Sediment DNA Research Articles

NahR, encoding a LysR-type transcriptional regulator, is highly conserved among naphthalene-degrading bacteria isolated from a coal tar waste-contaminated site and in extracted community DNA.

In Pseudomonas putida strain G7, a LysR-type positive transcriptional activator protein encoded by nahR is necessary for activation of two operons involved in naphthalene catabolism [Schell, M. A. & Poser, E. F. (1989). J Bacteriol 171, 837-846]. The role of an nahR homologue, NCIB-nahR, in another naphthalene-metabolizing bacterium, P. putida NCIB 9816-4 was verified. Targeted disruption of NCIB-nahR by homologous recombination resulted in a growth defect in the presence of naphthalene or salicylate as sole carbon and energy source. The nahR homologues and intergenic regions between nahR-like and nahG-like genes from P. putida NCIB 9816-4 and seven bacteria native to a naphthalene-rich coal tar contaminated site were amplified by PCR using degenerate primers. The amplified nahR homologues and the intergenic regions were cloned and sequenced. Alignment of the deduced amino acid sequences from NahR homologues revealed that NahR-like proteins showed only minor variations in all investigated naphthalene-degrading isolates. The intergenic regions, together with known NahR-binding sites showed the consensus NahR-protein-binding sites (5'-ATTCACGCTN(2)TGAT-3'). Surprisingly, amplified intergenic regions from naphthalene-degrading micro-organisms native to this study site were 100% identical to that of the pDTG1 plasmid (an archetypal naphthalene-catabolic plasmid from Pseudomonas putida NCIB 9816-4), but the nahR coding regions were not. DNA representing the uncultured microbial community was extracted from six sediment samples with varying coal tar exposure histories. PCR amplification of nahR from sediment DNA was observed in contaminated samples, but in uncontaminated samples only following laboratory incubation with naphthalene. The sediment-derived PCR products were sequenced and also found to be almost identical to known nahR genes. Thus, the structure and function of nahR-nahG regulatory genes appear to be highly conserved.

Quantification, base composition, and fate of extracellular DNA in marine sediments

The discovery of high concentrations of DNA in marine sediments unaccounted for by living biomass suggests the presence of a large fraction of extracellular DNA, which might play an important role in gene transfer via natural transformation as well as in phosphorous biogeochemical cycling. But a universally accepted procedure for extracellular DNA extraction is not available yet. In this study, we developed a new nuclease‐based procedure to extract extracellular DNA from marine sediments. Coastal sand and deep‐sea mud samples were collected to test the efficiency of extracellular DNA removal from different sediment types. Extracellular DNA concentrations were quantified at six sediment depths, and changes in base composition were investigated to gather information on extracellular DNA fate. The extraction procedure was highly specific and only extracellular DNA was hydrolyzed after nuclease treatment. Hydrolyzable DNA accounted for ≪10 to ≫70% of the total DNA pool, suggesting that extracellular DNA can only be partially degraded. Base composition changed vertically with depth in the sediment as deoxycytidine content increased and deoxyadenosine decreased with increasing depth. Integrating our results for the top 15 cm of the sediment, we calculate that more than 50% of extracellular DNA was recalcitrant to enzymatic degradation. This finding might explain why DNA accumulates in surface sediments and suggests that DNA might play a nonnegligible role in P biogeochemical cycle.

Application of the 5' fluorogenic exonuclease assay (TaqMan) for quantitative ribosomal DNA and rRNA analysis in sediments.

In this study, we report on the development of quantitative PCR and reverse transcriptase PCR assays for the 16S rRNA of Geobacter spp. and identify key issues related to fluorogenic reporter systems for nucleic acid analyses of sediments. The lower detection limit of each assay was 5 to 50 fg of genomic DNA or < or =2 pg of 16S rRNA. TaqMan PCR spectral traces from uncontaminated, amended aquifer sediments were significantly lower (P < 0.0002) than traces for the external standard curve. We also observed a similar, significant decrease in mean quencher emissions for undiluted extracts relative to those for diluted extracts (P < 0.0001). If PCR enumerations were based solely upon the undiluted sample eluant, the TaqMan assay generated an inaccurate result even though the threshold cycle (C(t)) measurements were precise and reproducible in the sediment extracts. Assay accuracy was significantly improved by employing a system of replicate dilutions and replicate analyses for both DNA and rRNA quantitation. Our results clearly demonstrate that fluorescence quenching and autofluorescence can significantly affect TaqMan PCR enumeration accuracy, with subsequent implications for the design and implementation of TaqMan PCR to sediments and related environmental samples.

