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PubMed2006

Faithful replication of foraging techniques along cultural transmission chains by chimpanzees and children.

Observational studies of wild chimpanzees (Pan troglodytes) have revealed population-specific differences in behavior, thought to represent cultural variation. Field studies have also reported behaviors indicative of cultural learning, such as close observation of adult skills by infants, and the use of similar foraging techniques within a population over many generations. Although experimental studies have shown that chimpanzees are able to learn complex behaviors by observation, it is unclear how closely these studies simulate the learning environment found in the wild. In the present study we have used a diffusion chain paradigm, whereby a behavior is passed from one individual to the next in a linear sequence in an attempt to simulate intergenerational transmission of a foraging skill. Using a powerful three-group, two-action methodology, we found that alternative methods used to obtain food from a foraging device ("lift door" versus "slide door") were accurately transmitted along two chains of six and five chimpanzees, respectively, such that the last chimpanzee in the chain used the same method as the original trained model. The fidelity of transmission within each chain is remarkable given that several individuals in the no-model control group were able to discover either method by individual exploration. A comparative study with human children revealed similar results. This study is the first to experimentally demonstrate the linear transmission of alternative foraging techniques by non-human primates. Our results show that chimpanzees have a capacity to sustain local traditions across multiple simulated generations.

Animals↗
PubMed1998

The path from the RNA world.

We describe a sequential (step by step) Darwinian model for the evolution of life from the late stages of the RNA world through to the emergence of eukaryotes and prokaryotes. The starting point is our model, derived from current RNA activity, of the RNA world just prior to the advent of genetically-encoded protein synthesis. By focusing on the function of the protoribosome we develop a plausible model for the evolution of a protein-synthesizing ribosome from a high-fidelity RNA polymerase that incorporated triplets of oligonucleotides. With the standard assumption that during the evolution of enzymatic activity, catalysis is transferred from RNA --> RNP --> protein, the first proteins in the "breakthrough organism" (the first to have encoded protein synthesis) would be nonspecific chaperone-like proteins rather than catalytic. Moreover, because some RNA molecules that pre-date protein synthesis under this model now occur as introns in some of the very earliest proteins, the model predicts these particular introns are older than the exons surrounding them, the "introns-first" theory. Many features of the model for the genome organization in the final RNA world ribo-organism are more prevalent in the eukaryotic genome and we suggest that the prokaryotic genome organization (a single, circular genome with one center of replication) was derived from a "eukaryotic-like" genome organization (a fragmented linear genome with multiple centers of replication). The steps from the proposed ribo-organism RNA genome --> eukaryotic-like DNA genome --> prokaryotic-like DNA genome are all relatively straightforward, whereas the transition prokaryotic-like genome --> eukaryotic-like genome appears impossible under a Darwinian mechanism of evolution, given the assumption of the transition RNA --> RNP --> protein. A likely molecular mechanism, "plasmid transfer," is available for the origin of prokaryotic-type genomes from an eukaryotic-like architecture. Under this model prokaryotes are considered specialized and derived with reduced dependence on ssRNA biochemistry. A functional explanation is that prokaryote ancestors underwent selection for thermophily (high temperature) and/or for rapid reproduction (r selection) at least once in their history.

Archaea↗
PubMed1993

Efficient insertion from an internal long terminal repeat (LTR)-LTR sequence on a reticuloendotheliosis virus vector is imprecise and cell specific.

To examine the fidelity and efficiency of integration from a covalently closed long terminal repeat (LTR)-LTR sequence in vivo, we isolated individual spleen necrosis virus proviruses that arose following infection of chicken embryo fibroblasts (CEFs) and sequenced the provirus-cell DNA junctions. Some but not all CEF preparations allowed efficient insertion from the internal sequence. Moreover, in contrast to integration from the normal ends of the viral DNA, which occurs with precision with respect to the viral DNA, insertion from the internal sequence was not precise. In particular, there were short deletions of variable size from the viral DNA and these proviruses were not flanked by short direct repeats. Although this imprecise insertion can be efficient in CEFs, such integration is very inefficient in two other cell types (D17 and QT47) that support the replication of reticuloendotheliosis viruses. Thus, it is possible that there is a cell-specific factor(s) in CEFs required for efficient but imprecise insertion or, alternatively, D17 and QT47 cells contain a factor that abrogates integration from an internal LTR-LTR junction. Virus particles released from CEFs do not efficiently use the LTR-LTR junction following infection of D17 cells. Therefore, if there is a CEF-specific factor required for insertion, it does not appear to be transferred through particles.

