Dr John N Anderson | DrJohnAnderson

John Anderson was born in Pittsburgh Pennsylvania and received his BS degree in biology at Slippery Rock College in Pennsylvania. He obtained his Doctor of Philosophy (Ph.D.) from Purdue University where he studied estrogen receptors with James H. Clark. He was then an ACS postdoctoral fellow with Robert T. Schimke at Stanford University where his work centered on gene isolation. He has been a full professor at Purdue University since 1986. He has a long standing research interest in the role of chromatin structure in the control of gene expression. Additional research topics include DNA structure, genomics and retroviral evolution. He teaches a graduate level course in endocrinology and courses in molecular biology for undergraduate students at Purdue. His most recent work has focused on the effects of DNA methylation in the control of nucleosome positioning and stability. He has published over 70 peer-reviewed research publications and several books for teaching college level biology.

Peer Reviewed Publications and Abstracts

An Unusual Property of the HIV Genome

Nucleotide Composition as a driving force in the evolution of retroviruses. J Mol Evol. 38: 506-532.

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. For example, the genomes of lentiviruses such as human immunodeficiency virus (HIV) are highly rich in A, less in G, and markedly deficient in C. Thus, proteins of HIV are rich in lysine and other polar amino acids encoded by A-rich codons and low in proline, which is encoded by C-rich codons. In contrast , the genomes of the E-type oncoviruse as exemplified by the human T cell leukemia virus (HTLV1) are depleted in A and rich in C and all major proteins encoded by these genomes are rich amino acids such as proline The extreme compositional differences extend into all major proteins of the viruses, from the hypervariable polypeptides that comprise the viral envelope to the conserved domain of reverse transcriptase 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. Once the viruses have adapted to different amino acid compositions, continued presence of the diverging viruses in the same cell would no longer be needed to maintain different phenotypes. Each virus would then possess a distinct mutational bias which would fix the patterns of amino acid substitution. These patterns would favor a degree of conservation of the phenotype in the viral progeny, thus promoting the concerted evolution of the species.

Fitzgerald, D. J., Bronson, E. C., Anderson, J. N. 1996. Compositional similarities between the human immunodeficiency virus and surface antigens of pathogens. AIDS Res Hum Retroviruses. 12: 99-106.

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 Gen Bank 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.

Albert, F. G., Bronson, E. C., Fitzgerald, D. J., Anderson, J. N. 1995. Circular structures in retroviral and cellular genomes. J Biol Chem. 270: 23570-23581

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 to 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.

Balzarini, J., Camarasa, M. J., Perez-Perez, M. J., San-Felix, A., Velazquez, S., Perno, C. F., De Clercq, E., Anderson, J. N., Karlsson, A. 2001. Exploitation of the low fidelity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase and the nucleotide composition bias in the HIV-1 genome to alter the drug resistance development of HIV. J Virol. 75: 5772-5777.

The RNA genome of the lentivirus human immunodeficiency virus type 1 (HIV-1) is significantly richer in adenine nucleotides than the statistically equal distribution of the four different nucleotides that is expected. This compositional bias may be due to the guanine-to-adenine (G3A) nucleotide hypermutation of the HIV genome, which has been explained by dCTP pool imbalances during reverse transcription. The adenine nucleotide bias together with the poor fidelity of HIV-1 reverse transcriptase markedly enhances the genetic variation of HIV and may be responsible for the rapid emergence of drug-resistant HIV-1 strains. We have now attempted to counteract the normal mutational pattern of HIV-1 in response to anti-HIV-1 drugs by altering he endogenous deoxynucleoside triphosphate pool ratios with antimetabolites in virus-infected cell cultures. We showed that administration of these antimetabolic compounds resulted in an altered drug resistance pattern due to the reversal of the predominant mutational flow of HIV (G3A) to an adenine-to-guanine(A3G) nucleotide pattern in the intact HIV-1-infected lymphocyte cultures. Forcing the virus to change its inherent nucleotide bias may lead to better control of viral drug resistance development