Affinity purification of DNA and RNA from environmental samples with peptide nucleic acid clamps.

Bispeptide nucleic acids (bis-PNAs; PNA clamps), PNA oligomers, and DNA oligonucleotides were evaluated as affinity purification reagents for subfemtomolar 16S ribosomal DNA (rDNA) and rRNA targets in soil, sediment, and industrial air filter nucleic acid extracts. Under low-salt hybridization conditions (10 mM NaPO(4), 5 mM disodium EDTA, and 0.025% sodium dodecyl sulfate [SDS]) a PNA clamp recovered significantly more target DNA than either PNA or DNA oligomers. The efficacy of PNA clamps and oligomers was generally enhanced in the presence of excess nontarget DNA and in a low-salt extraction-hybridization buffer. Under high-salt conditions (200 mM NaPO(4), 100 mM disodium EDTA, and 0.5% SDS), however, capture efficiencies with the DNA oligomer were significantly greater than with the PNA clamp and PNA oligomer. Recovery and detection efficiencies for target DNA concentrations of > or =100 pg were generally >20% but depended upon the specific probe, solution background, and salt condition. The DNA probe had a lower absolute detection limit of 100 fg of target (830 zM [1 zM = 10(-21) M]) in high-salt buffer. In the absence of exogenous DNA (e.g., soil background), neither the bis-PNA nor the PNA oligomer achieved the same absolute detection limit even under a more favorable low-salt hybridization condition. In the presence of a soil background, however, both PNA probes provided more sensitive absolute purification and detection (830 zM) than the DNA oligomer. In varied environmental samples, the rank order for capture probe performance in high-salt buffer was DNA > PNA > clamp. Recovery of 16S rRNA from environmental samples mirrored quantitative results for DNA target recovery, with the DNA oligomer generating more positive results than either the bis-PNA or PNA oligomer, but PNA probes provided a greater incidence of detection from environmental samples that also contained a higher concentration of nontarget DNA and RNA. Significant interactions between probe type and environmental sample indicate that the most efficacious capture system depends upon the particular sample type (and background nucleic acid concentration), target (DNA or RNA), and detection objective.

Spatial and Temporal Assessment of Diazotroph Assemblage Composition in Vegetated Salt Marsh Sediments Using Denaturing Gradient Gel Electrophoresis Analysis.

> Abstract Diazotroph assemblage compositions were assessed in rhizosphere sediments from the tall and short form Spartina alterniflora growth zones over an annual cycle. Sediment cores were collected for DNA extraction and nitrogenase (acetylene reduction) activity assays, and porewater samples were analyzed for several chemical parameters in March, June, September, and December 1997. These data were collected to determine if within- or between-zone differences in the diazotroph assemblage composition correlated with differences in key environmental variables or acetylene reduction activity. Acetylene reduction rates differed between zones and within a zone over an annual period. Soluble sulfide concentrations were higher in the short form S. alterniflora zone on all dates except those in June and differed within both zones on different sample dates. nifH sequences were recovered from rhizosphere sediment DNA by PCR amplification using nifH specific primers. These amplimers were analyzed using denaturing gradient gel electrophoresis (DGGE), and the resulting patterns were compared by neural network and linear discriminant analyses. Ten prominent amplimers, four of which were apparent heteroduplexes, were observed. DGGE banding profiles showed minor differences among sampling dates and between sample zones, but the overall banding pattern was remarkably consistent. This reflects overall similarity between the amplifiable diazotroph assemblages in the tall and short S. alterniflora growth zones and substantial seasonal stability in assemblage composition.http://link.springer-ny.com/link/service/journals/00248/bibs/38n2p157.html

Nucleic acid concentrations (DNA, RNA) in the continental and deep-sea sediments of the eastern Mediterranean: relationships with seasonally varying organic inputs and bacterial dynamics