Animals↗
PubMed2004

Increased G-->A transition frequencies displayed by primer grip mutants of human immunodeficiency virus type 1 reverse transcriptase.

A genetic screen based on the blue-white beta-galactosidase complementation assay designed to detect G-->A mutations arising during RNA-dependent DNA synthesis was used to compare the fidelity of mutant human immunodeficiency virus type 1 reverse transcriptases (RTs) with the mutations M230L and M230I with the wild-type enzyme, in the presence of biased deoxynucleoside triphosphate (dNTP) pools. The mutant RTs with the M230L and M230I changes were found to be 20 to 70 times less faithful than the wild-type RT in the presence of low [dCTP]/[dTTP] ratios but showed similar fidelity in assays carried out with equimolar concentrations of each nucleotide. Biased dNTP pools led to short tandem repeat deletions in the target sequence, which were also detectable with the assay. However, deletion frequencies were similar for all of the RTs tested. The reported data suggest that RT pausing due to the low dNTP levels available in the RT reaction mixture facilitates strand transfer, in a process that is not necessarily mediated by nucleotide misinsertion.

Base Sequence↗
PubMed1997

DNA synthesis arrest at C4'-modified deoxyribose residues.

Many genotoxic agents form base lesions that inhibit DNA polymerases. To study the mechanism underlying termination of DNA synthesis on defective templates, we tested the capacity of a model enzyme (Klenow fragment of Escherichia coli DNA polymerase I) to catalyze primer elongation across a series of C4' deoxyribose derivatives. A site with inverted C4' configuration or two different C4' deoxyribose adducts were introduced into the backbone of synthetic templates without modifying the chemistry of the corresponding bases. Inverted deoxyribose moieties may arise in cellular DNA as a product of C4' radical attack. We found that DNA synthesis by the Klenow polymerase was arrested transiently at the C4' inversion and was essentially blocked at C4' deoxyribose adducts. Major termination sites were located one position downstream of a C4' selenophenyl adduct and immediately 3' to or opposite a C4' pivaloyl adduct. Primer extension studies in the presence of single deoxyribonucleotides showed intact base pairing fidelity opposite all three C4' variants regardless of whether the Klenow fragment or its proofreading-deficient mutant was tested. These results imply that the coding ability of template bases is maintained at altered C4' deoxyribose moieties. However, their capacity to impede DNA polymerase progression indicates that backbone distortion and steric hindrance are important determinants of DNA synthesis arrest on damaged templates. The strong inhibition by C4' adducts suggests a potential target for new chemotherapeutic strategies.

DNA Polymerase I↗
PubMed2005

Spatial control of bacterial division-site placement.

The site of cell division in bacterial cells is placed with high fidelity at a designated position, usually the midpoint of the cell. In normal cell division in Escherichia coli this is accomplished by the action of the Min proteins, which maintain a high concentration of a septation inhibitor near the ends of the cell, and a low concentration at midcell. This leaves the midcell site as the only available location for formation of the division septum. In other species, such as Bacillus subtilis, this general paradigm is maintained, although some of the proteins differ and the mechanisms used to localize the proteins vary. A second mechanism of negative regulation, the nucleoid-occlusion system, prevents septa forming over nucleoids. This system functions in Gram-negative and Gram-positive bacteria, and is especially important in cells that lack the Min system or in cells in which nucleoid replication or segregation are defective. Here, we review the latest findings on these two systems.

Adenosine Triphosphatases↗
PubMed2004

P53 in cytoplasm may enhance the accuracy of DNA synthesis by human immunodeficiency virus type 1 reverse transcriptase.

