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J N Anderson

Publications and source records attributed to J N Anderson.

At least 37 records · Page 2Linked to original sources
PubMed1998

Direct cloning and analysis of DNA sequences from a region of the Chinese hamster genome associated with aphidicolin-sensitive fragility.

Fragile sites are reproducibly expressed and chemically induced decondensations on mitotic chromosomes observed under cytological conditions. They are classified both on the basis of the frequency with which they occur (rare and common) and in terms of the chemical agent used to induce expression in tissue culture cells. Aphidicolin-sensitive common fragile sites appear to be ubiquitous in humans and other mammals and have been considered as candidates of pathological importance. Recently DNA from FRA3B, the most highly expressed constitutive fragile site in the human genome, has been cloned although as yet the cause of the underlying fragility has not been identified. In this study we describe the isolation, using a direct cloning approach, of DNA from a region of the Chinese hamster genome associated with aphidicolin-inducible fragility. Cells of a human-hamster somatic cell hybrid were transfected with a pSV2HPRT vector while exposed to aphidicolin, an inhibitor of DNA polymerases alpha, delta and epsilon. FISH analysis of stable transfectant clones revealed that the ingoing plasmid DNA had preferentially integrated into fragile site-containing chromosomal bands. Plasmid rescue was used to recover DNA sequences flanking one such integration site in the hamster genome. We demonstrate by FISH analysis of metaphase cells induced with aphidicolin that the rescued DNA is from a region of fragility on Chinese hamster chromosome 2, distal to the DHFR locus. Analysis of the DNA sequences flanking the integration site revealed the overall A+T content of the 3,725 bp region sequenced to be 63.3%, with a highly [A].[T]-rich 156 bp region (86.5%) almost adjacent to the integration site. Computational analyses have identified strong homologies to Saccharomyces cerevisiae autonomous replicating sequences (ARS), polypyrimidine tracts, scaffold attachment site consensus sequences and a 24 bp consensus sequence highly conserved in eukaryotic replication origins, all of which appear to cluster around the [A].[T]-rich sequences. This domain also possesses structural characteristics which are common to both prokaryotic and eukaryotic origins of replications, in particular an unusually straight conformation of low thermal stability flanked either side by highly bent DNA segments. Further isolation and characterisation of DNA sequences from common fragile sites will facilitate studies into the underlying nature of these enigmatic regions of the mammalian genome, leading to a greater understanding of chromatin structure.

Animals↗
PubMed1996

Compositional similarities between the human immunodeficiency virus and surface antigens of pathogens.

The genome of the human immunodeficiency virus (HIV) is rich in A but not U and deficient in C but not G. This asymmetric nucleotide bias is the major factor in determining the unusual composition of HIV proteins. In this report, we have identified the cellular genes in the GenBank database that are compositionally similar to HIV in order to further understand the significance of the nucleotide bias of the viral genome. A total of 101 genes in the bacterial and invertebrate subdivisions of the database were found to have a base composition that is similar to the composition of the HIV genome. The identified cellular sequences represent a discrete subset of the database since 81 of the 101 entries code for antigens from pathogens and nearly all of these organisms infect humans. The amino acid compositions of these surface antigens are also similar to the unusual composition of HIV proteins, which are deficient in proline and rich in lysine and other polar residues encoded by A-rich codons. The similarities between the HIV proteins and the immunodominant antigens from other pathogens may indicate a common pathogenic strategy for the promotion of immune dysregulation.

Animals↗
PubMed1995

Circular structures in retroviral and cellular genomes.