Collings, C.K. Little, D. Davison, S. and Anderson J.N.: HIV Chromatin is a Preferred Target for Drugs that Bind in the DNA Minor Groove. 2019 PLOS ONE https://doi.org/10.1371/journal.pone.0216515

The HIV genome is rich in A but not G or U and deficient in C. This nucleotide bias controls HIV phenotype by determining the highly unusual composition of all majors HIV proteins. Since drugs that bind in the DNA minor groove disrupt nucleosomes on sequences that contain closely spaced oligo-A tracts which are prevalent in HIV DNA because of this bias, it was of interest to determine if these drugs exert this selective inhibitory effect on HIV chromatin. To test this possibility, nucleosomes were reconstituted onto five double-stranded DNA fragments from the HIV-1 pol gene in the presence and in the absence of several minor groove binding drugs (MGBDs). The results demonstrated that the MGBDs inhibited the assembly of nucleosomes onto all of the HIV-1 segments in a manner that was proportional to the A-bias, but had no detectable effect on the formation of nucleosomes on control cloned fragments or genomic DNA from chicken and human. Nucleosomes preassembled onto HIV DNA were also preferentially destabilized by the drugs as evidenced by enhanced nuclease accessibility in physiological ionic strength and by the preferential loss of the histone octamer in hyper-physiological salt solutions. The drugs also selectively disrupted HIV-containing nucleosomes in yeast as revealed by enhanced nuclease accessibility of the in vivo assembled HIV chromatin and reductions in superhelical densities of plasmid chromatin containing HIV sequences. A comparison of these results to the density of A-tracts in the HIV genome indicates that a large fraction of the nucleosomes that make up HIV chromatin should be preferred in vitro targets for the MGBDs. These results show that the MGBDs preferentially disrupt HIV-1 chromatin in vitro and in vivo and raise the possibility that non-toxic derivatives of certain MGBD might serve as a novel class of anti-HIV agents.

Drugs that Bind in the DNA Minor Groove

Albert, F. G., Eckdahl, T. T., Fitzgerald, D. J., Anderson, J. N. 1999. Heterogeneity in the actions of drugs that bind in the DNA minor groove. Biochemistry. 38: 10135-1014

Biophysical, and clinical studies of the drugs that bind in the DNA minor groove. However, the rsults presented in this investigation clearly show that 4,6-diamidino-2 phenylindole (DAPI) is superior to both of these drugs at negating the effects of intrinsic DNA curvature and anisotropic bendability as measured by electrophoretic and ligation analysis. In addition, DAPI was more effective than distamycin and Hoechst 33258 at inhibiting the assembly of nucleosomes onto synthetic and natural sequences that have multiple closely spaced oligo-AT sequences that serve as drug binding sites. Since these effects may be related to the biological action of the drugs, it was of interest to determine the mechanism that was responsible for the enhanced action of DAPI. The possibility that the differential drug potencies resulted from differential overall affinities of the ligands for A-tract molecules was considered, but drug binding studies suggested that this was not the case. It is also unlikely that the differential drug effects resulted from the binding of the drugs to different DNA sites since the oligo A/T binding sites for DAPI and Hoechst were centered on the same nucleotide positions as revealed by footprinting studies using exonuclease III, DNase I, and hydroxyl radical. However, the footprinting studies with DNase I did uncover a potentially important difference between the drugs. DAPI protected only the AT bp in the binding sites, while distamycin and Hoechst protected these bp as well as flanking Gs and Cs. These results permitted us to advance a preliminary model for the enhanced action DAPI. According to the model, the short length of DAPI and its absolute specificity for A/T bps with narrow minor grooves ensures that only particularly minor grooves that give rise to curvature and anisotropic bendability are occupied by the drug. Consequently, each helical deflection induced by an A-tract in the absence of the drug is countered by an opposite deflection induced by DAPI binding, thus effectively neutralizing intrinsic curvature and bending into the minor groove.