The tumor suppressor protein p53 displays 3' --> 5' exonuclease activity and can provide a proofreading function for DNA polymerases. Reverse transcriptase (RT) of human immunodeficiency virus (HIV)-1 is responsible for the conversion of the viral genomic ssRNA into the proviral DNA in the cytoplasm. The relatively low fidelity of HIV-1 RT was implicated as a dominant factor contributing to the genetic variability of the virus. The lack of intrinsic 3' --> 5' exonuclease activity, the formation of 3'-mispaired DNA and the subsequent extension of this DNA were shown to be determinants for the low fidelity of HIV-1 RT. It was of interest to analyse whether the cytoplasmic proteins may affect the accuracy of DNA synthesis by RT. We investigated the fidelity of DNA synthesis by HIV-1 RT with and without exonucleolytic proofreading provided by cytoplasmic fraction of LCC2 cells expressing high level of wild-type functional p53. Two basic features related to fidelity of DNA synthesis were studied: the misinsertion and mispair extension. The misincorporation of noncomplementary deoxynucleotides into nascent DNA and subsequent mispair extension by HIV-1 RT were substantially decreased in the presence of cytoplasmic fraction of LCC2 cells with both RNA/DNA and DNA/DNA template-primers with the same target sequence. The mispair extension frequencies obtained with the HIV-1 RT in the presence of cytoplasmic fraction of LCC2 cells were significantly lower (about 2.8-15-fold) than those detected with the purified enzyme. In addition, the productive interaction between polymerization (by HIV-1 RT) and exonuclease (by p53 in cytoplasm) activities was observed; p53 preferentially hydrolyses mispaired 3'-termini, permitting subsequent extension of the correctly paired 3'-terminus by HIV-1 RT. The data suggest that p53 in cytoplasm may affect the accuracy of DNA replication and the mutation spectra of HIV-1 RT by acting as an external proofreader. Furthermore, the decrease in error-prone DNA synthesis with RT in the presence of external exonuclease, provided by cytoplasmic p53, may partially account for lower mutation rate of HIV-1 observed in vivo.

Cell Line, Tumor↗
PubMed2002

Mutational analysis of HIV-1 long terminal repeats to explore the relative contribution of reverse transcriptase and RNA polymerase II to viral mutagenesis.

HIV-1 evolves rapidly, which is thought to result from one or more error-prone steps in the virus life cycle. Because HIV-1 reverse transcriptase (RT) does not possess 3'- to 5'-exonucleolytic proofreading activity and because RT has been shown to be error-prone in cell free systems, it should be an important contributor to the high rate of HIV-1 mutation. However, because RNA polymerase II (pol II) synthesizes viral RNA, it might also contribute significantly to HIV-1 mutagenesis. To assess the relative contributions of RT and RNA pol II to HIV-1 mutagenesis, a system was established to study the rate and nature of mutations in HIV-1 long terminal repeats (LTRs). Owing to the unique nature of replication at the ends of the viral genome, mutational analysis of retroviral LTRs provides an opportunity to evaluate the relative contribution of HIV-1 RT and RNA pol II to viral mutagenesis. Mutational analysis was performed on both LTRs of 215 proviruses, restricted to a single cycle of replication, employing single-stranded conformational polymorphism and DNA sequencing allowing direct identification of mutations in the absence of selection and within autologous viral sequences. A total of 21 independent mutations was identified. Ten mutations were observed in both LTRs, which could have been introduced by either RT or RNA pol II, whereas the other eleven mutations were only present in a single LTR and could only have been introduced by RT. This provides the first direct evidence that HIV-1 RT contributes significantly to HIV-1 mutagenesis and is likely to be the primary engine for HIV-1 mutagenesis. Moreover, mutations were observed at the U3-R border, but the nature of the mutations and their frequency differed from experiments performed using cell-free systems suggesting that other viral and/or cellular factors contribute to fidelity at the ends of the viral genome.

Base Sequence↗
PubMed2001

Fidelity and damage bypass ability of Schizosaccharomyces pombe Eso1 protein, comprised of DNA polymerase eta and sister chromatid cohesion protein Ctf7.