A computer program for predicting DNA bending from nucleotide sequence was used to identify circular structures in retroviral and cellular genomes. An 830-base pair circular structure was located in a control region near the center of the genome of the human immunodeficiency virus type I (HIV-I). This unusual structure displayed relatively smooth planar bending throughout its length. The structure is conserved in diverse isolates of HIV-I, HIV-II, and simian immunodeficiency viruses, which implies that it is under selective constraints. A search of all sequences in the GenBank data base was carried out in order to identify similar circular structures in cellular DNA. The results revealed that the structures are associated with a wide range of sequences that undergo recombination, including most known examples of DNA inversion and subtelomeric translocation systems. Circular structures were also associated with replication and transposition systems where DNA looping has been implicated in the generation of large protein-DNA complexes. Experimental evidence for the structures was provided by studies which demonstrated that two sequences detected as circular by computer preferentially formed covalently closed circles during ligation reactions in vitro when compared to nonbent fragments, bent fragments with noncircular shapes, and total genomic DNA. In addition, a single T-->C substitution in one of these sequences rendered it less planar as seen by computer analysis and significantly reduced its rate of ligase-catalyzed cyclization. These results permit us to speculate that intrinsically circular structures facilitate DNA looping during formation of the large protein-DNA complexes that are involved in site- and region-specific recombination and in other genomic processes.

Animals↗
PubMed1994

Sequence determinants of DNA bending in the ilvlH promoter and regulatory region of Escherichia coli.

Previous studies have shown that the promoter/regulatory region of the ilvlH operon displays intrinsic curvature, with the bend center located at position -120 relative to the transcription start site. In this report, a 57 bp sequence spanning the bend center was mutagenized in vitro in order to study the relationship between nucleotide sequence and curvature measured by electrophoresis. The strategy used for analyzing the results consisted of determining the strengths of the relationships between electrophoretic anomaly and predicted curvature calculated by computer programs that differ in wedge angle composition. The results revealed that programs which assume that bending occurs only at AA/TT display good predictive value, with correlation coefficients between electrophoretic anomaly and predicted curvature as high as 0.93. In contrast, a program which assumes that bending occurs at all 16 dinucleotide steps exhibited lower predictive value, while there were no significant relationships between the experimental data and curvature calculated by a program that was based on all non-AA/TT wedge values. These results show that the complete wedge model which incorporates values for all dinucleotide steps does not adequately describe the electrophoretic data in this report.

Acetolactate Synthase↗
PubMed1994

Conserved patterns of bending in satellite and nucleosome positioning DNA.

Intrinsic DNA curvature has previously been implicated in the condensation of satellite DNA in chromatin. In this article, electrophoretic methods and computer programs for predicting DNA structure from nucleotide sequence were used to determine if curvature is a conserved feature of satellite DNA. The results revealed that satellite sequences display wide variation in magnitude of intrinsic bending. Less than half of the satellites examined were strongly bent when compared with control DNA. However, a conserved pattern of bending was seen in all 57 satellite sequences that were studied. The pattern consisted of repeating units of two 50-60-base pair bending elements which were separated by a 20-30-base pair region of low curvature. This pattern resembles qualitatively the bending of DNA in the nucleosome where the helix is folded approximately two turns around the histone octomer with the turns interrupted by a less bent segment in the center of the particle. The distance between successive pairs of bending elements was also similar to the average spacing of nucleosomes in chromatin. Thus, the conserved structures could play some role in the positioning of nucleosomes along satellite chromatin. In order to strengthen the correlation between DNA structure and nucleosome positioning, sequences were examined which have been shown to position nucleosomes at single major sites. This analysis revealed a conserved pattern of DNA structure resembling the two peak units seen in satellites. In addition, the nucleotide sequence patterns responsible for the conserved patterns of bending were similar in both satellite and nucleosome positioning DNA. Likewise, nucleotide sequence patterns that are thought to direct the rotational orientation of DNA in the nucleosome were similar in both sequence sets. These results are considered in terms of a general model for the role of DNA bending and nucleotide sequence in the control of nucleosome positioning and chromatin condensation in eukaryotes.

Animals↗
PubMed1994

Nucleotide composition as a driving force in the evolution of retroviruses.

