Developing Multipollutant Exposure Indicators of Traffic Pollution: The Dorm Room Inhalation to Vehicle Emissions (DRIVE) Study.

Introduction: The Dorm Room Inhalation to Vehicle Emissions (DRIVE2) study was conducted to measure traditional single-pollutant and novel multipollutant traffic indicators along a complete emission-to-exposure pathway. The overarching goal of the study was to evaluate the suitability of these indicators for use as primary traffic exposure metrics in panel-based and small-cohort epidemiological studies. Methods: Intensive field sampling was conducted on the campus of the Georgia Institute of Technology (GIT) between September 2014 and January 2015 at 8 monitoring sites (2 indoors and 6 outdoors) ranging from 5 m to 2.3 km from the busiest and most congested highway artery in Atlanta. In addition, 54 GIT students living in one of two dormitories either near (20 m) or far (1.4 km) from the highway were recruited to conduct personal exposure sampling and weekly biomonitoring. The pollutants measured were selected to provide information about the heterogeneous particulate and gaseous composition of primary traffic emissions, including the traditional traffic-related species (e.g., carbon monoxide [CO], nitrogen dioxide [NO2], nitric oxide [NO], fine particulate matter [PM2.5], and black carbon [BC]), and of secondary species (e.g., ozone [O3] and sulfate as well as organic carbon [OC], which is both primary and secondary) from traffic and other sources. Along with these pollutants, we also measured two multipollutant traffic indicators: integrated mobile source indicators (IMSIs) and fine particulate matter oxidative potential (FPMOP). IMSIs are derived from elemental carbon (EC), CO, and nitrogen oxide (NOx) concentrations, along with the fractions of these species emitted by gasoline and diesel vehicles, to construct integrated estimates of gasoline and diesel vehicle impacts. Our FPMOP indicator was based on an acellular assay involving the depletion of dithiothreitol (DTT), considering both water-soluble and insoluble components (referred to as FPMOPtotal-DTT). In addition, a limited assessment of 18 low-cost sensors was added to the study to supplement the four original aims. Results: Pollutant levels measured during the study showed a low impact by this highway hotspot source on its surrounding vicinity. These findings are broadly consistent with results from other studies throughout North America showing decreased relative contributions to urban air pollution from primary traffic emissions. We view these reductions as an indication of a changing near-road environment, facilitated by the effectiveness of mobile source emission controls. Many of the primary pollutant species, including NO, CO, and BC, decreased to near background levels by 20 to 30 m from the highway source. Patterns of correlation among the sites also varied by pollutant and time of day. NO2 exhibited spatial trends that differed from those of the other single-pollutant primary traffic indicators. We believe this was caused by kinetic limitations in the photochemical chemistry, associated with primary emission reductions, required to convert the NO-dominant primary NOx, emitted from automobiles, to NO2. This finding provides some indication of limitations in the use of NO2 as a primary traffic exposure indicator in panel-based health effect studies. Roadside monitoring of NO, CO, and BC tended to be more strongly correlated with sites, both near and far from the road, during morning rush hour periods and often weakly to moderately correlated during other time periods of the day. This pattern was likely associated with diurnal changes in mixing and chemistry and their impact on spatial heterogeneity across the campus. Among our candidate multipollutant primary traffic indicators, we report several key findings related to the use of oxidative potential (OP)-based indicators. Although earlier studies have reported elevated levels of FPMOP in direct exhaust emissions, we found that atmospheric processing further enhanced FPMOPtotal-DTT, likely associated with the oxidation of primary polycyclic aromatic hydrocarbons (PAHs) to quinones and hydroxyquinones and with the oxidization and water solubility of metals. This has important implications in terms both of the utility of FPMOPtotal-DTT as a marker for exhaust emissions and of the importance of atmospheric processing of particulate matter (PM) being tied to potential health outcomes. The results from the personal exposure monitoring also point to the complexity and diversity of the spatiotemporal variability patterns among the study monitoring sites and the importance of accounting for location and spatial mobility when estimating exposures in panel-based and small-cohort studies. This was most clearly demonstrated with the personal BC measurements, where ambient roadside monitoring was shown to be a poor surrogate for exposures to BC. Alternative surrogates, including ambient and indoor BC at the participants' respective dorms, were more strongly associated with personal BC, and knowledge of the participants' mean proximity to the highway was also shown to explain a substantial level of the variability in corresponding personal exposures to both BC and NO2. In addition, untargeted metabolomic indicators measured in plasma and saliva, which represent emerging methods for measuring exposure, were used to extract approximately 20,000 and 30,000 features from plasma and saliva, respectively. Using hydrophilic interaction liquid chromatography (HILIC) in the positive ion mode, we identified 221 plasma features that differed significantly between the two dorm cohorts. The bimodal distribution of these features in the HILIC column was highly idiosyncratic; one peak consisted of features with elevated intensities for participants living in the near dorm; the other consisted of features with elevated intensities for participants in the far dorm. Both peaks were characterized by relatively short retention times, indicative of the hydrophobicity of the identified features. The results from the metabolomics analyses provide a strong basis for continuing this work toward specific chemical validation of putative biomarkers of traffic-related pollution. Finally, the study had a supplemental aim of examining the performance of 18 low-cost CO, NO, NO2, O3, and PM2.5 pollutant sensors. These were colocated alongside the other study monitors and assessed for their ability to capture temporal trends observed by the reference monitoring instrumentation. Generally, we found the performance of the low-cost gas-phase sensors to be promising after extensive calibration; the uncalibrated measurements alone, however, would likely not have led to reliable results. The low-cost PM sensors we evaluated had poor accuracy, although PM sensor technology is evolving quickly and warrants future attention. Conclusions: An immediate implication of the changing near-road environment is that future studies aimed at characterizing hotspots related to mobile sources and their impacts on health will need to consider multiple approaches for characterizing spatial gradients and exposures. Specifically and most directly, the mobile source contributions to ambient concentrations of single-pollutant indicators of traffic exposure are not as distinguishable to the degree that they have been in the past. Collectively, the study suggests that characterizing exposures to traffic-related pollutants, which is already difficult, will become more difficult because of the reduction in traffic-related emissions. Additional multi-tiered approaches should be considered along with traditional measurements, including the use of alternative OP measures beyond those based on DTT assays, metabolomics, low-cost sensors, and air quality modeling.