DNA polymerase eta (Poleta) functions in error-free bypass of ultraviolet light-induced DNA lesions, and mutational inactivation of Poleta in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum (XPV). Both Saccharomyces cerevisiae and human Poleta efficiently insert two adenines opposite the two thymines of a cyclobutane pyrimidine dimer. Interestingly, in the fission yeast Schizosaccharomyces pombe, the eso1(+) encoded protein is comprised of two domains, wherein the NH(2) terminus is highly homologous to Poleta, and the COOH terminus is highly homologous to the S. cerevisiae Ctf7 protein which is essential for the establishment of sister chromatid cohesion during S phase. Here we characterize the DNA polymerase activity of S. pombe GST-Eso1 fusion protein and a truncated version containing only the Poleta domain. Both proteins exhibit a similar DNA polymerase activity with a low processivity, and steady-state kinetic analyses show that on undamaged DNA, both proteins misincorporate nucleotides with frequencies of approximately 10(-2) to 10(-3). We also examine the two proteins for their ability to replicate a cyclobutane pyrimidine dimer-containing DNA template and find that both proteins replicate through the lesion equally well. Thus, fusion with Ctf7 has no significant effect on the DNA replication or damage bypass properties of Poleta. The possible role of Ctf7 fusion with Poleta in the replication of Cohesin-bound DNA sequences is discussed.

Acetyltransferases↗
PubMed1998

Fidelity of DNA ligation: a novel experimental approach based on the polymerisation of libraries of oligonucleotides.

Complete libraries of oligonucleotides were used as substrates for Thermus thermophilus DNA ligase, on a M13mp18 ssDNA template. A 17mer primer was used to start a polymerisation process. Ladders of ligation products were analysed by gel electrophoresis. Octa-, nona- and decanucleotide libraries were compared. Nonanucleotides were optimum for polymerisation and up to 15 monomers were ligated. The fidelity of incorporation was studied by sequencing 28 clones (2268 bases) of nonanucleotide polymers, 12 monomers in length. Of the ligated monomers, 79% were the correct complementary sequence. In a total of 57 (2.5%) mispaired bases, there was a strong bias to G.T, G.A, G.G and A.G mismatches. Of the mismatches, 86% were found to be purines on the incoming oligonucleotide, of which 71% were G. There is evidence for clustering of mismatches within specific 9mers and at specific positions within these 9mers. The most frequent mismatches were at the 5'-terminus of the oligonucleotide, followed by the central position. We suggest that sequence selection was imposed by the ligase and not just by base pairing interactions. The ligase directs polymerisation in the 3' to 5' direction which we propose is linked to its role in lagging strand DNA replication.

Bacteriophage M13↗
PubMed2005

PCR amplification, cloning, and construction of HIV-1 infectious molecular clones from virtually full-length HIV-1 genomes.

The development of mixtures of highly processive and high-fidelity thermostable DNA polymerases has enabled the routine recovery of DNA sequences in excess of 25 kb generated by polymerase chain reaction. This powerful tool has been instrumental in the ability to recover virtually full-length HIV-1 proviral DNA as a single, contiguous fragment. Such fragments allow for the clean interpretation of the genomic organization of HIV-1 provirus, as they are not confounded by molecular mosaicism that accrues to overlapping subgenomic amplification strategies. We detail here a robust procedure to produce virtually full-length, single contiguous 9.2-kb HIV-1 amplimers whose full-length infectious potential is reconstituted upon cloning into long terminal repeat-replacement vectors. Large numbers of HIV-1 proviral clones can now be quickly generated and screened to identify the fraction of the viral quasispecies with the highest capacity for viral replication. The methods used to construct long terminal repeat-replacement vectors, amplify HIV-1 provirus, reconstitute full-length provirus, and recover viral stocks will be illustrated using a circulating recombinant form 1 (CRF01_AE, formerly known as subtype E) primary isolate.

Cloning, Molecular↗
PubMed2005

A substitution in rous sarcoma virus integrase that separates its two biologically relevant enzymatic activities.

