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Biomedical subjects

B Fleckenstein

Publications and source records attributed to B Fleckenstein.

At least 181 records · Page 10Linked to original sources
PubMed1982

Structure of nonintegrated, circular Herpesvirus saimiri and Herpesvirus ateles genomes in tumor cell lines and in vitro-transformed cells.

Nonintegrated, circular DNA molecules of Herpesvirus saimiri and Herpesvirus ateles were found in five lymphoid cell lines originating from tumor tissues or established by in vitro immortalization of T lymphocytes. The arrangement of unique (L) and repetitive (H) DNA sequences in circular viral genomes was analyzed by partial denaturation mapping followed by visualization with an electron microscope. Three types of circular viral DNA structures were found. (i) The virus-producing cell line RLC, which is derived from an H. ateles-induced rabbit lymphoma, contains circular viral genomes which consist of a single L-DNA and a single H-DNA region, both the same length as in virion DNA. (ii) The circular viral genomes of the nonproducer cell lines H1591 and A1601, in vitro transformed by H. saimiri and H. ateles, respectively, have deletions in the unique L-DNA region and larger H-DNA regions. Cell line A1601 lacks about 8% of virion L-DNA, and H1591 cells lack about 40% of viral L-DNA information. (iii) The nonproducing H. saimiri tumor cell lines 1670 and 70N2 harbor viral genomes with two L-DNA and two H-DNA regions, respectively. Both types of circular molecules have a long and a short L-segment. The sequence arrangements of circular DNA molecules from H. saimiri-transformed cell lines were compared with those of linear virion DNA by computer alignment of partial denaturation histograms. The L-DNA deletion in cell line H1591 was found to map in the right half of the virion DNA. Comparison of the denaturation patterns of both L regions of cell lines 1670 and 70N2 identified the short L regions as subsets of the long L regions. Thus, circular viral DNA molecules of all four nonproducer cell lines represent defective genomes.

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PubMed1981

Episomal and nonepisomal herpesvirus DNA in lymphoid tumor cell lines.

Tumor cell lines derived from Herpesvirus saimiri (H. saimiri)- and Herpesvirus ateles (H. ateles)-induced lymphomas of New World primates and rabbits contain multiple copies of viral genomes. Partial denaturation mapping and blot hybridizations of episomal DNA from lymphoid tumor cell line No. 1670 showed that a 12.5md-fragment is missing which represents the EcoRI D- and H-fragments of virion L-DNA. However, the missing piece can be demonstrated in total cellular DNA by reassociation kinetics, possibly because it persists in integrated form. Both episomal and nonepisomal H-DNA are heavily methylated in a number of the lymphoid cell lines, and methylation may be reduced by conventional methylation inhibitors (S-adenosyl homocystein, SIBA) as well as by the tumor promoting phorbol ester TPA.

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PubMed1981

Relationship between Herpesvirus ateles-associated nuclear antigen (HATNA) and the number of virus genome equivalents in HVA-carrying lymphoid lines.

A DNA-binding antigen (HATNA) was demonstrated in 7 out of 14 cell lines carrying Herpesvirus ateles (HVA) by the acid-fixed nuclear-binding technique. The seven HATNA-positive lines had means of 95, 96, 103, 177, 240, 343 and 326 virus genome equivalents/cell. For the seven HATNA-negative lines, the figures were 4, 8, 10, 33, 39, 72 and 110. This indicates a relationship between the number of HVA genome equivalents/cell and the detectability of HATNA. This association was independent of the virus producer status of the lines.

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PubMed1979

Methylation of Herpesvirus saimiri DNA in lymphoid tumor cell lines.

Several continuous lymphoid cell lines have been established from tumors induced by Herpesvirus saimiri. At least a portion of the viral DNA in the marmoset lymphoid cell line 1670, which does not produce detectable virus, is present as covalently closed circular episomal DNA. The use of restriction endonuclease digestion, transfer to nitrocellulose filters, and hybridization of the virus-specific DNA has produced strong evidence that viral DNA sequences present in total 1670 cell DNA and in isolated episomes are extensively methylated. The restriction endonuclease Hpa II has the same recognition sequence as Msp I but, unlike Msp I, fails to cleave when the C of the C-G dinucleotide is methylated. Viral DNA sequences of 1670 cells are refractory to cleavage by Hpa II but not Msp I; greater than 80% of the Hpa II cleavage sites appear to be methylated. Similarly, viral DNA sequences of 1670 cells are refractory to cleavage by Sma I (C-C-C-G-G-G) and Sac II (C-C-G-C-G-G) but not Sac I, Pvu II, or Pst I, which lack the dinucleotide C-G in their recognition sequences. Methylation of mammalian DNA has been previously found exclusively at C residues in the dinucleotide C-G. H. saimiri DNA sequences of another nonproducer cell line, 70N2, also appeared to be extensively methylated, but analysis of total cell DNA extracted from three virus-producing lymphoid lines revealed no evidence of methylation of viral DNA sequences. It remains to be seen if methylation of viral DNA plays a role in the lack of complete expression of H. saimiri genome information in nonproducing lymphoid cell lines.