All complete retrovirus sequences in the GenEMBL database were examined with the goal of assessing possible relationships between the nucleotide composition of retroviral genomes, the amino acid composition of retroviral proteins, and evolutionary strategies used by retroviruses. The results demonstrated that the genome of each viral lineage has a characteristic base composition and that the variations between groups are related to retroviral phylogeny. By analogy to microbial species, we suggest that the variations arise from group-specific patterns of directional mutations where the bias can be exerted on any of the four nucleotides. It is most likely that the mutational patterns are introduced during reverse transcription, and a direct participation of reverse transcriptase in the process is suspected. A straightforward strategy was used to analyze the compositional relationship between nucleotides and encoded amino acids. The procedure entailed calculations of amino acid frequencies from nucleotide content and the comparison of the calculated values to the observed amino acid frequencies in retroviruses. The results revealed an excellent correspondence between variation in genomic base composition and variation in amino acid composition of proteins with the compositional differences extending into all major coding regions of the viruses. Because of the magnitude and dispersion of these effects, and because of the nonconservative nature of many of the substitutions between groups with different genomic biases, we suggest that the variations in protein composition driven by biased nucleotide frequencies are an important factor in shaping the characteristic phenotypes of the different viral lineages. A clue to the nature of the evolutionary forces that are responsible for the generation of nucleotide biases was provided by the observation that viruses with radically different base frequencies most often inhabit the same cell type. This observation, along with analysis of amino acid and nucleotide replacement patterns between and within reverse transcriptase sequences from the various groups, permitted us to advance a model for the evolution of retroviruses. According to the model, speciation could initiate when daughter virions from a single progenitor vary in the direction of their mutational bias. These variations would exert a pleiotropic effect on the frequencies of nucleotides in all viral genes and consequently on the frequencies of amino acids in the encoded proteins. The variants with the most extreme compositional differences would have a selective advantage because their different precursor requirements would enable them to occupy different ecological niches within a single cell.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

The time-dependent increase in the binding of benzo[a]pyrene to DNA through (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide in primary rat hepatocyte cultures results from induction of cytochrome P450IA1 by benzo[a]pyrene treatment.

The proportion and amount of benzo[a]pyrene (B[a]P) that binds to DNA through the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] increases with time of exposure to B[a]P in cell cultures derived from a number of species. Pretreatment of primary rat hepatocyte cultures for 12 h with 1 microgram B[a]P/ml medium increased the subsequent metabolism of [3H]B[a]P by 47% and [3H]B[a]P-DNA binding by 53% compared with acetone-pretreated hepatocytes. The amount of (+)-anti-BPDE bound to DNA in the B[a]P-pretreated hepatocytes increased 175%. B[a]P pretreatment also increased DNA-binding 2-fold in hepatocytes treated with [3H]7,8-dihydroxy-7,8-dihydro-B[a]P but had no effect on DNA binding in cells treated with anti-B[a]P-7,8-diol-9,10-epoxide. Western blotting showed that cytochrome P450IA1, which was not detectable prior to B[a]P treatment, was selectively increased by B[a]P treatment. A monoclonal antibody that specifically inhibits cytochrome P450IA1 reduced the binding of B[a]P to DNA by greater than 90% in microsomal preparations from B[a]P-pretreated hepatocytes. These results indicate that the time-dependent increase in the formation of (+)-anti-BPDE-DNA adducts results from an increase in the amount and proportion of B[a]P metabolized to this ultimate carcinogen by P450IA1 that is induced by the B[a]P treatment. The importance of P450IA1 induction by the B[a]P for its activation to this ultimate carcinogenic metabolite suggests that long-term exposure of cells to B[a]P could result in activation of a higher proportion of the B[a]P to the carcinogenic (+)-anti-BPDE.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1992

High selectivity of polyclonal antibodies against DNA modified by diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxides.

