A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase

A simple and rapid method for determining nucleotide sequences in single-stranded DNA by primed synthesis with DNA polymerase is described. It depends on the use of Escherichia coli DNA polymerase I and DNA polymerase from bacteriophage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by ionophoresis on acrylamide gels. The method was used to determine two sequences in bacteriophage φX174 DNA using the synthetic decanucleotide A-G-A-A-A-T-A-A-A-A and a restriction enzyme digestion product as primers.     

Mathematical model for determining the effects of intracytoplasmic inclusions on volume and density of microorganisms.

Procaryotic microorganisms accumulate several polymers in the form of intracellular inclusions as a strategy to increase survival in a changing environment. Such inclusions avoid osmotic pressure increases by tightly packaging certain macromolecules into the inclusion. In the present paper, a model describing changes in volume and density of the microbial cell as a function of the weight of the macromolecule forming the inclusion is derived from simple theoretical principles. The model is then tested by linear regression with experimental data from glycogen accumulation in Escherichia coli, poly-beta-hydroxybutyrate accumulation in Alcaligenes eutrophus, and sulfur accumulation in Chromatium spp. The model predicts a certain degree of hydration of the polymer in the inclusion and explains both the linear relationship between volume of the cell and weight of the polymer and the hyperbolic relationship between density of the cell and weight of the polymer. Other implications of the model are also discussed.     

Scaling of the equilibrium sedimentation distribution in dense DNA solutions

DNA molecules, several persistence lengths long in sedimentation equilibrium at speeds high enough to maintain fairly close packing, show a dense, sharply-bounded turbid phase and an isotropic phase (as with shorter fragments) and also an intermediate, somewhat turbid region. The concentration distribution in the isotropic phase is in satisfactory agreement with a simple extension of scaled particle theory in which semiflexible chains are equivalent to straight rods of the same length. The net intermolecular interactions, as inferred from the Zimm cluster integral, are purely repulsive. As in our previous study with short fragments, the results are compatible with a hard-core electrostatic radius, decreasing with increasing salt concentration. However, for the longer fragments it is necessary to infer either a slightly greater mass per unit length or a slightly smaller electrostatic radius for closest agreement with scaled particle theory. The properties of the solution at the boundary with the turbid, presumably strongly ordered phase are consistent with those found for shorter fragments and with theoretical scaling expectation for a hard, asymmetric particle.     

Rapid method for determining the rate of DNA synthesis and cellular proliferation

A new method has been developed for determination of DNA synthesis during cell proliferation. The method is based on the metabolism of [U-(13)C(6)]glucose to deoxyribose (DR) and then incorporation of [U-(13)C(5)]DR into newly synthesized DNA. Extracted cellular DNA is subjected to HCl hydrolysis (2 h at 100 degrees C), which converts DR into levulinic acid. The (13)C enrichment in DR is determined in the trimethylsilyl derivative of levulinate using gas chromatography-mass spectrometry. The method is rapid and sensitive. It can precisely determine (13)C enrichment below 1 at.% excess in as little as 4 ng DNA. We have used this method to determine the rate of cell proliferation in vitro and the level of DR in a given amount of DNA. The current approach has significant advantages over previously described methods and overcomes several difficulties related to the determination of DNA synthesis both in vivo and in vitro.     

Evaluation of a method for determining the free carboxyl group distribution in pectins

The distribution of free carboxyl groups in pectins has been investigated by a method that involves blocking the free carboxyl groups by glycolation, and hydrolysis of the methylesterified regions with a mixture of pectic enzymes. The hydrolysis products are separated from the glycolated regions on an ion exchange column and after deglycolation the oligomer size distribution is obtained by Sephacryl S-200 chromatography.The method was applied to five pectins with degrees of esterification in the range 5–70%. For two of the samples (an enzyme and an alkali de-esterified low methoxyl pectin) the degree of hydrolysis was significantly lower than would be predicted from the initial degree of esterification and thus for these materials the values obtained for the carboxyl group block sizes were considered to be a maximum rather than an accurate estimate.All the samples investigated had a significant proportion of free carboxyl regions with a degree of polymerisation greater than 10. With the possible exception of the pectate (degree of esterification 5%) none of the samples had a random distribution of carboxyl groups. This was considered to be a reflection of the distribution in the native pectin rather than indicating that chemical de-esterification was non-random. The large free carboxyl group block sizes was consistent with the egg-box model for low methoxyl pectin gelation. Larger blocks were found in the enzyme de-esterified pectin compared with the alkali and acid de-esterified material.     

A method for determining concentration profiles at the soil-root interface by thin slicing rhizospheric soil

Summary A method is described for the determination of concentration gradients in the vicinity of plant roots. Plants are grown in small containers in which the roots are separated from the soil by a screen of nylon cloth. Root hairs but not roots penetrate the screen into the soil. In order to investigate the rhizospheric soil, the soil is frozen by liquid nitrogen and sliced into layers about 0.06 mm thick by means of a refrigerated microtome.     

A method for determining DNA and chondrocyte content of articular cartilage

A novel and precise method was devised to study the DNA and chondrocyte content of articular cartilage. It involved the sequential digestion of cartilage matrix with hyaluronidase, trypsin, and collagenase to release the chondrocytes. A direct cell count and DNA assays were then performed on the cells. The concentration of cells was the quotient of the total number of cells and the weight of cartilage used. The DNA content of cartilage is identical to the amount of DNA in the chondrocytes. Our data also confirmed the earlier findings that cell density and DNA content of articular cartilage decreased gradually to a relatively constant level as animals matured to adulthood.     