Fitzgerald, D. J., Anderson, J. N. 1999. Selective nucleosome disruption by drugs that bind in the minor groove of DNA. J Biol Chem. 274: 27128-27138.

Previous studies have shown that drugs which bind in the DNA minor groove reduce the curvature of bent DNA. In this article, we examined the effects of these drugs on the nucleosome assembly of DNA molecules that display different degrees of intrinsic curvature. DAPI (4,6-diamidino-2-phenylindole) inhibited the assembly of a histone octamer onto a 192-base pair circular DNA fragment fromCaenorhabditis elegans and destabilized a nucleosome that was previously assembled on this segment. The inhibitory effect was highly selective since it was not seen with nonbent molecules, bent molecules with noncircular shapes, or total genomic DNA. This marked template specificity was attributed to the binding of the ligand to multiple oligo A-tracts distributed over the length of the fragment. A likely mechanism for the effect is that the bound ligand prevents the further compression of the DNA into the minor groove which is required for assembly of DNA into nucleosomes. To further characterize the effects of the drug on chromatin formation, a nucleosome was assembled onto a 322-base pair DNA fragment that contained the circular element and a flanking nonbent segment of DNA. The position of the nucleosome along the fragment was then determined using a variety of nuclease probes including exonuclease III, micrococcal nuclease, DNase I, and restriction enzymes. The results of these studies revealed that the nucleosome was preferentially positioned along the circular element in the absence of DAPI but assembled onto the nonbent flanking sequence in the presence of the drug. DAPI also induced the directional movement of the nucleosome from the circular element onto the nonbent flanking sequence when a nucleosome preassembled onto this template was exposed to the drug under physiologically relevant conditions.

Nucleosome Positioning Determinants

Fitzgerald, D. J., Dryden, G. L., Bronson, E. C., Williams, J. S., Anderson, J. N. 1994. Conserved patterns of bending in satellite and nucleosome positioning DNA. J Biol Chem. 269: 21303-21314. Fitzgerald, D. J., Anderson, J. N. 1998. Unique translational positioning of nucleosomes on synthetic DNAs. Nucleic Acids Res. 26: 2526-2535. Fitzgerald, D. J., Anderson, J. N. 1999. DNA distortion as a factor in nucleosome positioning. J Mol Biol. 293: 477-491.

Positioned nucleosomes are thought to be regulators of genome function but the role of DNA sequence and structure in the control of positioning is poorly understood. We examined the intrinsic curvature of DNA sequences that are known to position histone octamers at single translational sites as a first step to investigate this problem and discovered a conserved pattern of intrinsic DNA curvature that was proposed to direct the formation of nucleosomes to unique positions. The pattern consists of two 50-60 base pair regions of curved DNA separated by preferred lengths of non-curved DNA. The conserved pattern was also seen in all 57 satellite sequences present in the GeneBank database and the distances between successive pairs of curved elements in repeated arrays of satellite monomers were similar to the average spacing of nucleosomes in chromatin. To test the significance of the pattern, ten synthetic DNAs were constructed which contain two regions of curved DNA that are separated by non-curved regions of variable length. Translational mapping of in vitro reconstituted nucleosomes demonstrated that two of the fragments positioned nucleosomes at a single site while most of the remaining fragments positioned octamers at multiple sites spaced at 10 base intervals. In support of the curvature- based model, the positioning sequences contained non-curved central regions of the same lengths that were seen in natural positioning sequences and displayed an affinity for histone octamers comparable to the strongest known natural positioning sequences.