Population genetics of Schistosoma haematobium: development of novel microsatellite markers and their application to schistosomiasis control in Mali.

The recent implementation of mass drug administration (MDA) for control of uro-genital schistosomiasis has identified an urgent need for molecular markers to both directly monitor the impact of MDA, for example to distinguish re-infections from uncleared infections, as well as understand aspects of parasite reproduction and gene flow which might predict evolutionary change, such as the development and spread of drug resistance. We report the development of a novel microsatellite tool-kit allowing, for the first time, robust genetic analysis of individual S. haematobium larvae collected directly from infected human hosts. We genotyped the parasite populations of 47 children from 2 schools in the Ségou region of Mali, the first microsatellite study of this highly neglected parasite. There was only limited evidence of population subdivision between individual children or between the two schools, suggesting that few barriers to gene flow exist in this population. Complex relationships between parasite reproductive success, infection intensity and host age and gender were identified. Older children and boys harboured more diverse infections, as measured by the number of unique adult genotypes present. Individual parasite genotypes had variable reproductive success both across hosts, a pre-requisite for evolutionary selection, and, phenotypically, in hosts of different ages and genders. These data serve as a baseline against which to measure the effect of treatment on parasite population genetics in this region of Mali, and the tools developed are suitable to further investigate this important pathogen, and its close relatives, throughout their range.

Spermiogenesis defects in human: detection of transition proteins in semen from some infertile men.