In order to study temporal variations of the genetic material in the continental shelf and deep-sea sediments of the extremely oligotrophic Cretan Sea, samples were collected on seasonal basis from August 1994 to September 1995, with a multiple corer, at seven stations (from 40 to 1540 m depth). Surface sediments (0–1 cm) were sub-sampled and analyzed for nucleic acid content (DNA, RNA) and bacterial density. DNA concentrations in the sediments were high (on annual average, 25.0 μg g -1) and declined with increasing water depth, ranging from 3.5 to 55.2 μg g -1. DNA concentrations displayed wide temporal changes also at bathyal depths confirming the recent view of the large variability of the deep-sea environments. Also RNA concentrations decreased with increasing water depth (range: 0.4–29.9 μg g -1). The ratio of RNA to DNA did not show a clear spatial pattern but was characterized by significant changes between sampling periods. DNA concentrations were significantly correlated with protein and phytopigment concentrations in the sediment, indicating a possible relationship with the inputs of primary organic matter from the photic layer. Bacterial densities were generally high (range: 0.9–4.6×10 8 cells g -1) compared to other deep-sea environments and decreased with increasing water depth. Estimates of the bacterial contribution to the sedimentary genetic material indicated that bacterial-DNA accounted, on annual average, for a small fraction of the total DNA pool (4.3%) but that bacterial-RNA represented a significant fraction of the total sedimentary RNA (26%). Bacterial contribution to nucleic acids increased, even though irregularly, with increasing depth. In deep-sea sediments, changes in RNA concentrations appear to be largely dependent upon bacterial dynamics. Estimates of the overall living contribution to the DNA pools (i.e. microbial plus meiofaunal DNA) indicated that the large majority (about 90%) of the DNA in continental and deep-sea sediments of the eastern Mediterranean was detrital. The non-living DNA pools reach extremely high concentrations up to 0.41 g DNA m -2 cm -1. Thus, especially in deep benthic habitats, characterized by low inputs of labile organic compounds, detrital DNA could represent a suitable and high quality food source or a significant reservoir of nucleic acid precursors for benthic metabolism.

Novel division level bacterial diversity in a Yellowstone hot spring.

A culture-independent molecular phylogenetic survey was carried out for the bacterial community in Obsidian Pool (OP), a Yellowstone National Park hot spring previously shown to contain remarkable archaeal diversity (S. M. Barns, R. E. Fundyga, M. W. Jeffries, and N. R. Page, Proc. Natl. Acad. Sci. USA 91:1609-1613, 1994). Small-subunit rRNA genes (rDNA) were amplified directly from OP sediment DNA by PCR with universally conserved or Bacteria-specific rDNA primers and cloned. Unique rDNA types among > 300 clones were identified by restriction fragment length polymorphism, and 122 representative rDNA sequences were determined. These were found to represent 54 distinct bacterial sequence types or clusters (> or = 98% identity) of sequences. A majority (70%) of the sequence types were affiliated with 14 previously recognized bacterial divisions (main phyla; kingdoms); 30% were unaffiliated with recognized bacterial divisions. The unaffiliated sequence types (represented by 38 sequences) nominally comprise 12 novel, division level lineages termed candidate divisions. Several OP sequences were nearly identical to those of cultivated chemolithotrophic thermophiles, including the hydrogen-oxidizing Calderobacterium and the sulfate reducers Thermodesulfovibrio and Thermodesulfobacterium, or belonged to monophyletic assemblages recognized for a particular type of metabolism, such as the hydrogen-oxidizing Aquificales and the sulfate-reducing delta-Proteobacteria. The occurrence of such organisms is consistent with the chemical composition of OP (high in reduced iron and sulfur) and suggests a lithotrophic base for primary productivity in this hot spring, through hydrogen oxidation and sulfate reduction. Unexpectedly, no archaeal sequences were encountered in OP clone libraries made with universal primers. Hybridization analysis of amplified OP DNA with domain-specific probes confirmed that the analyzed community rDNA from OP sediment was predominantly bacterial. These results expand substantially our knowledge of the extent of bacterial diversity and call into question the commonly held notion that Archaea dominate hydrothermal environments. Finally, the currently known extent of division level bacterial phylogenetic diversity is collated and summarized.