Evaluation of an immunoenzymatic method for determining TSH and establishment of reference values]

In the present work we reported the results of the valuation of IMx Ultrasensitive hTSH assay which is a Microparticle Enzyme ImmunoAssay (MEIA) for quantitative determination of human stimulating hormone (hTSH) in the human serum or plasma. We have determined the method's precision, within run and between run, sensitivity and recovery. This method has been compared with another one (Immuno RadioMetric Assay). Also reference values have been calculated in the "normal" male and female population and shortly commented.     

Density-gradient sedimentation equilibrium of DNA and the effective density gradient of several salts

The various treatments of sedimentation equilibrium are compared on a theoretical and an experimental basis. Particular attention is paid to the polyelectrolyte nature of the problem and the choice of a neutral component. The effective density gradients of several cesium salts for DNA are measured. Two previous theories for the effective density gradient are shown to be equivalent, and the experimental values are interpreted with respect to these theories. It is clear t hat sedimentation equilibrium in a density gradient may be used for the determination of unambiguous molecular weights.     

Mathematical models of hierarchically structured cell populations under equilibrium with application to the epidermis

Abstract. There are three categories of keratinocytes in the germinative compartment of the epidermis – stem, transit-amplifying and post-mitotic. Their population structure is hierarchical. This means that stem cells differentiate into transit-amplifying cells which, after a few rounds of division, become post-mitotic cells. The cell processes of birth, differentiation, death and migration affect the composition and proliferation rate of the germinative compartment. These phenomena are quantified by various cell kinetic parameters. In this paper we derive equations that relate these parameters for different models of hierarchically structured cell populations in equilibrium. We include in the models asymmetric and symmetric division, variations in cell-cycle times, apoptosis and variation in the number of transit generations. We conclude that variation in cell-cycle times need only be considered if apoptosis is not negligible. If it is negligible, then only average cell-cycle times are needed. Unfortunately, it is impossible to predict the importance of apoptosis from the available experimental data. However, the strength of its effect is determined by the other parameters, especially the fraction of cycling stem cells. We show that variation in the number of transit generations can have a potentially large effect on cell birth rate. We also show that cell birth rate does not directly depend on the mean transit-amplifying cell-cycle time, only on the mean stem cell-cycle time. We argue that 'homogeneous cell population' equations should not be used to study hierarchical cell populations as has been done in the past. Finally we argue that stem cell parameters and transit-amplifying cell parameters should not be lumped together.     

Ovarian cycle in a temperate zone frog, Rana temporaria, with special reference to factors determining number and size of eggs

In the frog, Rana temporaria, the number of eggs in a clutch ranged from about 2500 to 9000/100g body mass and egg diameter ranged from 1.1 to 1.9 mm. There was an inverse relation between number and size of eggs. It is indicated that egg size depends upon the number of oocytes recruited to vitellogenic growth and that the number of oocytes recruited depends upon the number of small oocytes that constitutes the recruitment pool. The total amount of yolk deposited in the ovaries was independent of the number of eggs, i. e. clutch size. During an ovarian cycle, therefore, physiological mechanisms seem to regulate the amount of yolk deposited within the ovaries at large, rather than in the individual vitellogenic oocyte.     

A photographic microfluorimetric method for determining the DNA content in individual human chromosomes

A method for determination of DNA contents in individual human chromosomes has been elaborated based of the two step analysis: 1) identification of chromosomes by Q-banding; 2) photographic microfluorimetry of chromosomes after the Feulgen staining (a fluorescent variant using a Schiff-type reagent of Auramine-SO2). The DNA content in 24 human chromosomes was calculated in absolute values (fg). The data obtained are compared with the evidence published elsewhere and provided by different cytophotometric methods.     

Blood-based profiles of DNA methylation predict the underlying distribution of cell types

The potential influence of underlying differences in relative leukocyte distributions in studies involving blood-based profiling of DNA methylation is well recognized and has prompted development of a set of statistical methods for inferring changes in the distribution of white blood cells using DNA methylation signatures. However, the extent to which this methodology can accurately predict cell-type proportions based on blood-derived DNA methylation data in a large-scale epigenome-wide association study (EWAS) has yet to be examined. We used publicly available data deposited in the Gene Expression Omnibus (GEO) database (accession number GSE37008), which consisted of both blood-derived epigenome-wide DNA methylation data assayed using the Illumina Infinium HumanMethylation27 BeadArray and complete blood cell (CBC) counts among a community cohort of 94 non-diseased individuals. Constrained projection (CP) was used to obtain predictions of the proportions of lymphocytes, monocytes and granulocytes for each of the study samples based on their DNA methylation signatures. Our findings demonstrated high consistency between the average CBC-derived and predicted percentage of monocytes and lymphocytes (17.9% and 17.6% for monocytes and 82.1% and 81.4% for lymphocytes), with root mean squared error (rMSE) of 5% and 6%, for monocytes and lymphocytes, respectively. Similarly, there was moderate-high correlation between the CP-predicted and CBC-derived percentages of monocytes and lymphocytes (0.60 and 0.61, respectively), and these results were robust to the number of leukocyte differentially methylated regions (L-DMRs) used for CP prediction. These results serve as further validation of the CP approach and highlight the promise of this technique for EWAS where DNA methylation is profiled using whole-blood genomic DNA.