Semen samples from 60 infertile men were examined by flow cytometry following propidium iodide staining. Of these, 23 samples contained young haploid cells. Transition proteins (TP1 and/or TP2) were detected in 12 of these, using immunohistochemical staining. The presence of TPs in spermatids in semen indicates inhibition in the differentiation pathway from round spermatids to spermatozoa. Cells of this type were found in semen from patients with nonobstructive azoospermia, severe to extreme cases of oligozoospermia, asthenozoospermia and teratozoospermia.

Isolation and characterization of the first polymorphic microsatellite markers for Schistosoma haematobium and their application in multiplex reactions of larval stages.

The ability of microsatellite loci to reveal genetic diversity within the trematode Schistosoma haematobium is demonstrated for the first time. Nine novel polymorphic microsatellite markers were isolated and their viability assessed on 36 S. haematobium adult worm individuals from three geographical populations. Allelic diversity and gene diversity ranged from two to seven and from 0.29 to 0.76, respectively, suggesting high variability between individuals and between unrelated populations. Three primers also amplified Schistosoma mansoni and two Schistosoma japonicum. The results suggest these primers are useful for population genetic analyses of S. haematobium.

Flow cytometry of human semen: a preliminary study of a non-invasive method for the detection of spermatogenetic defects.

BACKGROUND: The pathway of spermatogenesis involves the conversion of diploid stem cells (spermatogonia) to tetraploid primary spermatocytes, followed by meiosis and two cell divisions, first forming diploid secondary spermatocytes and then haploid round spermatids. Differentiation of round spermatids results in spermatozoa containing condensed chromatin. It has long been known that semen from patients with non-obstructive azoospermia or oligospermia contains small numbers of immature germinal cells. In this article, a flow cytometric procedure is described for assessing defects in spermatogenesis by identifying the ploidy of those immature cells. METHODS: Cells in semen samples from 44 infertile patients and 14 controls were stained with propidium iodide, which displays red fluorescence when intercalated between bases in double-stranded DNA. The resulting cell suspension was examined by quantitative flow cytometry, with excitation by laser light (488 nm) and red fluorescence recorded on a logarithmic scale to allow easy differentiation between intensities of tetraploid, diploid and haploid round spermatids, and spermatozoa containing condensed chromatin. RESULTS: The flow cytometric method differentiated between cases of 'Sertoli cell-only' syndrome (complete absence of tetraploid and haploid cells) and cases where spermatogenesis was blocked in meiosis or in spermiogenesis. Flow cytometric histograms from semen samples from normozoospermic, oligozoospermic and azoospermic patients fell into patterns that correlated well with the results obtained from testis histology findings. CONCLUSIONS: The method described may serve as a simple, non-invasive and reliable assay to help clinicians counsel patients with severe male infertility before referring them for testicular surgery to locate spermatozoa for ICSI.

[Evaluation of sperm samples from infertile men by flow cytometry].

INTRODUCTION: Human spermatogenesis begins at adolescence and continues throughout life. This process includes morphologic, cytologic and biological changes, leading to the formation of mature spermatozoa. Male infertility may be caused by several reasons, including oligozoospermia at variable degrees and complete absence of mature spermatozoa. Routine spermatogram, measuring sperm counts, motility and morphology, might not provide complete information in the evaluation of these cases. This study is aimed to evaluate the possible use of flow cytometry in the identification of different sperm cell populations in sperm samples obtained from infertile men, and in determining the different cell types in various groups of infertile men. MATERIALS AND METHODS: Sperm samples from normal and infertile men (the latter were azoospermic or oligoteratozoospermic OTA) underwent flow cytometry analysis, after preparation with TNE buffer and staining with Propidium Iodide. The separation of germinal cells into different populations, according to their DNA content and chromatin condensation, was evaluated. The WINMDI (http://fac.-scripps.edu, J. Trotter) software was used for data analysis. RESULTS: Flow cytometric analysis enabled identification of several cell populations in sperm samples, including haploid, diploid and tetraploid cells. Certain cellular distribution patterns were observed in sperm samples from infertile men: mature haploid cells, diploid cells, domination of tetraploid or non-mature haploid cells, and combination of these patterns. These patterns appeared in a statistically different manner among fertile and infertile men; the median value of mature haploid cells was higher in normal men (91%, compared to 85% in the OTA group and 0% in the azoospermic men), while the median value of diploid and tetraploid cells was higher in azoospermic men (72% and 8.5% respectively, compared to only 1% and 0% in normal men). CONCLUSIONS: These findings suggest that flow cytometry of sperm samples may serve as a non-invasive tool for investigations of male infertility and for identification of appropriate candidates for interventional treatment.