DNA recovery and PCR quantification of catechol 2,3-dioxygenase genes from different soil types

With the objective of monitoring xenobiotic degrading bacteria in soil, a method for rapid extraction of DNA from soil, amenable to amplification by PCR, was developed. The method was based on lysis by freeze-thawing and subsequent addition of sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide and proteinase K. The extraction method required 2 h and was tested on six different soils differing in organic content, water holding capacity and pH, including ones from which DNA extraction is difficult. DNA yields from the soils ranged from 6.1 to 54.0 μg of DNA per g soil. The efficiency and reproducibility of the DNA extraction method were evaluated by competitive PCR. The organic content in the soils was a major factor affecting the amount of obtained DNA amenable for amplification by PCR. A PCR primer-pair was designed on the basis of the known nucleotide sequences of several catechol 2,3-dioxygenase genes. The specificity of the primer-pair was demonstrated on different sequenced catechol 2,3-dioxygenase genes and on site-specific bacterial isolates from polycyclic aromatic hydrocarbon (PAH)-contaminated soil. The concentration of catechol 2,3-dioxygenase DNA in PAH-contaminated sediment undergoing an ex situ compost process was quantified by competitive PCR over a period of 16 weeks. The concentration of PAHs and catechol 2,3-dioxygenase DNA in the soil samples, was found to correlate.

Acid extraction of tritium label from bacterial DNA in clay sediment

During the process of bacterial production measurements in sediment with tritiated thymidine, the extraction of labelled DNA is often serious problem, particulary with clay sediments. When the efficiency of labelled DNA recovery with hydrochloric acid at different concentrations was tested, it was found that 0.5 M HCl at 95°C for 16 h gave good recovery (≈80%) with low relative standard deviation variation (1%) for sediments rich in fine silt-clay.

Possible Use of Rna:Dna Ratio for Detecting Oil Induced Disturbance: A Field Report

Abstract The effects of oil contamination on the metabolic state of suspended participate and sedimented organic matter were investigated through the analysis of the RNA:DNA ratio on samples collected from January 1991 to January 1992 in the Ligurian Sea (northwestern Mediterranean). the ratio of RNA to DNA appeared to be highly sensitive to hydrocarbon stress since the oil disturbance resulted in an evident decline in the RNA:DNA ratio relative to the pre-pollution conditions. Such a pattern was unusual since it occurred in a period when we would expect a seasonal increase in bacterial activity. Differences are seen when comparing the temporal trend of DNA in suspended particulates and in sediments. However, the effects of oil on the RNA:DNA ratio were similar. Oiled samples showed a RNA:DNA ratio lower than 1 whereas RNA concentrations exceeded DNA content in undisturbed samples. Bacterial DNA accounted for a significant fraction (average 26%) of total DNA in sediments. It is likely that changes in RNA:DNA ratio in oiled sediments are due to the disturbance of bacterial communities. Consistent with the meiofaunal response to oil contamination, the hydrocarbons had only temporary and limited effects and RNA:DNA values recovered to pre-pollution conditions after a few weeks. the possible use of the RNA:DNA ratio in pollution monitoring is discussed.

Identification of H-ras Mutations in Urine Sediments Complements Cytology in the Detection of Bladder Tumors

Urinary cytology has long been used as a noninvasive screen for the detection of urinary tract cancer but is limited by the generation of false positive and false negative results. More recently, molecular changes associated with urothelial neoplastic progression have been identified in DNA from urine sediments, demonstrating an alternative approach for identifying neoplastic change in the bladder. The purpose of this prospective study was to determine the value of detection of H-ras (also known as HRAS) mutations in urine sediment DNA as a clinical indicator of tumor presence, recurrence, and/or progression. Urine sediments were collected from 100 patients presenting with bladder tumors, with follow-up samples collected from 19 patients. DNA extracted from urine sediments was analyzed for changes in exon 1 of the H-ras gene, using single-strand conformation polymorphism (SSCP) analysis. A representative number of aberrant H-ras/SSCP migrating bands were excised and sequenced to confirm the presence of a mutation. Human bladder specimens were obtained from patients (93 of the 100 patients initially and 18 of the 19 patients studied by follow-up) and histologically evaluated for tumor content and grade. Mutations in exon 1 of the H-ras gene were detected in urine sediments from 44% (44 of 100) of the patients; concordant results were obtained by cytologic analysis, where 33% (31 of 93) of the patients displayed positive cytology. Analysis of the distribution of abnormalities with tumor grade revealed greater detection of low-grade (1-2) lesions using ras analysis (47%) compared with cytology (16%). In contrast, cytology was more effective in identifying the presence of carcinoma in situ. Combined results from these two approaches substantially increased the sensitivity of tumor detection, resulting in the identification of tumors in 60% of patients. Identification of H-ras mutations in DNA from urine sediments facilitates the detection of low-grade bladder tumors and, in combination with cytology, increases the overall tumor detection from 33% to 60%. Preliminary results in patient follow-up suggest that detection of H-ras mutations may have some clinical utility in detecting the presence of abnormal cells in the absence of an overt lesion following cystoscopy or positive cytology.