Retroviral integrase prepares viral DNA for integration by removing 2 nucleotides from each end of unintegrated DNA in a reaction referred to as processing. However, it has been known since the processing assay was first described that avian integrases frequently nick 3 nucleotides, as well as 2 nucleotides, from viral DNA ends when reaction mixtures contain Mn2+. We now report that specificity for the biologically relevant "-2" site is enhanced when the serine at amino acid 124 of Rous sarcoma virus (RSV) integrase is replaced by alanine, valine, glycine, lysine, or aspartate. The protein with a serine-to-aspartate substitution exhibited especially high fidelity for the correct site, as evidenced by a ratio of -2 nicks to -3 nicks that was more than 40-fold greater than that for the wild-type enzyme in reactions with Mn2+. Even with Mg2+, the substituted proteins exhibited greater specificity than the wild type, especially the S124D protein. Moreover, this protein was more efficient than the wild type at processing viral DNA ends. Unexpectedly, however, the S124D protein was significantly impaired at catalyzing the insertion of viral DNA ends in reactions with Mn2+ and joining was undetectable in reactions with Mg2+. Thus, the S124D protein has separated the processing and joining activities of integrase. Similar results were found for human immunodeficiency virus integrase with the analogous substitution. No proteins with comparable properties have been described. Moreover, RSV virions containing integrase with the S124D mutation were unable to replicate in cell cultures. Together, these data suggest that integrase has evolved to have submaximal processing activity so that it can also catalyze DNA joining.

Amino Acid Substitution↗
PubMedDate not supplied

A method for describing and evaluating naturopathic whole practice.

CONTEXT: Even though complementary and alternative medicine (CAM) is generally practiced as distinct systems of medicine, almost all CAM research has focused on single therapies. In order to more adequately evaluate the effectiveness of these medical systems, studies that evaluate the outcome of intact whole systems are needed. One challenge lies in defining the whole medical system (and any medical system it is compared to) in a way that ensures treatment fidelity. OBJECTIVE: This paper presents a proposed method to measure treatment fidelity (treatment criteria) in studies of the naturopathic medical system. DESIGN: Illustrative example of the theory-based development and post-hoc "testing" of treatment criteria against an existing database of actual treatments prescribed by a random sample of naturopathic physicians. MAIN OUTCOME MEASURES: Treatment criteria for 3 conditions--menopausal symptoms, bowel dysfunction, and fatigue/fibromyalgia--and their comparison to actual treatments prescribed. RESULTS: A set of meaningful, measurable treatment criteria based on the naturopathic practice principles were defined that could have generated the majority (82%-93%) of treatment prescriptions given at visits for these conditions. Several of the treatment criteria components are common across the 3 conditions studied, and might be appropriate for all visits to doctors of naturopathy (NDs). Others are specific to each condition. In addition to ensuring model validity, these criteria help identify critical components of care, enable study replication, provide a measure of quality of care, and are one step toward allowing CAM to be studied as it is generally practiced-as distinct systems of medicine. SETTING: Work was performed at Bastyr University and the University of Arizona.

Clinical Protocols↗
PubMed2004

Rationale for maintenance of the M184v resistance mutation in human immunodeficiency virus type 1 reverse transcriptase in treatment experienced patients.

The M184V substitution in HIV-1 RT develops rapidly following initiation of therapy with 3TC and confers high-level phenotypic resistance to this drug both in vitro and in vivo. Interestingly, the presence of M184V is also associated with alteration of several mechanisms relating to RT function that include decreased RTprocessivity, reduced nucleotide-dependent primer unblocking, increased fidelity, hypersensitization to other NRTIs, impaired viral fitness, and delayed appearance of mutations in RT that are responsible for resistance to thymidine analogues (i.e. thymidine-associated mutations or TAMs). In addition, M184V may affect viral transmission and immunological response. Collectively, these factors might explain the residual antiviral effect and clinical benefit observed with continued use of 3TC in combination therapy regimens following the emergence of M184V.

Amino Acid Substitution↗
PubMed2005

Eukaryotic translesion synthesis DNA polymerases: specificity of structure and function.

