Search PubMed⌕ Search

Biomedical subjects

W Doerfler

Publications and source records attributed to W Doerfler.

At least 181 records · Page 10Linked to original sources
PubMed1984

Identification and localization of reiterated sequences in the Choristoneura fumiferana MNPV genome.

The genome of Choristoneura fumiferana nuclear polyhedrosis virus (CfMNPV) contained reiterated sequences interdispersed in four locations. These regions, termed RS, were found in EcoRI fragments A, F, E and B. The sequences were identified by hybridization of the fragment EcoRI-A to a Southern blot of EcoRI-digested viral DNA. Further confirmation and more precise localization of the RS sequences was obtained by hybridization of nick-translated 32P-labeled EcoRI-E fragment to Southern blots of viral DNA digested with EcoRI, BamHI, XbaI and Bg/II. Hybridization of 32P-labeled EcoRI-E to HindIII blots of viral DNA revealed the presence of a 'ladder' consisting of eight fragments. The three fragments of the ladder with the lowest sizes represented the HindIII fragments, O, PQ and R. The other five fragments were submolar in amount, in that they could not be seen in ethidium bromide-stained gels and probably represented minor virus variants that arose after passage of virus in larvae. Each variant was distinguished from the others by an additional insertion of 210 bp into the EcoRI-B fragment of the genome.

Chromosome Mapping↗
PubMed1984

DNA methylation of three 5' C-C-G-G 3' sites in the promoter and 5' region inactivate the E2a gene of adenovirus type 2.

The E2a gene of human adenovirus type 2 (Ad2) encodes the 72-kilodalton DNA-binding protein. We previously described perfect inverse correlations between the methylation of all 5' C-C-G-G 3' sequences in the Ad2 E2a gene in virus-transformed hamster cells containing viral DNA sequences in an integrated state and the extent to which this gene is expressed. We subsequently showed that in vitro methylation of all 14 5' C-C-G-G 3' sequences in the cloned E2a gene by prokaryotic Hpa II DNA methyltransferase leads to transcriptional inactivation after microinjection into Xenopus laevis oocytes. The unmethylated cloned E2a gene is expressed in these cells. We report here the construction of partly methylated clones of the E2a gene. In the promoter (5')-methylated construct, three 5' C-C-G-G 3' sequences at the 5' end of the subclone were methylated. One of these sites is located 215 base pairs (bp) upstream (bp 26,169 of Ad2 DNA), and two sites are located 5 and 23 bp downstream from the cap site (bp 25,931 and 25,949 of Ad2 DNA) of the E2a gene. This construct was transcriptionally inactive upon microinjection into nuclei of X. laevis oocytes. In the gene (3')-methylated construct, 11 5' C-C-G-G 3' sequences in the main part of the transcribed gene region were methylated in vitro. This construct was transcribed in X. laevis oocytes, and at least some of the Ad2-specific RNA synthesized was initiated at the same sites as in Ad2-infected human KB cells. Both mock-methylated constructs were transcribed into Ad2-specific RNA in X. laevis oocytes. These results demonstrate that DNA methylations at or close to the promoter and 5' end of the E2a gene cause transcriptional inactivation. Perhaps only one methyl group would be adequate for inactivation; in vivo methylation of more than one cytosine may be a form of safeguard or redundancy.

Adenoviruses, Human↗
PubMed1984

Mapping of Early and Late Transcripts Encoded by the Autographa californica Nuclear Polyhedrosis Virus Genome: Is Viral RNA Spliced?

Early and late transcripts were mapped on the Autographa californica nuclear polyhedrosis virus genome by Northern blotting and hybridization with the cloned viral EcoRI fragments. At least 11 early and about 90 late RNAs were compared with over 32 polypeptides synthesized by in vitro translation of hybrid-selected RNA. The latter method, of course, had its limitations also and did not guarantee that all viral RNAs could be detected in this way. A comparison of cytoplasmic and total cellular RNAs showed no clear-cut differences in their size distributions. We found that there were more RNA classes than corresponding proteins encoded by them and mapped by in vitro translation. By using the Berk-Sharp method and analyses of DNA-RNA hybrids by one-dimensional or two-dimensional neutral and alkaline gel electrophoreses, we were unable to adduce evidence for RNA splicing in this viral system. Minor splices, particularly at sites close to the termini of RNA molecules, could not be excluded.