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PubMed1978

Endogenous New World primate type C viruses isolated from owl monkey (Aotus trivirgatus) kidney cell line.

A type C virus (OMC-1) detected in a culture of owl monkey kidney cells resembled typical type C viruses morphologically, but was slightly larger than previously characterized mammalian type C viruses. OMC-1 can be transmitted to bat lung cells and cat embryo fibroblasts. The virions band at a density of 1.16 g/ml in isopycnic sucrose density gradients and contain reverse transcriptase and a 60-65S RNA genome composed of approximately 32S subunits. The reverse transcriptase is immunologically and biochemically distinct from the polymerases of othe retroviruses. Radioimmunoassays directed to the interspecies antigenic determinants of the major structure proteins of other type C viruses do not detect a related antigen in OMC-1. Nucleic acid hybridization experiments using labeled viral genomic RNA or proviral cDNA transcripts to normal cellular DNA of different species show that OMC-1 is an endogenous virus with multiple virogene copies (20-50 per haploid genome) present in normal owl monkey cells and is distinct from previously isolated type C and D viruses. Sequences related to the OMC-1 genome can be detected in other New World monkeys. Thus, similar to the Old World primates (e.g., baboons as a prototype), the New World monkeys contain endogenous type C viral genes that appear to have been transmitted in the primate germ line.

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PubMed1978

Herpesvirus ateles DNA and its homology with Herpesvirus saimiri nucleic acid.

Analysis of the structural organization of Herpesvirus ateles DNA shows that two types of viral DNA molecules are encapsidated in virions: (i) M-genomes, which contain 74% light sequences (L-DNA, 38% guanine plus cytosine) and 26% highly repetitive heavy sequences (H-DNA, 75% guanine plus cytosine), and (ii) defective H-genomes, which consist exclusively of repetitive H-DNA. The structure of M-genomes from H. ateles consists of an L-DNA region of about 70 x 10(6) daltons inserted between H-DNA termini of variable length. M-genomes with a shorter H-DNA region at one end of the molecule have a long stretch of H-DNA at the other end, resulting in a total molecular weight of 89.8 +/- 8.5 x 10(6). Thus it resembles the structure of M-genomes of H. saimiri. H-DNA of the two independent H. ateles isolates, strains 810 and 73, reveals different patterns after cleavage with restriction endonuclease Sma I. H-DNA of H. ateles 810 appears to consist of identical tandem repeat units with a molecular weight of 1,035,000; the H-DNA repeat unit of strain 73 is shorter (930,000 molecular weight). Corresponding DNA sequences of the two H. ateles strains (810 and 73) are completely homologous in cross-hybridizations. However, a discrete nucleotide sequence divergence between these virus strains is detected by measuring melting temperatures (T(m)) of DNA hybrid molecules. Some homology exists between H. ateles and H. saimiri DNA. Hybridization of L-DNA from H. ateles with L-DNA from H. saimiri shows about a 35% homology between the respective L-DNA sequences; the resulting heteroduplex molecules show a decrease of T(m) by 13.5 degrees C, corresponding to about a 9% mismatching in cross-hybridizing parts of L-regions. Very little homology is found between H-DNA of H. ateles and H. saimiri.

Base Sequence↗
PubMed1978

Episomal viral DNA in herpesvirus saimiri-transformed lymphoid cell lines.

Structural analysis of episomal viral genomes from two herpesvirus saimiri (HVS)-transformed tumour cell lines (No. 1670 and 70N2) showed that both types of episomes have a higher molecular weight than linear virion DNA. The arrangement of unique (L) and repetitive (H) DNA in No. 1670 episomes was studied by partial denaturation mapping. Part of the L-sequences present in linear virion DNA was found to be missing, part was found to be duplicated in the episomes. The episomal L-DNA regions were correlated with the known physical gene maps of linear HVS DNA.

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PubMed1977

The presence of Herpesvirus Saimiri genomes in virus-transformed cells.