A detailed study was then carried out to identify the features that were responsible for high affinity and unique translational positioning activity. Nucleosomes assembled onto positioning fragments of different lengths shared a common upstream border suggesting that the positioning signals were located on the upstream half of these nucleosomal DNAs. In this region, the compressed minor grooves of the A-tracts did not assume the typical rotational orientation of facing the histone octamer. This unusual orientation was showed to be required for unique positioning since positioning activity was lost upon the insertion of 4 bp between the upstream tracts and the pseudo-dyad region. A permanganate hypersensitive site was also found in this region 1.5 turns from the pseudo-dyad at a site known to display DNA distortion in the nucleosome. The sequence of the hypersite contained a TA step flanked by an oligo-pyrimidine tract and the rotational orientation of the reactive TA step in the nucleosomal DNAs was such that the minor groove faces the histone octamer. Substitutions were made in the region of the hypersite and the resulting constructs tested for affinity for histone octamers and translational positioning. The results revealed that a single base change in the TA step and a few changes in the adjacent tract were sufficient to dramatically reduce affinity and positioning activity in a manner that appeared to be correlated with the presence of a permanganate hypersite. In addition, the rotational orientation of the sequence was shown to be important for function since altering the orientation of the site in a positioning fragment reduced positioning activity and octamer affinity while altering the orientation of the sequence in a nonpositioning fragment had the opposite effects. The 5S rDNA positioning sequence from L. variegatus also contained a permanganate hypersite at 1.5 turns from the pseudo-dyad and other natural positioning sequences were enriched in the sequence motifs that give rise to permanganate hypersensitivity in this location. These results suggest a model in which translational positioning is due to a concerted action between the stabilizing forces associated with the hypersite sequences occupying specific sites within the central three turns of nucleosomal DNA and destabilizing forces which appear when the upstream A- tracts with outward facing minor grooves occupy particular translational positions.

Fernandez, A. G., Anderson, J. N. 2007. Nucleosome positioning determinants. J Mol Biol. 371: 649- 663

A previous report demonstrated that one site in a nucleosome assembled onto a synthetic positioning sequence known as Fragment 67 is hypersensitive to permanganate. The site is required for positioning activity and is located 1.5 turns from the dyad, which is a region of high DNA curvature in the nucleosome. Here, the permanganate sensitivity of the nucleosome positioning Fragment 601 was examined in order to expand the dataset of nucleosome sequences containing KMnO(4) hypersensitive sites. The hyperreactive T residue in the six sites detected as well as the one in Fragment 67 and three in the 5 S rDNA positioning sequence were contained within a TA step. Seven of the ten sequences were of the form CTAGPuG or the related sequence TTAAPu. These motifs were also found in the binding sites of several transcriptional regulatory proteins that kink DNA. In order to assess the significance of these sites, the 10 bp positioning determinant in Fragment 67 was removed and replaced by the nine sequences from the 5 S rDNA and Fragment 601. The results demonstrated that these derivative fragments promoted high nucleosome stability and positioning as compared to a control sequence that contained an AT step in place of the TA step. The importance of the TA step was further tested by making single base-pair substitutions in Fragment 67 and the results revealed that stability and positioning activity followed the order: TA>TG>TT>/=TC approximately GG approximately GA approximately AT. Sequences flanking the TA step were also shown to be critical for nucleosome stability and positioning. Nucleosome positioning was restored to near wild-type levels with (CTG)(3), which can form slipped stranded structures and with one base bulges that kink DNA. The results of this study suggest that local DNA structures are important for positioning and that single base-pair changes at these sites could have profound effects on those genomic functions that depend on ordered nucleosomes.

Collings CK, Fernandez AG, Pitschka CG, Hawkins TB, Anderson JN (2010) Oligonucleotide Sequence Motifs as Nucleosome Positioning Signals. PLoS ONE 5(6): e10933. doi:10.1371/journal.pone.0010933 pages1-18

