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Biomedical subjects

M T Muller

Publications and source records attributed to M T Muller.

At least 55 records · Page 3Linked to original sources
PubMed1994

Association between the p170 form of human topoisomerase II and progeny viral DNA in cells infected with herpes simplex virus type 1.

Endogenous host topoisomerase II acts upon herpes simplex virus type 1 (HSV-1) DNA in infected cells (S.N. Ebert, S.S. Shtrom, and M.T. Muller, J. Virol. 56:4059-4066, 1990), and cleavage is directed exclusively at progeny viral DNA while parental DNA is resistant. To evaluate the possibility that HSV-1 induces topoisomerase II activity which could account for the preferential cleavage of progeny viral DNA, we assessed topoisomerase II cleavage activity on cellular and viral DNA substrates before and after the initiation of viral DNA replication. We show that cleavage of a host gene in mock-infected cells was similar to that observed in HSV-1-infected cells, regardless of whether viral DNA replication had occurred. In addition, quantitative measurements revealed comparable amounts of topoisomerase II activity in infected and mock-infected cells; thus, HSV-1 neither induces nor encodes its own type II topoisomerase and cleavages in vivo are due to a preexisting host topoisomerase. Human cells contain two isozymes of topoisomerase II (p170 and p180), encoded by separate genes. Through the use of isozyme-specific antibodies, we demonstrate that only p170 was found to be cross-linked to HSV-1 DNA even though both forms were present at nearly constant levels in HSV-1-infected cells. Immunofluorescence revealed that by 6 h postinfection, p170 becomes redistributed and localized to sites of active viral DNA synthesis. The data suggest that p170 gains preferential access to replicated viral DNA molecules, which explains why topoisomerase II activity is concentrated on progeny DNA.

Amino Acid Sequence↗
PubMed1993

[Hearing impairment in nursing home patients not easily screened when relying on nurses' and patients' opinions].

OBJECTIVE: To assess the diagnostic value of the opinion of the nursing staff and the patients themselves about hearing impairment of somatic nursing home patients. DESIGN: Descriptive. SETTING: Nursing Home and Rehabilitation Centre Amstelhof in Amsterdam. METHOD: All newly admitted patients in the period October 1989 to mid-May 1990 were asked to give an opinion about their hearing; the nursing staff were asked for an opinion as well. The results of these two measurements were compared with the results of the screening audiogram, used as the gold standard. For both the nursing staff opinion and the patients' opinion sensitivity, specificity and predictive values were calculated. RESULTS: 79 patients were included in this study. The sensitivity of the opinion of the nursing staff was 54%, the specificity 80%. The predictive values of the positive and negative test results were 59% and 76% respectively. The sensitivity of the patients' opinion was 68% and the specificity 80%. The predictive value of the positive test result was 65% and that of the negative test result 82%. CONCLUSION: Neither the opinion of the nursing staff, nor the opinion of the patients were valid methods to identify hearing-impaired nursing home patients. We recommended paying more attention to tracing hearing impairment in nursing home patients: the nursing staff should be trained and a screening audiogram should be made of all patients admitted.

Aged↗
PubMed1992

Eukaryotic topoisomerase II cleavage of parallel stranded DNA tetraplexes.

A guanine-rich single-stranded DNA from the human immunoglobulin switch region was shown by Sen and Gilbert [Nature, (1988) 334, 364-366] to be able to self-associate to form a stable four-stranded parallel DNA structure. Topoisomerase II did not cleave the single-stranded DNA molecule. Surprisingly, the enzyme did cleave the same DNA sequence when it was annealed into the four-stranded structure. The two cleavage sites observed were the same as those found when this DNA molecule was paired with a complementary molecule to create a normal B-DNA duplex. These cleavages were shown to be protein-linked and reversible by the addition of salt, suggesting a normal topoisomerase II reaction mechanism. In addition, an eight-stranded DNA molecule created by the association of a complementary oligonucleotide with the four-stranded structure was also cleaved by topoisomerase II despite being resistant to restriction endonuclease digestion. These results suggest that a single strand of DNA may possess the sequence information to direct topoisomerase II to a binding site, but the site must be base paired in a proper manner to do so. This demonstration of the ability of a four-stranded DNA molecule to be a substrate for an enzyme further suggests that these DNA structures may be present in cells.