Herpesvirus saimiri (H. salmiri) -transformed cells contained both types of viral DNA, unique L-DNA and highly repetetive H-DNA. DNA from spleen and lymph-node autopsies of two tumor-bearing marmoset monkeys contained 0.14-0.75% viral L-DNA -AND 0.115-1.08% H-DNA. This amount of H-DNA would be equivalent to the presence of 14-130 M-genomes per diploid tumor tissue cell. Six virus-transformed lymphoid cell lines, two of them virus-producing, contained 0.69-2.27% H-DNA and more than 0.72-1.95% L-DNA. These concentrations of H-DNA sequences correspond to 83-274 M-genome copies per lymphoid tissue culture cell. The majority of viral genomes in transformed non-producer lymphoid cell lines appeared to be defective, since part of the L-sequences present in virions were found to be deleted in the genome copies of transformed cells. There was a relative excess of repetitive H-sequences in all transformed cells in regard to the ratio of H-DNA/L-DNA in M-genomes of H. saimiri virions.

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PubMed1977

Episomal viral DNA in a Herpesvirus saimiri-transformed lymphoid cell line.

The lymphoid cell line #1670 has been derived from the infiltrated spleen of a tumor-bearing marmoset monkey infected with Herpesvirus saimiri. The cells contain both types of H. saimiri DNA, unique light (L-) DNA (36% cytosine plus guanine) and repetitive heavy (H-) DNA (71% cytosine plus guanine), without producing infectious virus. Viral DNA was found to persist in these cells as nonintegrated circular DNA molecules. Closed circular superhelical viral DNA molecules were isolated by three subsequent centrifugation steps: (i) isopycnic centrifugation in CsCl, (ii) sedimentation through glycerol gradients, and (iii) equilibrium centrifugation in CsCl-ethidium bromide. The isolated circles had a molecular weight of 131.5 +/- 3.6 x 10(6). This is significantly higher than the molecular weight of linear DNA molecules isolated from purified H. saimiri virions (about 100 x 10(6)). Partial denaturation mapping of circular molecules from #1670 lymphoid cells showed uniform arrangement of H- and L-DNA sequences in all circles. All denatured molecules contained two L-DNA regions (molecular weights of 54.0 +/- 1.8 x 10(6) and 31.5 +/- 1.3 x 10(6)) and two H-DNA regions (molecular weight of 25.6 +/- 1.9 x 10(6) and 20.0 +/- 0.8 x 10(6)) of constant length. Maps of both L-regions suggested that the sequences of the shorter L-DNA region were a subset of those of the longer region. The sequences of both L-regions had the same orientation. Circular molecules from H. saimiri-transformed lymphoid cell line #1670 appeared to represent defective genomes, containing only 75% of the genetic information present in L-DNA of H. saimiri virions.

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PubMed1976

Structure of Herpesvirus saimiri genomes: arrangement of heavy and light sequences in the M genome.

Herpesvirus saimiri contains two species of DNA molecules. (i) The M genome is composed of 70% light (L) DNA (36% cytosine plus guanine; density in CsCl, 1.695 g/ml), which consists of unique sequences, and 30% heavy (H) DNA (71% cytosine plus guanine; density, 1.729 g/ml). (ii) The H genome contains heavy sequences exclusively. H sequences in M and H genomes cross-hybridize completely and are cleaved identically by restriction endonuclease R-Sma I into four classes of fragments with molecular weights of about 360,000, 300,000, 130,000 and 40,000, respectively. H sequences are chains of identical repeat units in tandem arrangement. The molecular weight of each repeat unit is about 830,000. L sequences have no cleavage site for endo R-Sma I H sequences are terminally arranged at both ends of the M genome, as seen by electron microscopy after partial denaturation. The length of the individual heavy ends varies between 21 mum and less than 1 mum, whereas the light region is uniform in size (35.3+/-0.35 mum). As a rule, molecules with a long heavy end at one side have a short heavy end at the other side, thus giving rise to a limited size heterogeneity. Orientation of M DNA molecules by the denaturation map of the light region shows that the longer heavy end may be located at the left or at the right side of the M genome.

Base Sequence↗
PubMed1975

Repetitive sequences in complete and defective genomes of Herpesvirus saimiri.

Two types of Herpesvirus saimiri genomes can be isolated from purified virions: (i) the M genome is a double-stranded, liniear DNA molecule with a mean contour length corresponding to 89 times 10-6 daltons. The M genome contains about 70% of unique sequences (light DNA, 36% guanine plus cytosine) and 30% reiterated sequences (heavy DNA, 71% guanine plus cytosine). (ii) the H genome is composed of heavy DNA only and is more heterogeneous in size. The sequences in the H genome are up to 40-fold reiterated, indicating defectiveness of this type of genome. The repetitions in the H genome and the M genome cross-hybridize almost completely and have identical kinetic complexity (2.8 times 10-6 daltons). DNA infectivity studies by using the calcium phosphate and the DEAE-dextran method gave further evidence that H genomes are defective: no infectious virus was recovered from permissive cells treated with heavy DNA, whereas M genome-infected cells developed cytopathic changes after 11 to 56 days. Defective H genomes were present in the progeny virus two passages after transfection.

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