To gain a better understanding of the sequence patterns that characterize positioned nucleosomes, we first performed an analysis of the periodicities of the 256 tetranucleotides in a yeast genome-wide library of nucleosomal DNA sequences that was prepared by in vitro reconstitution. The approach entailed the identification and analysis of 24 unique tetranucleotides that were defined by 8 consensus sequences. These consensus sequences were shown to be responsible for most if not all of the tetranucleotide and dinucleotide periodicities displayed by the entire library, demonstrating that the periodicities of dinucleotides that characterize the yeast genome are, in actuality, due primarily to the 8 consensus sequences. A novel combination of experimental and bioinformatic approaches was then used to show that these tetranucleotides are important for preferred formation of nucleosomes at specific sites along DNA in vitro. These results were then compared to tetranucleotide patterns in genome-wide in vivo libraries from yeast and C. elegans in order to assess the contributions of DNA sequence in the control of nucleosome residency in the cell. These comparisons revealed striking similarities in the tetranucleotide occurrence profiles that are likely to be involved in nucleosome positioning in both in vitro and in vivo libraries, suggesting that DNA sequence is an important factor in the control of nucleosome placement in vivo. However, the strengths of the tetranucleotide periodicities were 3–4 fold higher in the in vitro as compared to the in vivo libraries, which implies that DNA sequence plays less of a role in dictating nucleosome positions in vivo. The results of this study have important implications for models of sequence-dependent positioning since they suggest that a defined subset of tetranucleotides is involved in preferred nucleosome occupancy and that these tetranucleotides are the major source of the dinucleotide periodicities that are characteristic of positioned nucleosomes.

The Nucleosome is a Functional Target for DNA Methylation

Collings, C.K. Waddell, P.J. and Anderson J. N. 2013 Effects of DNA methylation on Nucleosome Stability Nucleic Acids Research 41:2918-2931

Methylation of DNA at CpG dinucleotides represents one of the most important epigenetic mechanisms involved in the control of gene expression in vertebrate cells. In this report, we conducted nucleosome reconstitution experiments in conjunction with high-throughput sequencing on 572 KB of human DNA and 668 KB of mouse DNA that was unmethylated or methylated in order to investigate the effects of this epigenetic modification on the positioning and stability of nucleosomes. The results demonstrated that a subset of nucleosomes positioned by nucleotide sequence was sensitive to methylation where the modification increased the affinity of these sequences for the histone octamer. The features that distinguished these nucleosomes from the bulk of the methylation-insensitive nucleosomes were an increase in the frequency of CpG dinucleotides and a unique rotational orientation of CpGs such that their minor grooves tended to face toward the histones in the nucleosome rather than away. These methylation-sensitive nucleosomes were preferentially associated with exons as compared to introns while unmethylated CpG islands near transcription start sites became enriched in nucleosomes upon methylation. The results of this study suggest that the effects of DNA methylation on nucleosome stability in vitro can recapitulate what has been observed in the cell and provide a direct link between DNA methylation and the structure and function of chromatin.

Collings, Clayton K. and Anderson, John N., 2017 Links between DNA methylation and nucleosome occupancy in the human genome, Epigenetics & Chromatin 2017 10:18 DOI:10.1186/s13072-017-0125-5

Previous studies have suggested that DNA methylation directly enhances the stability of nucleosomes in vitro. However, the relationship between DNA methylation and nucleosome occupancy is poorly understood. In this study, we implemented a bioinformatics approach that combines MNase-seq and NOMe-seq data to study links between DNA methylation and nucleosome occupancy throughout the human genome. Using this approach, we demonstrated that increasing mCpG density is correlated with nucleosome occupancy and that in mCpG-rich nucleosomes, methylation levels are greater in the core than in the adjacent linker DNA. These nucleosomal DNA methylation patterns were detected not only in total genomic DNA but also within most subgenomic regions. Prominent exceptions to the positive correlation between mCpG density and nucleosome occupancy included CpG islands marked by H3K27me3 and CpG-poor heterochromatin marked by H3K9me3, and these modifications, along with DNA methylation, characterize the major silencing mechanisms of mammalian chromatin. Thus, the density of methylated CpG dinucleotides may be an important factor in regulating nucleosome occupancy levels in the human genome.

Structure, Function and Evolution of Bent and Anti-Bent DNA

Anderson, J. N. 1986. Detection, sequence patterns and function of unusual DNA structures. Nucleic Acids Res. 14: 8513-8533.