Journal Article↗
PubMed1984

Transcription of overlapping sets of RNAs from the genome of Autographa californica nuclear polyhedrosis virus: a novel method for mapping RNAs.

The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) contains a double-stranded, supercoiled circular genome of 126 to 129 kilobase pairs in length. In cultured Spodoptera frugiperda insect cells the virus replications, and early and late phases of viral genome expression are discernible. We previously mapped 5 early and at least 32 late different viral polypeptides on the viral genome (H. Esche, H. Lübbert, B. Siegmann, and W. Doerfler, EMBO J. 1:1629-1633, 1982). However, at the same time we located 11 early and more than 90 late different size-classes of viral RNA on the AcNPV genome. Evidence for extensive RNA splicing in this virus system has not been adduced, although tiny splices cannot yet be ruled out (H. Lübbert and W. Doerfler, J. Virol. 50:497-506, 1984). The large number of AcNPV transcripts and the apparent lack of splicing have raised tantalizing questions about the mechanisms involved in the expression of AcNPV DNA and its regulation. It is also unknown how the widely differing numbers of RNAs and polypeptides can be correlated. For this reason, we have started to analyze in detail the map locations of some of the RNA size-classes in three different segments of the viral genome. For this purpose a novel method has been devised which will prove useful for the analyses of transcriptional patterns in complex viral genomes. The EcoRI fragments J, O through F, and Q, comprising viral DNA segments between 81.8 and 86.4, 32.6 and 41.0, and 88.2 and 89.7 map units, respectively, were investigated. Surprisingly, overlapping sets of viral RNAs of various lengths and with apparently common 3' termini in EcoRI fragments J (seven size-classes) and O through F (four size-classes) or with common 5' termini in EcoRI fragment Q (two size-classes) have been detected. At present, the functional significance of this mode of transcription is unknown. EcoRI fragment Q of AcNPV DNA encodes a 10,000-molecular-weight polypeptide which is expressed abundantly late after infection. The function of this protein has not yet been elucidated. The promoter and 5' part of the gene for the 10,000-molecular-weight polypeptide have been sequenced, and we have shown that at least two RNAs of different lengths are transcribed in this region and initiated at one site of three nucleotides. Studies on the expression of the AcNPV genome have revealed interesting properties not commonly found in other eucaryotic systems.

Animals↗
PubMed1983

Integration of viral DNA into the genome of the adenovirus type 2-transformed hamster cell line HE5 without loss or alteration of cellular nucleotides.

Hamster cell line HE5 has been established from primary LSH hamster embryo cells by transformation with adenovirus type 2 (Ad2) (1). Each cell contains two to three copies of integrated Ad2 DNA (2, 3). We cloned and sequenced the sites of junction between viral and cellular DNAs. The terminal 10 and 8 nucleotides of Ad2 DNA were deleted at the left and right sites of junction, respectively. The integrated viral DNA had an internal deletion between map units 35 and 82 on the Ad2 genome. At the internal site of deletion, the remaining viral sequences were linked via a GT dinucleotide of unknown origin. From HE5 DNA, the unoccupied sequence corresponding to the site of insertion was also cloned and sequenced. Part of this sequence was shown to be expressed as cytoplasmic RNA in HE5 and primary LSH hamster embryo cells. The viral DNA had been inserted into cellular DNA without deletions, rearrangements or duplications of cellular nucleotides at the site of insertion. Thus, insertion of Ad2 DNA appeared to have been effected by a mechanism different from that of bacteriophage lambda in Escherichia coli and from that of retroviral genomes in vertebrates. It was conceivable that the terminal viral protein (4) was somehow involved in integration either on a linear or a circularized viral DNA molecule.

Adenoviruses, Human↗
PubMed1983

Excision of amplified viral DNA at palindromic sequences from the adenovirus type 12-transformed hamster cell line T637.

