Use of EDTA to prevent spontaneous cell-to-cell transformation provides a more accurate estimation of gene transfer frequencies

Summary Spontaneous cell-to-cell transformation between naturally competent bacteria on selective media resulted in an overestimation of the transferability of genetic information. EDTA effectively prevented transformation on selective media whereas DNaseI did not reliably inhibit cell-to-cell transformation. An improved method to estimate gene transfer frequencies is described.     

Regeneration of Triticum aestivum apical explants after microinjection of germ line progenitor cells with DNA

A highly efficient method of regenerating fertile, phenotypically normal plants from shoot apex cultures of T. aestivum was developed. The hypodermal layer (L2) of the vegetative apex containing germ line precursor cells could be located with bright field microscopy and targeted for microinjection. Fluorescently labelled dextrans were used as markers to develop a microinjection procedure which did not disrupt nuclear or cytoplasmic structure. This procedure was used to inject plasmid DNA into L2 cells. Capillary microinjection did not shear the plasmid DNA and delivery of DNA was confirmed by polymerase chain reaction analysis of DNA isolated from injected apices. The significance of these findings in relation to the development of cereal transformation systems will be discussed.     

Improved Histology for the Chick-Quail Chimera

A simple modification of nuclear staining after acid hydrolysis has been made which provides easy identification of quail nuclear markings in a chick-quail chimera. This method also improves the histologic detail normally seen with hematoxylin and eosin when compared to the more commonly used Feulgen reaction. Embryonic tissues can be fixed in Zenker's or Helly's solution and the sections obtained are hydrolyzed in acid (3.5 N HCl at 37 C for 40-50 min). After acid hydrolysis the sections are stained with hematoxylin and eosin rather than Schiff reagent and fast green. The interphase nuclei of chick cells show homogeneous or mottled purplish blue staining, while quail nuclei contain a dark blue spot. This staining corresponds to the reddish purple staining of the quail's heterochromatin seen adjacent to the nucleolus in the standard Feulgen stain. This new technique facilitates identification of quail cell types in the chick host and provides superior histology of the chick tissues by demonstrating cytoplasmic detail.     

Analysis of Physarum proteins throughout the cell cycle by two-dimensional PAGE

The accumulation of several hundred proteins during the nuclear division cycle of Physarum polycephalum was measured by digital image processing of silver-stained two-dimensional (2D) polyacrylamide gels. In contrast to previous studies, we have used an organism with a naturally synchronous cell cycle, so there are no uncertainties concerning synchronization artifacts or cell-sorting artifacts, and we have measured the specific amounts of each protein rather than the rate of synthesis. Since one-dimensional SDS-PAGE shows no significant fluctuations in the most abundant plasmodial proteins, we have loaded 2D gels so that proteins of low-to-moderate abundance appear in the linear range of the silver stain standard curve. Only five proteins showed reproducible, measureable fluctuations during the cell cycle. One of these proteins was tubulin. Full quantitative information was obtained by analysing the digital images of silver-stained gels by a general image processing system.     

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers a potential drug delivery system

Synthetic ¹²⁵I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [¹²⁵I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.