This review focuses on eukaryotic translesion synthesis (TLS) DNA polymerases, and the emphasis is on Saccharomyces cerevisiae and human Y-family polymerases (Pols) eta, iota, kappa, and Rev1, as well as on Polzeta, which is a member of the B-family polymerases. The fidelity, mismatch extension ability, and lesion bypass efficiencies of these different polymerases are examined and evaluated in the context of their structures. One major conclusion is that, despite the overall similarity of basic structural features among the Y-family polymerases, there is a high degree of specificity in their lesion bypass properties. Some are able to bypass a particular DNA lesion, whereas others are efficient at only the insertion step or the extension step of lesion bypass. This functional divergence is related to the differences in their structures. Polzeta is a highly specialized polymerase specifically adapted for extending primer termini opposite from a diverse array of DNA lesions, and depending upon the DNA lesion, it contributes to lesion bypass in a mutagenic or in an error-free manner. Proliferating cell nuclear antigen (PCNA) provides the central scaffold to which TLS polymerases bind for access to the replication ensemble stalled at a lesion site, and Rad6-Rad18-dependent protein ubiquitination is important for polymerase exchange.

Amino Acid Motifs↗
PubMed2006

Base excision repair fidelity in normal and cancer cells.

In mammalian cells, base excision repair (BER) is the major repair pathway involved in the removal of non-bulky damaged nucleotides. The fidelity of BER is dependent on the polymerization step, where the major BER DNA polymerase (Pol beta) must incorporate the correct Watson-Crick base paired nucleotide into the one nucleotide repair gap. Recent studies have indicated that expression of some Pol beta variants or changes in expression of wild-type Pol beta protein, frequently found in cancer cells, can lead to DNA repair synthesis errors and confers to cells a mutator phenotype.

Animals↗
PubMed2005

A sliding-clamp toolbelt binds high- and low-fidelity DNA polymerases simultaneously.

This report demonstrates that the beta sliding clamp of E. coli binds two different DNA polymerases at the same time. One is the high-fidelity Pol III chromosomal replicase and the other is Pol IV, a low-fidelity lesion bypass Y family polymerase. Further, polymerase switching on the primed template junction is regulated in a fashion that limits the action of the low-fidelity Pol IV. Under conditions that cause Pol III to stall on DNA, Pol IV takes control of the primed template. After the stall is relieved, Pol III rapidly regains control of the primed template junction from Pol IV and retains it while it is moving, becoming resistant to further Pol IV takeover events. These polymerase dynamics within the beta toolbelt complex restrict the action of the error-prone Pol IV to only the area on DNA where it is required.

DNA Polymerase III↗
PubMed1999

Replication block by an enediyne drug-DNA deoxyribose adduct.

Under anaerobic conditions neocarzinostatin chromophore, an enediyne antibiotic, forms a covalent drug-DNA adduct on the 5' carbon of deoxyribose at a specific single site in a 2-nucleotide bulge, rather than strand cleavage, by a mechanism involving general base-catalyzed intramolecular drug activation to a reactive radical species. We have taken advantage of the selectivity of this reaction to prepare a single-stranded oligonucleotide containing a single drug adduct at a T residue and to study its effect on the template properties of the oligonucleotide in replicative synthesis, as followed by 5'-32P-labeled primer extension by several DNA polymerases. With the Klenow fragment of Escherichia coli DNA polymerase I, synthesis stops at the base immediately 3' to the adduct. The same enzyme, but lacking 3' to 5' exonuclease activity, permits synthesis to proceed by one additional nucleotide. This effect is enhanced when Mn2+ is substituted for Mg2+. T4, herpes simplex virus, and cytomegalovirus DNA polymerases all act like Klenow polymerase. Sequenase (exo-minus T7 DNA polymerase) is qualitatively similar to exo-minus Klenow polymerase but is more efficient in inserting a nucleotide opposite the lesion. With the small-gap-filling human DNA polymerase beta, which lacks intrinsic exonucleolytic activity, primer extension proceeds to the nucleotide opposite the lesion. However, when a gap was created opposite the lesion, polymerase beta adds as many as two additional nucleotides 5' to the adduct site. The fidelity of base incorporation opposite the lesion was not impaired, in contrast with adducts on DNA bases.

Antibiotics, Antineoplastic↗