A computer-aided measuring system for the characterization of yeast populations combining 2D-image analysis, electronic particle counter, and flow cytometry

An integrated measuring system was developed that directly compares the shape of size distributions of Saccharomyces cerevisiae populations obtained from either microscopic measurements, electronic particle counter, or flow cytometer. Because of its asymmetric mode of growth, a yeast population consists of two different subpopulations, parents and daughters. Although electronic particle counter and flow cytometer represent fast methods to assess the growth state of the population as a whole, the determination of important cell cycle parameters like the fraction of daughters or budded cells requires microscopic observation. We therefore adapted a semiautomatic and interactive 2D-image processing program for rapid and accurate determination of volume distributions of the different sub-populations. The program combines the capacity of image processing and volume calculation by contour-rotation, with the potential of visual evaluation of the cells. High-contrast images from electron micrographs are well suited for image analysis, but the necessary air drying caused the cells to shrink to 35% of their hydrated volume. As an alternative, hydrated cells overstained with the fluorochrome calcofluor and visualized by fluorescence light microscopy were used. Cell volumes calculated from length, and diameter measurements with the assumption of an ellipsoid cell shape were underestimated as compared to volumes derived from 2D-image analysis and contour rotation, because of a deviating cell shape, especially in the older parent cells with more than one bud scar. The bimodal volume distribution obtained from microscopic measurements was identical to the protein distribution measured with the flow cytometer using cells stained with dansylchloride, but differed significantly from the size distribution measured with the electronic particle counter. Compared with the flow cytometer, 2-D image analysis can thus provide accurate distributions with important additional information on, for instance, the distributions of subpopulations like parents, daughters, or budded cells.     

Two new species of Viola (Violaceae) from the Intermountain West,U.S.A.

Viola lithion andV. franksmithii are rock-dwelling endemics that belong to the North American perennial, caulescent, simple-leaved, blue-, purple-, or white-flowered group of species.Viola lithion, from the White Pine and Pilot mountain ranges of eastern Nevada and adjacent Utah, belongs to sectionChamaemelanium subsectionCanadenses. It most closely resembles the reniform-leaved, Olympic MountainV. flettii, together with which it possibly shares an ancestry with the widespread and allopatricV. canadensis. Viola franksmithii, restricted to the Logan Canyon drainage of the Bear River Range in northern Utah, belongs to sectionRostellatae subsectionRosulantes. It comes closest morphologically toV. howellii of the Coast Range, but likely evolved from the widespreadV. adunca or a common ancestor.     

Polymorphism and inheritance of gliadin polypeptides in T. monococcum L.

Summary More than 80 different gliadin electrophoretic patterns (spectra) have been found in 109 accessions of the diploid wheat Triticum monococcum. Each pattern consists of 15–20 gliadin bands. Some patterns are clearly related and might arise from one another through single mutations in the gliadin-coding loci. From the analysis of 15 grains of each, only 61 accessions were found to be uniform; others consisted of two or more grain variants differing in their gliadin spectrum. An analysis of F₂ grains from three crosses between different accessions showed that groups (blocks) of components are jointly and codominantly inherited. Two independent major Gli loci were established. The close resemblance of the composition of some blocks of T. monococcum to some of those in polyploid wheats indicates that one locus in each T. monococcum genotype is located on chromosome 1A (Gli-A1) and the other on 6A (Gli-A2). However, the blocks of T. monococcum include more bands than corresponding (equivalent) blocks of polyploid wheats. Two out of 275 F₂ grains of the cross k-14244 x k-20409 were found to have gliadin spectra which can be explained as a result of intralocus recombination. Also, a second gliadin-coding locus on chromosome 1A was found in the cross k-46140 x k-46753. This locus recombines with the main Gli-A1 locus with a frequency of about 22% and was clearly analogous to the additional Gli locus found earlier on chromosome 1A of certain polyploid wheats.     

A statistical comparison of various factors on embryogenic proliferation,morphogenesis and regeneration in Lolium temulentum cell suspension colonies

Cell suspension colonies from four embryogenic Lolium temulentum lines were selected and plated individually in 25 embryoid maturation treatments which varied in various factors reported to stimulate embryogenesis or improve regeneration. Using a numerical scoring system to compare the cultures against a control, treatments were identified which increased growth, suppressed morphogenesis or encouraged premature shoot formation.No treatment significantly improved the proportion of colonies with globular or mature embryoids, but some prevented maturation and increased the proportion with translucent embryogenic proliferation. Other treatments accelerated maturation causing increased de-differentiation of embryogenic tissues. These treatments also tended to discourage the differentiation of discreet embryoids.Colonies were later transferred en masse to a regeneration medium and scored using another numerical system. Embryoid maturation conditions were then identified which increased or suppressed subsequent shoot regeneration. The two scoring systems enabled cultures of the four lines to be characterised in detail and identified somatic variation in embryogenic development, morphogenesis and de-differentiation.     