Interaction of marine sediment with DNA and DNA availability to nucleases

A method for the determination of DNA in sediments is described. The comparison between DNA extraction profiles from natural sediment samples and from precleaned sediments (concentrated nitric acid and subsequently 1 100 °C), to which known amounts of DNA were added, demonstrates that the sand particles participate in the delay of extraction of DNA we observed. Experiments with nuclease mixtures show that DNA by its interaction with the sediment is protected considerably against degradation by nucleases.

Absence of measles proviral DNA in systemic lupus erythematosus.

MEASLES VIRUS usually causes an acute self-limiting infection in humans and a productive cytolytic infection in susceptible tissue culture cells. However, measles-like virus has been isolated from the brains of patients with subacute sclerosing panencephalitis (SSPE), a chronic progressive neurological disorder of children1. Also, persistent measles infection has been implicated in the pathogenesis of several chronic diseases of unknown aetiology, including multiple sclerosis2 and systemic lupus erythematosus (SLE)3. Manipulation of the conditions of infection can result in persistently infected tissue culture cells, which have been proposed as a model system for studying the molecular mechanisms responsible for cytolytic virus persistence in man4–6. There is experimental evidence for at least four mechanisms leading to the induction of persistent measles infections: the production of defective virus particles which interfere with the replication of complete virus7, the generation of mutant virus strains with the capacity for non-cytolytic replication8, and infection of certain cell types (for example, neurones) restricted in their capacity to support all steps in virus replication9. Zhdanov et al.10–13 have proposed that measles virus persists in cells by a mechanism similar to that shown for retroviruses, that is, the synthesis of a DNA copy of the viral genome and the insertion of the proviral DNA into the host cell chromosome. This would be a novel mode of replication, as the replication of measles virus in cytolytic infections involves a double-stranded RNA intermediate. Using tritium-labelled viral RNA probes in molecular hybridisation assays, Zhdanov et al. have demonstrated the presence of measles virus-related DNA in chick embryo cells11 and human lymphoid cells13 persistently infected with measles virus. Further, they detected measles virus-related DNA in leukocytes, lymph nodes, kidney and urine cell sediment from five SLE patients12. Negative results were obtained using cells from appropriate control tissues. The obligative reverse transcriptase was found in the persistently infected cell lines and SLE tissues, and concomitant retrovirus infection was proposed as a prerequisite for persistent measles infection. Thus, these studies not only provide insights into the molecular mechanisms of measles virus persistence but suggest aetiological relationships for a fatal disease of unknown cause. However, we report here that, using a more sensitive nucleic acid hybridisation assay, we were unable to confirm the presence of measles virus-related DNA in either persistently infected cells or cells from patients with SLE.

Integration of adenovirus type 2 DNA in productively infected cells: results of sequential hybridization experiments.

In human KB cells permissively infected with adenovirus type 2, the high mol. wt. forms of virus DNA have been characterized. These size classes of virus DNA sediment at greater than 100S and 40 to 90S in alkaline sucrose density gradients. Considerable evidence from a series of earlier communications supports the notion that the high mol. wt. forms of virus DNA represent virus DNA sequences covalently linked to cellular DNA. 3H-labelled high mol. wt. adenovirus type 2 DNA from productively infected cells can be shown to hybridize to virus DNA fixed to filters. In the present paper we demonstrate that on alkali elution of the DNA from the filters used in the first step of the hybridization experiment, the labelled DNA re-hybridizes to cellular DNA in the second step of a sequential hybridization experiment. The order of performing the two successive hybridization experiments can be reversed and very similar results are obtained. These data provide conclusive evidence for the covalent linkage of virus and cellular DNA sequences in KB cells productively infected with adenovirus type 2.