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 polyacrylamlde gels. Fragments from yeast centromeres migrated faster in polyacrylamlde than predicted from their base composition and size and this property was attributed to a nonrandon 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 A 2–6 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 adenovlrus genome. Each sequence is ˜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.

Eckdahl, T. T., Anderson, J. N. 1987. Computer Modelling of DNA structures involved in chromosome maintenance. Nucleic Acids Res. 15: 8531-8545

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 electrophoretlc 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.

Eckdahl, T. T., Anderson, J. N. 1990. Conserved DNA structures in origins of replication. Nucleic Acids Res. 18: 1609-1612.

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.

Eckdahl, T. T., Bennetzen, J. L., Anderson, J. N. 1989. DNA structures associated with autonomously replicating sequences from plants. Plant Molecular Biology. 12: 507-516.

DNA fragments capable of conferring autonomous replicating ability to plasmids inSaccharomyces cerevisiae were isolated from four different plant genomes and from the Ti plasmid ofAgrobacterium tumefaciens. The DNA structure of these autonomously replicating sequences (ARSs) as well as two from yeast were studied using retardation during polyacrylamide gel electrophoresis and computer analysis as measures of sequence-dependent DNA structures. Bent DNA was found to be associated with the ARS elements. An 11 bp ARS consensus sequence required for ARS function was also identified in the elements examined and was flanked by unusually straight structures which were rich in A+T content. These results show that the ARS elements from genomes of higher plants have structural and sequence features in common with ARS elements from yeast and higher animals.

Eckdahl, T. T., Anderson, J. N. 1988. Bent DNA is a conserved structure in an adenovirus control region. Nucleic Acids Res. 16: 2346.

Williams, J. S., Eckdahl, T. T., Anderson, J. N. 1988. Bent DNA functions as a replication enhancer in Saccharomyces cerevisiae. Mol Cell Biol. 8: 2763-2769.

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.

VanWye, J. D., Bronson, E. C., Anderson, J. N. 1991. Species-specific patterns of DNA bending and sequence. Nucleic Acids Res. 19: 5253-5261.

Nucleotlde sequences in the GenEMBL database were analyzed using strategies designed to reveal specles speclflc 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 electrophoretlc 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 etectrophoretic 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)>10 and (A-T)>5tracts are prevalent throughout the untranslated DNA of D.dlscodium and P.falclparum, 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.

Wang, Q., Albert, F. G., Fitzgerald, D. J., Calvo, J. M., Anderson, J. N. 1994. Sequence determinants of DNA bending in the ilvlH promoter and regulatory region of Escherichia coli. Nucleic Acids Res. 22: 5753-5760.

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 mutagenlzed In vitro In order to study the relationship between nucleotlde 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 dinucleotlde 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 dinucleotlde steps does not adequately describe the electrophoretic data In this report.

Chromatin Proteins

Bloom, K. S., Anderson, J. N. 1978. Fractionation and characterization of chromosomal proteins by the hydroxyapatite dissociation method. J Biol Chem. 253: 4446-4450.

Vanderbilt, J. N., Anderson, J. N. 1983. Monoclonal antibodies to tissue-specific chromatin proteins. J Biol Chem. 258: 7751-7756.

Vanderbilt, J. N., Anderson, J. N. 1985. Monoclonal antibodies as probes for the complexity, phylogeny, and chromatin distribution of high mobility group chromosomal proteins 1 and 2. J Biol Chem. 260: 9336-9345.

Brown, J. W., Anderson, J. A. 1986. The binding of the chromosomal protein HMG-2a to DNA regions of reduced stabilities. J Biol Chem. 261: 1349-1354.

Babin, J. K., Anderson, J. N. 1988. Isolation and analysis of hepatoma nuclear proteins using monoclonal antibodies. Cancer Res. 48: 5495-5502.

Steroid Hormone Action at the Level of Chromatin

Bloom, K. S., Anderson, J. N. 1978. Fractionation of hen oviduct chromatin into transcriptionally active and inactive regions after selective micrococcal nuclease digestion. Cell. 15: 141-150.