Animals↗
PubMed1991

A predictive model for DNA recognition by the herpes simplex virus protein ICP4.

The herpes simplex virus (HSV) type 1 immediate early protein ICP4 is an essential regulatory enzyme that binds DNA directly in order to stimulate or repress gene expression. The degree of transaction is related to the locations and affinities of the ICP4 binding sites. A number of binding sites have been identified; some sites showed obvious homology to one another, and these were called consensus ICP4 binding sites. Other binding sites did not appear to be related, and these were termed non-consensus sites. We hypothesized, however, that a single model could describe all ICP4 binding sites, given the appropriate characterizations of sites. We performed statistical analyses on a set of ICP4 binding sites and found that the bases important for defining binding were located within a 13 base region. Missing contact analyses on several high-affinity binding sites revealed the same 13 base region as important for critical protein-DNA contacts. From these data we derived the consensus sequence RTCGTCNNYNYSG, where R is purine, Y is pyrimidine, S is C or G, and N is any base. In addition, we found that a better profile for ICP4 binding sites involves use of a matrix of base proportions from the binding site data; sites are analyzed by calculating the Matrix Mean score. We show that this Matrix Mean model could accurately predict the locations of novel ICP4 binding sites. Finally, we analyzed the entire HSV-1 genome for potential ICP4 binding sites and speculate about what these results suggest for the role of ICP4 in viral gene regulation.

Amino Acid Sequence↗
PubMed1991

Aggregates of oligo(dG) bind and inhibit topoisomerase II activity and induce formation of large networks.

DNA cleavage by eukaryotic type II DNA topoisomerase (EC 5.99.1.3) was strongly inhibited by an oligonucleotide containing 10 dGua residues. Catalytic activities of topoisomerase II, as measured by relaxation and decatenation reactions, were also inhibited by oligo(dG)10. Inhibition was specific to oligo(dG)10; other oligonucleotides, nucleotides, or single-stranded DNAs tested did not influence the activity of topoisomerase II. Oligo(dG)10 did not inhibit other activities such as restriction enzymes. Although the enzyme neither binds nor cleaves oligo(dG)10, inhibition can be explained by the finding that topoisomerase II binds tightly with aggregated oligo(dG) structures (estimated to contain between 20 and 30 molecules of monomeric oligo(dG)10) that form spontaneously prior to addition of enzyme. These aggregated oligo(dG)-topoisomerase complexes are large networks that can be pelleted by a 20-min centrifugation step in a Microfuge. Western blotting with a monoclonal antibody confirmed that topoisomerase II is trapped in these pellets. The ability of the enzyme to form large DNA-protein networks could be a biochemical mechanism by which topoisomerase II might promote or participate in chromosome condensation in vivo prior to mitosis.

Animals↗
PubMed1990

Eukaryotic topoisomerase II preferentially cleaves alternating purine-pyrimidine repeats.

Alternating purine-pyrimidine sequences (RY repeats) demonstrate considerable homology to the consensus sequence for vertebrate topoisomerase II (Spitzner and Muller (1988) Nucleic Acids Res. 16: 1533-1556). This is shown below and positions that can match are underscored. RYRYRYRYRYRYRYRYRY = alternating purine-pyrimidine 18 bp RNYNNCNNGYNGKTNYNY = topoisomerase II consensus sequence (R is purine, Y is pyrimidine, K is G or T.) Topoisomerase II cleavage reactions were performed (in the absence of inhibitors) on a plasmid containing a 54 base RY repeat and the single strong cleavage site mapped to the RY repeat. Analysis of this DNA on sequencing gels showed that the enzyme cleaved a number of sites, all within the 54 base pair RY repeat. Topoisomerase II also made clustered cleavages within other RY repeats that were examined. Quantitative analysis of homology to the consensus sequence, as measured by the match of a site to a matrix of base proportions from the consensus data base (the matrix mean), showed that both the locations and the frequencies of cleavage sites within RY repeats were proportional to homology scores. However, topoisomerase II cleaved RY repeats preferentially in comparison to non-RY sites with similar homology scores. The activity of the enzyme at RY repeats appears to be proportional to the length of the repeat; additionally, GT, AC and AT repeats were better substrates for cleavage than GC repeats.