Polyclonal antibodies were developed in New Zealand White rabbits against DNA modified with diastereomeric benzo[c]phenanthrene-3,4-diol-1,2-epoxide (B[c]PhDE)-1 (4-hydroxyl and epoxide cis) and B[c]PhDE-2 (4-hydroxyl and epoxide trans). Antiserum developed against B[c]PhDE-2-DNA was stereoselective. In competitive ELISA assays using wells coated with 160 fmol B[c]PhDE-2-DNA adducts, B[c]PhDE-2-DNA gave 50% inhibition at 200 fmol adducts/well. B[c]PhDE-1-DNA required a 10-fold higher amount of adducts/well to give 50% inhibition. Benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA and 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA caused only a 30% inhibition even at the highest doses tested (greater than 4000 fmol adducts/well). For antiserum developed against B[c]PhDE-1-DNA, 50% inhibition required 570 fmol B[c]PhDE-1-DNA adducts in wells coated with 100 fmol B[c]PhDE-1-DNA adducts. 7,12-Dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA and B[c]PhDE-2-DNA were also effective competitors: they caused 50% inhibition at 1900 and 1800 fmol adducts/well respectively. In contrast, benzo[a]pyrene-7,8-diol-9,10-epoxide-2-DNA gave no inhibition at the highest dose of competitor tested (4050 fmol adducts/well). Antisera from three rabbits immunized with B[c]PhDE-2-DNA demonstrated similar antigen specificities. The properties of these antisera differ from those reported previously for antibodies developed against benzo[a]pyrene-DNA in that they show selectivity for DNA modified by specific hydrocarbon diolepoxides, in one case for B[c]PhDE-2-DNA and in the other for B[c]PhDE-DNA or 7,12-dimethylbenz[a]anthracene-3,4-diol-1,2-epoxide-1-DNA. The specificity of these antisera will facilitate analysis of the modification of DNA by different polycyclic aromatic hydrocarbon diolepoxides.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed1991

Species-specific patterns of DNA bending and sequence.

Nucleotide sequences in the GenEMBL database were analyzed using strategies designed to reveal species-specific patterns of DNA bending and DNA sequence. The results uncovered striking species-dependent patterns of bending with more variations among individual organisms than between prokaryotes and eukaryotes. The frequency of bent sites in sequences from different bacteria was related to genomic A + T content and this relationship was confirmed by electrophoretic analysis of genomic DNA. However, base composition was not an accurate predictor for DNA bending in eukaryotes. Sequences from C. elegans exhibited the highest frequency of bent sites in the database and the RNA polymerase II locus from the nematode was the most bent gene in GenEMBL. Bent DNA extended throughout most introns and gene flanking segments from C.elegans while exon regions lacked A-tract bending characteristics. Independent evidence for the strong bending character of this genome was provided by electrophoretic studies which revealed that a large number of the fragments from C.elegans DNA exhibited anomalous gel mobilities when compared to genomic fragments from over 20 other organisms. The prevalence of bent sites in this genome enabled us to detect selectively C.elegans sequences in a computer search of the database using as probes C.elegans introns, bending elements, and a 20 nucleotide consensus sequence for bent DNA. This approach was also used to provide additional examples of species-specific sequence patterns in eukaryotes where it was shown that (A) greater than or equal to 10 and (A.T) greater than or equal to 5 tracts are prevalent throughout the untranslated DNA of D.discodium and P.falciparum, respectively. These results provide new insight into the organization of eukaryotic DNA because they show that species-specific patterns of simple sequences are found in introns and in other untranslated regions of the genome.

Algorithms↗
PubMed1990

Conserved DNA structures in origins of replication.

According to the model of Bramhill and Kornberg, initiation of DNA replication in prokaryotes involves binding of an initiator protein to origin DNA and subsequent duplex opening of adjacent direct repeat sequences. In this report, we have used computer analysis to examine the higher-order DNA structure of a variety of origins of replication from plasmids, phages, and bacteria in order to determine whether these sequences are localized in domains of altered structure. The results demonstrate that the primary sites of initiator protein binding lie in discrete domains of DNA bending, while the direct repeats lie within well-defined boundaries of an unusual anti-bent domain. The anti-bent structures arise from a periodicity of A3 and T3 tracts which avoids the 10-11 bp bending periodicity. Since DNA fragments which serve as replicators in yeast also contain these two conserved structural elements, the results provide new insight into the universal role of conserved DNA structures in DNA replication.