Evidence for biological rhythm in spermatogenesis in the pubertal hamster (Mesocricetus auratus): a flow cytometric study.

The number of cells in the S-phase fraction of the cell cycle reflects proliferative activity. Using flow cytometry histograms and the Phoenix M+ cell cycle program, the percent of cells in the S-phase fraction was measured in single cell suspensions prepared from testes of hamsters of different ages. A cyclical pattern with a period of 9 days, superimposed on another rhythm with a 38 day period was observed (p < 0.01) during hamster maturation and it disappeared after the second spermatogenic wave, where the S phase values reached a plateau. It was concluded that maturing animals passed through a stage in which testicular biological rhythm was involved. Therefore it was concluded that it takes approximately two spermatogenic waves before the proliferation rate in the testis reached a steady state.

Evaluation of damage to the testicular cells of golden hamsters caused by experimental cryptorchidism using flow cytometry and confocal microscopy.

Artificial unilateral cryptorchidism was performed in golden hamsters which were then held for different periods of time. The non-operated side was used as a control. At various times from 4 to 15 days, hamsters were killed, testes were removed and weighed, single cell suspensions were prepared for flow cytometry analysis and seminiferous tubules were fixed for confocal microscopy. Using DNA staining by propidium iodide or acridine orange followed by flow cytometry analysis, a marked decrease in the haploid condensed cell fraction was detected at the beginning stages of experimental cryptorchidism. In correlation with flow cytometry results, spermiogenic arrest at stages IX and X of seminiferous epithelium was detected in these animals by confocal microscopy and there were no mature forms of haploid cells in the cryptorchid testis. In the testis with more severe damage, there were almost no haploid cells in the seminiferous tubules of cryptorchid animals. In addition, a significant decrease in tetraploid cell fraction and an increase in S-phase fraction was obtained in severe cases. This may be explained by cell arrest before entrance into meiosis. Destruction of tubule structure and cell arrangement were also observed by confocal microscopy in such cases. In conclusion, flow cytometry, combined with confocal analysis, added useful information about spermatogenesis disturbances in cryptorchid testis and it may be used as diagnostic tools in other cases of spermatogenic disorders.

Meiosis in the golden hamster: a confocal microscopy and flow cytometric analysis.

In this study, confocal microscopy and flow-cytometry were utilized to follow meiosis in hamster spermatogenesis. Confocal microscopy was used as an analytical tool to observe spermatocytes inside the tubules following meiotic progression consecutively at defined spermatogenic stages. To study spermatocyte differentiation, the structure of the synaptonemal complex was studied in detail at various stages of hamster spermatogenesis using the antibody against SC3 (the protein of axial/lateral element). The synaptonemal complex was observed from the leptotene stage until the first meiotic division with maximal staining in mid-pachytene spermatocytes, suggesting a role for SC3 at this postrecombinational stage. In addition, 3-dimensional (3D) images of synaptonemal complex were observed, providing information about spatial distribution of the chromosomes within the nuclei of spermatocytes at different stages of meiosis. Changes in spermatocyte sizes and DNA condensation allowed assessment of meiosis by flow cytometry. Changes in chromatin condensation at different stages of hamster meiosis were followed, revealing decondensation from early to late pachytene stages. The analysis also allowed a comparing of chromatin status of mitotic and meiotic chromosomes, confirming the less compact structure of the latter, possibly connected to increased transcriptional activity during meiosis.

