James L. Bolen

Effective generation of transgenic pigs and mice by linker based sperm-mediated gene transfer.

BackgroundTransgenic animals have become valuable tools for both research and applied purposes. The current method of gene transfer, microinjection, which is widely used in transgenic mouse production, has only had limited success in producing transgenic animals of larger or higher species. Here, we report a linker based sperm-mediated gene transfer method (LB-SMGT) that greatly improves the production efficiency of large transgenic animals.ResultsThe linker protein, a monoclonal antibody (mAb C), is reactive to a surface antigen on sperm of all tested species including pig, mouse, chicken, cow, goat, sheep, and human. mAb C is a basic protein that binds to DNA through ionic interaction allowing exogenous DNA to be linked specifically to sperm. After fertilization of the egg, the DNA is shown to be successfully integrated into the genome of viable pig and mouse offspring with germ-line transfer to the F1 generation at a highly efficient rate: 37.5% of pigs and 33% of mice. The integration is demonstrated again by FISH analysis and F2 transmission in pigs. Furthermore, expression of the transgene is demonstrated in 61% (35/57) of transgenic pigs (F0 generation).ConclusionsOur data suggests that LB-SMGT could be used to generate transgenic animals efficiently in many different species.

Changes in phospholipid composition in hibernating ground squirrel,Citellis lateralis, and their relationships to membrane function at reduced temperatures

The hearts of hibernating mammals have the capacity to function at reduced temperatures of 1 C in contrast to hearts of nonhibernating mammals which undergo ventricular fibrillation during deep hypothermia. Thus, all vital membrane functions continue in hibernators. This suggests that there may be special features of their lipid and protein composition. Analysis of the hearts of the hibernating ground squirrel,Citellus lateralis, in the active (37 C) and hibernating state (1–4 C) reveals differences in the percent composition of the phospholipid classes. There is a decrease in the percentage of diacyl choline, ethanolamine, serine phosphoglycerides, and diphosphatidyl glycerol and an increase in the percentage of lysophosphatidyl choline, lysophosphatidyl ethanolamine, lysodiphosphatidyl glycerol, and phosphatidic acid in the hibernating state. There is also an increase in the molar amount of phosphorus/100 g fresh wt of tissue during hibernation. The most striking difference in phospholipid composition between active and hibernating heart tissue is the 3.5-fold increase in lysoglycerophosphatides. These compounds are implicated as contributing factors controlling membrane fluidity during hibernation and upon which the physiological state of hibernation may be contingent.

SELF-ORGANIZATION IN BIOLOGICAL TISSUES: ANALYSIS OF ASYNCHRONOUS AND SYNCHRONOUS PERIODICITY, TURBULENCE AND SYNCHRONOUS CHAOS EMERGENT IN COUPLED CHAOTIC ARRAYS

Large discrete arrays of chaotic attractors, coupled by diffusion, organize into asynchronous periodic spiral waves, synchronous periodic bands, turbulent fields or synchronous chaos as a function of coupling strength and array size. Self-organization of periodic spirals in both two and three dimensional arrays of nonexcitable systems appears to require the early establishment of an antipodal phase relationship between the few cells that will form the vortex. Cells within or close to the vortex maintain low z amplitude, near limit cycle trajectories, with stable, well-defined phase relationships. In periodic banding structures, initial antipodal phase seeds evolve to isochrons that form nested periodic trajectories. The likelihood that biological systems are fundamentally oscillatory and chaotic is discussed.

Autogenous formation of spiral waves by coupled chaotic attractors.

When large arrays of strange attractors are coupled diffusively through one of the variables, chaotic systems become periodic and form large archimedean spirals or concentric bands. This observation may have importance for many applications in the field of deterministic chaos and seems particularly relevant to the question of the formal temporal structure of the biological clock in metazoan organisms. In particular, although individual cellular oscillators, as manifested in the cell cycle, may have deep basins of attraction and appear to be more or less periodic, we suggest that cells oscillate with chaotic dynamics in the ultradian domain. Only when large aggregates of these cells are tightly coupled can a precise circadian clock emerge. For changing coupling strength or parameter values, period increase occurs through quantal or integral multiple increments of the fundamental. All calculations were implemented on a 386AT, using a Mercury MC6400 floating point processor.