Uptake and fate of the DNA of adenovirus type 2 in KB cells

The uptake of adenovirus DNA by KB cells growing in monolayers has been studied. Between 3% and 9% of the DNA of adenovirus type 2 added to a culture of KB cells becomes associated with the cells. Uptake is linear for approximately 2 hr. Between 2 and 6 hr, part of the DNA initially adsorbed is shed into the adsorption medium and subsequently partly readsorbed. By both cell-fractionation experiments and autoradiography in combination with electron microscopy it can be shown that the viral DNA reaches the nucleus starting 30 min-2 hr postinfection. At 24 hr postinfection, 70% of the DNA taken up has reached the nucleus. Viral DNA is not detected in lysosomes or cytoplasmic vesicles. Thus, direct penetration of the cytoplasmic membrane appears the most likely mechanism for the uptake of viral DNA. The viral DNA is cleaved endonucleolytically both in the cytoplasm and the nucleus. The fragments of viral DNA sediment at a rate of 19–23 S. Approximately 19.6% of the DNA in the nucleus becomes associated with cellular DNA and is perhaps integrated into the cell genome. The viral DNA that is not taken up by KB cells but remains in the medium is also fragmented endonucleolytically. The fate of viral DNA in KB cells was studied after infection with viral DNA and with type 2 adenovirions. A comparison of the results shows that cleavage of the viral DNA occurs later and that association of viral DNA with cellular DNA is more extensive after infection with adenovirions.

Evidence for the presence of RNA in the purified virions of vaccinia virus.

Vaccinia virus, strain WR, was propagated in HeLa cells, L mouse fibroblats, or primary chicken embryo fibroblasts in the presence of [5- (3)H]uridine. Carefully purified virions were found to contain significant amounts of labeled trichloroacetic acid-precipitable material which was rendered acid soluble when digested with pancreatic RNase or hydrolyzed in alkali. Controlled degradation of virions with Nonidet P-40 and 2-mercaptoethanol demonstrated that 65 to 80% of the [5- (3)H]uridine-labeled molecules resided in the viral core. When the total nucleic acids were extracted from viral cores prepared from virions propagated in HeLa cells, 30 to 50% of the total incorporated [5- (3)H]uridine was found in RNA; in L mouse fibroblasts, 40 to 50%; in primary chicken embryo fibroblasts, 50 to 60%. The RNA molecules do not appear to be covalently linked to the viral DNA genome but sediment in sodium dodecyl sulfate-sucrose gradients as 8 to 10S species relative to ribosomal RNA.

The anatomy of the T5 bacteriophage DNA molecule

The T5 bacteriophage DNA molecule, unlike the molecules from T2, P1, λ and T7 bacteriophage, contains interruptions at specific locations in both of its polynucleotide chains. When the molecule is denatured, it gives rise to polynucleotide chains which sediment in at least four different zones. By contrast, denatured T2, P1, λ and T7 DNA sediment as unimodal zones. The lengths of the polynucleotide chains -which comprise these zones were calculated through use of a relationship established for determining the relative lengths of polynucleotide chains from their rate of sedimentation in alkaline sucrose gradients. The positions of the interruptions along the molecule are under the genetic control of the phage and apparently are not a property of the host. The phenomenon is seen in another phage, PB, which is closely related to T5. The interruptions in the molecule are probably gaps in the polynucleotide chains. This conclusion was reached because the chains are not intact when the molecule is denatured in formamide, a mild organic denaturing agent. A number of models were proposed for ways in which the six polynucleotide chains could be arranged to form the intact duplex molecule. Two reannealing experiments were performed which confirmed some general features which all of the models had in common. The existence of gaps in the T5 DNA molecule helps to explain some earlier observations on the structure of the molecule and suggests interpretations for other results concerning its biological function.

Sedimentation Rate as a Measure of Molecular Weight of DNA

Zone centrifugation of mixtures of two labeled DNA's at low concentrations in density gradients of sucrose permits accurate measurement of relative sedimentation rates. The individual rates are constant during the run. Measurements with DNA's from phages T2, T5, and lambda conform to the relation D(2)/D(1) = (M(2)/M(1))(0.35), where D and M refer to distances sedimented and molecular weights of the DNA pair. The results show that high molecular weight DNA's sediment artificially fast in the optical centrifuge, owing to a hitherto unknown effect of molecular interactions. The molecular weight of lambda DNA is 31 million, measured either from sedimentation rate or from tests of fragility under shear.