Procarbazine effects on spermatogenesis in golden hamster: a flow cytometric evaluation.

The response of hamster testis to the administration of 450mg/kg procarbazine (PCB) over a period of 4 weeks was evaluated. Flow cytometry was used to investigate changes in cell populations in testicular single cell suspensions and to correlate these changes with those observed in histological sections. PCB caused significant decrease in testicular and epididymal weight and a drastic reduction in haploid cells and spermatogenic arrest, demonstrating variation among the test animals. The results obtained confirm previous observations concerning detrimental effects of PCB upon spermatogenesis in species such as the rat and mouse, though its effect on hamster testis is milder and does not include the germinal stem cells. The histological evaluation of the testis showed a good correlation with flow cytometric evaluation, emphasizing the usefulness of this method in providing quantitative and rapid results.

Spermatogenesis in the golden hamster: the role of c-kit.

c-kit is related to the family of transmembrane tyrosine kinase receptors. Mutations in genes for either c-kit or its ligand, Steel factor, result in infertility, but the role of c-kit/SCF system in spermatogenesis is not well understood. In this study Western blot analysis together with confocal microscopy were used to follow c-kit expression in hamsters during the first spermatogenic wave in mature animals and in old age. Three antibodies raised against different domains of c-kit were tested on Western Blot. Confocal microscopy was performed after incubation of fixed seminiferous tubules with tested antibodies followed by binding of FITC-labeled secondary antibody. Longitudinal sections of seminiferous tubule were observed by confocal microscopy to determine in which stages of spermatogenesis and in which cell types c-kit was found. C-kit bands of 80,140, and 150 kDa were observed on Western blot, indicating that c-kit is a name related to several proteins sharing some common domains. Only the band of 150 kDa correlated with positive staining of c-kit in tubules using confocal microscopy. We term this protein c-kit150T (150 kDa, testis). We demonstrated that c-kit150T appeared in differentiating hamster spermatogonia at stages VII-VIII of adult spermatogenesis and at day 13-14 during the first spermatogenic wave. It remained attached to the cell until late pachytene. This suggests that c-kit may play a role in preparing the germinal cells to enter meiosis. In order to evaluate the effect of aging on the number of germ cells, B2 spermatogonia/Sertoli cell ratio was calculated in the group of young animals (5-7 months) compared to this ratio in older ones (20-26 months). A significant decrease (P < 0.01) in the number of B2 spermatogonia in the group of old hamsters as compared to young ones was seen. The calculated value for the B2 spermatogonia/Sertoli cell ratio was 5.6 +/- 0.7 in young animals and 3.8 +/- 1.2 in the 20-26 months ones. In addition, decrease in the intensity of staining for c-kit was detected in the old hamsters. These may be the reasons for subfertility in old age and in other cases of testicular disorders.

Probing biopolymers with the atomic force microscope: a review.

This short review presents an overview of atomic force microscopy (AFM) of biopolymers and specific examples of some of the biopolymers that have been analyzed by AFM. These specific examples include extracellular polymeric substances on the surfaces of bacterial biofilms, condensed DNA, DNA constructs, and DNA-protein interactions. In addition, two examples are presented for AFM analyses of proteins: laminin flexing its arms in solution and neurofilaments entropically brushing away the space around themselves.

Inhibition of Papaya latex papain by photosensitive inhibitors. 1-(4,5-dimethoxy-2-nitrophenyl)-2-nitroethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene.