Base Sequence↗
PubMed1990

Amblyomma americanum: identification of tick salivary gland antigens from unfed and early feeding females with comparisons to Ixodes dammini and Dermacentor variabilis.

Salivary gland antigens involved in host resistance to tick feeding by Amblyomma americanum (lone star tick) have been identified. Gland extracts from unfed and partially fed 12-, 48-, 72-, 96-, and 120-hr females and their corresponding midgut tissues were analyzed by immunoblotting with sera from naturally immune and hyperimmune sheep and rabbits. Polypeptides at 90, 75, 58, 45, 33, and 23 kDa from the salivary glands of A. americanum females were consistently observed with antibodies from both sheep and rabbits. No antigens unique to tick midgut tissue were detected with immune sera. Female Dermacentor variabilis and Ixodes dammini shared 90- and 45-kDa salivary gland antigens with A. americanum, and these may represent conserved polypeptides. We speculate that some of the salivary gland antigens represent components of tick cement, while others are playing some other yet undetermined role in tick feeding.

Animals↗
PubMed1990

Topoisomerase II cleavage of herpes simplex virus type 1 DNA in vivo is replication dependent.

The genome of herpes simplex virus type 1 contains a large number of recognition sites for eucaryotic DNA type II topoisomerase. Topoisomerase II sites were identified by means of the consensus sequence described previously (J.R. Spitzner and M.T. Muller, Nucleic Acids Res. 16:5553-5556, 1988) and then confirmed by sequencing DNA cleavages introduced by purified topoisomerase II. In vivo, host topoisomerase II also introduced double-stranded DNA breaks in the viral genome at sites predicted by the consensus sequence. Host topoisomerase II acted on all immediate-early genes as well as on genes from other temporal classes; however, cleavages were not detected until 4 to 5 h postinfection and were most intense at 10 h postinfection. Topoisomerase II cleavages were not detected when viral DNA replication was prevented with phosphonoacetic acid. These data indicate that, although progeny viral genomes are acted upon by host topoisomerase II, this enzyme either does not act on parental viral genomes before DNA replication or acts on them with such low efficiency that cleavages are beyond our limit of detection. The findings suggest that host topoisomerase II is involved in aspects of viral replication at late times in the infectious cycle.

Animals↗
PubMed1989

Application of a degenerate consensus sequence to quantify recognition sites by vertebrate DNA topoisomerase II.

A consensus sequence has been derived for vertebrate topoisomerase II cleavage of DNA (Spitzner, J. R. and Muller, M. T. (1988) Nucleic Acid. Res. 16, 5533-5556). An independent sample of 65 topoisomerase II sites (obtained in the absence of topoisomerase II inhibitors) was analyzed and found to match the consensus sequence as well as enzyme sites determined in the presence of the anti-tumor drug 4'-(9-acridinyl-amino)-methanesulfon-m-anisidide (m-AMSA). As originally described, conventional application of the consensus sequence afforded accuracy in the prediction of the locations but not the frequencies of topoisomerase II cleavages. In the present report, we describe a new method which quantitatively discriminates sites from nonsites, called the 'matrix mean' method (the mean match of a site to the matrix of base proportions from the original consensus sequence derivation). Furthermore, we derived a second method, called the 'unique score' model, which predicts frequency of topoisomerase II activity at a cleavage site. In the unique score method both DNA strands of a site are examined to determine the total number of the consensus positions that match on at least one strand of a potential site. From the new data base of 65 topoisomerase II sites, cleavages were scored for relative cleavage strength. Linear regression analysis showed a significant (p less than 0.01) correlation between the unique score and cleavage strength. The study was extended to show that the unique score model accurately and quantitatively predicts topoisomerase II sites either in the absence or presence of m-AMSA using the same consensus sequence.

Amsacrine↗
PubMed1989

Characterization of ixodid tick salivary-gland gene products, using recombinant DNA technology.