In the DNA of the adenovirus type 12 (Ad12)-transformed hamster cell line T637 approximately 20-22 viral DNA molecules per cell are covalently linked to cellular DNA. Spontaneously arising morphological revertants of T637 cells have lost the bulk of the viral DNA. We have been able to mimic the excision event of viral DNA, as it occurs during reversion, by autoincubation of isolated nuclei from T637 cells. The same Ad12 DNA sequences, which had been deleted in morphological revertants, proved highly sensitive to endogenous nucleases in isolated nuclei of T637 cells. Viral DNA sequences, which persisted in the revertants, are resistant to endogenous nucleases in isolated T637 nuclei. All attempts to clone the nuclease-sensitive sites of Ad12 DNA in cell line T637 have so far failed. After denaturation and renaturation of T637 DNA followed by treatment with S1 nuclease, large fold-back structures of DNA have been found. These snap-back structures were derived from precisely those viral DNA restriction fragments which were uncloneable. The fragments containing palindromic sequences were both highly sensitive to endogenous nucleases in isolated T637 nuclei and were absent from the DNA of all revertant cell lines. Moreover, the palindromic sequences are susceptible to the phage T4-specific endonuclease VII which specifically attacks cruciform structures in DNA. The peculiar structures at the termini of integrated Ad12 DNA molecules are highly sensitive to endogenous nucleases in isolated nuclei. These nucleases may be related to the reversion event.

Adenoviruses, Human↗
PubMed1983

Can DNA methylation regulate gene expression?

The E2a region of the Ad2 genome encodes the Ad2-specific DBP. An inverse correlation between the level of DNA methylation at the 5'-CC*GG-3' sites of the E2a region and the extent of expression of DBP has been demonstrated in Ad2-transformed hamster cell lines (Vardimon et al. 1980). Four different leaders are used in the transcription of the E2a region in cells productively infected with Ad2. The leader located at coordinate 75 on the viral genome is used early after infection and the other three leaders are used late after infection (Chow et al. 1979). The analysis of the integration patterns of the viral DNA in the Ad2-transformed cell lines has revealed that the early leader is deleted in the cell lines which do not express the DBP (Vardimon and Doerfler 1981). The late leader located at coordinate 72 on the viral genome is present. The region encoding that late leader has been subcloned, and the cytoplasmic RNA from the cell line which expresses the DBP has been analyzed. It has been shown that the late leader is used in transformed cells. Hence the absence of the early leader cannot be the immediate reason for the lack of expression of the DBP. Correlations between DNA methylation and the absence of gene expression may indicate that methylation regulates gene expression or that methylation is the consequence of lacking gene expression. In order to decide between these alternatives an in vitro system has been employed. The HindIII A fragment of the Ad2 DNA which encodes the DBP has been methylated in vitro by the HpaII DNA methyltransferase. Methylated or unmethylated HindIII A fragment has been microinjected into the nuclei of Xenopus laevis oocytes. Unmethylated HindIII A fragment has been found to be expressed as specific viral RNA, whereas no viral RNA can be found in oocytes microinjected with methylated HindIII A fragment. The possibility of a nonspecific inhibitory factor in the methylated DNA preparation has been ruled out by the simultaneous microinjection of sea urchin histone gene DNA together with the methylated HindIII A fragment. Histone genes are expressed, while the expression of the methylated viral gene is blocked. By using the single-strand-specific endonuclease S1 we have shown that in Xenopus laevis oocytes initiation of transcription of the E2a region starts exactly at the same site as in Ad2 productively infected cells. These results provide direct evidence for the notion that DNA methylation at specific sites is involved in the regulation of gene expression.

5-Methylcytosine↗
PubMed1983

Quantitative determination of 5-methylcytosine in DNA by reverse-phase high-performance liquid chromatography.

A method to separate the four major bases (cytosine, guanine, thymine and adenine) and the two minor modified bases (5-methylcytosine and 6N-methyladenine) in DNA has been developed. For optimal separation, several different buffer systems are available for isocratic elution. The 12 5-methylcytosine (5-mC) residues in the plasmid pBR322 can be determined with a deviation of less than 3% of the expected value and have been used for internal standardization. Formic acid hydrolysis of bases and probably of DNA does not lead to the deamination of cytosine or 5-mC and thus can be used routinely for DNA hydrolysis. Adenovirus or baculovirus DNA does not contain detectable amounts of 5-mC. The distribution of 5-mC in hamster cell DNA appears to be nonrandom in that different 5'-CpG-3'-containing restriction sites are methylated to different extents.