1-(4,5-Dimethoxy-2-nitrophenyl)-2-nitroethene (1) was shown to be an irreversible inhibitor of papain (EC 3.4.22.2), causing a complete inhibition (120 min preincubation, pH 8.0), assuming that it attached to Cys-25 at the active site of the enzyme (while a short preincubation time caused activation). Only partial inhibition of papain was achieved, however, with 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2), a compound synthesized in this work, which is also an irreversible inhibitor of papain. Since both compounds 1 and 2, and in each case of the inhibited enzyme, were 2-nitrobenzyl derivatives, they and the modified enzyme were expected to be photosensitive. Indeed, irradiation of the inhibited enzyme in the presence of mercaptoethanol resulted in a full recovery of the enzyme activity following inactivation with compound 1 (similar to our previous finding with beta-galactosidase) and up to 67% recovery following inhibition with compound 2.

Photoreversible modulators of Escherichia coli beta-galactosidase. 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene and 1,1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene.

Beta-galactosidase (EC 3.2.1.23) is known to be inhibited by some thiol reagents. 1-Benzoyl-1-cyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (1) was shown to be an irreversible inhibitor, while 1, 1-dicyano-2-(4,5-dimethoxy-2-nitrophenyl)-ethene (2) was demonstrated as a positive irreversible modulator causing a rise of up to 186% in beta-galactosidase activity. Compound 2 is, however, an irreversible inhibitor of the cysteine proteinase papain (preceding paper). Kinetic values of beta-galactosidase at pH 8.3 with o-nitrophenyl beta-D-galactopyranoside (ONPG) as the substrate and for compounds 1 and 2 were determined and in view of model experiments, it was assumed that both compounds possibly reacted with the thiol side chain of Cys in the active site inducing allosteric changes in the enzyme. Since the enzyme, modified by compound 1 or 2, was a 2-nitrobenzyl derivative, near-UV irradiation resulted in a recovery of up to 91% and a reduction of the enzyme's activity to 90%, respectively.

Chromatin condensation during spermiogenesis in the golden hamster (Mesocricetus aureus): a flow cytometric study.

DNA-staining of hamster testis cell suspensions followed by flow cytometry demonstrated appearance of the first haploid cells at 23 days post partum (dpp) and of condensed chromatin (in elongated spermatids and spermatozoa) at 33-34 dpp. Mature spermatozoa were first observed in the caput epididymis at 36-37 dpp, thus completing the first spermatogenic wave. Testicular cell suspensions from animals from 23 to 38 dpp were stained with acridine orange, and flow cytometer gating was adjusted to include only the haploid cells. Acridine orange intercalated into double-stranded DNA to produce green fluorescence. The decrease in green fluorescence intensity from 23 until 37 dpp was caused by changes in the binding of DNA to basic proteins in such a fashion as to impede the access of the dye to the DNA double helix. When the green fluorescence values (of the most advanced spermatids) were plotted against the age of the hamsters (in dpp) or the corresponding steps of spermiogenesis, the decrease in fluorescence could be seen to occur in three phases. The inflection point between the first and second phases was observed at about spermiogenesis step 7, consistent with the hypothesis that this represents removal of histone from the chromatin. The second phase presumably represents the period in which transition proteins are bound to the DNA. At approximately steps 15 or 16 a further inflection point was seen where protamines replaced the transition proteins. The red fluorescence produced when acridine orange bound to RNA in spermatids, increased early in spermiogenesis and decreased dramatically at 34 dpp, consistent with the fact that elongating spermatids discard the bulk of their cytoplasm during the maturation process.

Spermatogenesis in the golden hamster during the first spermatogenic wave: a flow cytometric analysis.