Ticks secrete an array of lesion-maintenance factors into the host via the salivary glands while feeding, some of which elicit an immune response by the host that adversely affects the ability of the tick to feed and reproduce. Our approach to characterizing these factors has been to make expression libraries from mRNA of salivary glands (from unfed and 3-day-feeding Amblyomma americanum females) which will serve as sources of the genes (clones) that code for them. Thus far, we have detected 10 positive clones in primary screens using polyspecific antiserum from rabbits hyperimmunized to 3-day-feeding tick salivary glands. We also report making a cDNA library from whole unfed females, and a genomic library from whole unfed ticks, which will serve as additional sources of genetic information for characterizing salivary-gland secretory products. Immunoblots of salivary glands from A. americanum females feeding for various intervals (unfed, and 12, 48, 72, and 96 h) revealed the presence of several prominent polypeptides (90 & 45 kDa) when probed with the same rabbit antiserum that was used to screen the expression library. Ixodes dammini had several immunogens in common with A. americanum at 96 h (90, 45, 43 and 23 kDa). We plan to use monospecific antiserum raised to antigens detected in our immunoblots (e.g. 90 kDa) to further screen the expression libraries, in addition to using the polyspecific antiserum already in hand. We discuss the future use of the salivary-gland genes for characterizing secretory products which facilitate attachment to the host (cement) and maintain the lesion during the lengthy feeding interval.

Animals↗
PubMed1989

DNA binding and gene regulation by the herpes simplex virus type 1 protein ICP4 and involvement of the TATA element.

We report the results of fine mapping the sequences responsible for negative regulation of immediate-early (IE) gene 3 by its own gene product, ICP4. Affinity-purified ICP4 binds the transcriptional start site of IE gene 3 and protein-protein interactions induce a secondary mobility shift that footprints exactly as the primary complex. Since these DNA-protein complexes contain ICP4, it is likely that the two differ only in stoichiometry of protein. Additional data show that the DNA-binding domain recognized by ICP4 can be embedded as a cassette in foreign DNA and that native ICP4 will recognize and bind the resulting DNA. In two different immediate-early promoters, the ICP4 binding site can be located either 3' or 5' of the TATA box; however, the ICP4 site is rotationally displaced from the transcription factor IID (TFIID) site by a roughly one-half helical turn, suggesting that ICP4 and TFIID are on the opposite helical face when bound at their respective sites. In the IE1 and IE3 promoters, binding of ICP4 causes an alteration in the helical geometry of the minor groove of the TATA region as visualized by copper footprinting. In contrast, TATA hypersensitivity was not detected in the glycoprotein D promoter (an early gene promoter containing the ICP4 site separated from TATA by eight helical turns) or in an artificial IE3 promoter construct in which the TATA-A4 separation was increased from 2.5 to roughly 5 helical turns. Such stereospecific and distance-dependent conformational alterations in the TATA box under the influence of ICP4 binding may be important in the repression of immediate-early genes.

DNA↗
PubMed1988

Single-strand DNA cleavages by eukaryotic topoisomerase II.

A new purification method for eukaryotic type II DNA topoisomerase (EC 5.99.1.3) is described, and the avian enzyme has been purified and characterized. An analysis of the cleavage reaction has revealed that topoisomerase II can be trapped as a DNA-enzyme covalent complex containing DNA with double-stranded and single-stranded breaks. The data indicate that DNA cleavage by topoisomerase II proceeds by two asymmetric single-stranded cleavage and resealing steps on opposite strands (separated by 4 bp) with independent probabilities of being trapped upon addition of a protein denaturant. Single-strand cleavages were directly demonstrated at both strong and weak topoisomerase II sites. Thus, a match to the vertebrate topoisomerase II consensus sequence (sequence; see text) (N is any base, and cleavage occurs between -1 and +1) [Spitzner, J.R., & Muller, M.T. (1988) Nucleic Acids Res. 16, 5533-5556)] does not predict whether a cleavage site will be single stranded or double stranded; however, sites cleaved by topoisomerase II that contain two conserved consensus bases (G residue at +2 and T at +4) generally yield double-strand cleavage whereas recognition sites lacking these two consensus elements yield single-strand cleavages. Finally, single-strand cleavages with topoisomerase II do not appear to be an artifact caused by damaged enzyme molecules since topoisomerase II in freshly prepared, crude extracts also shows the property of single-strand cleavages.