Base Sequence↗
PubMed1988

Isolation and analysis of hepatoma nuclear proteins using monoclonal antibodies.

Monoclonal antibodies were prepared to nuclear nonhistone proteins from a 2-aminoacetyl fluorine-induced transplantable rat hepatocellular carcinoma. These antibodies recognized a total of six distinct antigens as revealed by molecular weight analysis. Studies of antigen specificity with respect to various tissues, tumors, cultured cells, and oncodevelopmental stages indicated that these nuclear species could be divided into two categories. Four antigens were classified as tumor related since they were significantly enriched in tumor tissue as compared to tissues of the normal adult rat. The remaining two antigens were detected only in tumors and transformed cells; one, only in certain hepatomas. Thus, these antigens were classified as tumor specific. As an initial step toward elucidating the function of these proteins, each antigen was isolated by immunoaffinity chromatography, radioiodinated in situ, and analyzed for the ability to bind DNA. Three antigens were positive for DNA binding, and one of these was selectively released from tumor nuclei with the transcriptionally active chromatin upon digestion with micrococcal nuclease. The implications of these results for the possible functional contribution of the six tumor antigens to transformation is discussed.

2-Acetylaminofluorene↗
PubMed1988

Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae.

Previous studies have demonstrated that bent DNA is a conserved property of Saccharomyces cerevisiae autonomously replicating sequences (ARSs). Here we showed that bending elements are contained within ARS subdomains identified by others as replication enhancers. To provide a direct test for the function of this unusual structure, we analyzed the ARS activity of plasmids that contained synthetic bent DNA substituted for the natural bending element in yeast ARS1. The results demonstrated that deletion of the natural bending locus impaired ARS activity which was restored to a near wild-type level with synthetic bent DNA. Since the only obvious common features of the natural and synthetic bending elements are the sequence patterns that give rise to DNA bending, the results suggest that the bent structure per se is crucial for ARS function.

Base Sequence↗
PubMed1987

Computer modelling of DNA structures involved in chromosome maintenance.

Sequence-dependent DNA bending of synthetic and natural molecules was studied by computer analysis. Modelling of synthetic oligonucleotides and of 107 kb of natural sequences gave results which closely resembled published electrophoretic data, demonstrating the powerful predictive capacity of the procedure. The analysis was extended to the study of DNA structures involved in chromosome maintenance. Centromeric DNAs from yeast were found to have sequences in their functional elements which cause them to be unusually straight. Autonomous replicating sequences were found to have two structural domains, one consisting of unusually straight sequences surrounding the consensus and the other of bending elements in flanking DNA. In addition to a structural homology, centromeric and autonomous replicating sequences share common sequence elements. These observations show that computer modelling of natural sequences is a viable approach to the study of the biological implications of alternative DNA structures.

Base Sequence↗
PubMed1986

Detection, sequence patterns and function of unusual DNA structures.

Unusual DNA structures were detected by an electrophoretic procedure in which DNA fragments were separated according to size on agarose gels and then by shape on polyacrylamide gels. Fragments from yeast centromeres migrated faster in polyacrylamide than predicted from their base composition and size and this property was attributed to a nonrandom distribution of oligomeric A tracts that exhibited minima at 10-11 base intervals. Fragments from seven loci in 107 kb of DNA migrated anomalously slow and these fragments contained blocks of A2-6 in a 10-11 base periodicity which is indicative of bent DNA. The most pronounced bent sequences were found within yeast ARS1 and centered at 245 and 240 bp from the left and right ends of the adenovirus genome. Each sequence is approximately 150 bp away from a replication origin and the adenovirus sequences are within 50 bp of enhancers. Nuclear matrix attachment sites, which are also adjacent to enhancers, contain sequences characteristic of bent DNA. These results suggest that bent structures reside at the base of DNA loops in chromosomes.

Base Sequence↗
PubMed1985

Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2.