Some comments on coordinate-free and scale-invariant methods in morphometrics

The unusual strategy for comparing biological shapes is to use some kind of superimposition of the two forms under study and then look at the "residuals" as the shape change. In this paper, I take a careful look at this general strategy and point out some subtle but inherent and important pitfalls. Additionally an alternative approach based on Euclidean Distance Matrix representation is presented. It is applicable to two- as well as three-dimensional objects.     

An alternate method of calculating the heat of growth of Escherichia coli K-12 on succinic acid

The DeltaH(f) (0) unit weight of a complex substance such as a biological macromolecule is almost always obtained by means of combustion analysis. In theory, this can also be done by summing the DeltaH(f) (0) values for the monomers comprising the macromolecule plus the enthalpic energies involved in their polymerization. The enthalpy of formation of one unit-carbon formula weight of dried Escherichia coli K-12 cells was determined by summing the values of the enthalpies of formation of the quantities of monomers in the major classes of macromolecules substances comprising the cellular biomass and the enthalpic energies involved in their polymerizations. To this value was added the enthalpy of formation of the cellular ions in their aqueous standard states, per unit-carbon formula weight of cellular substance and the enthalpy change with respect to the ionization of the protein amino acid side chains. If it is assumed that the cellular fabric is insoluble and that the ions are soluble, the sum of the enthalpies of formation of all the cellular components should closely approximate the enthalpy of formation of one unit-carbon formula weight equivalent of living cells. Using this value, a calculation of the enthalpy change accompanying anabolism shows this latter to be effectively zero, indicating that the heat of growth (anabolism plus catabolism) is equal to that calculated for catabolism alone. This conclusion is in accord with those of several investigators who have used manometry or direct calorimetry.     

Extrachromosomal human immunodeficiency virus type-1 DNA can initiate a spreading infection of HL-60 cells

In this report, we describe a human immunodeficiency virus type-1 (HIV-1)-infected promyelocytic cell line, OM, derived from HL-60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV-1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV-1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to less than 10% HIV-1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV-1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV-1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV-1 genome was less than 0.1%, indicating nonfaithful transmission of an unintegrated HIV-1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV-1 DNA can contribute to the spread of HIV-1 infection and give rise to cells which have stably integrated HIV-1 provirus.     

Projection of Monte Carlo and molecular dynamics trajectories onto the normal mode axes: human lysozyme

A method is presented to describe the internal motions of proteins obtained from molecular dynamics or Monte Carlo simulations as motions of normal mode variables. This method calculates normal mode variables by projecting trajectories of these simulations onto the axes of normal modes and expresses the trajectories as a linear combination of normal mode variables. This method is applied to the result of the molecular dynamics and the Monte Carlo simulations of human lysozyme. The motion of the lowest frequency mode extracted from the simulations represents the hinge bending motion very faithfully. Analysis of the obtained motions of the normal mode variables provides an explanation of the anharmonic aspects of protein dynamics as due first to the anharmonicity of the actual potential energy surface near a minimum and second to trans-minimum conformational changes.     

Microsite differentiation in a Mediterranean oak savanna

Abstract. Microscale variation of vegetation was studied in a Mediterranean oak savanna dominated by annual plants at Neve Ya'ar Experimental Station, in the lower Galilee of Israel. Species presence/absence data were recorded in 20 cm x 20 cm quadrats on a 500-quadrat transect. These data were ordinated with Detrended Correspondence Analysis. Soil nitrate, phosphorous, potassium, pH, soil moisture and salinity were determined at 2-m intervals on the 100-m transect, at monthly intervals from November 1981 to April 1982. Litter cover and canopy shade were surveyed in October 1981 and April 1982. A sharp rise in available nitrate occurred in November, following the first winter rains. Patterns in herbaceous vegetation, as expressed by sample ordination scores, correlated best with litter cover in October. This suggested that vegetation pattern in this community may be strongly influenced by environmental conditions at the time of seedling germination and establishment.