virus-infected cells. Differences in the composition of intra- and extracellular virions were not recognized. Differences might also be sought in late expression or assembly of progeny virions or both. The data indicated that the virions released from the infected cells differed from those retained in the nucleus with respect to their specific infectivities. Active mechanisms of virus release have not yet been investigated.

Adenoviruses, Human↗
PubMed1985

Adenovirus types 2 and 5 functions elicit replication and late expression of adenovirus type 12 DNA in hamster cells.

Human adenovirus type 12 (Ad12) cannot replicate in hamster cells. There is a complete block of viral DNA replication and of the expression of late viral genes. Early viral functions are expressed. In contrast, hamster cells are permissive for human adenovirus type 2 (Ad2). Some of the Ad12-specific functions are insufficient to support viral replication in hamster cells, or else cellular functions are missing or inhibitory for Ad12 replication. It was shown that the block in the replication and late expression of the Ad12 genome in hamster cells could, at least in part, be complemented by Ad2 and adenovirus type 5 (Ad5) functions. When hamster cells were coinfected with Ad2 (or Ad5) and Ad12, both Ad2 (Ad5) and Ad12 DNA replicated. Ad2 (Ad5) virions were produced in double-infected hamster cells. The assembly of intact Ad12 virions was not detectable by the techniques used here. The analysis was further refined by Ad12 superinfecting Ad2- or Ad5-transformed cells which carried in an integrated form defined fragments of the Ad2 or Ad5 genome. Persistence and continued expression of the left terminus of the Ad2 or Ad5 DNA in these cells has been documented and helped to support replication and late expression of Ad12 DNA. It remains to be determined which of the E1 functions of Ad2 or Ad5 were responsible for the helper effect. Investigations on the biochemical mechanism of this complementation will entail studies on very complex viral and possibly cellular functions involved in the control of viral gene expression.

Adenoviruses, Human↗
PubMed1985

Lack of evidence for methylation of parental and newly synthesized adenovirus type 2 DNA in productive infections.

Methylations of highly specific sites in the promoter and 5' regions of eucaryotic genes have been shown to shut off gene activity and thus play a role in the long-term regulation of gene expression. It was therefore of interest to investigate whether site-specific DNA methylations could also play a role in adenovirus DNA in productive infections. It has been reported earlier that adenovirion DNA is not detectably methylated (U. Günthert, M. Schweiger, M. Stupp, and W. Doerfler, Proc. Natl. Acad. Sci. USA 73:3923-3927, 1976). In the present study, evidence for the methylation of cytidine residues in 5'-CCGG-3' and 5'-GCGC-3' sequences or the methylation of adenine residues in 5'-GATC-3' and 5'-TCGA-3' sequences in intranuclear adenovirus type 2(Ad2) DNA isolated and analyzed early (5 h) or late (24 h) after infection could not be obtained. In Ad2 DNA, 22.5% of all 5'-CG-3' dinucleotides reside in 5'-CCGG-3' and 5'-GCGC-3' sequences. Intranuclear viral DNA was examined by restriction endonuclease cleavage by using HpaII, MspI, HhaI, DpnI, or TaqI and Southern blot hybridizations. The HindIII fragments of Ad2 DNA served as hybridization probes. The data rendered it very unlikely that free intracellular adenovirus DNA in productively infected cells was extensively methylated. Thus, DNA methylation was not a likely element in the regulation of free adenovirus DNA expression in productively infected cells.

Adenoviruses, Human↗
PubMed1984

Deletion of cellular DNA at site of viral DNA insertion in the adenovirus type 12-induced mouse tumor CBA-12-1-T.

The adenovirus type 12 (Ad12)-induced mouse tumor CBA-12-1-T contains greater than 30 copies of viral DNA integrated into cellular DNA. One of the sites of linkage between the left terminus of Ad12 DNA and mouse DNA was cloned, mapped and sequenced by using conventional techniques. The preinsertion sequence was also cloned from normal CBA/J mouse DNA and sequenced. The sequence data and blotting analyses demonstrated that at the site of linkage nine nucleotide pairs of viral DNA and at least 1500 to 1600 nucleotide pairs of cellular DNA were deleted. Up to the site of linkage, the cellular DNA sequence in CBA-12-1-T tumor DNA and the preinsertion sequence in CBA/J mouse cells were identical. The site of Ad12 DNA integration was found to be located close to a site of transition from unique to repetitive cellular DNA sequences. The nucleotide sequence at the site of linkage and at the preinsertion site revealed palindromic stretches of 5 and 10 nucleotides pairs, respectively. Scattered patch homologies (8-10 nucleotide pairs long) were observed between adenoviral and cellular DNAs. A hypothetical model for DNA arrangements at the site of recombination is presented.

Adenoviridae↗