Cell‐cycle‐dependent expression of human melanoma membrane antigen analyzed by flow cytometry

Knowledge of tumor antigenic expression is crucial to the design of therapeutic strategy. A murine monoclonal antibody (BE4) against a human melanoma membrane antigen, was used to study the in vitro expression of this antigen. By membrane immunofluorescence, BE4 reacted against 5 of 8 melanoma lines as compared to zero of 13 other cell populations. Using flow cytometry, the antigenic M14 CEM melanoma cells consisted of 40% to 60% of the total cell population. Dual‐parameter measurements of DNA content and membrane antigen demonstrated that the nonantigenic cells were predominantly in G0/G1 phase, whereas the antigenic cells were distributed throughout the cell cycle. Within one passage, the sorted and recultured nonantigenic population demonstrated a similar proportion of antigenic cells as the unsorted original population. It was concluded that the expression of human melanoma antigen was cell‐cycle‐dependent. Understanding factors that turn off the expression of antigen in G0/G1 phase may lead to better immunotherapeutic strategies. Cancer 55:1276‐1283, 1985.

Amplification and damping of deterministic noise in coupled cellular arrays

Abstract Large scale spatiotemporal organization and enhanced periodicity are seen when a population of chaotic Rossler attractors is coupled through one or more of the variables. The resulting periodicity has been analyzed by means of return maps and by the projection of these maps as state variable dispersion histograms. Self-mapping of cells in the array, within dispersion criteria, yielded evidence of reverse bifurcation sequences, F ...8,4,2,1 , expressed in the behavior of most of the 900 to 25 600 individual cells. Self-mapping is seen in spiral and periodic banded fields, but not in turbulent fields or in synchronous chaos. Coupling of arrays of periodic attractors leads to dispersion in self-mapping similar to that seen in the coupled chaotic arrays. Fixed patterns representing the bifurcation sequences that occur in these arrays were overlaid on the spiral field portraits.

Signal Processing and the Design of Microarray Time-Series Experiments

Recent findings of a genome-wide oscillation involving the transcriptome of the budding yeast Saccharomyces cerevisiae suggest that the most promising path to an understanding of the cell as a dynamic system will proceed from carefully designed time-series sampling followed by the development of signal-processing methods suited to molecular biological datasets. When everything oscillates, conventional biostatistical approaches fall short in identifying functional relationships among genes and their transcripts. Worse, based as they are on steady-state assumptions, such approaches may be misleading. In this chapter, we describe the continuous gated synchrony system and the experiments leading to the concept of genome-wide oscillations, and suggest methods of analysis better suited to dissection of oscillating systems. Using a yeast continuous-culture system, the most precise and stable biological system extant, we explore analytical tools such as wavelet multiresolution decomposition, Fourier analysis, and singular value decomposition to uncover the dynamic architecture of phenotype.

Heterogeneous amino acid transport rate changes in an E. coli unsaturated fatty acid auxotroph.

Abstract Amino acid transport rates in an E. coli unsaturated fatty acid auxotroph were non-uniformly affected by enrichment of membrane lipids in various unsaturated fatty acids. Proline and threonine transport rates were depressed much more than lysine and asparagine rates by trans unsaturated acids. Myristoleate and linolenate enrichment also produced non-uniform but lesser rate reductions. Although changes in the relative numoer of effective transport catalysts could account for these findings, comparisons of proline and lysine transport rates over a broad temperature range indicated that non-uniform alterations in transport catalyst reaction rates account at least partly for the activity changes associated with membrane lipid alterations.