Animals↗
PubMed1988

A consensus sequence for cleavage by vertebrate DNA topoisomerase II.

Topoisomerase II, purified from chicken erythrocytes, was reacted with a large number of different DNA fragments and cleavages were catalogued in the presence and absence of drugs that stabilize the cleavage intermediate. Cleavages were sequenced to derive a consensus for topoisomerase II that predicts catalytic sites. The consensus is: (sequence; see text) where N is any base and cleavage occurs at the indicated mark between -1 and +1. The consensus accurately predicts topoisomerase II sites in vitro. This consensus is not closely related to the Drosophila consensus sequence, but the two enzymes show some similarities in site recognition. Topoisomerase II purified from human placenta cleaves DNA sites that are essentially identical to the chicken enzyme, suggesting that vertebrate type II enzymes share a common catalytic sequence. Both viral and tissue specific enhancers contain sites sharing strong homology to the consensus and endogenous topoisomerase II recognizes some of these sites in vivo.

Animals↗
PubMed1988

The nuclear scaffold exhibits DNA-binding sites selective for supercoiled DNA.

Binding of exogenous DNA to the nuclear scaffold was investigated using a plasmid DNA (pBR322, EcoRI site deleted) of various topological forms and nuclear subfractions with different levels of nuclear DNA depletion. When supercoiled DNA was incubated with histone-depleted nuclei (nuclear halo), a dose-dependent binding of the DNA occurred, whereas no binding was observed with relaxed and linear forms of DNA. The bound DNA was released upon linearization with BamHI or digestion of the scaffolding structure with proteinase K. Extensive digestion of the halo with micrococcal nuclease generated additional sites which bind both relaxed and linear DNA. In the presence of a large excess of calf thymus DNA, these sites were effectively blocked and the specificity to supercoiled DNA was restored. The binding of all forms of DNA was abolished by heat-denatured DNA. There was no detectable change in linking number of the scaffold-associated supercoils. Competitive binding was observed between supercoiled DNAs with unrelated sequences, indicating that no specific nucleotide sequence is required for the binding. RNA was found to be a weak competitor. A DNA binding assay performed on electrophoretic blots of solubilized nuclear scaffold revealed a protein component with apparent molecular weight of 120,000 which retained selective binding to supercoils. These results suggest that the nuclear scaffold possesses DNA-binding sites for torsionally strained domains of chromatin and that an integral protein factor is involved in the binding. Implications of the findings are discussed in connection with proposed functions of the nuclear scaffold and topoisomerase II.

Animals↗
PubMed1988

Stabilization of type I topoisomerase-DNA covalent complexes by actinomycin D.

The activity of the endogenous DNA topoisomerase type I (EC 5.99.1.2) can be quantified in situ by determining how efficiently the enzyme is trapped in a covalent complex with DNA upon lysis of nuclei with detergents. In this way, we can measure relative levels of topoisomerase binding to DNA at native sites in chromatin. Since the majority of topoisomerase I is localized in the nucleolus at rRNA genes, we have evaluated how low levels of actinomycin D, which terminate transcription of rRNA genes, affect the activity of topoisomerase I. In vivo, as well as in vitro with purified topoisomerase I, we have found that drug treatment extends the half-life of the covalent topoisomerase-DNA complex. Actinomycin D stabilizes the nicked intermediate in the cleavage and resealing reaction but otherwise does not significantly alter the strand-passing ability of topoisomerase I. Sequence-specific cleavages by topoisomerase I were stimulated by actinomycin D at identical sequences recognized by the enzyme in the absence of drug. The localization of topoisomerase I in the nucleolus, coupled with the observation that transcription in this organelle is highly sensitive to actinomycin D and camptothecin treatment, leads us to propose that topoisomerase I contributes to actinomycin D inhibition of transcription.

Cell Line↗