5-Methylcytosine↗
PubMed1983

Autographa californica nuclear polyhedrosis virus (AcNPV) DNA does not persist in mass cultures of mammalian cells.

Autographa californica nuclear polyhedrosis virus (AcNPV) is one of the most extensively studied baculoviruses. We have investigated whether AcNPV or its DNA can replicate and/or persist in cultures of mammalian cells. Human HeLa cells or primary human embryonic kidney cells, simian CV1 cells, hamster BHK21 (B3) cells or Muntiacus muntjak cells growing in monolayer cultures were used in these studies. Cells were inoculated with AcNPV at multiplicities ranging from 0.1 to 100 PFU/cell. Subsequently, the inoculated cells were investigated for virus production and for the replication and the persistence of viral DNA. Extracts of inoculated cells were also screened for the occurrence of AcNPV-specific RNA. AcNPV does not multiply in any of the cell lines studied. Viral DNA replication or transcription could not be detected by blotting and nucleic acid hybridization experiments using nick-translated, cloned viral probes. Furthermore, there was no evidence for the persistence of viral DNA or of fragments of viral DNA in mass cultures of mammalian cells. A puzzling homology between pBR322 plasmid DNA and human, simian, and hamster DNAs was detected. Since mammalian cells can take up and integrate any foreign DNA at very low frequency, it cannot be ruled out by the approach chosen that a very small number of cells might have incorporated and fixed viral DNA in their genomes. As this caveat is always pertinent for any population of cells exposed to foreign DNA, this reservation does not appear to be of particular significance in safety considerations when working with baculoviruses or any virus for that matter.

Animals↗
PubMed1983

Proof of recombination between viral and cellular genomes in human KB cells productively infected by adenovirus type 12: structure of the junction site in a symmetric recombinant (SYREC).

In previous work we have described a symmetric recombinant (SYREC1) between Ad12 DNA and human KB cell DNA. This recombinant DNA molecule has been generated during productive infection and is encapsidated into virions. From the DNA of a similar symmetric recombinant (termed SYREC2) between the left terminus of Ad12 DNA and human KB cellular DNA, the site of linkage between the two DNAs was cloned and sequenced. It was demonstrated that the first 2081 Ad12 nucleotides counting from the left viral terminus are conserved and linked to a sequence of GC-rich (70.4% G + C) KB cell DNA which occurs about 20 times per cellular genome. Except for a common 5'-CTGGC-3' pentanucleotide between the Ad12 DNA and KB cell DNA sequences, extensive patch homologies were not apparent at the site of junction. Similarly, comparisons of the deleted Ad12 DNA sequence and the cellular sequence replacing it did not reveal patch homologies. The 304 bp abutting the Ad12 terminus were shown to hybridize to KB cell DNA. These results provided definitive proof for the occurrence of recombinants between viral and cellular DNAs in human cells productively infected by Ad12 as previously shown by less direct experiments (Burger and Doerfler, 1974; Schick et al., 1976). Across the site of junction, an open reading frame exists which extends the truncated 54-kDal protein of the E1b region of Ad12 DNA for another 66 amino acids encoded by KB cellular DNA. This sequence is terminated by two UGA translational termination signals. The hypothetical protein has not yet been isolated.

Adenoviridae↗
PubMed1983

Expression of the chloramphenicol acetyltransferase gene in mammalian cells under the control of adenovirus type 12 promoters: effect of promoter methylation on gene expression.