In the present study propidium iodide was used as a fluorescent dye to stain DNA of cells of hamster testicular origin and fluorescent intensities were analyzed by flow cytometry. We used hamster testicular cells from the first spermatogenic wave to observe the consecutive appearance of the different types of cells during puberty. At 12 days postpartum (dpp) diploid cells (including spermatogonia) predominated and some tetraploid cells were also present. Tetraploid spermatocytes increased dramatically by 21 dpp. The first haploid cells appeared at 21 dpp but substantial numbers were first present at 23 dpp. Immature haploid cells predominated at 32 dpp. Elongating condensing spermatids appeared at 34 dpp and spermatozoa began to leave the testis to enter the epididymidis at 36-38 dpp marking the end of the first round of spermatogenesis. Using acridine orange staining flow cytometry, chromatin condensation was followed by measuring fluorescence decrease from early round spermatids to spermatozoa obtained from the initial segment and from the cauda epididymides. The major portion of sperm chromatin condensation (88-90%) in the hamster occurred in the testis and only 10-12% occurred during epididymal sperm maturation. Spermatozoa in the initial segment of the epididymidis of the hamster contained a small amount of RNA that was no longer present in sperm of the cauda epididymidis, indicating that RNA was lost during epididymal sperm maturation in this species. Mol. Reprod. Dev. 55:205-211, 2000.

Recent highlights from atomic force microscopy of DNA.

Abstract Seven recent highlights are presented from atomic force microscopy (AFM) of DNA in this lab. The first two involve advances in the observation of enzymatic reactions in near-physiological solutions. E. coli RNA polymerase was observed to process along its DNA template in a series of time-lapse images [S. Kasas, et al., Biochemistry 36, 461 (1997)], and a new small-cantilever atomic force microscope (AFM) imaged DNA degradation by DNase I at rates as fast as two seconds per image. The next five highlights involve structural observations of DNA and DNA-protein complexes, including DNA condensed for gene delivery, sequence-dependent DNA condensation, an AFM assay for RNA polymerase, and AFM evidence for a yeast kinetochore complex that may be involved in holding together sister chromatids during cell division.

Polymerase activities and RNA structures in the atomic force microscope.

The structures of the reaction products are the basis for novel polymerase assays using the atomic force microscope (AFM). Polymerases are the enzymes involved in transcription and replication of DNA. Rapid semiquantitative estimates of the activity of DNA polymerases such as Sequenase, Taq polymerase, and AMV reverse transcriptase and RNA polymerases (RNAP) such as Escherichia coli RNAP were obtained from AFM images of the nucleic acids after polymerase reactions. DNA polymerases were assayed via replication of the single-stranded φX-174 virion. RNAP was assayed via transcription, using a rolling circle DNA template that produces long strands of RNA. In some cases, AFM was better than agarose gel electrophoresis for assaying DNA polymerase activity, since aggregation prevented the DNA from entering the agarose gel. Extended molecules of single-stranded RNA synthesized with the rolling circle DNA template showed varied conformations and degrees of stretching. Some structural differences were observed between two RNAs-a ribozyme concatamer and an RNA with 90% purines.

DNA toroids: stages in condensation.

The effects of polylysine (PLL) and PLL-asialoorosomucoid (AsOR) on DNA condensation have been analyzed by AFM. Different types of condensed DNA structures were observed, which show a sequence of conformational changes as circular plasmid DNA molecules condense progressively. The structures range from circular molecules with the length of the plasmid DNA to small toroids and short rods with approximately 1/6 to 1/8 the contour length of the uncondensed circular DNA. Single plasmid molecules of 6800 base pairs (bp) condense into single toroids of approximately 110 nm diameter, measured center-to-center. The results are consistent with a model for DNA condensation in which circular DNA molecules fold several times into progressively shorter rods. Structures intermediate between toroids and rods suggest that at least some toroids may form by the opening up of rods as proposed by Dunlap et al. [(1997) Nucleic Acids Res. 25, 3095]. Toroids and rods formed at lysine:nucleotide ratios of 5:1 and 6:1. This high lysine:nucleotide ratio is discussed in relation to entropic considerations and the overcharging of macroions. PLL-AsOR is much more effective than PLL alone for condensing DNA, because several PLL molecules are attached to a single AsOR molecule, resulting in an increased cation density.