Monoclonal antibodies were prepared against the high mobility group (HMG) proteins 1, 2a, and 2b from hen erythrocyte chromatin. One antibody that recognized multiple sites along HMG-1, -2a, and -2b reacted strongly with HMG proteins from all vertebrates tested. In contrast, five antibodies that detected unique epitopes on chicken HMG-1 and -2a recognized antigenic sites that exhibited restricted phylogenic distributions. The differential reactivity of these antibodies on vertebrate proteins was in agreement with traditional taxonomy in that the avian HMGs were most closely related to those from reptiles and less related to those from mammals, amphibians, bonyfish, and especially the jawless fish. Mononucleosomes generated by mild digestion of erythrocyte chromatin with micrococcal nuclease were highly enriched in HMG-2a. One antigenic determinant located within the N-terminal domain of HMG-2a was freely accessible to its antibody when the protein was bound to these mononucleosomes. In contrast, two antibodies that recognized determinants in the central region of HMG-2a exhibited little chromatin binding activity. The masking of the central domain by DNA binding was presumably not responsible for these results because all three determinants were available for antibody binding when HMG-2a was bound to DNA in vitro. Therefore, the central region of HMG-2a may be masked from antibody binding by protein-protein interactions in chromatin.

Amphibians↗
PubMed1985

Benzo[a]pyrene-DNA adduct formation in target and activator cells in a Wistar rat embryo cell-mediated V79 cell mutation assay.

To determine the relationship of the benzo[a]pyrene (BaP)-DNA adducts formed in the activator cells of a cell-mediated mutation assay to the adducts formed in the target cells and to mutation induction, irradiated second passage Wistar rat embryo (WRE) cells and V79 Chinese hamster lung cells were exposed to [3H]BaP for 5, 24 and 48 h under the conditions of a cell-mediated mutation assay. The V79 target cells were separated from the WRE activator cells by an immunoseparation procedure; the resulting V79 cell pellet contained less than 7% WRE cells. The percentage of the BaP-DNA adducts containing cis vicinal hydroxyl groups and the h.p.l.c profile of individual adducts in the V79 target cells were similar to those of the WRE activator cells for each time point. The transfer of reactive BaP metabolites from the activator cells to the target cell DNA was detectable after only 5 h of exposure to BaP, however, exposure for this length of time did not result in significant mutation induction. The (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BaPDE)-deoxyguanosine (dGuo) adduct was essentially absent after 5 h of exposure, but the amount of this adduct increased at longer times of exposure as did the mutation frequency. The complex mixture of BaP-DNA adducts formed in the WRE cells was also present in the V79 cells after all three times of exposure to BaP, a result which demonstrates the value of this cell-mediated mutation assay for investigating the role of species-specific differences in the activation of BaP. The correlation between the increase in mutation induction and the relative amount of the (+)-anti-BaPDE-dGuo adduct present in V79 cell DNA suggests the importance of this adduct in mutation induction by BaP.

Animals↗
PubMed1984

Flow kinetics of fibronectin secretion by livers of young preweanling rats.

Fibronectin is a high molecular weight adhesive glycoprotein present both on the cell surface (cellular fibronectin) and circulating free in the plasma (plasma fibronectin). To investigate the cellular origins of plasma fibronectin, monoclonal antibodies prepared against purified plasma fibronectin of the rat were used in both in vivo and in vitro pulse-chase experiments comparing livers of animals of different ages. For in vitro analyses, slices were pulsed with 3H-leucine and purified fractions of endoplasmic reticulum, Golgi apparatus and plasma membrane were prepared at various times post-chase and characterized morphologically. The total protein was solubilized with NP-40, and incorporation into fibronectin was determined from PAGE analysis of immunoprecipitates. Incorporation was greatest in 5-day-old animals, representing greater than 0.3% of the total radioactivity and was least in adults animals. Label appeared in fibronectin associated with the plasma membrane within 60 min. At earlier times, labeled fibronectin appeared first in the endoplasmic reticulum (30 min) and then in the Golgi apparatus (45 min). The findings demonstrate synthesis of fibronectin by rat liver and evidence for participation of liver endomembranes in its secretion.

Animals↗