Bloom, K. S., Anderson, J. N. 1979. Conformation of ovalbumin and globin genes in chromatin during differential gene expression. J Biol Chem. 254: 10532-10539

Bloom, K. S., Anderson, J. N. 1982. Hormonal regulation of the conformation of the ovalbumin gene in chick oviduct chromatin. J Biol Chem. 257: 13018-13027.

.Vanderbilt, J. N., Bloom, K. S., Anderson, J. N. 1982. Endogenous nuclease. Properties and effects on transcribed genes in chromatin. J Biol Chem. 257: 13009-13017.

Anderson, J. N., Vanderbilt, J. N., Lawson, G. M., Tsai, M. J., O'Malley, B. W. 1983. Chromatin structure of the ovalbumin gene family in the chicken oviduct Biochemistry

Folger, K., Anderson, J. N., Hayward, M. A., Shapiro, D. J. 1983. Biochemistry. 22: Nuclease sensitivity and DNA methylation in estrogen regulation of Xenopus laevis vitellogenin gene expression. J Biol Chem. 258: 8908-8914..

Anderson J.N. 1984 The Effect of Steroid Hormones on Gene Transcription. In: Goldberger R.F., Yamamoto K.R. (eds) Biological Regulation and Development. Springer, Boston, MA

Estrogen Receptors and Estrogen Action

Anderson, J., Clark, J. H., Peck, E. J., Jr. 1972. Oestrogen and nuclear binding sites. Determination of specific sites by ( 3 H)oestradiol exchange. Biochem J. 126: 561-567.

Anderson, J. N., Clark, J. H., Peck, E. J., Jr. 1972. The relationship between nuclear receptor-estrogen binding and uterotrophic responses. Biochem Biophys Res Commun. 48: 1460-1468.

Clark, J. H., Anderson, J., Peck, E. J., Jr. 1972. Receptor-estrogen complex in the nuclear fraction of rat uterine cells during the estrous cycle. Science. 176: 528-530.

Anderson, J. N., Peck, E. J., Jr., Clark, J. H. 1973. Nuclear receptor estrogen complex: accumulation, retention and localization in the hypothalamus and pituitary. Endocrinology. 93: 711-717.

Anderson, J. N., Peck, E. J., Jr., Clark, J. H. 1973. Nuclear receptor-estrogen complex: relationship between concentration and early uterotrophic responses. Endocrinology. 92: 1488-1495.

Clark, J. H., Anderson, J. N., Peck, E. J., Jr. 1973. Estrogen receptor-anti-estrogen complex: atypical binding by uterine nuclei and effects on uterine growth. Steroids. 22: 707-718.

Clark, J. H., Anderson, J. N., Peck, E. J., Jr. 1973. Nuclear receptor-estrogen complexes of rat uteri: concentration-time-response parameters. Adv Exp Med Biol. 36: 15-59.

Anderson, J. N., Peck, E. J., Jr., Clark, J. H. 1974. Nuclear receptor estradiol complex: a requirement for uterotrophic responses. Endocrinology. 95: 174-178.

Anderson, J. N., Peck, E. J., Jr., Clark, J. H. 1974. Nuclear receptor-estrogen complex: in vivo and in vitro binding of estradiol and estriol as influenced by serum albumin. J Steroid Biochem. 5: 103-107.

Clark, J. H., Peck, E. J., Jr., Anderson, J. N. 1974. Oestrogen receptors and antagonism of steroid hormone action. Nature. 251: 446-448.

Anderson, J. N., Peck, E. J., Jr., Clark, J. H. 1975. Estrogen-induced uterine responses and growth: relationship to receptor estrogen binding by uterine nuclei. Endocrinology. 96: 160-167.

Dryden, G. L., Anderson, J. N. 1977. Ovarian hormone: lack of effect on reproductive structures of female Asian musk shrews. Science. 197: 782-784.

John N. Anderson

John N. Anderson

Professor Department of Biological Sciences - Purdue University

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