The effect of DNA methylation at specific promoter sites on gene expression was tested by using a sensitive and quantitative assay system. The plasmid pSVO CAT contains the prokaryotic gene chloramphenicol acetyltransferase (CAT) and a HindIII site in front of it for experimental promoter insertion. Upon insertion into pSVO CAT, the E1a and protein IX gene promoters from adenovirus type 12 (Ad12) DNA were capable of mediating CAT expression upon transfection in mouse cells. In many viral and nonviral eukaryotic genes, DNA methylation at highly specific sites in the promoter region can attain a regulatory function in gene expression. One of the important sites is the 5' C-C-G-G 3' sequence. The CAT-promoting activity of the early simian virus 40 promoter in plasmid pSV2 CAT is refractory to methylation by the Hpa II or Hha I DNA methyltransferase at 5' C-C-G-G 3' or 5' G-C-G-C 3' sequences, respectively, because this promoter lacks such sites. The CAT coding sequence of this plasmid carries four Hpa II and no Hha I sites. Methylation of the Hpa II sites in the coding region does not affect expression. The E1a promoter of Ad12 DNA comprising the leftmost 525 base pairs of the viral genome carries two 5' C-C-G-G 3' and three 5' G-C-G-C 3' sites upstream from the leftmost "TATA" signal. Methylation of the Hpa II or Hha I sites incapacitates this promoter. The promoter of protein IX gene of Ad12 DNA contains one 5' C-C-G-G 3' and one 5' G-C-G-C 3' site downstream and two 5' G-C-G-C 3' sites greater than 300 base pairs upstream from the TATA motif and probably outside the promoter. The protein IX promoter is not inactivated by methylation of these sites. These data demonstrate that critical 5' methylations in the promoter region decrease or eliminate transcription; methylations of sites too far upstream or probably any sites downstream from the TATA site do not affect expression.

Acetyltransferases↗
PubMed1983

Loss of viral genomes from hamster tumor cells and nonrandom alterations in patterns of methylation of integrated adenovirus type 12 DNA.

The insertion stability and DNA methylation patterns of integrated adenovirus type 12 (Ad12) genomes were investigated in Ad12-induced tumors and in tumor cell lines established from them as a function of time of passage under culture conditions. Upon subcultivation of cells from some of the tumors, the viral genomes were eliminated, apparently in a stepwise process with segments of the left termini of Ad12 DNAs persisting the longest. Morphological variants of these tumor cells lost all viral DNA and yet retained the oncogenic phenotype. All 13 independently isolated clones from one revertant line were devoid of Ad12 DNA. It could not be ruled out that very short sequence elements of viral DNA, such as promoters or enhancing sequences, could have persisted in these variants. The extent of viral DNA methylation was minimal in Ad12-induced tumors, although the viral genome was not extensively expressed, if at all. Upon passage in culture, the levels of viral DNA methylation increased. It was interesting that establishment of the final methylation pattern of integrated Ad12 DNAs required many cell generations after the fixation of foreign DNA in the host genome. The shift in methylation was nonrandom. The late parts of the inserted viral genomes became methylated more extensively than did the early gene segments.

Adenoviruses, Human↗
PubMed1982

Nucleotide sequence at the site of junction between adenovirus type 12 DNA and repetitive hamster cell DNA in transformed cell line CLAC1.

The hamster cell line CLAC1 originated from a tumor induced by injecting human adenovirus type 12 (Ad12) into newborn hamsters. Each cell contained about 12 copies of viral DNA colinearly integrated at two or three different sites. We have cloned and sequenced a DNA fragment comprising the site of junction between the left terminus of Ad12 DNA and cellular DNA. The first 174 nucleotides of Ad12 DNA were deleted at the site of junction. Within 40 nucleotides, there were one tri-, two tetra-, one penta-, and one heptanucleotide which were identical in the 174 deleted viral nucleotides and the cellular sequence replacing them. In addition, there were patch-type homologies ranging from octa- to decanucleotides between viral and cellular sequences. There is no evidence for a model assuming adenovirus DNA to integrate at identical cellular sites. The cellular DNA sequence corresponding to the junction fragment was cloned also from BHK21 (B3) hamster cells and sequenced. Up to the site of linkage with viral DNA, this middle repetitive cellular DNA sequence was almost identical with the equivalent sequence from CLAC1 hamster cells. Taken together with the results of previously published analyses (11, 12), the data suggest a model of viral (foreign) DNA integration by multiple short sequence homologies. Multiple sets of short patch homologies might be recognized as patterns in independent integration events. The model also accounts for the loss of terminal viral DNA sequences.

Adenoviruses, Human↗